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==== Front Med HistMed HistMDHMedical History0025-72732048-8343Cambridge University Press Cambridge, UK 0009110.1017/mdh.2012.91S0025727312000919Book Review Book ReviewBook ReviewKichigina Galina University of Toronto, Canada 1 2013 57 1 142 144 Henze Charlotte E. , Disease, Health Care and Government in Late Imperial Russia , BASEES/Routledge Series on Russian and East European Studies ( London and New York : Routledge , 2011 ), 232 pages, $158.00, hardback, ISBN: 978-0-415-54794-9. Published by Cambridge University Press2013The Author ==== Body Disease as an important tool for economic, cultural and political analysis has long been recognised by historians. In particular, historians of social medicine have paid much attention to cholera epidemics in nineteenth-century England, France and Germany. Although a number of studies on public health in pre-revolutionary Russia have dealt with cholera epidemics and various facets of its socio-political impact, Charlotte Henze’s book is the first to concentrate entirely on the history of cholera in Saratov throughout the nineteenth to the early twentieth century. The choice of the locale is rightfully justified since Saratov, an important shipping port on the Volga, one of Russia’s major trade routes, with its socially, culturally and ethnically diverse population, including a large German community, experienced all major cholera pandemics of 1823–1914. With this central focus, Henze is able to construct a social, political and public health history of the city of Saratov. She uses the cholera outbreak of 1892 in Saratov as a means of exploring living conditions and medical and administrative infrastructures in the city on the Volga. She applies the same approach to address broader issues of Russia’s socio-economic developments at the age of modernisation associated with rapid urbanisation, increasing migration of impoverished rural population and growing social tension. The book is divided into five chapters. The first chapter traces the history of cholera in Russia before 1892, focusing on the multiple outbreaks during 1823–59. It also analyses anti-epidemic policies after Russia’s defeat in the Crimean war, when Russia entered the reform era that drastically changed the existing economic and social structure of the country. Chapter 2 documents Saratov’s appalling sanitary conditions and inadequate public health provision and administration, which in large measure were responsible for disaster in coping with the cholera epidemics of 1892. Chapter 3 provides detailed coverage of this epidemic. It analyses anti-epidemic measures, set up by the central government to combat cholera arrival to Russia, discussing briefly the reception of Robert Koch’s discovery of vibrio-cholerae. It also examines the responses of local administrative and medical authorities, as well as the notoriously famous ‘cholera riots’ and underlying social constraints and conflicts. Chapter 4 is devoted to cholera’s impact on Saratov, the most important being the growing self-identity and self-confidence of local physicians. Improvements in the sphere of city renewal and public health care are attributed to economic factors rather than to direct consequences of the cholera outbreak. The subject of chapter 5 is cholera’s return to Saratov in the early 1900s. The city was better prepared for the epidemics in terms of medical and public health care as well as administrative logistics. The new outbreak revealed the new realities of anti-cholera combat at the age of bacteriology, and old social contradictions of the coming turbulent 1905. The book convincingly covers Saratov’s cholera history. References to similar developments in combating cholera in Western Europe are valuable and highlight the peculiarities of the Russian situation. Although the severity of the sixth pandemic in Russia is undisputable, extensive areas of Greece, the Balkans and the Ottoman Empire were also severely affected during the first decade of the twentieth century. The Italian wave of 1910–11 was quite heavy in Venice, Aquila, Palermo and Naples, so we cannot say there was none recorded in Europe after 1892. A more elaborate comparative perspective is welcome. The book touches upon some important political and social issues; however, it contains little that adds to our knowledge or alters our understanding of the processes that eventually led to the unprecedented social and political upheavals of 1905 and 1917, which ended in the collapse of imperial Russia. Does the cholera epidemic of 1892 therefore provide an appropriate criterion for assessing the viability of the autocratic regime, a thesis which Henze has particularly emphasised? Another reiterating thesis is Russia’s confrontation with modernity and the ultimate inability of the autocratic regime to cope with challenges such as cholera outbreaks. This is, I believe, an overstatement of the case, and overtones some of the complexity mapped out in the text, returning us to a rather standard treatment of Russia’s development during the late imperial period. Lastly, important advances in Russian military medicine in combating epidemic diseases including cholera translated to the civil population remain unexplored and need to be addressed if the government strategies to prevent epidemics are to be fully understood. Overall, the study is useful insofar as it contributes to Russia’s history of cholera and is stimulating for provoking discussion on some important episodes in the history of late imperial Russia, and has undoubtedly confirmed the importance of examining the impact of individual disease and the issues surrounding public health as a means of exploring key debates in social and political history. Given the dearth of scholarly studies of epidemics and the health care system in Russia, this volume is particularly noteworthy.	0	PMC3566737	NO-CC CODE	2021-01-04 22:18:23	yes	Med Hist. 2013 Jan; 57(1):142-144
==== Front Int J NanomedicineInt J NanomedicineInternational Journal of Nanomedicine1176-91141178-2013Dove Medical Press 10.2147/IJN.S33589ijn-8-1525Original ResearchNanostructured self-assembling peptides as a defined extracellular matrix for long-term functional maintenance of primary hepatocytes in a bioartificial liver modular device Giri Shibashish 1Braumann Ulf-Dietrich 23Giri Priya 13Acikgöz Ali 14Scheibe Patrick 35Nieber Karen 6Bader Augustinus 11 Department of Cell Techniques and Applied Stem Cell Biology, Center for Biotechnology and Biomedicine (BBZ), University of Leipzig, Leipzig, Germany2 Institute for Medical Informatics, Statistics, and Epidemiology (IMISE), University of Leipzig, Leipzig, Germany3 Interdisciplinary Center for Bioinformatics (IZBI), University of Leipzig, Leipzig, Germany4 Klinikum St Georg, Leipzig, Germany;5 Translational Center for Regenerative Medicine (TRM Leipzig), Leipzig, Germany6 Department of Pharmacology for Natural Sciences, Institute of Pharmacy, University of Leipzig, Leipzig, GermanyCorrespondence: Shibashish Giri Department of Cell Techniques and Applied Stem Cell Biology, Center for Biotechnology and Biomedicine, University of Leipzig, Deutscher Platz 5, D-04103 Leipzig, Germany, Tel+49 34 1973 1353, Fax+49 34 1973 1329, Email [email protected] 2013 18 4 2013 8 1525 1539 © 2013 Giri et al, publisher and licensee Dove Medical Press Ltd2013This is an Open Access article which permits unrestricted noncommercial use, provided the original work is properly cited.Much effort has been directed towards the optimization of the capture of in vivo hepatocytes from their microenvironment. Some methods of capture include an ex vivo cellular model in a bioreactor based liver module, a micropatterned module, a microfluidic 3D chip, coated plates, and other innovative approaches for the functional maintenance of primary hepatocytes. However, none of the above methods meet US Food and Drug Administration (FDA) guidelines, which recommend and encourage that the duration of a toxicity assay of a drug should be a minimum of 14 days, to a maximum of 90 days for a general toxicity assay. Existing innovative reports have used undefined extracellular matrices like matrigel, rigid collagen, or serum supplementations, which are often problematic, unacceptable in preclinical and clinical applications, and can even interfere with experimental outcomes. We have overcome these challenges by using integrated nanostructured self-assembling peptides and a special combination of growth factors and cytokines to establish a proof of concept to mimic the in vivo hepatocyte microenvironment pattern in vitro for predicting the in vivo drug hepatotoxicity in a scalable bioartificial liver module. Hepatocyte functionality (albumin, urea) was measured at days 10, 30, 60, and 90 and we observed stable albumin secretion and urea function throughout the culture period. In parallel, drug metabolizing enzyme biomarkers such as ethoxyresorufin-O-deethylase, the methylthiazol tetrazolium test, and the lactate dehydrogenase test were carried out at days 10, 30, 60, and 90. We noticed excellent mitochondrial status and membrane stability at 90 days of culture. Since alpha glutathione S-transferase (GST) is highly sensitive and a specific marker of hepatocyte injury, we observed significantly low alpha GST levels on all measured days (10, 30, 60, and 90). Finally, we performed the image analysis of mitochondria-cultured hepatocytes at day 90 in different biophysical parameters using confocal microscopy. We applied an automatic algorithm-based method for 3D visualization to show the classic representation of the mitochondrial distribution in double hepatocytes. An automated morphological measurement was conducted on the mitochondrial distribution in the cultured hepatocytes. Our proof of concept of a scalable bioartificial liver modular device meets FDA guidelines and may function as an alternative model of animal experimentation for pharmacological and toxicological studies involving drug metabolism, enzyme induction, transplantation, viral hepatitis, hepatocyte regeneration, and can also be used in other existing bioreactor modules for long-term culture for up to 90 days or more. Keywords image analysis3D visualizationbioreactorFDA guidelinesprimary hepatocyteshepatotoxicity ==== Body Introduction Despite the tremendous efforts in hepatic tissue engineering,1 there is a lack of defined long-term functional maintenance of primary hepatocytes. Much encouraging effort has been made to optimize the capturing of the in vivo hepatocytes microenvironment for functional maintenance of primary hepatocytes in a micropatterned co-culture module,2 bioreactor-based liver module,3–5 microfluidic 3D chip,6 and other innovative approaches.7–9 The most encouraging reports relied on undefined extracellular matrices like Matrigel, rigid collagen, or serum supplementations, which are often problematic and unacceptable in preclinical and clinical applications. Moreover, animal-derived extracellular matrices can give rise to immunological conditions and zoonosis. The paracrine fashion secretion pattern of several soluble factors from feeder cells for primary cells cultures10 is more suitable, but it is much more difficult to test the effect of the exogenous factors on the functional maintenance of primary hepatocytes. However, most experimental approaches aimed at the long-term functional maintenance of primary hepatocytes in a culture from 1 week to several weeks (42 days) have been reported by various investigators,2–4 including us.11–13 However, to the best of our knowledge, there is no report for the long-term functional maintenance of primary hepatocytes up to 90 days within a defined hepatic microenvironment to provide a better strategy as an alternative to in vivo animal experimentation. US Food and Drug Administration (FDA) rules require the survival potential of primary hepatocytes of a minimum of 14 days and a maximum of 90 days for in vivo toxicological experiments.14 We have designed nano-structured self-assembling peptides as a defined extracellular matrix (ECM) for the long-term functional maintenance of primary hepatocytes in a defined bioartificial liver modular device. Our bioartificial liver modular device in its ex vivo form aims to mimic the in vivo liver for the next generation of ex vivo drug screening and many other liver related experiments. Our bioartificial liver device faithfully mimics the in vivo liver15 in various ways11,17 and was previously evaluated in a novel porcine hepatectomy model16 and viral infection.18 The present proof of concept study of the bioartificial liver modular device could replace the millions of animals that are currently sacrificed in preclinical testing and could open up a new vista for the production of safer pharmaceuticals. The integration of nanostructured self-assembling peptides into a bioartificial liver modular device to generate 3D cell interactions with a special combination of cytokines and growth factors is the focus of this study. Fundamentally, in vivo hepatocytes interact with their complex surrounding environment through exposure to a network of cytokines, chemokines, and growth factors19 to perform and regulate hepatic cellular functions. The ECM is one of the key components of the hepatic microenvironment and serves as a reservoir for several soluble factors that cells can use for various functions. In vivo hepatocytes interact with soluble signals via the ECM that controls the diffusion and activity of soluble factors that interact with the surrounding environment. Since the in vivo ECM is in the nanorange, to create such a nanoscale ECM for hepatocytes culture is challenging. Furthermore, cells acquire complex 3D geometries for intricate interactions with adjacent cells, cytokines, and growth factors.20 The nanoscale ECM can surround each hepatocyte cell (10–20 μM) in 3D rather than the partial attachment observed in the conventional 2D scaffold. Such a nanoscale scaffold allows each hepatocyte cell to have its own position in direct contact and to interact with the network of cytokines, chemokines, and growth factors in the cultivation media. The long-term maintenance of the hepatocyte function and optimization of the culture conditions under a defined hepatic microenvironment is one of the most significant challenges in developing an effective bioartificial liver model in addition to the drug discovery process. Current trends of in vitro hepatic toxicological models do not reflect the in vivo hepatic microenvironments. According to a 2006 survey report of pharmaceutical companies, hepatotoxicity was ranked first in terms of adverse events and it remains the most common reason for the restriction or withdrawal of a drug from the market by the FDA. Although there are many reasons underlying drug-induced hepatotoxicity, one of the most important is the limitation of existing in vitro cellular hepatocyte models. Short-term culture very often suffers from rapid loss of hepatic cellular activity due to 2D interaction, unlike the in vivo milieu. Consequently, it is rather difficult to use these short-term models for long-term experiments involving drug metabolism, virus infection, carcinogenesis, and other such studies. To integrate a defined and highly biocompatible substrate into a bioartificial liver system that could support the long-term maintenance of hepatocyte function is one of the prominent challenges in developing effective bioartificial liver support as well as drug discovery. We have used a self-assembling peptide as a defined substrate or ECM for ex vivo long-term functional maintenance of primary hepatocytes under 3D defined culture conditions in a bioartificial modular device. These self-assembling peptides comprise a RAD motif, which is a 16-residue peptide composed of alternating hydrophilic arginine, hydrophobic alanine, and hydrophilic aspartic acid (RADARADARADARADA). The most common well-known motif cell adhesion sequence is RGD (arginine-glycine-aspartate). It is a tri-peptide and its sequence can be found in proteins of the ECM or cell adhesion proteins. The RGD peptide motif was discovered to be a major element in the recognition system for essential cell adhesion.21 Although the RGD peptide motif is the most common element to use for cell adhesion and culture, only some RGD-containing proteins can support cell adhesion22 despite the large number of RGD-containing proteins. Therefore, in vitro cultured cells may not come into contact with these specific RGD-containing proteins during cell adhesion and expansion and will eventually be lost. This is because the RGD sequences are not freely available for in vitro cultured cells during cell adhesion and expansion. Another disadvantage of these conventional RGD-containing extracellular matrices is that sometimes the RGD motif itself may not be compatible with integrin binding.23 Therefore, here we integrated self-assembling peptides that contain the RAD motif as a defined substrate for cell adhesion as they are more compatible for cell attachment three-dimensionally. Interestingly, the RAD motif in self-assembling peptides mimics the known cell adhesion properties of the RGD motif.24 Previously, the RAD motif self-assembling peptide had been used for in vitro 3D culture for a wide range of cells. We have reported 3D scaffolding and signaling in bioartificial liver modules for up to 35 days under exposure of various growth factors, cytokines, and hormones, but here we have optimized and standardized a novel in vitro 3D defined culture system in a bioarticial liver (BAL) module up to 90 days of culture to fulfill the FDA guidelines for general toxicological studies. To the best of our knowledge, we are the first group to use self-assembling peptides for ex vivo long-term functional maintenance of primary hepatocytes in a clinically relevant bioartificial liver module. Hepatocyte functionality (albumin, urea) was measured at days 10, 30, 60, and 90. We evaluated the potential for hepatotoxicity by analyzing drug metabolizing enzyme biomarkers such as ethoxyresorufin-O-deethylase, the methylthiazol tetrazolium (MTT) test, and the lactate dehydrogenase (LDH) test at days 10, 30, 60, and 90. Liver failure is linked by the common pathway of mitochondrial failure.25 Thus, we aimed to perform image analysis of mitochondria of single or double hepatocyte cells. Currently, there are few methods26 to quantify the mitochondrial image, but we have shown a 3D visualization, which revealed a classic representation of the mitochondrial distribution around the liver cell. The idea of recreating a 3D defined hepatic microenvironment has become very challenging owing to the establishment of the structural and functional complexity of in vivo hepatocytes in an ex vivo bioartificial liver module. This optimized 3D defined culture system in bioartificial liver modules could be useful for physiological and pathophysiological states of the liver, pharmacological and toxicological screening of drug candidates, and generation of clinical grade hepatocyte cells. Materials and methods Cell culture Hepatocytes were isolated from male Sprague-Dawley rats (weighing 200–250 g) by the two-step collagenase perfusion method, as previously described.27 Pure hepatocytes were obtained by Percoll gradient separation. Cell viability was assessed by trypan blue exclusion and hepatocytes with a viability of greater than 85%–90% were used. Hepatocytes were cultured in Williams’ E medium supplemented with L-glutamine 2 mM, penicillin 100 U/mL, streptomycin 100 μg/mL, dexamethasone 1 μM, insulin 0.2 U/mL, and glucagon 4 ng/mL without fetal bovine serum. The cells were seeded at a density of 2.5 × 105 cells/cm2 in a RAD peptides coated bioreactor under a defined combination of growth factors and cytokines (Williams’ E medium + Activin A + WNT 3a + HGF + FGF-4 + EGF + Oncostatin M + FGF-10 + BMP-4 + Dex + RA all from Sigma-Aldrich Chemie Gmbh [Munich, Germany] to mimic the in vivo hepatic microenvironment)28 and were incubated at 37°C in a humidified atmosphere containing 5% CO2 and 20% O2 (v/v). The fundamental design of our flat membrane bioreactor11 and the minibioreactor13 is similar. Funtional maintenance of primary hepatocytes was conducted three times. Self-assembling peptides coated procedure in bioartificial liver module The self-assembling peptide was discovered from a segment in a yeast protein by Zuotin29,30 and is now commercially available under the name PuraMatrix™ (BD Biosciences, San Jose, CA, USA). The viscosity of the self-assembling peptides stock solution of our aliquots (1.5 mL microtube) decreased by vortexing for 30 minutes in a bath sonicator. If air bubbles were present, the aliquots were centrifuged at high speed for a few seconds. Then, a 0.5% solution of self-assembling peptides was prepared by diluting it with sterile water. In order to create the nanoscaffold in the six-well bioreactor, 1.2 mL of 0.5% (v/v) self-assembling peptides (300 (iL for a 24-well bioreactor, 1 mL for a six-well bioreactor, and 75 μL for a 96-well format bioreactor) was uniformly distributed over each well and then 2.4 mL of Williams’ Medium E was added very carefully to each well of the six-well bioreactor. To promote gelation, we put the bioreactor in an incubator for 1 hour. After the nanostructure hydrogel was assembled, we carefully changed the medium using a wide top micropipette. Aspirator use was avoided because of the risk of destroying the nanostructured hydrogel. The medium was changed (300 μL per well for 24 wells) twice over a period of 1 hour to equilibrate the gel at physiological pH and finally the bioreactor was put in an incubator overnight with the medium. After that we seeded the desired cell density. The culture medium (250 μL) was replaced with fresh medium every 78 hours, and the supernatant was stored at −20°C for LDH, albumin, and urea. RNA isolation Total RNA was isolated from the cells using the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. RNA concentration was quantified and their purity was determined by standard spectrophotometric methods. RNA samples were stored at −80°C. cDNA synthesis and reverse transcription (RT) polymerase chain reaction (PCR) Total RNA was isolated from the cultured cells using an RNeasy MinElute Cleanup Kit (Qiagen) and then 8 μg of the RNA was reverse transcribed into cDNA from cultured rat hepatocytes, prepared using the Fastlane Cell cDNA kit (Qiagen) according to the manufacturer’s instructions. Quantitative real-time PCR was performed using SYBR Green PCR Master Mix (Applied Biosystems, Carlsbad, California). Primers for rat albumin and CYPA31 and GAPDH genes were selected (rat CYP3A1-specific primer set: forward, 5′ GCCATCACGGACACAGAAATA 3′; reverse, 5′ GAACGTGGGTGACAGTAAGGCT 3′; rat albumin forward, 5′ ATACACCCAGAAAGCACCTC 3′; reverse, 5′ CACGAATTGTGCGAATGTCAC 3′; rat housekeeping gene forward 5′ CAG TTC CAC CCA CCT CAG AT 3′ and reverse 5′ TTT TGG GCT CCT TCA GAG TG 3′). Primer (Sigma-Aldrich) concentrations were optimized before use. SYBR Green Master Mix (1×) was used with 1 μM of forward and reverse primers in a total volume of 12 μL that also included 1 μL of cDNA. All PCR reactions were performed in duplicate. PCR amplification was performed as follows: 95°C for 10 minutes, 40 cycles of 95°C for 15 seconds, 60°C for 10 seconds, and 68°C for 1 minute on a Mastercycler Realplex (Eppendorf, Hamburg, Germany). The PCR products were then analyzed using the electrophoresis of 2% agarose gels stained with ethidium bromide for visualization. The housekeeping gene (GAPDH) was used as an internal standard to determine the relative levels of albumin, albumin and CYP3A1 gene expression. Biochemical assays Albumin and urea were measured by enzyme-linked immunosorbent assay, as previously described.27 LDH activity was determined in a colorimetric enzymatic assay (Cytotoxicity Detection Kit, Cat No 1644793; Roche, Mannheim, Germany). For induction of CYP3A1 expression, dexamethasone (as a 10-mM solution in dimethyl sulfoxide; final concentration: 10 μM) was added to the replacing medium on various days: day 10, day 30, day 60, and day 90. Cell viability was then assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide MTT assay (Sigma-Aldrich). The protocol of MTT assay ethoxyresorufin-O-deethylase is given in our previous report.31 α-glutathione S-transferase (GST) was measured in the culture supernatant as a marker for hepatic damage by a commercially available enzyme immunoassay method (Biotrin Rat Alpha GST EIA; Biotrin, Dublin, Ireland). Statistical analysis All experimental results are shown as mean ± standard error, and analyzed for significance using Wilcoxon’s test for non-paired examination. P-values of less than 0.05 were judged to be statistically significant. Image analysis of confocal microscopy of mitochondria We analyzed mitochondrial structural status by confocal microscopy by using MitoTracker (Life Technologies), a dye used for fluorescent mitochondrial markers. This was simply incubated in submicromolar concentrations (dilution with culture medium: 1:1000) of the MitoTracker probe in cultured hepatocytes and directly observed with the fluorescent microscope. In general, MitoTracker probes are cell-permeant mitochondrion-selective dyes that diffuse passively across the plasma membrane and accumulate in active mitochondria and stain mitochondria with a bright red, fluorescein-like fluorescence. Visualization and quantification of mitochondrial distribution Mitochondria play a central part in cellular survival32,33 and apoptotic death. With time, liver cells malfunction and die because of a lack of energy. A central factor in liver cell survival is the decay of the mitochondria in cells. The survival/apoptosis of cells is accompanied by an increase/decrease in the numbers of mitochondria per cell. So, from the experimentalist point of view, it is important to know the mitochondrial distribution around the liver cell nucleus. When cells come from in vivo to in vitro, they require an in vivo-like microenvironment. In long-term culture, cells are unable to get the appropriate in vivo-like microenvironment. Under this stress condition, mitochondria are unable to survive and they swell and finally burst. Currently there are few methods to quantify the mitochondria, but apart from these methods, we have explained 3D visualization which shows a classic representation of mitochondrial distribution around liver cells. Experimental outcomes have been observed using confocal microscopy and microscopy of cultured hepatocytes has been stained using MitoTrackers. High quality fluorescence images were captured for the subsequent quantitative analysis of mitochondria using the computer algebra system Mathematica (v 8; Wolfram Research, Inc, Champaign, IL, USA), which offers a variety of image processing functionalities. Data-set and an image processing pipeline In this section we describe the data set (Figure 1), followed by the image processing pipeline according to Figure 2. Three-dimensional volume data sets of mitochondria in rat liver cells expressing MitoTrackers were acquired by the digitization of liver cell cultures using confocal microscopy with a 10 × objective. Respective 2D image stacks have dimensions of 1024 × 1024 × n voxels, and n is adapted according to cell extent. Each 2D image in a stack is in 24 bit color depth which is converted into scalar 8 bit for processing. We have noticed some artifacts in the middle of the nucleus region of a 2D image. There is some a priori knowledge about an acceptable minimum mitochondrial site, and apart from this, the selected 2D slide has been taken from a 3D stack and the artifacts shadow comes from the other slide. 3D visualization results The ability of a confocal microscope to create sharp optical sections makes it possible to build 3D renderings of the specimen. Data collected from a series of optical sections imaged at short and constant intervals along the optical axis are used to create some 3D visualization. Results are shown in Figure 1A and BB and are realized as shaded 3D surface rendering. In the data, however, despite reflecting a 3D topology, the cell appears to be implausibly squeezed, so that the visible mitochondria enclosing the nucleus (not stained) occur along a ring instead of a sphere. That is why for further quantitative assessments we decided to stick to selected single images taken centrally from the volume data set. For this work we rather take the 3D visualizations of the location of mitchondria around nucleous. Image processing pipeline 2D Confocal laser scanning microscopy (CLSM) image data (Figure 2A), has a certain degree of noise due to image capture conditions; eg, due to the statistical characteristic of light, scattering, etc. In order to reduce this effect we applied a smoothing operation implemented as an edge preserving total variation filtering,34 assuming Poisson noise. The results are shown in Figure 2B. Due to a particularly high variability of fluorescence intensity and contrast, for mitochondria segmentation, we used an iterative active-contour level-set segmentation in its variant proposed by Chan and Vese,35 as shown in Figure 2C. For further processing we required a number of cell masks according to the respective number of cells. We applied a dilation operation,36 ie, a Minkowski-sum of binary image and a circular area structuring element, to have the mitochondria segments merged and to get some connected segments according to the number of cells. Connected component labeling37 was accomplished to allow for small element (artifact) removal. Overall, cell masks were then computed according to the connected segment’s convex hulls,38 as shown in Figure 2D. Further, we needed to take the difference of the masks and label region shown in Figure 2D, then needed to do another connected component labeling to sort out small noncentral rests (Figure 2E). We then needed to compute the convex hulls of the large inner regions to get the inner masks of cell nuclei; output is shown in Figure 2F. Further, we subtracted the nuclei masks from the outer masks to obtain representative ring-shaped mitochondrial distribution regions, as shown in Figure 2G. Since we were interested in the mitochondrial distribution around the nuclei, we focused on the characteristics of the margin thickness. The thickness measurement was taken along the course of a closed circular line as was obtained by skeletonization39 of the ring, while distances were computed using Euclidean distance transform39 applied on the ring (Figure 2H). Results Morphological analysis of long-term culture Cultured hepatocytes were continuously observed under phase-contrast microscopy at day 10, day 30, day 60, and day 90. All hepatocytes showed well-defined cell membranes, nuclei and nucleoli, bile canaliculi, and distinct cytoplasm (Figure 3A). Hepatocytes cultured in the bioartificial liver module adopted an in vivo like polygonal and established extensive cell-to-cell contracts with natural organization. Stability of albumin and urea secretion at various days The albumin secretions were 26.4 ±5.1 mg/mL, 24.2 ± 6.3 mg/mL, 21.8 ± 3.3 mg/mL, and 25.4 ± 1.3 mg/mL at day 10, day 30, day 60, and day 90, respectively. These results showed stable albumin secretion throughout the culture period (Figure 3B). The urea secretions were 46.4 ± 2.1 mg/mL, 39.2 ± 2.3 mg/mL, 34.8 ± 8.3 mg/mL, and 40.4 ± 2.3 mg/mL at day 10, day 30, day 60, and day 90, respectively (Figure 3C). These results showed stable albumin and urea secretion throughout the culture period. So, under the appropriate 3D hepatic culture conditions, cultured hepatocytes might recover from the stress of the isolation procedure from the in vivo liver. If one can count per cell, the present showed that the albumin and urea performance per cell was almost similar to that in the in vivo liver. Hepatocyte cell number at day 10, day 30, day 60, and day 90 Cell death was assessed by MTT assay in primary hepatocytes and it was found that there is no significant cell death on these days. Mitochondria metabolism such as the reduction of a tetrazolium salt (MTT) was used here. Although primary hepatocytes did not proliferate significantly during long-term maintenance, they remained excellent and viable up to 90 days, as demonstrated by the MTT assay. There was no significant difference in cell viability between day 30, day 60, and day 90 (Figure 3D). The number of mitochondria is higher at the end day of the culture period (day 90) Since mitochondria is the main target of cellular stress, apoptosis, and ageing, in these conditions the shape of the mitochondria changes and becomes swollen. The mitochondria of primary hepatocytes cultured in the 3D hepatic defined microenvironment in the bioartificial liver module were very well shaped and uniformly distributed throughout the cytoplasm, which was confirmed by confocal microscopy at day 90 (Figure 4). It was interesting to find a high number of mitochondria present in culture hepatocytes at day 90 which was noticed by confocal microscopy using MitoTracker red. The mitochondria were found throughout the cytoplasm. Gene expression of albumin and CYP3A1 RT-PCR analysis revealed that the albumin and CYP3A1 gene expression was also stable throughout the culture period (Figure 3E and F). The mRNAs of albumin and CYP3A1 were detected at day 10, day 30, day 60, and day 90 which was also significantly high. The albumin mRNA and CYP3A1 were significantly expressed in the hepatocytes cultured in the bioartificial liver modules for up to 90 days. We confirmed that the defined hepatic microenvironment was highly important to maintain such hepatic gene expression throughout the period. Stable hepatocyte membrane stability up to 90 days The LDH test is frequently used to check for tissue damage as elevated levels of LDH may indicate liver damage. The LDH release of cells is an index of their viability. It is a sign of the integrity of the plasma membrane since the LDH activity outside the cell is measured. The LDH releases were 2.4 ± 1.1 U/L, 4.2 ± 6.3 U/L, 2.8 ± 3.3 U/L, and 2.4 ± 1.3 U/L at day 10, day 30, day 60, and day 90, respectively (Figure 3G). These findings demonstrate that this present study has major advantages over the conventional in vitro culture regarding the cellular integrity. Negligible amount of α-GST throughout culture period The release of α-GST either from in vitro hepatocytes culture or in vivo liver is a more sensitive parameter than the release of conventionally used liver enzymes (aspartate aminotransferase, alanine aminotransferase, and LDH) in the assessment of early hepatocellular damage. α-GST is a cytosolic enzyme mostly located in hepatocytes with a uniform distribution in the liver. Accumulating evidence from numerous clinical and experimental studies has shown that α-GST is an early and sensitive biomarker for hepatocyte injury. Herein, we showed the α-GST was 110 ± 1.5 μg/L, 90 ± 6.8 μg/L, 78 ± 4.3 μg/L, and 100.4 ± 7.3 μg/L at day 10, day 30, day 60, and day 90, respectively (Figure 3H). This highly sensitive method also supports that this 3D culture system in the bioartificial module provides ideal conditions for long-term functional maintenance. Image analysis results We have analyzed the mitochondria margins using the image processing pipeline in 2D, as was explained above. The results for the specimen with two cells, already depicted in Figure 1A and B, are given in Figure 4. Curves show the course of the mitochondria margin thickness drawn as a function of the normalized perimeter (obtained in counter-clockwise direction). Since cells appear arbitrarily rotated, curves were periodically shifted so that their minimum is placed at the zero position. Another example with three cells is given in Figure 5. Further characteristic numbers derived from the curves are given in Table 1 for all specimens; ie, mean, standard deviation, and median. Additionally, a simple measure named “area coverage,” addressing an approximation of the mitochondria density, is given (see table caption for details). Discussion To the best of our knowledge, this is the first report for the long-term functional maintenance of primary hepatocytes up to 90 days under a defined 3D hepatic microenvironment in a bioartificial liver module device that mimics the state of the art liver. We optimized our bioartificial liver module model for long-term culture for up to at least 90 days to meet the FDA guidelines,14 which recommend that the duration of a toxicity assay of a drug should be a minimum of 14 days, to a maximum of 90 days for a general toxicity assay. Repeated drug dose toxicity testing in animals for 28 and 90 days is used to evaluate chronic toxic effects to find out the observed primary toxic effect on various organ systems, including the liver, from minimal dose to maximum dose tolerance. Drug screening data obtained from unnatural conventional 2D systems is often confusing and matrigel, collagen based systems are often associated with unphysiological substances, while animal models are expensive, time consuming, and present ethical dilemmas. Herein, we reported a challenging, alternative, novel, non-animal approach in a bioartificial liver modular platform to replace animal testing for assessing the chronic toxic effects of drug candidates to find out the observed primary toxic effects before further trials to the later clinical stage. The 3D defined hepatocyte culture system presents the great challenge of maintaining hepatocyte cells for the extended period of time of up to 90 days that is required to assess the chronic toxic drug screening effects. The present proof of concept experiment generated great challenges with a new strategy (self-assembling peptides, growth factors, cytokines, enhanced oxygenation, and 3D signaling) for the long-term maintenance of primary hepatocytes in a bioartificial liver module using a defined microenvironment. We hope that this 3D interaction of growth factors and cytokines in nanostructured self-assembling peptides in a bioartificial liver modular device might add significant value to other existing toxicology screening devices as well as to the pharmaceutical industrial methodology, enabling more accurate toxicological assays and increasing the predictive accuracy during drug candidate screening. This 3D signaling can be useful in other existing micropatterned co-culture modules,2 bioreactor based liver modules,3–5 microfluidic 3D chips,6 and other innovative approaches. It is a formidable challenge to establish an in vitro hepatocyte cellular model to allow the long-term functional maintenance of primary hepatocytes up to 90 days, which can be useful for basic studies of hepatocytes physiology, drug metabolism, enzyme induction, transplantation, viral hepatitis, hepatocyte regeneration, and other purposes. After 80 years of reliance on animal testing that gives unforeseen and unrealistic results, it is time to explore the modular device to replace animals and provide relevant outcomes for safer drugs. This nanostructured self-assembling peptide was originally found in Zuotin.41,42 It contains alternating hydrophobic and hydrophilic residues which are characterized by a stable sheet structure that undergoes self-assembly into nanofibers similar to those in other biological protein self-assembly.29 The surface of an in vivo hepatocyte cell is composed of thousands of membrane proteins, cell receptors, different lipids, proteins, and carbohydrates. All of these complex forms are arranged in 3D and perform cellular physiological functions. Every growth factor or cytokine has its specific membrane receptors out of thousands of membrane receptors. Here we utilized a nanostructured self-assembling peptide to create such a 3D microenvironment in an ex vivo model in a bioartificial liver modular device so that the exposed growth factors or cytokines were able to freely search out their receptors on the membrane of hepatocytes three-dimensionally, and to recapitulate in vivo milieu rather than two-dimensionally in existing conventional in vitro models. Growth factors and cytokines may not be able find their appropriate membrane receptors in flattened 2D culture, so are unable to recapitulate in an in vivo microenvironment.43,44 Therefore, when hepatocytes are removed from their in vivo microenvironment and isolated hepatocytes are cultured in 2D conditions, they will lose their hepatospecific functions quickly. This is because these hepatocytes become more flattened. The use of short peptides is always more advantageous than naturally derived proteins for cell adhesion. This is because short peptides provide a lot less variables than naturally derived proteins.46 Further, self-assembling peptide scaffolds facilitate the slow and sustained release of active cytokines that are extremely important for long-term culture.44 We used a self-assembling peptide nanoscaffold built from a peptide that was discovered from a segment of the yeast protein Zuotin.28,29 Zuotin is a member of a new class of peptides that preferentially bind to the left-handed Z-DNA binding protein in Saccharomyces cerevisiae.28 The world’s most important yeast, S. cerevisiae, has been a very useful fungus in baking and brewing since ancient times. Furthermore, S. cerevisiae yeast is completely harmless for healthy people. When hepatocytes are isolated from the in vivo liver and put in ex vivo conditions, hepatocytes search in the in vivo microenvironment during the initial vitro culture conditions. Generally, if hepatocytes do not get into the in vivo milieu, then finally the in vitro hepatocytes try to search for the neighbor cell to communicate for the exchange of many molecules to survive and function. Therefore, cultured hepatocytes get more elongated and stretched. Herein, we provide a hypothetical diagram (Figure 6) for how hepatocytes suffer and behave in unnatural conventional 2D culture conditions and lose hepatospecific functions and die quickly. The whole cell membrane of the seeded hepatocyte cells are partially polarized to interact with unrealistic stimulation by growth factors, cytokines, nutrients, and signals of the surrounding environment. This is because one side of the hepatocyte cell body will be in direct contact with the rigid ECM which creates unnatural cell interaction with the surrounding ECM. Half or less of the hepatocyte membrane receptor is available to interact for exposure with growth factors, cytokines, nutrients, and signals in the 2D system. Thus, cells in a 2D conventional culture are partially polarized which can seriously impair cellular communication, the transport of oxygen and nutrients, the removal of wastes, and cellular metabolism. In contrast, in a 3D nanorange microenvironment using the nanostructured self-assembling peptides reported here, all functional motifs on the nanostructured self-assembling peptides encircle the whole hepatocyte cell body in all dimensions where all growth factors, cytokines, nutrients, and signals can interact three-dimensionally just like in an in vivo hepatic microenvironment. In the 3D interaction these cytokines or growth factors freely search out their receptors three-dimensionally, rather than two-dimensionally as in existing conventional in vitro models. These types of defined, nanostructured, self-assembling peptides could be highly valuable for both the development of the defined 3D culture system as well as effective bioartificial liver configuration. Different bioreactor configurations have been developed to obtain a BAL device but none of them reported the long-term survivability and functionality of primary hepatocytes. However, the long-term functional maintenance of primary hepatocytes of up 90 days requires an appropriate in vivo microenvironment. This has not been developed so far because the scaffold and cells do not match the complete profile of the in vivo microenvironment. The actual microenvironment is comprised of a collection of cells, the ECM/scaffold, cytokines, and growth factors, which form the basis of normal tissue architecture and function. A large number of research studies have shown that using collagen to fix hepatocytes in the manner of sandwich configuration could create a matrix environment close to that seen in vivo and reported encouraging hepatic function up to a few weeks. Although the double layer of collagen sandwich configuration cellular model has been widely accepted for various hepatic studies, it is still a monolayer attachment culture. The 3D signaling in an in vitro cellular model with special reference to three-dimensional interactions of growth factors, cytokines, and hormones either in a conventional monolayer or sandwich configuration is inherently asymmetric and does not reflect an authentic in vivo environment. Virtually, in native liver tissue, hepatocytes reply in a 3D hepatic environment with a nanorange ECM to provide adequate oxygen and solution factor transport. Further, the liver lobule is a functional unit of a whole liver that consists of hepatocytes that are arranged into hepatic cords separated by the sinusoidal space called the space of Disse (10–15 μM). The fundamental concept of in vivo hepatocytes is that each hepatocyte has direct contact with the space of Disse for the uptake of nutrients, growth factors, cytokines, hormones, oxygen, and other things. So, surrounding the in vivo hepatocyte is the nanorange environment. Thus, the substrate or ECM used during in vitro culture should be smaller than the hepatocytes cells, so that the scaffold can bind three-dimensionally. The average size of the hepatocytes is 10–20 μM. It is widely believed that when the size of the ECM is larger than the cells, the cells cannot be surrounded by the biomaterial scaffold. Most conventional biomaterials used for hepatocyte cell cultures are in the microscale range, where upon attachment the cells still exist in a 2D topography, which is very common in conventional cell cultures in a bioartificial liver device and culture plates. Great difficulty is encountered if the cells cannot be attached in an in vivo 3D topology where the signaling, as well as the diffusion, is inherently asymmetric in traditional 2D culture and is the current main limitation for long-term in vitro culture. Therefore, a nanostructured self-assembling peptide is essential to create an authentic 3D microenvironment by holding hepatocytes of all dimensions. Oxygen supplies during primary hepatocyte culture are a critical issue since conventional cultures are often under oxygen-deficient culture conditions and are thus forced into anaerobic metabolic states. Fundamentally, the oxygen consumption does not only depend on hepatocellular uptake rates but is also limited by culture medium thickness as well as ambient oxygen concentration. However, despite this oxygen supply limitation, hepatocytes generally tolerate hypoxia due to their extraordinary capacity to satisfy energy requirements by anaerobic glycolysis. This hypoxia situation leads to an inefficient utilization of glucose since the conversion of glucose to lactate leads to the generation of 2 mol ATP/mol glucose compared to 38 mol ATP/mol glucose during oxidative phosphorylation.47 Enhanced oxygenation is important for the in vitro liver cellular microenvironment5 and the BAL model.48 Around half a century ago, Stevens reported that in vitro liver cells obtain 4% of their oxygen requirement and degenerate rapidly.49 The oxygen supply in an in vivo liver is 2000 nmol/mL, but in vitro hepatocyte cells get an oxygen supply of less than 200 nmol/mL,50 which is a significant current limitation and neglected area, particularly in primary hepatocyte culture. In our construction, our bioreactor offers a maximal oxygen supply of 90 μmol per 1.77 cm2 via direct delivery of oxygen to the cells from the bottom of the device.13 Our bioreactor also allows direct contact of every individual liver cell with the oxygen supply, for enhanced oxygenation that is closer to the in vivo liver.5,11,12 While the available image data was obtained from CLSM, basically providing the image series for our image analysis, we have restricted ourselves to 2D image-based assessments on single images taken centrally from the stacks. The reason was two-fold: first the cells considerably adhere to the ground, leading to heavy distortions so that the cells appear to be strongly flattened. Thus, even dedicated efforts to analyze the 3D morphology (with respect to the mitochondria in this work) remains of limited use (Figure 1). While the mitochondria should be expected to be distributed on a spherical hull, what we see is some degenerate torus-like spatial distribution, an obvious phenomenon which undermines a reasonable 3D morphometry. The second reason in part is a consequence of the first. Due to the distortions, single mitochondria pieces appear even closer to each other than under physiological conditions. This effect, however, worsens the conditions for the following image processing and analysis, since mitochondria pieces in particular appear to be densely packed. In turn, this also affects any segmentation capabilities; ie, the separated depiction of mitochondria pieces. However, there is another reason why a strict 3D analysis does not appear worthwhile at this point: position dependent blurring of the noisy images, an effect, which is inherent to CLSM, requiring effective image restoration, is always a challenge. It can basically be tackled using a variety of image deconvolution techniques.51 This effort, however, was not undertaken here, due to the strong cell distortions discussed above. Interestingly,52 we did not apply image deconvolution, but in spite of this outline, a 3D image analysis processing chain ended up with a couple of 3D based morphometric numbers with respect to single mitochondria pieces. With respect to the blurring and artifacts in our image data, a similar single mitochondria assessment approach did not appear feasible. High quality fluorescence images were captured for the subsequent quantitative analysis of mitochondria using the Mathematica software, which offers a variety of image processing functionality. It has been found that in liver cells, total mitochondrial volume is distributed into many mitochondrial elements. Also, it shows that a healthy cell in a bioreactor has a very good level of mitochondrial volume around the nucleus. Previously, we have investigated the automatic algorithms-based image analysis of various biomedical images that enable the quantitative analysis and 3D visualization of medical images of numerous modalities such as microscopy histological structure. The main highlights of the present study are that this optimized 3D defined culture system in bioartificial liver modules could be useful for drug metabolism, enzyme induction, transplantation, viral hepatitis, and hepatocyte regeneration. This proof of concept may be convenient for other existing bioartificial devices and hepatotoxicity assessments. Nanostructured self-assembling peptides are an excellent substrate for 3D culture and long-term culture. 2D/3D visualization of mitochondria revealed healthy shapes and distributions at 90 days of culture. This bioartificial liver module might be an alternative to animal experimentation. This proof of concept may meet the regulatory guidelines as well as pharmaceutical good manufacturing practice guidelines as all conditions are absolutely defined. Instead, we decided to do the mitochondria growth analyses that refer to representative single images taken from the image stacks. Applying our image processing pipeline in 2D detailed above, we could obtain a new comprehensive description of determined mitochondria margins, mainly using distance transforms and skeletons. The method was exemplified in this work and needs to be assessed in a comparative study. Conclusion We have developed a defined, cost effective, efficacious, and scalable ex vivo bioartificial modular device for performing long-term functional maintenance of primary hepatocytes to meet the regulatory guidelines and provide alternative animal experimentation with special reference to chronic dose screening of drug candidates that can also reduce the number of animals used in in vivo testing. The long-term functional maintenance of primary hepatocytes in this clinically relevant modular device opens up a new avenue not only for rapid drug screening testing from sub-acute to chronic exposure but also has clinical implications for the bioartificial liver support system. This concept may be useful for other existing bioartificial liver systems to make them more efficient, especially for the long-term functional maintenance of housed primary hepatocytes in these systems. Herein, we proved that using nanostructured, self-assembling peptides and 3D interaction of growth factors and cytokines, it is possible to mirror the in vivo liver microenvironment. This 3D interaction is very effective to recapitulate the physiological functions of in vivo hepatocytes after the isolation of hepatocytes from the in vivo liver. Pharmaceutical companies have been under high pressure to develop safer drugs because a number of approved drugs entering the market have failed in the last decade. Many pharmaceutical and biotechnology companies are now searching for effective strategies to improve the preclinical stage to recognize unsuccessful drug candidates early in the drug discovery process. The pharmaceutical and biotechnology industries are increasingly seeking an ex vivo cellular model that mimics the state of the art liver which could replace the use of animals in drug screening tests in the early stage of drug candidate selection. A modular novel bioreactor-based bioartificial liver support system was designed and constructed using self-assembling peptides and a special combination of growth factors, cytokines, and hormones in order to simplify the tedious operation of artificial liver treatment and to improve the applicability of the system in the bioartificial liver system and in the in vitro hepatic model to produce safer drugs. Additionally, we have shown the 3D visualization of well-shaped mitochondria of single/double hepatocyte cells at day 90, since mitochondria is directly proportional to the survivability of hepatocytes. With the proof of concept, in the future, it is hoped that several basic hepatocyte physiological disease-screening models, such as those that are induced pluripotent stem cells-based, can be integrated into a modular device platform to realize a complete in vivo liver for different genetic background populations. This modular optimized device has great potential to predict in vivo toxicity and is very much suitable to measure metabolism and the toxicity of drugs with a very good correlation to the in vivo situation. It will minimize the gap between the in vivo situation and in vitro situation. This proof of concept study in a bioartificial liver modular device could replace the millions of animals that are currently sacrificed in preclinical testing and open up a new vista for new, safer pharmaceutical products. Acknowledgments The authors wish to thank Frank Struck for supplying the primary hepatocytes. Special thanks also go to Ingo Schäfer for his confocal microscopy. The funding for this project was provided by the Medicine Faculty of the University of Leipzig, TRM Leipzig, and IZBI Leipzig, Leipzig, Germany. Disclosure The authors report no conflicts of interest in this work. Figure 1 Mitochondria data sets depicted in 3D visualization using shaded surface rendering. Note: A supplementary 3D movie is available to view: http://youtu.be/Alc_72FM9Ho. Figure 2 Proposed image processing pipeline towards 2D mitochondrial distribution assessment. (A) Confocal laser scanning microscopy image (mitochondria staining); (B) total-variation filtering; (C) binarization, dilation, labeling; (D) artifact removal, separation, convex hulls (cell masks); (E) cell masks minus mitochondria regions, labeling; (F) nuclei region isolation, convex hull of cell nuclei regions; (G) subtraction of convex hulls (D and F), ring-shaped mitochondria regions; (H) Euclidean distance mapping, skeletonization of mitochondria regions. Notes: The single steps basically do not require any specific interaction; for example, the number of cells can be implicitly determined by means of size considerations referring to the label images. What the pipeline eventually provides is some chain of mitochondria margin measurements obtained along the skeleton positions. Figure 3 (A) Morphological analysis (scale bar 100 μM) at various days. Green arrows indicate bile duct and red arrows indicate nuclei. (B) Albumin secretion. (C) Urea secretion. (D) MTT test. (E) Gene expression of albumin. (F) Gene expression of CYP3A1. These gene expressions are normalized against the Housekeeping gene (GAPDH) as an internal standard. (G) LDH test; (H) oc-GST test at various days (day 10, day 30, day 60, and day 90). (I) EROD activity. Notes: Results are presented as the mean ± SD from three independent experiments. P < 0.05 was considered to be statistically significant. Abbreviations: α-GST, alpha glutathione S-transferase; EROD, ethoxyresorufin-O-deethylase; LDH, lactate dehydrogenase; MTT, methyl thiazol tetrazolium; SD, standard deviation. Figure 4 The first mitochondria margin measurement example plots describe the course of the determined mitochondria margin’s thickness (counterclockwise cycle). The cyan curve represents the upper right cell (LI)’s mitochondria margin, and the magenta curve represents that of the lower left cell (L2). Curves are plotted so that their global minimum is placed at the abscissa’s origin. Figure 5 The second mitochondria margin measurement example. Notes: The plots illustrate the margin thicknesses (counterclockwise cycle). The red curve represents the lower left cell (L3)’s mitochondria margin, the green curve represents that of the upper cell (L4), and the blue curve represents that of the lower right cell (L5). Curves are plotted so that their global minimum is placed at the abscissa’s origin. Figure 6 A hypothetical diagram showing how primary hepatocyte suffers in conventional 2D culture (A) in comparison to 3D culture (B) with special reference cytokines and growth factor interaction in exposure media during in vitro culture. (C) The difference of oxygenation in bioreactor and conventional culture plates with special reference to the distance between cultured hepatocyte and humidified oxygen. Notes: Partially polarized or unpolarized hepatocytes of conventional tissue culture plastic dishes do not facilitate autocrine and paracrine soluble signals from their surrounding environments and finally lead to apoptosis. But under 3D culture, hepatocytes are polarized and able to respond to both paracrine and autocrine soluble signals three dimensionally and maintain their functional property on a long-term basis. 2D conventional culture is partially polarized which can seriously impair cellular communication, the transport of oxygen and nutrients, the removal of wastes, and cellular metabolism. By contrast, in a 3D nanorange microenvironment, using the nanostructured self-assembling peptides reported here, all functional motifs on the nanostructured self-assembling peptides encircle the whole hepatocyte cell body in all dimensions where all growth factors, cytokines, nutrients, and signals can interact three-dimensionally, just like in an in vivo hepatic microenvironment. Table 1 Characteristic numbers obtained from the mitochondria margin measurements (see also Figure 4) Cell Mitochondria margin thickness Area coverage [%] Mean [μM] Standard dev [μM] Median [μM] L1 5.23 2.69 4.95 53.14 L2 6.10 1.74 6.37 40.85 L3 5.47 2.02 5.15 39.16 L4 6.66 3.04 6.05 31.80 L5 6.69 2.40 6.92 25.53 Notes: Mean, standard deviation, and median were determined from the curves depicted in Figure 5. The area coverage refers to the respective portion of segmented mitochondria; eg, Figure 4C, covered by the determined ring-shaped mitochondria regions, eg, Figure 4G. 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==== Front Ther Clin Risk ManagTher Clin Risk ManagTherapeutics and Clinical Risk ManagementTherapeutics and Clinical Risk Management1176-63361178-203XDove Medical Press 10.2147/TCRM.S43811tcrm-9-171Original ResearchVitamin D reduces falls and hip fractures in vascular Parkinsonism but not in Parkinson’s disease Sato Yoshihiro 1Iwamoto Jun 2Honda Yoshiaki 1Amano Nobuko 31 Department of Neurology, Mitate Hospital, Tagawa, Japan2 Institute for Integrated Sports Medicine, Keio University School of Medicine, Tokyo, Japan3 Department of Food and Nutrition, Tezukayama University, Nara, JapanCorrespondence: Yoshihiro Sato, Department of Neurology, Mitate Hospital, 3237 Yugeta, Tagawa 826-0041, Japan, Tel +81 947 44 0924, Fax +81 947 46 3090, Email [email protected] 2013 22 4 2013 9 171 176 © 2013 Sato et al, publisher and licensee Dove Medical Press Ltd.2013This is an Open Access article which permits unrestricted noncommercial use, provided the original work is properly cited.Purpose Vitamin D supplementation is suggested to reduce the risk of falls in older institutionalized or ambulatory individuals by 20%. The present study was undertaken to address the reduced risk, by vitamin D supplementation, of falls and hip fractures in patients with vascular Parkinsonism (VP) and Parkinson’s disease (PD). Patients and methods In the open-label-study, 94 elderly patients with VP and 92 age-matched patients with PD were followed for 2 years. All patients received 1200 IU ergocalciferol daily. The number of falls per person and incidence of hip fractures were compared between the two groups. Results At baseline, serum 25-hydroxyvitamin D (25-OHD) levels were in the deficient range (<25 nmol/L) in all patients, and vitamin D treatment enhanced serum 25-OHD and 1,25-dihydroxyvitamin D levels in both groups. Improved muscle strength of lower extremities was observed in both groups. There was significant difference between the two groups in the number of falls per subject during the 2 years (1.9 ± 0.5 in the PD group and 0.8 ± 0.4 in the VP group, P < 0.001). Hip fractures occurred in seven of 88 in the PD group and one in 90 of the VP group during the 2-year study period (P = 0.035). Conclusion It is suggested that vitamin D decreases falls and hip fractures in VP by increasing muscle strength but not in PD. fallhip fractureParkinson’s diseasevascular Parkinsonismvitamin D ==== Body Introduction Recent advances in diagnosis and treatment have prolonged survival in elderly patients with Parkinson’s disease (PD), and patients’ physical states have become increasingly important in PD management. Previous studies1–5 demonstrated a high incidence of falls and hip fractures in PD patients, particularly in elderly women.3 Prolonged survival may contribute to the decreased bone mineral density (BMD) and increased risk of fractures seen in the PD population. Several reports have documented low BMD of the lumbar vertebrae,5 hip joint,6 or second metacarpal7,8 in PD patients, with severe osteoporosis being more prevalent at higher Hoehn and Yahr stages.5,7 We previously demonstrated that 25-hydroxyvitamin D (25-OHD) deficiency (less than 25 nmol/L) due to sunlight deprivation induces compensatory hyperparathyroidism, which further contributes to reduced BMD in PD patients, particularly those who are functionally dependent.7 However, when serum 25-OHD was in an insufficient range (25–47 nmol/L), immobilization-induced hypercalcemia inhibited parathyroid hormone (PTH) secretion.7 Compensatory hyperparathyroidism associated with deficient 25-OHD levels and immobilization induce increased bone resorption and contribute to reduced BMD and occurrence of hip fractures.8,9 On the other hand, patients with vascular Parkinsonism (VP) are similar in terms of abnormal bone and calcium metabolism and have a high risk of falls and hip fractures (Sato et al, unpublished data, 2003). Gait and balance disorders causing falls are common in PD and VP, but fall pathophysiology is still poorly understood. Prevention of a hip fracture, which is likely to offset gains from rehabilitation and preclude new gains, is extremely important in PD and VP. It is suggested that vitamin D supplementation reduces the risk of falls in older institutionalized or ambulatory individuals.10 Previously, the moderate protective effect of vitamin D on a fracture risk was attributed primarily to BMD changes.11 Vitamin D may increase muscle strength by improving atrophy of type II muscle fibers, which may lead to decreased falls and hip fractures.12 We previously found that PD patients had remarkably low serum 25-OHD levels,8 and many of them had a concentration of less than 25 nmol/L. Many such patients also had very low serum levels of 1,25-dihydroxyvitamin D (1,25-[OH]2D), and immobilization-induced hypercalcemia may be responsible for inhibition of renal synthesis of 1,25-[OH]2D.8 In the present study, we focused on vitamin D treatment in PD and VP for the effective prevention of falls and hip fracture. We conducted a 2-year case control study to evaluate the efficacy of ergocalciferol therapy in reducing the risk of falls in elderly patients with PD and VP. Materials and methods Study population The study was approved by the local ethics committee of Mitate Hospital, Tagawa, Japan, and informed consent was obtained from all subjects in the presence of a witness. This study compared the occurrence of falls in the two groups (PD and VP) administered ergocalciferol. Consecutive studies included 92 elderly patients with PD and 94 elderly patients with VP followed in the neurology clinic of Mitate Hospital, which provides comprehensive long-term outpatient care for neurology. Patients matched for sex and age with diagnoses of PD and VP were included in the study. No PD patients underwent deep brain stimulation surgery as there is no equipment for this surgery in Mitate or surrounding hospitals. The diagnostic criteria of VP were as follows: (1) Parkinsonism, defined as bradykinesia, and at least one of the following: rest tremor, rigidity, or postural instability; (2) cerebrovascular disease, defined as evidence of relevant cerebrovascular disease by brain imaging or the presence of focal signs or symptoms that are consistent with stroke; (3) a relationship between (1 and 2): an acute or delayed progressive onset of Parkinsonism (within 1 year) after stroke with evidence of infarcts that increase the basal ganglion motor output or decrease the thalamocortical drive directly, or an insidious onset of Parkinsonism with extensive subcortical white matter lesions, bilateral symptoms at the onset, and the presence of early shuffling gait or early cognitive dysfunction.13 Patients of both groups were selected by detailed examination of case notes of patients to match patients with respect to age, sex, and the motor part of the Unified Parkinson’s Disease Rating Scale III (UPDRS III).14 Exclusion criteria included inability to walk and previous history of hip fracture. Other exclusion criteria included peripheral neuropathy, renal insufficiency (serum creatinine concentration ≥ 1.5 mg/dL), hepatic insufficiency, or cardiac failure. Patients were excluded if they had received any drugs known to alter vitamin D metabolism, such as anticonvulsants, calcium, or vitamins D and K, during the 12 months preceding the study. No attempt was made to alter subjects’ diet or activity during the study. At baseline, we determined body mass index (BMI), and UPDRS III.14 Sunlight exposure during the preceding year was assessed and graded as less than 15 minutes per week or longer.15 A physical therapist who was blinded to information concerning the patients evaluated the muscle strength of the lateral and medial rotators of the hip, and flexion and extension of thigh with hip and knee flexed 90° using the British Medical Research Council (MRC) scale.16 The British MRC scale defines a score of 0 as no contraction of the tested muscle while a score of 5 represents normal power. The total points for muscle strength of the four different movements of the hip joint were calculated for each patient. The muscle strength was evaluated at baseline, and 1 and 2 years later. Patients who fell at least once in the 3 months before recruitment were defined as “fallers.” Falls were defined as incidents in which a subject fell due to an unexpected loss of balance. The number of falls per subject was also recorded during the 2-year follow-up period. Falls were registered by means of monthly “fall calendars.” The participants were instructed to complete the calendar daily, marking an “X” for each fall on the date that the fall occurred. If the patient suffered dementia, the calendar was completed by family members. All patients received a daily dose of 1200 IU ergocalciferol. We used ergocalciferol as a part of the synthesized vitamin powder commercially available in Japan. Ergocalciferol was administered twice per day with breakfast and dinner. Doses of ergocalciferol were not adjusted for each patient and no dose adjustments were made at any time during the study. Patients were not allowed to take any other drugs that could affect bone and calcium metabolism. Adherence to study medication was assessed by pill count of returned tablets. Follow-up assessment of the patients was performed by two physicians who did not participate in the initial randomization. Both groups were observed for 2 years. General medical evaluation and serum indices of bone metabolism were assessed upon study entry and after 2 years. Four patients in the PD group and four in the VP group dropped out or withdrew from the study due to noncompliance, loss to follow-up, intercurrent illness, or death. Thus, a total of 178 patients (88 in the PD group and 90 in the VP group) completed the trial. A blood sample was obtained from each patient after an overnight fast. Blood samples were analyzed for ionized calcium, intact PTH, and 25-OHD and 1,25-[OH]2D as described previously.17 The biochemical data, at the start and 1 and 2 years later, of the patients who completed the cohort were analyzed. Statistical analysis All statistical analyses were performed using the StatView J 5.0 software package (Abacus Concepts, Berkeley, CA, USA). Values are given as the mean ± SD unless otherwise indicated. Group differences of the categorical data were tested by χ2 analyses or Fisher’s exact method. The unpaired t-test was used to determine the differences between the two groups. Spearman’s rank correlation coefficients were calculated to determine the relationships between UPDRS III or strength scale of hip muscle and 25-OHD levels. The two groups were compared with respect to their laboratory values by using Wilcoxon rank-sum test. The difference in the incidence of hip fractures between the two groups during the 2 years was tested by Fisher’s exact test. P-values of less than 5% were considered statistically significant. Results Demographic and baseline clinical characteristics of study subjects Characteristics of the study population are given in Tables 1 and 2. Patient characteristics, number of falls, MRC hip strength, sunlight exposure, and laboratory values did not differ between the two groups at baseline. Mean serum 25-OHD concentrations were 22 nmol/L. Serum ionized calcium levels were high, while PTH and 1,25-[OH]2D concentrations were low as compared to the reference range of normal Japanese population.8 When the two patient groups were analyzed together, ionized calcium concentrations correlated negatively with UPDRS III (r = −0.412, P < 0.0001) and PTH (r = −0.574, P < 0.0001). There was no correlation between serum 25-OHD and PTH (r = 0.070, P = 0.49). When the PD and VP groups were analyzed together, 25-OHD concentrations correlated positively with UPDRS III (r = 0.255, P = 0.0122) and strength scale of hip muscle (r = 0.568, P < 0.0001). Fallers and hip fracture incidence Table 2 summarizes time-dependent changes in the frequency of fallers who fell at least once in 3 months. The numbers after 12 and 24 months remained unchanged in PD patients and decreased in VP patients (P < 0.001). There was a significant difference between the two groups in the number of falls per subject during the 2 years (1.9 ± 0.5 in the PD group and 0.8 ± 0.4 in the VP group, P < 0.001). Hip fractures caused by falls occurred in seven cases in the PD group, and in one case in the VP group, during the 2-year study period (P = 0.035). The number of hip fractures per 1000 patient-years was 21 and 152 for the VP and PD groups, respectively. Muscle strength and serum indices of bone metabolism During the 2-year period, significant increase of muscle strength was observed in both groups. During the 2-year period, serum 25-OHD levels had increased to the normal range in both groups. In both groups, serum PTH concentration increased but remained low as compared to the reference range, while serum ionized calcium concentration decreased but remained high as compared to the reference range. Serum 25-[OH]2D levels had increased in both groups (Table 2). Discussion Prevention of fractures is one of the important issues in the management of PD and VP patients. There are multiple factors for falls in PD, including postural instability as well as psychological and physical complications.21,22 The high incidence of hip fractures in elderly PD and VP patients may be attributed to frequent falls and osteoporosis due to hypovitaminosis D and disuse.1,2,7 The present study demonstrated that vitamin D reduced the number of falls in VP but did not affect falls in PD during the 2 years. As a result, hip fracture incidence may be low in VP and high in PD. Fall incidence in PD did not increase during the 2 years (data not shown), despite PD being a progressive disease. The study suggests that falls in PD are not caused by hypovitaminosis D but caused by PD specific extrapyramidal system (EPS) abnormalities. This is the first study that documents a reduction in falls among frail VP patients but not in PD patients with a single medication over 2 years. Previous studies on the relationship between vitamin D and muscle strength in elderly subjects demonstrated the beneficial effect in relation to muscle strength and balance. One such study demonstrated the effectiveness of vitamin D in restoring musculoskeletal function in institutionalized elderly women.23 Also, 2-month treatment with vitamin D and calcium was found to decrease both body sway and falls in ambulatory elderly women.24 It has been demonstrated that serum 25-OHD levels are low in elderly fallers25 and muscle strength is higher in the ambulatory elderly with higher 25-[OH]2D levels.26 In the present study, by administering vitamin D, we found improvement in muscle strength in VP and PD patients who had deficient levels of serum 25-OHD before the therapy. Severe vitamin D deficiency is common in PD and VP9 and type II fiber atrophy is one of the characteristics of vitamin D deficient myopathy.27 We observed improvement in muscle strength in VP and PD patients to whom vitamin D had been administered. The effect of vitamin D on muscle strength may be explained by its direct effects on muscle tissues.28 These effects may be mediated by de novo protein synthesis, affecting muscle cell growth.29 Because this effect on muscle tissues seems to result in clinical improvement even after a short-term intervention,12,30 it is of major clinical interest if vitamin D may be effective for the prevention of falls and thus fractures in elderly people. Indeed, a study showed that in vitamin D-deficient subjects, severely impaired muscle function may be present even before biochemical signs of bone disease develops.31 Despite the similar effectiveness of vitamin D on muscle strength in both PD and VP, the reason for the different incidence of falls between the two groups is unclear. We postulate that falls in PD are not caused by hypovitaminosis D-induced muscular weakness, but are caused by PD-specific EPS abnormalities, while vitamin D deficiency causing muscular weakness rather than EPS disorder causes falls in VP. Kalra et al reviewed 25 articles about differentiating VP from idiopathic PD and concluded there were no accepted international diagnostic criteria for VP.32 Although the applied diagnostic criteria for VP in the present study is not a universally accepted international standard, we believe that the criteria is better than the other 24 articles describing criteria of VP. Hip fracture is a serious complication in VP, leading to surgical treatment that may be complicated by pulmonary embolism, fat embolism syndrome, pneumonia, urinary tract infections, and deep vein thrombosis. Also, a bedridden state after surgery is not uncommon.33 Thus, ergocalciferol administration for VP is of benefit in the prevention of hip fracture and the necessity for surgical treatment leading to potential complications and a bedridden state. On the other hand, open-label-study and absent data of BMD and muscle biopsy and/or electrophysiology before and after the treatment are the limitations of the study. However, our previous study12 in patients following stroke with hypovitaminosis D showed increases in the relative number and size of type II muscle fibers and improved muscle strength in the vitamin D-treated (1000 IU ergocalciferol daily) group over 2 years. Therefore, we believe vitamin D may increase muscle strength by improving atrophy of type II muscle fibers, which may lead to decreased falls and hip fractures. In future studies, randomized controlled trials measuring BMD and performing muscle biopsy and/or electrophysiology should be considered. Also, we did not study age-matched controls for proper comparison and to demonstrate the effect of vitamin D treatment. This is another study limitation not indicating how much the risk of falling was reduced in the cohorts study. We did not assess autonomic neuropathy or visual problems in the present study, which is an additional study limitation. Conclusion Vitamin D supplementation in VP patients with low serum vitamin D causes decreased risk for falls, while such a phenomenon is not observed in PD patients. Treatment with ergocalciferol may be safe and effective in restoring muscular strength, which may reduce falls and the risk of fractures in VP. Disclosure The authors report no conflicts of interest in this work. Table 1 Demographic and baseline clinical characteristics of the patients with Parkinson’s disease and vascular Parkinsonism at study entry Characteristic Parkinson’s disease (n = 92) Vascular Parkinsonism (n = 94) P-value* Age (years) 73.6 ± 5.9 73.9 ± 6.2 0.80 Men (n = 105), n (%) 49 (53) 56 (60) 0.65† Duration of illness (years) 4.8 ± 2.9 5.0 ± 3.1 0.68 Hoehn and Yahr stage18 3.2 ± 1.2 3.3 ± 1.6 0.75 UPDRS III (motor function score)14 53.4 ± 12.8 55.8 ± 15.1 0.71 Faller, n (%)‡ 33 (36) 32 (34) 0.86† Strength scale of hip muscle§ 3.8 ± 1.4 3.9 ± 1.6 0.64 Sunlight exposure/week, n (%) >15 minutes 4 (4) 3 (3) <15 minutes 12 (13) 15 (16) None 76 (83) 76 (81) 0.80† Notes: Values are mean ± standard deviation. * Unpaired t-test; † Fisher’s exact test; ‡ patients who fell at least once in the 3 months before recruitment or study period were defined as “fallers;” § the British Medical Research Council scale defines a score of 0 as no contraction of the four tested muscles while a score of 5 represents normal power of the hip muscle.16 The values are the average point of four muscles. Abbreviation: UPDRS III, Unified Parkinson’s Disease Rating Scale III. Table 2 Falls and biochemical tests in Parkinson’s disease and vascular Parkinsonism groups at baseline and after 1 and 2 years of follow-up Biochemical indices and group Follow-up Baseline 1 year 2 years Faller, n (%)\|\| Parkinson’s disease 33 (36) 30 (33)* 31 (35),‡ Vascular Parkinsonism 32 (34) 13 (14)† 14 (16)† Strength scale of hip muscle¶ Parkinson’s disease 3.8 ± 1.4 4.4 ± 1.2† 4.5 ± 1.6† Vascular Parkinsonism 3.9 ± 1.6 4.5 ± 1.5† 4.4 ± 1.3† Ionized calcium (mmol/L) Parkinson’s disease 1.32 ± 0.06 1.33 ± 0.07§ 1.33 ± 0.08§ Vascular Parkinsonism 1.31 ± 0.08 1.31 ± 0.09 1.32 ± 0.10 Intact parathyroid hormone (ng/L) Parkinson’s disease 32.1 ± 12.1 33.0 ± 9.9§ 36.3 ± 4.5§ Vascular Parkinsonism 30.5 ± 11.5 35.6 ± 6.0 34.2 ± 3.9 25-hydroxyvitamin D (nmol/L) Parkinson’s disease 23.0 ± 10.7 56.2 ± 6.2† 58.9 ± 7.0†,§ Vascular Parkinsonism 22.7 ± 9.7 54.9 ± 8.2† 59.7 ± 8.7† 1,25-dihydroxyvitamin D (pmol/L) Parkinson’s disease 81.1 ± 28.1 98.8 ± 26.8† 102.7 ± 22.6†,§ Vascular Parkinsonism 87.1 ± 29.1 104.8 ± 25.7 108.2 ± 21.1† Notes: Values are mean ± SD. P < 0.001 versus vascular Parkinsonism; † P < 0.001 for the comparison with the baseline value; ‡ not significant for the comparison with the baseline value; § not significant versus vascular Parkinsonism; \|\| patients who fell at least once in the 3 months before recruitment or study period were defined as “fallers;” ¶ the British Medical Research Council scale defines a score of 0 as no contraction of the four tested muscles while a score of 5 represents normal power of the hip muscle.16 The reference ranges of a healthy elderly person:19,20 ionized calcium, 1.22–1.27 mmol/L; intact parathyroid hormone, 35–52 ng/L; 25-hydroxyvitamin D, 47.2–62.2 nmol/L; 1,25-dihydroxyvitamin D, 102.4–147.7 pmol/L. ==== Refs References 1 Aita JF Why patients with Parkinson’s disease fall JAMA 1982 247 4 515 516 7054557 2 Chiu KY Pun WK Luk KDK Chow SP Sequential fractures of both hips in elderly patients – a prospective study J Trauma 1992 32 5 584 587 1588646 3 Grisso JA Kelsey JL Strom BL Risk factors for falls as a cause of hip fracture in women. The Northeast Hip Fracture Study Group N Engl J Med 1991 324 19 1326 1331 2017229 4 Johnell O Melton LG Atkinson EJ O’Fallon WM Kurland LT Fracture risk in patients with parkinsonism: a population-based study in Olmsted Country, Minnesota Age Ageing 1992 21 1 32 38 1553857 5 Kao CH Chen CC Wang SJ Chia LG Yeh SH Bone mineral density in patients with Parkinson’s disease measured by dual photon absorptiometry Nucl Med Commun 1994 15 3 173 177 8190408 6 Taggart H Crawford V Reduced bone density of the hip in elderly patients with Parkinson’s disease Age Ageing 1995 24 4 326 328 7484491 7 Sato Y Kikuyama M Oizumi K High prevalence of vitamin D deficiency and reduced bone mass in Parkinson’s disease Neurology 1997 49 5 1273 1278 9371907 8 Sato Y Kaji M Tsuru T Oizumi K Risk factors for hip fracture among elderly patients with Parkinson’s disease J Neurol Sci 2001 82 2 89 93 11137512 9 Sato Y Honda Y Iwamoto J Kanoko Y Satoh K Abnormal bone and calcium metabolism in immobilized Parkinson’s disease patients Mov Disord 2005 20 12 1598 1603 16114020 10 Bischoff-Ferrari HA Dawson-Hughes B Willett WC Effect of vitamin D on falls: a meta-analysis JAMA 2004 291 16 1999 2006 15113819 11 Dawson-Hughes B Harris SS Krall EA Dallal GE Effect of calcium and vitamin D supplementation on bone density in men and women 65 years of age or older N Engl J Med 1997 337 10 670 676 9278463 12 Sato Y Iwamoto J Kanoko T Satoh K Low-dose vitamin D prevents muscular atrophy and reduces falls and hip fractures in women after stroke: a randomized controlled trial Cerebrovasc Dis 2005 20 3 187 192 16088114 13 Zijlmans JC Daniel SE Hughes AJ Révész T Lees AJ Clinicopathological investigation of vascular parkinsonism, including clinical criteria for diagnosis Mov Disord 2004 19 6 630 640 15197700 14 Fahn S Elton R Members of UPDRS Development Committee Unified Parkinson’s disease rating scale Fahn S Marsden C Calne D Goldstein M Recent Developments in Parkinson’s Disease New York Macmillan 1987 2 153 163 15 Komar L Nieves J Cosman F Rubin A Shen V Lindsay R Calcium homeostasis of an elderly population upon admission to a nursing home J Am Geriatr Soc 1993 41 10 1057 1064 8409150 16 Medical Research Council of the United Kingdom Aids to the Examination of the Peripheral Nervous System London Pendragon House 1978 17 Sato Y Honda Y Iwamoto J Risedronate and ergocalciferol prevent hip fracture in elderly men with Parkinson disease Neurology 2007 68 12 911 915 17372126 18 Hoehn MM Yahr MD Parkinsonism: onset, progression and mortality Neurology 1967 6 17 427 442 6067254 19 Sato Y Asoh T Kaji M Oizumi K Benefical effect of intermittent cyclical etidronate thirapy in hemiplegic patients following an acute stroke J Bone Miner Res 2000 15 12 2487 2494 11127214 20 Sato Y Kanoko T Yasuda H Satoh K Iwamoto J Beneficial effect of etidronate therapy in immobilized hip fracture patients Am J Phys Med & Rehabil 2004 83 4 298 303 15024332 21 Landers MR Backlund A Davenport J Fortune J Schuerman S Altenburger P Postural instability in idiopathic Parkinson’s disease: discriminating fallers from nonfallers based on standardized clinical measures J Neurol Phys Ther 2008 32 2 148 149 18978672 22 Dibble LE Lange M Predicting falls in individuals with Parkinson disease: a reconsideration of clinical balance measures J Neurol Phys Ther 2008 32 2 56 61 18645292 23 Gallagher JC Fowler SE Detter JR Sherman SS Combination treatment with estrogen and calcitriol in the prevention of age-related bone loss J Clin Endocrinol Metab 2001 86 8 3618 3628 11502787 24 Pfeifer M Begerow B Minne HW Abrams C Nachtigall D Hansen C Effects of a short-term vitamin D and calcium supplementation on body sway and secondary hyperparathyroidism in elderly women J Bone Miner Res 2000 15 6 1113 1118 10841179 25 Stein MS Wark JD Scherer SC Falls relate to vitamin D and parathyroid hormone in an Australian nursing home and hostel J Am Geriatr Soc 1999 47 10 1195 1201 10522952 26 Bischoff HA Stahelin HB Urscheler N Muscle strength in the elderly: its relation to vitamin D metabolites Arch Phys Med Rehabil 1999 80 1 54 58 9915372 27 Russell JA Osteomalacic myopathy Muscle Nerve 1994 17 6 578 580 8196699 28 Costa EM Blau HM Feldman D 1,25-dihydroxyvitamin D3 receptors and hormonal responses in cloned human skeletal muscle cells Endocrinology 1986 119 5 2214 2220 3021437 29 Boland R Role of vitamin D in skeletal muscle function Endocr Rev 1986 7 4 434 447 3536463 30 Eastwood JB Stamp TC De Wardener HE Bordier PJ Arnaud CD The effect of 25-hydroxyvitamin D3 in the osteomalacia of chronic renal failure Clin Sci Mol Med 1997 52 5 499 508 862343 31 Stern G Thonchin M Smith R Muscular weakness in metabolic bone disease Neurology 1973 20 6 480 483 32 Kalra S Grosset DJ Benamer HT Differentiating vascular parkinsonism from idiopathic Parkinson’s disease: a systematic review Mov Disord 2010 25 2 149 156 20077476 33 Rogmark C Carlsson A Johnell O Sernbo I A prospective randomised trial of internal fixation 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Functional outcome for 450 patients at two years J Bone Joint Surg Br 2002 84 2 183 188 11922358	23637537	PMC3639218	NO-CC CODE	2021-01-04 22:39:09	yes	Ther Clin Risk Manag. 2013 Apr 22; 9:171-176
==== Front Adv Med Educ PractAdv Med Educ PractAdvances in Medical Education and Practice1179-7258Dove Medical Press 10.2147/AMEP.S21201amep-2-173Original ResearchImpact of an educational program on nursing students’ caring and self-perception in intensive clinical training in Jordan Khouri Rawda Al Hussein Bin Talal University, Princess Aisha Bint Al Hussein College Of Nursing, Ma’an, JordanCorrespondence: Rawda Khouri, Al Hussein Bin Talal University, Princess Aisha Bint Al Hussein College of Nursing, PO Box 20, Ma’an, Jordan, Tel +96 27 95722133, Fax +96 23 2179050, Email [email protected] 29 6 2011 2 173 185 © 2011 Khouri, publisher and licensee Dove Medical Press Ltd.2011This is an Open Access article which permits unrestricted noncommercial use, provided the original work is properly cited.Background Framing and development of clinical skills in nursing students during their clinical practice is critical because this can shape their future caring skills. Professional caring empowers patients and contributes to their well-being and health. Education may enhance the capacity of nurses to be effective caring practitioners. Their study program encourages caring behavior in nursing students, consequently affecting their professional self-perception. Methods The present study investigated the effect of an educational program on caring behavior and professional self-perception in nursing students using a controlled pre/post test study design. The study sample consisted of 50 nursing students undertaking their final year in 2010–2011. Subjects were randomly assigned to either an experimental or a control group. The study was conducted in two critical care units affiliated to the Ma’an and Queen Rania hospitals in the south of Jordan. The instruments utilized were the Caring Dimensions Inventory, Nursing Students Attitude Observational Checklist, and Professional Self-Concept of Nurses Instrument. Results The study findings favor the effect of the educational program because there was increased knowledge and understanding of caring theory and related concepts, a more holistic approach to care, enhanced caring practices, and improved self-perception in the study group compared with the control group during different periods of assessment. The study group showed significantly better caring perception in psychological, technical, and professional terms than the control group during different periods of assessment. There was a significant positive trend of overall professional self-perception for the study group compared with the control group. Conclusion Nursing curricula should incorporate concepts and principles that guide students in developing caring, safe, competent, and professional behavior. Nursing students must seek educational opportunities to acquire knowledge for role preparation, to participate in knowledge generation, and for personal and professional development. caringnursing studentsclinical trainingeducational programself-perceptionself-identity ==== Body Introduction Nurses are expected to provide high-quality care in health and illness, and to empower their patients by moving them toward an independent self-regulated healthy life.1,2 Nursing students need to develop their abilities in order to view professional caring from a different perspective and translate new knowledge into action. However, the newly graduated nurse suffers from low levels of self-confidence and professional self-worth that can make the difference between continuing with nursing or leaving the profession.3 Nurses are torn between the human caring model of nursing that attracted them to the profession and the task-orientated biomedical model and institutional demands that consume their practice time.4 Moreover, the dynamics of relational, human-to-human caring practices and comprehensive therapeutic modalities for caring and healing seem to be eclipsed by the daily routines, mechanics, and demands of the economic, management, physical, and technological aspects of health care. The necessary changes needed for renewal and transformation seem to be dependent on human dimensions and skills that result in transforming patterns and depths of communication, relationships, and healing modalities.4–6 Caring is viewed as a central but difficult concept in nursing.5,7 The concept of caring is considered by many to be central to the practice of nursing and, indeed, some consider caring and nursing to be almost synonymous.8–10 Caring has been seen as the essence of nursing and as a process of interaction and communication.11,12 Arthur and Randle13 view the concept of professional caring as something that empowers the patient and contributes to well-being and health. Lack of professional caring results in reduced wellbeing and health. Caring is a complex concept and has to be expanded to include professionalism. Caring means being open to and perceptive of others, being genuinely concerned about patients, being morally responsible, being truly present for patients, being dedicated, and having the courage to be appropriately involved as a professional nurse.14 Caring is a process that involve feelings together with professional knowledge, competence, skills, and nursing actions.15 Larson and Ferketich16 stated that, for nursing to achieve true professional recognition and the esteem it deserves, an understanding of the makeup of its members and their views of themselves as professionals would be valuable. If self-perception on the part of nurses can be confidently strengthened, then further research that focuses on exploring relationships with job satisfaction, retention, and stress becomes realistic. Resolution of this conflict requires nursing to reconnect with the foundations of professional nursing and its theoretical, knowledgeable, ethical, and philosophical principles. However, to resolve practice dilemmas, abstract conceptualizations of what nursing is must translate into the concrete realm of what nursing does and must guide integrative professional clinical judgment for those actions within the context of a system and culture in crisis and conflict. The new emphasis is on a change of consciousness, a focused intentionality towards caring and healing relationships and modalities, a shift towards a spiritualizing of health versus a limited medicalized view.17–19 Thus, the intensive clinical training course is a critical period for nurses, is the first contact with the profession, and might alleviate any anxiety resulting from inability of the recent graduate to translate nursing theory into practice.9,20,21 Graduate framing and development during their learning is critical because this might shape their future way of caring. Fahrenwald et al22 defined professional identity in nursing as the values and beliefs held by the nurse that guides his/her thinking, actions, and interaction with patients. It refers to the nurse’s concept of what it means to be and act as a nurse. It is often addressed in terms of related concepts, ie, professionalism, perception of the role of the nurse, and professional self-perception. Values are inherent in developing and sustaining professional identity and are expressed in actions in relation to others.23 Professional identity refers to the commonality of the nursing profession and to the special way the nurse utilizes this commonality within the nursing profession. The need to strengthen the professional identity of the nurse has attracted attention in recent years. Such endeavors must focus upon the educational and administrative demands of support in an effort to make professional personal development and growth of nurses possible. This could mainly be realized through the development of personal self-care abilities and increased possibilities for nurses to share their experiences with other nurses in a narrative and reflective way. Professional group supervision may have certain potential.22,24 An educational supervision program directed toward professional self-perception is needed to address low recruitment and retention. Considering the importance of nursing services in any health system, programs need to be designed to strengthen and empower nurses.6,25,26 Educational programs and supervision may enhance the nursing student’s capacity to become an effective caring practitioner, which reflects on professional identity.27 Clinical supervision is a very important concept in nursing because of the potential benefits it can bring to patient care and nurses themselves, both individually and as a profession. Simonson27 mentioned that with increasing emphasis on work-based learning, one of the many strategies designed to support students and professionals is supervision. Professional group supervision is a useful means of strengthening the professional identity of nurses. Walsh and Dolan7 stated that reflection is important because it equips nurses to meet various practical problems and deal with them intelligently, which are necessary requirements for all nurses. Lee-Hsieh12 defined supervision as a support mechanism for practicing professionals within which they can share clinical, organizational, developmental, and emotional experiences with another professional in a secure, confidential environment in order to enhance knowledge and skills. Clinical supervision is both necessary and beneficial. It can be advantageous to individual practitioners and professional groups in enhancing practice and accountability, and promoting professional development.13,28 Bankert and Kozel15 stressed the value of clinical supervision in the development of professional expertise and quality of care. The literature is replete with references to clinical group supervision, but its outcomes have not been sufficiently investigated.29,30 Clinical group supervision is well described in empirical studies of care in older people and mental health nursing.31–33 However, only three published studies on clinical supervision were found to be related to intensive care.17,34,35 It seems reasonable to believe that focusing on clinical group supervision will facilitate the understanding of and prerequisites for the provision of care in an intensive care unit. It has been claimed that clinical supervision, if implemented effectively, will bring benefits as diverse as improved patient care through increased skills and knowledge,36,37 including reduction in stress levels and complaints as well as an increase in staff morale.20 Increasingly, research is identifying other benefits, including increased knowledge and awareness of possible solutions to clinical problems, increased confidence, reduced emotional strain and burnout, increased participation in reflective practice,38,39 and better self-awareness.33,40 The benefits identified have implications not only for practitioners but also for patient care and employing organizations. Arthur et al41 emphasized that professional identity would be developed by interaction with other nurses and through internalization of knowledge, skills, norms, and the culture of the nursing profession. He also explained that in order for individuals to create their own professional identity, they should initiate relationships at work and participate in professional activities within an organization. Barker11 added that nurses have a responsibility to educate other nurses to help establish their professional identities. Nurses as educators and role models are essential to the process. By monitoring the process of nursing education and administration, and conducting further research in this area, we can greatly improve the quality of care, retention of nurses, and the professionalization of nursing. The interventional educational program investigated in this study aimed to develop the nursing student’s ability to become a caring practitioner. It was also intended to enforce and empower caring behavior which consequently affects professional self-perception. Raising awareness of personal orientation provides an opportunity for change, which is essential for professional development of nursing. The present study was designed to investigate the effect of this educational program on nursing student’s caring behavior and professional self-perception. Methods and materials This research was designed to test the hypotheses that nursing students who participate in an educational program will score higher in their knowledge about caring and professionalism than those who do not participate in such a program, and that nursing students who participate in the program will score higher on aspects of caring and develop more positive professional self-perception than those who do not participate in such a program. A controlled pre/post test study design was utilized to show the impact of education on caring behavior and professional self-perception. The study program was conducted in two clinical areas in Jordan where nursing students are trained, ie, the critical care units of Ma’an and Queen Rania Hospitals. The study sample comprised 50 nursing students, representing all those undertaking their final year in 2010–2011 at our university. Both groups were selected in a random fashion. Participants was further randomly assigned to either an experimental group or a control group. Demographic data sheet A demographic data sheet was developed by the researcher to record the background characteristics of the nursing students, including age, type of education before joining the faculty (secondary school degree or technical institute degree), and any work experience prior to entering nursing training. Caring dimensions inventory The Caring Dimensions Inventory (CDI) was developed by Watson et al.42–44 This instrument was used to gather data on nursing students’ perception of what represents caring in nursing. It includes 25 items divided into four dimensions, ie, technical/professional (nine items), psychosocial (10 items), involvement (three items), and altruism (three items). The content of the CDI spans a range of nursing actions, including instrumental, affective, and professional activity. The scoring system was on a five-point Likert scale from 5 (“strongly agree”) to 1 (“strongly disagree”). The validity and reliability of the CDI has been reported by Watson and Lea.42 The reliability of the CDI, measured by internal consistency, was acceptable, with a Cronbach’s alpha of 0.96. The validity of the contrasted groups of nursing students was established. Professional self concept instrument The Professional Self Concept Instrument (PSCNI) was developed by Arthur45,46 to study professional self-perception in a population of nursing students. The 16 items ask nurses to rate their degree of agreement on three scales examining nurses’ views toward their work. Scale one professional practice has three subscales, ie, leadership, flexibility, and skill, while scales two and three measure satisfaction and communication, respectively. A high score indicates a positive attitude or belief and a low score indicates a negative attitude or belief. Individual scores and group scores were then obtained for the components of the CaPSTI, ie, professional self-perception (including professional practice, satisfaction, and communication). A four-point Likert scale was used, with a score of 1 reflecting a low opinion of self as a professional and a score of 4 a more positive professional self-view. Conceptually, the PSCNI relies on the assumption that attitudes toward self as a professional exist on a continuum from positive to negative, and that these can be measured using an interval scale, such as the Likert. Furthermore, certain attitudes toward oneself as a professional will increase or decrease the likelihood of certain patterns of behavior. This relies on the assumption that cognitive/affective and psycho motor functions are inextricably linked. The instrument has proven to be reliable and flexible in this context. Factor analytic studies on the PSCNI have supported the construct validity of the subscale structure. Cronbach’s alpha was 0.89 for internal consistency. Acceptable internal consistency measures were reported for the subscales in two separate studies in Australia and Canada,47 in which the Cronbach’s alpha estimates for the professional practice subscale were 0.85 and 0.89, and for the satisfaction subscale were 0.82 and 0.82. The communication subscale was weaker at 0.40 and 0.59 in these studies. Observational checklist An observational checklist was developed by the researcher to measure the caring and professional behavior of nursing students. It included 30 items divided into two subscales, ie, caring behavior (12 items) and professional behavior (18 items). The scoring system was as follows: 1 (never), 2 (rarely), 3 (usually), and 4 (always). Reliability of the tool was 0.96. The alpha coefficient was 0.83. Content validity was confirmed by a panel of six experts, and any necessary modifications were done by omitting items that could not be measured or performed by female nursing students, and replacement items were added as advised. Knowledge test questionnaire A knowledge test questionnaire comprising 40 multiple questions was developed by the researcher and used to measure nursing students’ knowledge regarding both the concepts of caring and professional behavior. Reliability of the tool was 0.88. The alpha coefficient was 0.86. Content validity was confirmed by a panel of ten experts and modifications were made by omitting two difficult questions and addition of two questions as advised. Pilot study A pilot study was conducted in ten subjects to test the clarity and validity of the content of the study tools. Necessary modifications were done by adding or omitting questions and increasing the time needed to respond to each questionnaire. Procedure Prior to implementation of the training course, permission was obtained from the nursing supervisors of the selected units. This was intended to facilitate data collection and to explain the purpose of the study. At the beginning of the course, nursing students were invited to participate in the study. The researcher explained the study purpose and procedures to the randomly selected sample. Student nurses were further informed that participation was voluntary and that study findings would be presented group wise and no individual would be recognized. A knowledge test was then provided to students in the study and control groups as well as the CDI and professional self-concept scales to assess perceptions about the two concepts. Observations of caring and professional behavior were carried out while the subjects were caring for their patients utilizing the designed observational check lists. The educational sessions were held twice a week for the study group, while the control group received only the usual course orientation. Six sessions were held, lasting an hour each. As the level of knowledge of the group increased, the content of sessions developed in both depth and range of concepts covered. Contents of the educational sessions were perception of caring and nursing (session 1), caring theories and values (session 2), holistic approach to caring (session 3), concept of professionalism (session 4), ethics and professionalism (session 5), and communication, interpersonal relationship, and assertiveness (session 6). These sessions were followed by supervision of the nursing students in their clinical areas covering different work shifts in order to provide each student with support, as he/she became involved in new nursing situations and implemented out his/her new knowledge. This was also intended to bridge the gap between theory and practice by providing a role model. The supervision focused on conceptualizing nursing in terms of activities of daily practical work and on interpersonal skills emphasizing nurses as caring professionals. Groups of four or five nursing students met twice weekly. These small groups allowed students to learn how other students practiced. Students also had the opportunity to receive input and feedback from each other, particularly when they were feeling overwhelmed by a certain situation or family. The group members helped each other look at the situation in new ways (reframing), suggested trying certain interventions, and provided support to the student who was trying out a new intervention. Sometimes, the group provided the student with a setting in which to seek support with the difficult experiences that they had encountered, such as the death of a patient. The program thus encouraged reflection and increased self-awareness. This type of clinical teaching is seen as an opportunity to unite theory and practice. It encourages students to be receptive to patients and places value on contextual experiences. An examination of contextual experience shows that care is relational and encourages connections with others. Care is sustained through relationships that give voice to nursing students and patients. This process of giving voice makes caring more visible to others. This visibility allows nurses to celebrate caring occasions and validates worth of caring in nursing. Thus, instead of only attending lectures on course content, nursing students shared their personal stories about caring experiences with their colleagues, how they felt, and what the experiences had meant for them. This type of interactive experience strategy provided meaningful insights for them and formed the basis for dialog and sharing of meaning and values. Eight supervised sessions were held and the overall program period lasted 2 months, including both the theoretical component and the supervised sessions. To assess the impact of the program on caring behavior and professional self-perception of nursing students, the previous instruments were utilized immediately after the program and 3 months later. Statistical analysis Statistical analyses were done utilizing SPSS version 11.5 (SPSS Inc, Chicago, IL). A P value <0.05 was considered to be statistically significant. The analysis proceeded in phases. In the first phase, a descriptive profile of the study and control groups was done. The Chi-square statistic and t-test were used to determine if there were any statistically significant baseline differences between the groups. In the next phase, the effects of group and time on study outcomes were examined using repeated-measures analysis of variance. The investigators looked at the significant effect of the interaction term, which was group by time. If this interaction term was statistically nonsignificant, the independent effects of time and group were evaluated separately. The independent sample t-test was also carried out to test the difference between group means at each given time point. Results Regarding demographic characteristics, as shown in Table 1, 50 students were randomized into the study and control groups (n = 25 each). The majority of students were aged 21–23 years, with a mean age of 21 years. Their educational background reflected that of nursing students in general, with the majority (88.3%) being secondary school graduates. The rest comprised students with technical education. Almost half of the participants had previous working experience (14 in the study group versus 17 in the control group) for less than 2 years, with a mean career duration of 1.2 years. There were no statistically significant differences between the study groups at baseline for any of the socio demographic characteristics. It should be noted that all students undertook a 4-week general orientation program before actual commencement of training. To show the impact of the program on the study outcomes, repeated-measures analysis of variance was used to assess the effectiveness of the proposed program by examining differences in changes in study outcomes across time between the groups. Group and time were treated as the independent between-subject and within-subject variables, respectively. Table 2 shows comparison of the total mean scores for the knowledge test at the three time points (baseline, immediately following the program, and 3 months later) between the two groups. The total knowledge score was not significantly different between the groups at baseline. The interaction term for time by group was statistically significant (F = 35.33, at P < 0.001), and so was the main effect of group (F = 28.71, at P < 0.001), indicating that the program had a consistent effect on improving overall knowledge in the study group, which exceeded that of the control group subjects across the study period. The study group showed significantly better knowledge immediately after the program than at follow-up. For caring, as shown in Table 3, the product term (for group by time) was statistically significant (F = 113.19, P < 0.000), and was the main effect for the intervention group, which further supports the beneficial effect of the program in improving the caring perception. High scores on the CDI indicate agreement that nursing action is considered to be caring. Differences in scores on the CDI subscales were further investigated using the independent Student’s t-test to see if any scores changed significantly between the two groups at the different assessment points. Mean scores for the CDI subscales changed significantly between study time points, according to t-test results. The characteristics of the subscale scores strongly endorsed the psychosocial aspects of nursing, as well as professional and technical aspects, as shown in Table 4. Looking at the data 3 months after conclusion of the program, it was clear that students in the interventional group perceived caring in more psychosocial, technical, and professional terms than did the students in the control group. There were significant changes in the scores for a range of subscales as supported by multivariate analysis of variance, suggesting that nursing students had improved ideals about caring and nursing 3 months after the interventional program. Involvement was also endorsed strongly by students in the interventional group compared with those in the control group, whereas altruism was less well endorsed in the interventional group. By inspecting the univariate t-test results, it was found that the groups showed a statistically significant difference at the different study time points. The baseline means and standard deviations for the CDI subscales are reported in Table 4. Repeated-measures analysis of variance also showed a statistically significant positive trend of endorsing overall professional self-perception for the interventional group compared with the control group (F = 20.6, P < 0.000). Time was also shown to have a statistically significant effect (F = 9.64, P < 0.000). With the negative item scores reversed, the maximum possible score on the PSCNI was 97. Table 5 shows the spread of results for the PSCNI at the different assessment time points. Concerning the PSCNI dimensions, inspection of the product term for group by time and the independent effect for group alone and time alone, showed that the study group demonstrated better outcomes, as seen in Table 6. These results collectively favor the effect of the interventional program. Comparison of scores for the dimensions on the PSCNI showed a relative difference in terms of mean scores. The dimensions soliciting the strongest positive response, in descending order, were professional practice, being more flexible, satisfaction, being skillful, and leadership. Table 7 shows attitude means, standard deviations, and repeated-measures analysis of variance for both groups at baseline and at each study assessment point thereafter, with both groups having similar attitudes at baseline. It is clear from Table 7 that the product terms for group by time for caring and professional self-perception attitudes between the interventional and control groups were all significant, and that the interventional group demonstrated better attitudes. Again, time had a significant effect on all the outcomes studied in both the interventional and control groups. The t-test of the mean difference between groups at each successive time point is also shown. These results collectively favor the effect of the studied education program in improving attitudes toward caring and professional self-perception over time. Because the researcher was interested in the retained effect from the program, correlations were done between scores on both the CDI and PSCN at baseline and at follow-up. Results of this analysis are shown on Table 8 and Table 9. This analysis yielded statistically significant results. Among the study groups, Table 8 shows that knowledge change scores were positively correlated with satisfaction. Caring was correlated with psychosocial, technical/professional, altruism, involvement, and leadership changes. Moreover, professional self-perception was significantly correlated with skill changes. As shown in Table 9, the control group showed a significant correlation between caring and psychosocial and technical/professional change scores. Professional self-perception changes were correlated with changes in attitude about skill, leadership, flexibility, and autonomy. Discussion Our rapidly changing health care system is challenging the nursing profession to confront ethical, moral, and legal dilemmas and to define itself, its services, and appropriate roles, including advocacy for the needs of clients. Acceptance of responsibility and accountability for one’s own actions as well as maintenance of continuous competence characterize professional nurses.48 Nursing is a professional discipline with both arts and science components. As an art, nursing involves the implementation of caring strategies to promote well-being, including intuition, creativity, compassion, nursing therapeutics, communication skills, patient advocacy, and empowerment. Nursing education guides and encourages caring behavior, preparing the students for their ever changing role in nursing practice. It is the goal of nursing educational programs to provide students with multiple models of professional activities to aid in their development of professional identity.8 The present study investigated the effect of an interventional education program on caring behavior and professional self-perception among nursing students, and was based on the hypothesis that nursing students who participate in an educational program will score higher for knowledge about caring and professionalism than those who do not participate in such a program. This hypothesis is supported by the study results, showing statistically significant differences between the intervention and control groups regarding knowledge of caring and professionalism. These results are consistent with those of Suchitra and Lakshmi Devi49 who found an increased number of subjects scoring good or excellent in their post education knowledge questionnaire, and commented that education has a positive impact on retention of knowledge. In the same domain, Zeiger50 added that continuing education programs in nursing beyond basic preparation designed to promote and enrich knowledge, improve skills, and develop attitudes for enhancement of nursing practice, and the profession of nursing, is advanced because its practitioners are enriched by dissemination of new skills and knowledge through educational programs. The results of the present study also demonstrated significantly better knowledge in the study group immediately after implementation of the program than 6 months afterwards. This result is again consistent with the report by Suchitra and Lakshmi Devi,49 who found that a nurse’s overall knowledge declined with the passage of time. From the researcher’s point of view, while there was an improvement in knowledge following the educational program, there is always a need for further improvement in knowledge levels, including knowledge retention based on memory and the ability to recall. The study results favor the effect of the educational program on improvement of student’s knowledge in comparison with the control group which received nothing except general clinical orientation given at the beginning of the course for all nursing students in different clinical areas. The study was also based on another hypothesis that nursing students who participate in an interventional education program will score higher on aspects of caring and would develop more positive professional self-perception than those who do not participate in such a program. This hypothesis is supported by the study results showing significant improvement in caring perception and a positive trend of endorsing overall professional self-perception for the interventional group compared with the control group during different periods of assessment after implementation of the program. In this respect, Zeiger50 asserted that both training and supervision are essential in developing core skills and a positive professional identity. With supervision students will acquire a solid professional identity that includes commitment to professional growth through continued learning. On the same issue, Davis and North51 emphasized that nursing education develops students as people and citizens, in addition to being practitioners. During the educational sessions, faculty members guided students to use their strengths and inner resources, which are essential to the development of caring, moral, and ethical nurses who demonstrate integrity as people, citizens, and nurses. From the researcher’s point of view, during the supervision sessions, nursing students received guidance and direction, and gained much knowledge and experience from our practical hands-on approach. This favors the effect of our program in the interventional group compared with the control group. When caring subscales were compared between the interventional group and the control group during different periods of assessment after the program, significant changes were evident for mean scores between the two groups, with the interventional group perceiving caring in more psychological, technical, and professional terms than the control group. This result is consistent with that reported by Moore et al52 who emphasized the importance of educational programs in enhancing caring behavior in nurses. They added that programs should stress the importance of learning to value the types of behaviors and interactions that the patients consider to be quality of care and systematically incorporate these interactions in performance and measures of care. Moreover, Moore et al,52 when assessing the caring behavior of skilled maternity care providers, demonstrated that there is still a long way to go to ensure that the level of care that all women expect and deserve is routinely available to them as a routine element of care. Moore et al asserted the importance of behavior change strategies to increase caring behavior on the part of caregivers. In the same domain, Suchitra and Lakshmi Devi49 stated that the development of a caring relationship between caregivers and patient reinforces the holistic approach, and that caring is the basic element in the development of a therapeutic relationship and provides a balance between the “high touch” human response and the “high tech” nature of today’s health care environment. Moreover, Brunton and Beaman17 in their study of perception of caring behavior in nursing after introducing an educational program, highlighted the importance of the emotional aspects of caring, that included appreciating the patient as a human being, showing respect for the patient, being sensitive to the patient, being honest, talking to the patient, listening attentively and treating patient information confidentially, and maintaining patient privacy. They also added that placing “caring” practice high on the agenda and integrating sensitization about caring issues into all aspects of care provider’s training is key. When professional self-perception subscales were investigated in the interventional and study groups, the interventional group demonstrated better outcomes during different periods of assessment after implementation of the program in relation to their professional practice, being more flexible, satisfaction, being skillful, as well as their leadership behavior. The results of the present study also show that the interventional group demonstrated better caring and professional attitudes than the control group. In this respect, Martin and Ashton3 stated that educational programs enhance the development of professional values and value-based behavior. Understanding the values that patients and other health professionals bring to the therapeutic relationship is critically important to providing quality of patient care. During the educational program, nursing students are prepared for numerous dilemmas that will arise in practice and are able to make and assist others in making decisions within a professional ethical framework. Martin and Ashton3 asserted that by introducing educational programs, nurse’s graduate professionalism is enhanced by demonstration of care values and by applying principles of altruism, excellence, caring, ethics, respect, communication, leadership, and accountability. Although the control students received no relevant intervention during the program, they had a slight increase in mean scores on some subscales of caring and professionalism during different periods of assessment, but these were still lower than those of the study group. This result could be the result of several factors, given that students in the control group were supervised by their clinical instructors in the faculty of nursing, and underwent periodic evaluation by their clinical instructors for communication, accountability and responsibility, leadership, self-awareness, caring behavior, and professional conduct, and thus their behavior changed and they demonstrated a better outcome. From the researcher’s point of view, frequent interaction between nursing students and their tutors in the interventional group offered opportunities for students to learn more professional roles. Recommendations In light of the findings of this study, the researcher recommends that the nursing curriculum should incorporate concepts and principles that guide students in developing caring, safe, competent, and professional behaviors. The emphasis of the curriculum should be to ensure that all our nursing graduates are able to maximize their growth, and develop and expand their unique qualities. Faculty members and clinical instructors must act as role models and facilitate learning by providing an environment that promotes holistic care, inquiry, critical thinking, accountability, and more autonomous and professional behavior. Nurses serving as mentors as well as members of the multidisciplinary health care team, so it is essential to foster caring and professional behavior among newly graduated nurses. Nursing students should seek educational opportunities to acquire knowledge for role preparation, to participate in knowledge generation, and for personal and professional development. Finally, additional research should include replication of this study in baccalaureate nursing programs, as well as baccalaureate programs in other regions. Conclusion The study findings favor the effect of the interventional program because there were statistically significant differences between the intervention and control groups regarding their mean scores on knowledge about caring and professionalism. The interventional group showed significantly better knowledge test scores than the control group during the different periods of assessment. There were also statistically significant differences between the interventional and control groups regarding caring outcomes. The interventional group showed significantly improved caring perception in more psychological, technical, and professional terms than the control group during different periods of assessment. In addition, statistically significant differences were found in mean scores regarding professional self-perception between the two groups, with a significant positive trend of overall professional self-perception for the interventional group compared with the control group. Finally, the interventional group demonstrated better caring and professional self-perception attitudes than the control group during the different periods of assessment. Disclosure The author reports no conflicts of interest in this work. Table 1 Socio demographic characteristics of the study participants (n = 50) Characteristic Intervention group Control group X2, * n % n % Age ≤21 years 20 70.0 16 60.0 0.66 (0.59) 22–24 years 5 30.0 9 40.0 Marital status Not married 23 93.3 24 96.7 0.35 (1.0) Married 2 6.7 1 3.3 Prior education Secondary school 22 90.0 21 86.7 0.16 (1.0) Technical education 3 10.0 4 13.3 Previous work experience Yes 14 46.7 12 56.7 0.60 (0.61) No 11 53.3 13 43.3 Table 2 Knowledge outcomes for intervention and control groups at different study time points (n = 50) Variable Control group Intervention group ta-(P value) M SD M SD Knowledge Baseline 5.93 1.76 6.57 2.22 1.22 (0.23) Post program 5.93 1.48 12.3 2.39 11.89 (0.000) 3 months 6.13 2.81 10.77 2.82 6.37 (0.000) F-test for repeated measure (P value) Time 43.98 (P < 0.000) Group 110.75 (P < 0.000) GTb 42.47 (P < 0.000) Notes: a t-test of comparison between study and control groups at each time point; b F-test for product of time by group; P < 0.05; P < 0.01; * P < 0.001. Abbreviation: SD, standard deviation. Table 3 Caring outcomes for intervention and control groups at different study time points (n = 50) Variable Control group Intervention group ta-(P value) M SD M SD Caring total score Baseline 94.65 93.3 95.53 5.93 0.68 (0.499) Post program 93.27 2.82 111.15 4.57 18.25 (0.000) 6 months 97.07 2.84 117.47 2.35 30.34 (0.000) F-test for repeated measures (P value) Time 150.38 (P < 0.000) Group 457.37 (P < 0.000) GTb 113.19 (P < 0.000) Notes: a t-test of comparison between study and control groups at each time point; b Product of time by group; P < 0.05; P < 0.01; * P < 0.001. Abbreviation: SD, standard deviation. Table 4 Caring subscale outcomes for intervention and control groups at different study time points (n = 50) Variable Control group Intervention group ta-(P value) M SD M SD Psychosocial Baseline 37.63 1.75 38.97 2.68 2.28 (0.026) Post program 37.53 1.85 44.55 2.34 12.89 (0.000) 3 months 38.76 1.45 46.70 1.21 23.35 (0.000) F (bP value) 52.1 (P < 0.000) Technical professional Baseline 35.67 2.58 35.65 2.39 −0.10 (0.92) Post program 34.50 1.36 41.63 2,25 14.86 (0.000) 3 months 35.10 1.61 42.3 1.71 16.85 (0.000) F (bP value) 53.51 (P < 0.000) Altruism Baseline 11.87 0.86 11.83 1.02 −0.14 (0.89) Post program 11.50 0.82 13.93 0.93 1.66 (0.000) 3 months 11.70 0.54 14.00 0.87 12.32 (0.000) F (bP value) 41.397 (P < 0.000) Involvement Baseline 9.40 1.74 9.13 1.94 −0.37 (0.47) Post program 9.73 1.20 11.03 1,098 4.38 (0.000) 3 months 11.60 10.69 14.47 0.73 12.12 (0.000) F (bP value) 19.39 (P < 0.000) Notes: a t-test of comparison between study and control groups at each time point; b Product of time by group; * P < 0.05; P < 0.01; * P < 0.001. Abbreviation: SD, standard deviation. Table 5 Professional self-perception outcomes for intervention and control groups at different study time points (n = 50) Variable Control group Intervention group ta-(P value) M SD M SD Professional self-perception total Baseline 77.63 4.79 81.23 6.32 2.49 (0.016) Post program 78.93 4.10 83.47 4.05 4.31 (0.000) 3 months 74.90 2.25 84.47 1.57 23.10 (0.000) F-test for repeated measures (P value) Time 9.64 (0.000) Group 96.92 (0.000) GTb 20.63 (0.000) Notes: a t-test of comparison between study and control groups at each time point; b Product of time by group; P < 0.05; P < 0.01; * P < 0.001. Abbreviation: SD, standard deviation. Table 6 Professional self-perception subscale outcomes for intervention and control groups at different study time points (n = 50) Variable Control group Intervention group ta-(P value) M SD M SD Skill Baseline 14.97 1.65 15.90 5.65 0.87 (0.39) Post program 15.13 1.50 17.10 1.52 5.05 (0.000) 3 months 13.83 1.39 18.37 0.99 14.49 (0.000) F (bP value) 13.87 (P < 0.000) Leadership Baseline 12.03 1.38 12.33 1.49 0.81 (0.422) Post program 12.23 1.01 12.77 1.28 1.796 (0.078) 3 months 10.90 0.712 12.40 0.724 8.09 (0.000) F (bP value) 5.32 (P = 0.007) Flexibility Baseline 21.50 1.28 21.93 1.41 1.25 (0.218) Post program 21.53 1.63 24.80 1.79 7.38 (0.000) 3 months 19.90 1.35 25.60 1.38 16.19 (0.000) F (bP value) 47.97 (P = 0.000) Satisfaction Baseline 18.73 0.83 19.77 0.82 4.87 (0.000) Post program 19.33 2.04 18.80 1.67 −1.11 (0.272) 3 months 18.83 2.41 20.07 2.24 2.04 (0.046) F (bP value) 5.04 (P = 0.010) Communication Baseline 10.30 1.84 11.00 2.51 1.23 (0.222) Post program 10.75 1.51 10.00 1.86 −1.60 (0.115) 3 months 9.53 1.042 8.33 0.92 −4.72 (0.000) F (bP value) 7.27 (P = 0.001) Professional practice Baseline 48.50 3.15 50.17 6.13 1.32 (0.191) Post program 48.90 2.75 54.67 3.49 7.12 (0.000) 3 months 44.63 2.11 56.67 1.75 23.44 (0.000) F (bP value) 31.88 (0.000) Notes: a t-test of comparison between study and control groups at each time point; b Product of time by group; * P < 0.05; P < 0.01; * P < 0.001. Abbreviation: SD, standard deviation. Table 7 Caring and professional self-perception attitudes for intervention and control groups at different study time points (n = 50) Variable Control group Intervention group ta-(P value) M SD M SD Competence attitude Baseline 13.00 1.17 13.75 1.78 1.79 (0.078) Post program 16.40 1.07 21.13 1.55 13.78 (0.000) 3 months 17.50 1.37 22.13 1.07 14.66 (0.000) F (bP value) 61.09 (0.000) Communication attitude Baseline 10.57 1.55 11.03 1.30 1.27 (0.211) Post program 12.17 0.91 14.67 1.21 9.02 (0.000) 3 months 10.83 0.95 14.13 1.01 13.03 (0.000) F (bP value) 29.40 (0.000) Autonomy attitude Baseline 3.30 0.65 2.97 1.03 −1.50 (0.140) Post program 4.77 0.78 5.83 0.34 6.78 (0.000) 3 months 5.30 0.75 6.57 0.97 5.65 (0.000) F (bP value) 17.29 (0.000) Leadership attitude Baseline 10.27 1.08 10.47 1.43 0.61 (0.544) Post program 12.87 1.41 16.27 1.01 10.73 (0.000) 3 months 13.07 1.36 17.07 1.93 9.28 (0.000) F (bP value) 37.86 (0.000) Professional-self attitude total Score Baseline 37.13 2.85 38.17 3.77 1.19 (0.236) Post program 46.20 2.30 57.90 2.77 17.81 (0.000) 3 months 46.70 2.45 59.90 3.19 17.98 (0.000) F (bP value) 113.03 (0.000) Caring attitude total score Baseline 26.13 2.03 28.40 6.28 1.88 (0.065) Post program 30.13 1.31 39.47 2.24 19.72 (0.000) 3 months 31.47 2.27 41.90 2.26 17.82 (0.000) F (bP value) 19.45 (0.000) Notes: a t-test of comparison between study and control groups at each time point; b Product of time by group; * P < 0.05; P < 0.01; * P < 0.001. Abbreviation: SD, standard deviation. Table 8 Partial correlation matrix for outcome measures change scores six months from the program for the study group (n = 30) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 1. Knowledge – 2. Psychosocial 0.17 – 3. Technical/professional −0.01 0.42* – 4. Altruism 0.24 0.04 0.45* – 5. Involvement 0.11 0.20 0.14 −0.06 – 6. Skill −0.01 −0.00 −0.07 −0.13 0.09 – 7. Leadership −0.03 0.20 0.39* 0.24 0.11 −0.15 – 8. Flexibility 0.13 0.15 0.18 −0.03 −0.02 0.09 0.12 – 9. Satisfaction −0.38* −0.26 −0.01 −0.18 −0.41* 0.20 −0.22 0.12 – 10. Communication 0.33 0.18 0.06 0.26 0.52** −0.07 0.09 −0.11 −0.46* – 11. Attitude competence 0.32 −0.09 −0.08 0.06 0.16 −0.06 0.13 −0.06 −0.41* 0.18 – 12. Attitude communication −0.16 −0.15 0.15 0.01 −0.22 0.06 −0.13 0.11 −0.09 −0.31 −0.03 – 13. Attitude autonomy −0.13 0.13 0.12 −0.19 0.31 0.11 −0.10 0.04 −0.21 0.06 −0.05 0.02 – 14. Attitude leadership −0.02 −0.14 0.12 0.10 0.01 0.06 0.14 −0.06 0.05 −0.20 −0.04 −0.01 0.34 – 15. Attitude caring total score −0.01 −0.23 −0.37* 0.21 −0.22 −0.01 −0.09 −0.04 0.02 0.06 0.41* −0.04 −0.19 0.19 – 16. Attitude professional self total score 0.03 −0.13 0.14 0.03 0.12 0.07 0.06 −0.01 −0.27 −0.13 0.41* 0.37* 0.57* 0.73 0.22 – 17. Caring total score (CDI) 0.16 0.72 0.83** 0.45* 0.49** −0.03 0.38* 0.14 −0.35 0.35 −0.01 −0.07 0.18 0.03 −0.31 0.06 – 18. Professional self-perception total score −0.01 0.06 0.09 −0.02 0.12 0.85** 0.13 0.40* 0.34 0.11 −0.12 −0.10 0.01 0.02 −0.02 −0.07 0.11 – Notes: ** Correlation is significant at the 0.01 level (two-tailed); * Correlation is significant at the 0.05 level (two-tailed). Table 9 Partial correlation matrix for outcome measures change scores 3 months from the program for the control group ( n = 25) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 1. Knowledge – 2. Psychosocial −0.32 – 3. Technical/professional −0.20 0.33 – 4. Altruism 0.19 0.22 0.002 – 5. Involvement 0.04 −0.28 −0.21 −0.13 – 6. Skill 0.04 −0.07 −0.30 0.09 0.09 – 7. Leadership 0.11 −0.27 −0.15 0.05 −0.28 0.33 – 8. Flexibility −0.22 0.03 −0.13 −0.15 −0.01 −0.11 0.17 – 9. Satisfaction −0.41 0.31 0.02 0.21 0.03 −0.14 −0.12 0.03 – 10. Communication −0.14 −0.03 −0.06 0.05 −0.22 0.09 −0.10 0.24 0.31 – 11. Attitude competence 0.34 0.01 −0.44* 0.05 0.33 0.06 −0.43* 0.03 −0.17 −0.12 – 12. Attitude communication −0.01 0.05 −0.29 0.03 −0.17 0.16 0.25 0.06 0.02 0−0.01 0.12 – 13. Attitude autonomy −0.01 0.17 0.26 0.06 −0.17 −0.39* −0.23 −0.38* 0.04 −0.02 −0.04 −0.36 – 14. Attitude leadership 0.18 0.15 0.04 0.27 −0.10 0.11 −0.02 −0.02 −0.17 −0.03 0.12 0.31 0.18 – 15. Attitude caring total score −0.31 0.32 −0.17 −0.26 −0.11 −0.02 0.04 0.11 0.21 0.11 0.13 0.31 0.07 −0.01 – 16. Attitude professional self total score 0.26 0.15 −0.24 0.20 −0.01 0.05 −0.18 −0.08 −0.15 −0.08 0.60 0.55 0.21 0.77 0.21 – 17. Caring total score −0.26 0.71 0.79 0.29 0.04 −0.19 −0.34 −0.11 0.24 −0.12 −0.17 −0.23 0.22 0.13 −0.05 −0.05 – 18. Professional self-perception total score −0.04 −0.13 −0.30 0.01 −0.05 0.82 0.62 0.60 −0.12 0.13 −0.10 0.21 −0.49 0.06 0.05 −0.07 −0.15 – Notes: Correlation is significant at the 0.01 level (two-tailed); * Correlation is significant at the 0.05 level (two-tailed). ==== Refs References 1 Andersson EP The perceptions of student nurses and their perceptions of professional nursing during the nurse training programme J Adv Nurs 1993 18 808 815 8514938 2 Cowin L Measuring nurses’ self-concept West J Nurs Res 2001 23 313 325 11291434 3 Martin P Ashton C The Essentials of Baccalaureate Education for Professional Nursing Practice Washington DC American Association of Colleges of Nursing 2008 4 Watson R Lea A Perceptions of caring among nurses: the influence of age and sex J Clin Nurs 1998 7 97 9510714 5 Kyle TV The concept of caring: a review of the literature J Adv Nurs 1995 21 506 514 7745205 6 Randle J Bullying in the nursing profession J Adv Nurs 2003 43 395 401 12887358 7 Walsh M Dolan B Emergency nurses and their perceptions of caring Emerg Nurse 1999 7 24 31 8 Kosowski MM Clinical learning experiences and professional nursecaring: a critical phenomenological study of female baccalaureate nursing students J Nurs Educ 2002 34 235 242 7790976 9 Lea A Watson R Perceptions of caring among nurses: the relationship to clinical area J Clin Nurs 1995 8 617 10786537 10 Lea A Watson R Deary IJ Caring in nursing: a multivariate analysis J Adv Nurs 1998 28 662 671 9756236 11 Barker P Reflections on caring as a virtue ethic within an evidence-based culture Int J Nurs Stud 200;37 329 336 12 Lee-Hsieh J Kuo CL Tseng HF Application and evaluation of a caring code in clinical nursing education J Nurs Educ 2005 44 177 184 15862051 13 Arthur D Randle J The professional self-concept of nurses: a review of the literature from 1992–2006 Aust J Adv Nurs 2007 24 60 64 17518168 14 Sarmiento TP Laschinger HK Iwasiw C Nurse educators’ workplace empowerment, burnout, and job satisfaction: testing Kanter’s theory J Adv Nurs 2004 46 135 143 15 Bankert EG Kozel VV Transforming pedagogy in nursing education: a caring learning environment for adult students Nurs Educ Perspect 2005 26 227 229 16175914 16 Larson PJ Ferketich SL Patients’ satisfaction with nurses’ caring during hospitalization West J Nurs Res 1993 15 690 703 8284928 17 Brunton B Beaman M Nurse practitioners’ perceptions of their caring behaviors J Am Acad Nurse Pract 2000 12 451 456 11930383 18 Watson J Assessing and Measuring Caring in Nursing and Health Science New York, NY Springer 2002 19 Watson R Deary IJ Lea A A longitudinal study of into the perceptions of caring among student nurses using multivariate analysis of the caring dimensions inventory J Adv Nurs 1999 30 1080 1089 10564407 20 Ironside P Diekelmann N Hirschmann M Learning the practices of knowing and connecting: the voices of students J Nurs Educ 2005 44 153 155 15862047 21 Leininger MM Caring: a Central Focus of Nursing and Health Care Services Detroit, MI Wayne State University Press 1988 22 Fahrenwald NL Bassett SD Tschetter L Carson PP White L Winterboer VJ Teachingcore nursing values J Prof Nurs 2005 21 46 51 15682160 23 Omdahl BL O’Donnell C Emotional contagion, empathic concern and communicative responsiveness as variables affecting nurses’ stress and occupational commitment J Adv Nurs 1999 29 1351 1359 10354229 24 Oermann M Professional Nursing Practice: a Conceptual Approach Philadelphia, PA JB Lippincott Company 1998 25 Diekelmann N Schooling, learning and teaching: toward a narrative pedagogy 2010 Available at: http://www.son.wisc.edu/diekelmann/research/slt/index.html . 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Training Year 2008–2009 The big picture, Understanding the Consortium 2008 51 Davis R North I Nursing Program, Student Handbook New York, NY National League for Nursing Accreditation Commission 2005 52 Moore M Armbruster D Graeff J Copeland R Assessing the: Caring” Behaviors Of Skilled Maternity Care Providers During Labor and Delivery: experience from Kenya and Bangladesh Washington, DC 2002	23745088	PMC3661257	NO-CC CODE	2021-01-04 22:45:05	yes	Adv Med Educ Pract. 2011 Jun 29; 2:173-185
==== Front J Environ Sci Health BJ Environ Sci Health BlesbJournal of Environmental Science and Health. Part. B, Pesticides, Food Contaminants, and Agricultural Wastes0360-12341532-4109Taylor & Francis 10.1080/03601234.2012.636575Research ArticleAssays of dioxins and dioxin-like compounds in actually contaminated soils using transgenic tobacco plants carrying a recombinant mouse aryl hydrocarbon receptor-mediated β-glucuronidase reporter gene expression system Inui Hideyuki 12Gion Keiko 1Utani Yasushi 2Wakai Taketo 2Kodama Susumu 23Eun Heesoo 4Kim Yun-Seok 45Ohkawa Hideo 1261 Research Center for Environmental Genomics, Kobe University, Kobe, Hyogo, Japan2 Graduate School of Science and Technology, Kobe University, Kobe, Hyogo, Japan3 Division of Drug Metabolism and Molecular Toxicology, Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba-ku, Sendai, Miyagi, Japan4 Chemical Analysis Research Center, National Institute for Agro-Environmental Sciences, Tsukuba, Ibaraki, Japan5 Water Analysis & Research Center, Yeonchuk, Daedeok, Daejeon, South Korea6 Integrated Institute for Regulatory Science, Waseda University, Shinjuku, Tokyo, JapanAddress correspondence to Hideo Ohkawa, Kobe University, Visiting Researcher, Waseda University, Kashio-dai 14-14, Kita-ku, Kobe 651-1255, Japan; E-mail: [email protected] 3 2012 4 2012 47 4 233 239 14 7 2011 Copyright © Taylor & Francis Group, LLC2012This is an open access article distributed under the Supplemental Terms and Conditions for iOpenAccess articles published in Taylor & Francis journals, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.The transgenic tobacco plant XD4V-26 carrying the recombinant mouse aryl hydrocarbon receptor XD4V-mediated β-glucuronidase (GUS) reporter gene expression system was used for assay of dioxins and dioxin-like compounds consisting of polychlorodibenzo-p-dioxins, polychlorinated dibenzofurans, and coplanar polychlorinated biphenyls (Co-PCBs) in actually contaminated soils. The transgenic tobacco plant XD4V-26 showed a significant dose-dependent induced GUS activity when cultured on MS medium containing PCB126 [toxic equivalency factor (TEF) = 0.1]. In contrast, PCB169 and PCB180, which have 0.03 of TEF and unassigned TEF values, respectively, did not significantly induce GUS activity under the same conditions as with PCB126. When the tobacco plants were cultivated for up to 5 weeks on actually contaminated soils with dioxins and dioxin-like compounds collected from the periphery of an incinerator used for disposal of life and industrial wastes, GUS activity in the leaves was dose-dependently increased. The plants clearly detected 360 pg-TEQ g−1 of dioxins and dioxin-like compounds in this assay. There was a positive correlation between GUS activity and TEQ value of dioxins and dioxin-like compounds in the plants. This assay does not require any extraction and purification processes for the actually contaminated soil samples. Biochemical assaydioxinaryl hydrocarbon receptortransgenic plantspolychlorinated biphenyl ==== Body Introduction Polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), and coplanar polychlorinated biphenyls (Co-PCBs), the so-called dioxins and dioxin-like compounds, are highly lipophilic and persistent in the environment. These compounds widely contaminated the environment. These were deposited in sediments in aquatic environments, and then highly accumulated at the tops of food chains, including humans. This contamination mainly first occurred at the sites of incinerators of life and industrial wastes. Therefore, it is important to continuously monitor dioxins and dioxin-like compounds in the periphery of incinerators from the standpoint of risk assessment and management. PCDDs, PCDFs, and Co-PCBs each consist of a number of congeners with different numbers and positions of chlorines attached on two benzene rings and are represented by a toxic equivalency factor (TEF), which was updated by the World Health Organization in 2005.[1] The TEF is a relative toxicity value based on the value 1 of the most toxic 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD). A mixture of these congeners is represented by the total toxic equivalency (TEQ), which is the sum of concentration of each of the congeners multiplied by its TEF. High-resolution gas chromatography/high-resolution mass spectrometry (HRGC/HRMS) is used to identify and quantify residues of PCDDs, PCDFs, and Co-PCBs in environmental samples. This method is sensitive and accurate for measuring extremely low amounts of the residues in a variety of environmental samples. However, extraction and purification of these compounds from samples are imperative and result in a high cost of analysis. In contrast, biochemical assays based on molecular mechanisms of modes of actions of chemicals are suitable for rapid screening of a number of different kinds of samples and are advantageous for assessing the toxicity of these residues in mammals. Most of the biochemical assays estimate toxicity levels of dioxins and dioxin-like compounds in recombinant mammalian hepatoma cells expressing aryl hydrocarbon receptors (AhRs), since AhRs are primarily responsible for toxicity in mammals towards these compounds.[2,3] However, like instrumental analyses, these methods require several steps for extraction and purification of the samples. The transgenic tobacco plants carrying a gene encoding the recombinant AhR, XDV, consisting of the ligand-binding domain of mouse AhR, the DNA-binding domain of bacterial repressor protein LexA, and the transactivation domain of the virus VP16 as well as β-glucuronidase (GUS) reporter gene were genetically engineered. These transgenic tobacco plants showed a significantly increased GUS activity when treated with the AhR ligands such as indigo, β-naphthoflavone, and 3-methylcholanthrene (MC).[4] The transgenic tobacco plants seemed to be useful for a biochemical assay of dioxins and dioxin-like compounds toxic to mammals. The assay did not need any extraction and purification of chemicals, since the plants took up these chemicals in medium through their developed roots by passive diffusion. In this study, the transgenic tobacco plant XD4V-26 was examined for practical assays of PCDDs, PCDFs, and Co-PCBs in actually contaminated soils collected from the periphery of an incinerator used for disposal of life and industrial wastes, since the transgenic plants showed low background activity and dose- and time-dependent GUS activity induced in response to MC. Materials and Methods Chemicals The compounds3,3′,4,4′,5-Pentachlorobiphenyl (PCB126; TEF = 0.1), 3,3′,4,4′,5,5′-hexachlorobiphenyl (PCB169; TEF = 0.03), and 2,2′,3,4,4′,5,5′-heptachlorobiphenyl (PCB180; TEF value was not assigned) were purchased from AccuStandard Inc. (New Heaven, CT, USA). They were each dissolved in dimethyl sulfoxide (DMSO) for application to Murashige and Skoog (MS) medium. The final concentration of DMSO was 0.1 % in MS medium. The compounds 4-Methyl-umbelliferyl-β-D-glucuronide (4MUG) for a substrate of GUS and 4-methyl-umbelliferone (4MU) as a product of GUS reaction were purchased from Nacalai Tesque (Kyoto, Japan). Plants Tobacco plants (Nicotiana tabacum cv. Samsun NN) were transformed by the use of Agrobacterium tumefaciens carrying the plasmid pGP?XD4VGUS as previously described.[4] The resultant transgenic tobacco strain XD4V-26 carries the gene encoding recombinant AhR, which consists of the DNA-binding domain of bacterial LexA [amino acids (AA) 1 to 202], the ligand-binding domain of mouse AhR (a.a. 83 to 494), and the transactivation domain of virus VP16 (a.a. 413 to 490), as well as the gene encoding the reporter GUS. The transgenic tobacco plant XD4V-26 was aseptically and separately incubated in MS medium containing each of the PCB congeners in a growth chamber at 25 °C under 16-h light/8-h dark cycle conditions or grown on the soils contaminated with PCDDs, PCDFs, and Co-PCBs in a closed greenhouse under natural day-length light conditions. Soils Weathered contaminated soils with PCDDs, PCDFs, and Co-PCBs (5100 pg-TEQ g−1) were collected from the periphery of an incinerator used for disposal of life and industrial wastes in Japan. Table 1 shows concentrations of PCDD, PCDF, and Co-PCB congeners in the collected soils. The soils were diluted with uncontaminated soils (1.1 pg-TEQ g−1) purchased from Suntory Flowers Ltd. (Tokyo, Japan) prior to assays. The uncontaminated soils were also used as the control soils. Table 1. Concentrations of PCDD, PCDF, and Co-PCB congeners in the contaminated soils containing 5100 pg-TEQ g−1. PCDD, PCDF, and Co-PCB TEF Concentration (pg g−1) TEQ (pg-TEQ g−1) PCDDs 1,3,6,8-TetraCDD 0 1005.06 0 1,3,7,9-TetraCDD 0 346.84 0 2,3,7,8-TetraCDD 1 22.71 22.71 1,2,3,7,8-PentaCDD 1 476.47 476.47 1,2,3,4,7,8-HexaCDD 0.1 983.23 98.32 1,2,3,6,7,8-HexaCDD 0.1 1235.29 123.53 1,2,3,7,8,9-HexaCDD 0.1 1155.27 115.53 1,2,3,4,6,7,8-HeptaCDD 0.01 17355.54 173.56 1,2,3,4,5,6,7,8-OctaCDD 0.0003 23222.10 6.97 Total PCDDs 79735.53 – PCDFs 1,2,7,8-TetraCDF 0 186.37 0 2,3,7,8-TetraCDF 0.1 111.29 11.13 1,2,3,7,8-PentaCDF 0.03 1193.18 35.80 2,3,4,7,8-PentaCDF 0.3 2565.58 769.67 1,2,3,4,7,8-HexaCDF 0.1 4962.89 496.29 1,2,3,6,7,8-HexaCDF 0.1 6015.95 601.59 1,2,3,7,8,9-HexaCDF 0.1 2237.86 223.79 2,3,4,6,7,8-HexaCDF 0.1 12378.06 1237.81 1,2,3,4,6,7,8-HeptaCDF 0.01 48617.88 486.18 1,2,3,4,7,8,9-HeptaCDF 0.01 9901.31 99.01 1,2,3,4,5,6,7,8-OctaCDF 0.0003 83064.51 24.92 Total PCDFs 326048.35 – Co-PCBs 3,3′,4,4′-TetraCB(#77) 0.0001 110.46 0.01 3,4,4′,5-TetraCB(#81) 0.0003 27.45 0.01 3,3′,4,4′,5-PentaCB(#126) 0.1 919.70 91.97 3,3′,4,4′,5,5′-HexaCB(#169) 0.03 936.83 28.11 2,3,3′,4,4′-PentaCB(#105) 0.00003 480.92 0.01 2,3,4,4′,5-PentaCB(#114) 0.00003 126.31 0.00 2,3′,4,4′,5-PentaCB(#118) 0.00003 941.48 0.03 2′,3,4,4′,5-PentaCB(#123) 0.00003 129.42 0.00 2,3,3′,4,4′,5-HexaCB(#156) 0.00003 938.53 0.03 2,3,3′,4,4′,5′-HexaCB(#157) 0.00003 557.97 0.02 2,3′,4,4′,5,5′-HexaCB(#167) 0.00003 1436.44 0.04 2,3,3′,4,4′,5,5′-HeptaCB(#189) 0.00003 1340.37 0.04 2,2′,3,3′,4,4′,5-HeptaCB(#170) 0 5238.69 0 2,2′,3,4,4′,5,5′-HeptaCB(#180) 0 940.58 0 Total Co-PCBs 7945.89 – Total TEQ 5123.54 Fluorometric GUS assay of transgenic tobacco plants Axillary buds of the transgenic tobacco plants were cultured on MS medium containing 3.0 % (w/v) sucrose, 0.7 % (w/v) agar, and 0.1 to 1000 ng mL−1 PCB126, PCB169, or PCB180. After two weeks, either the second or third leaf from the top of the plant, or both, were used for fluorometric GUS assay as previously described.[5] Soluble fractions were prepared from leaves and incubated with 4MUG After stopping GUS reaction, fluorescence at 360 nM excitation and 450 nM emission was measured with a microplate reader (MTP-100F; CORONA, Katsuta, Japan). GUS activity was determined from a standard curve of the fluorescence of 4MU, and data were presented as means ± standard deviation (SD). Axillary buds were also aseptically cultured on MS medium for about a month. Approximately 10 cm high plants were individually transferred into 1/5000-acre pots filled with the soils contaminated with dioxins and dioxin-like compounds or the uncontaminated soils. The surface of the soils was covered with the uncontaminated soils or aluminum foil. The tobacco plants were grown in a closed greenhouse for several weeks, and then either the second or third leaf from the top of the plants, or both, were assayed for GUS activity as described above. HRGC/HRMS analysis of PCDDs, PCDFs, and Co-PCBs in tobacco plants As previously described, PCDDs, PCDFs, and Co-PCBs were extracted from the aerial parts of the tobacco plants.[6] Congeners of PCDDs, PCDFs, and Co-PCBs were each quantified by HRGC/HRMS (HP6890/Micromass Autospec-Ultima, Micromass Ltd., Manchester, UK) under the same conditions as previously reported.[7] Statistical analysis Statistical analysis used Student's t-test for GUS activity in the transgenic tobacco plants in the response to PCB congeners in the transgenic tobacco plants as well as differences between uncontaminated and contaminated soils. Pearson's correlation coefficient was used for analysis of correlation. Results GUS activity in the transgenic tobacco plant XD4V-26 cultured on MS medium containing PCB congeners The transgenic tobacco plant XD4V-26 was cultured on MS medium containing each of PCB126 (TEF = 0.1), PCB169 (TEF = 0.03), and PCB180 (not assigned TEF value) for 2 weeks, and then leaves were subjected to GUS assay. As shown in Figure 1, a dose-dependent increased GUS activity was clearly observed when the tobacco plants were treated with PCB126, with significant differences at 100 and 1000 ng mL−1 (P < 0.01 and P < 0.05, respectively, relative to no treatment with PCB126, Student's t-test). The GUS activity at 1000 ng mL−1 was 7.4 times higher than that in the plants treated with DMSO alone. In contrast, no dose-dependent increased GUS activity was observed when treated with PCB169 and PCB180. Background GUS activity was detected in tobacco plants treated with DMSO and was significantly decreased by the treatment with increasing doses of PCB169 and PCB180 (P < 0.05). Thus, the present study indicated that the transgenic tobacco plant XD4V-26 showed induced GUS activity in response to the agonist PCB126 and reduced GUS activity in response to the antagonists PCB169 and PCB180. Fig. 1. GUS activity in leaves of the transgenic tobacco plant XD4V-26 cultured for 2 weeks on MS medium containing PCB126, PCB169, or PCB180. Values are means ± SDs (PCB126, n = 9–12; PCB169, n = 6–8; PCB180, n = 3–9). Significant difference (Student's t-test): ∗∗, p < 0.01; ∗, p < 0.05. GUS activity and uptake of PCDDs, PCDFs, and Co-PCBs in the transgenic tobacco plant XD4V-26 cultured on the actually contaminated soils The transgenic tobacco plant XD4V-26 was cultured in pots containing the soils actually contaminated with PCDDs, PCDFs, and Co-PCBs. The GUS activity in their leaves was significantly induced after 5 weeks of culture on the soils containing 360 pg-TEQ g−1 of these congeners (Fig. 2a). On the other hand, the GUS activity in the tobacco plants cultured on the uncontaminated soils containing 1.1 pg-TEQ g−1 was not induced so high. The transgenic tobacco plant XD4V-26 was also cultured in the soils containing 510 and 5100 pg-TEQ g−1 for 33 days. The leaves were subjected to assay of GUS activity, and the aerial parts were analyzed in HRGC/HRMS for PCDD, PCDF, and Co-PCB congeners. The GUS activity in the tobacco plants cultured on the soils containing 5100 pg-TEQ g−1 was twice higher than that in the plants cultured on the soils of 510 pg-TEQ g−1 (Fig. 2b). The concentrations of PCDDs, PCDFs, and Co-PCBs in the plants cultured on the soils containing 5100 pg-TEQ g−1 were higher than those of the plants cultured on the soils of 510 pg-TEQ g−1 (Fig. 2c). Particularly, the concentrations of 1,2,3,4,5,6,7,8-octaCDD and 2,3′,4,4′,5-pentaCB (PCB118) in the plants were the highest, followed by 2,3,3′,4,4′-pentaCB (PCB105) and others (Fig. 3). Fig. 2. Time-dependent (a) and dose-dependent (b) GUS activity, and concentrations of PCDD, PCDF, and Co-PCB congeners (c) in the transgenic tobacco plant XD4V-26 cultured on the actually contaminated soils. The transgenic plant XD4V-26 was cultured on the soils containing 510 or 5100 pg-TEQ g−1 for 33 days. The induction rate is expressed as the GUS activity in the transgenic plants cultured on the contaminated soils divided by the activity in the plants cultured on the uncontaminated soils. Values are means ± SDs (a: uncontaminated, n = 11; dioxins 360 pg-TEQ g−1, n = 16–17; b: uncontaminated, n = 4; 510 pg-TEQ g−1, n = 8; 5100 pg-TEQ g−1, n = 8; c: 510 pg-TEQ g−1, n = 8; 5100 pg-TEQ g−1, n = 7). Significant difference (Student's t-test): ∗∗, P < 0.01 versus uncontaminated soils (a, b) and 510 pg-TEQ g−1 (c). Fig. 3. Concentrations of PCDD, PCDF, and Co-PCB congeners in the transgenic tobacco plant XD4V-26 cultured on the contaminated soils containing 510 and 5100 pg-TEQ g−1 for 33 days. Values are means ± SDs (510 pg-TEQ g−1, n = 8; 5100 pg-TEQ g−1, n = 7). Correlation between GUS activity and TEQ values of PCDDs, PCDFs, and Co-PCBs taken up into the transgenic tobacco plant XD4V-26 There was a significant positive correlation [0.681 (Person's correlation coefficient), P < 0.01, Fig. 4] between the GUS activity and TEQ values of PCDDs, PCDFs, and Co-PCBs in the tobacco plant. In contrast, the transgenic tobacco plants grown on the uncontaminated soils that contained low level of PCDDs, PCDFs, and Co-PCBs including toxic and non-toxic congeners, accumulated as indicated by 0.5 to 1.0 pg-TEQ g−1, although the GUS activity was very low. These results suggested that the induced GUS activity may be due to TEF values of the congeners, although the uptake of congeners of PCDDs, PCDFs, and Co-PCBs in the plants seemed to be under passive diffusion mechanism. The effects of certain congeners, in which a TEF value was not assigned, were examined on GUS activity in the transgenic plants. The relative amounts are defined as the amounts of PCDD and PCDF congeners without TEF value in the plants divided by the amounts of PCDD and PCDF congeners with TEF values of 0. These relative amounts were negatively correlated with GUS activity [-0.486 (Person's correlation coefficient), P < 0.05, data not shown]. These results suggested that relatively low GUS activity in the transgenic plants grown in the uncontaminated soils seemed to be due to antagonistic effects of certain congeners in which TEF values were not assigned. Fig. 4. Correlation between GUS activity and TEQ value in the transgenic tobacco plant XD4V-26 cultured on the actually contaminated soils containing 510 (open circle) and 5100 (closed circle) pg-TEQ g−1 for 33 days. Pearson's correlation coefficient; r = 0.681. Discussion The transgenic tobacco plant XD4V-26 carrying the recombinant mouse AhR-mediated GUS reporter gene expression system exhibited a dose-dependent GUS activity towards PCB126 (TEF = 0.1), but not towards PCB169 (TEF = 0.03) and PCB180 (TEF was not assigned.). It suggested that the plant took up PCB126, which activated the recombinant mouse AhR XD4V, and then induced GUS activity. Uptake of PCB169 and PCB180 also occurred, but those compounds did not contribute to induce GUS activity because of very low TEF value and almost no toxicity towards mammals, respectively. Instead, background GUS activity, probably due to endogenous AhR ligands such as indoleacetic acid,[8] was suppressed by PCB169 and PCB180. Concurrent treatment with PCB126 and PCB180 tended to suppress GUS activity induced by PCB126 (data not shown). It was reported that certain congeners with low or not assigned TEF values have shown antagonistic activity toward AhR in rat primary hepatocytes: PCB153 (TEF was not assigned), decreased the induction of CYP1A1 by the treatment with 2,3,7,8-TCDD, but PCB77 (TEF = 0.0001) and PCB156 (TEF = 0.00003) did not.[9] It was also reported that di-, tri-, and tetra-ortho-substituted PCBs suppressed the activation of AhR by PCBs with no or one ortho chlorine substitution.[10] It was reported that certain persistent organic pollutants (POPs), including o,p'-dichloro-diphenyl-trichloroethane (DDT) and dieldrin, were antagonistic toward AhRs.[11, 12] In transgenic Arabidopsis plants carrying genes encoding a recombinant guinea pig AhR and the GUS reporter, p,p-DDT, p,p’-dichloro-diphenyl-dichloroethane (p,p-DDE), and p,p-dichloro-diphenyl-dichloroethylene (p,p-DDD) decreased the GUS activity induced by MC.[13] Antagonism between the agonist MC and dieldrin or p,p’-DDT was also observed in an in vitro assay using an Ah immunoassay.[13] The present assay responded to both agonists and antagonists among PCB congeners. Therefore, the GUS activity in the assay may reflect the relative toxicity level of a mixture of PCB congeners. It was suggested that the relative toxicity level of a mixture of congeners of PCDD, PCDF, and Co-PCB should not be simply determined by TEQ as the sum of concentrations of congeners multiplied by their TEF values, since TEQ does not count antagonistic effects of certain congeners. When the transgenic tobacco plant XD4V-26 was cultured on the soils containing dioxins and dioxin-like compounds for 5 weeks, the GUS activities in the leaves were time- and dose-dependently increased. The transgenic plant was thus able to monitor the levels of both 360 pg-TEQ g−1 and 5100 pg-TEQ g−1. The environmental standard (less than 1000pg-TEQ g−1) for dioxins and dioxin-like compounds in soils in Japan was possibly detected in the transgenic plant XD4V-26 within 5 weeks of planting. However, the GUS activity of plants grown in 5100 pg-TEQ g−1 was not 10 times higher than that of plants grown in 510 pg-TEQ g−1. This suggests that the tobacco plants may not be able to accumulate very high concentrations of these congeners and may reach the plateau between 510 and 5100 pg-TEQ g−1. Moreover, the different levels of uptake of the congeners in the plants may be due to the mass and growth stages of the plants. The amounts of uptake of dioxins and dioxin-like compounds in the tobacco plants by passive diffusion were not so large as compared with that of zucchini plants.[6,14] It was reported that root exudates such as low-molecular-weight organic acids [15, 16] and biosurfactants [17, 18] may be involved in the uptake of hydrophobic compounds such as POPs. By the use of these compounds, the transgenic tobacco plants seem to increase the uptake of dioxins and dioxin-like compounds in soils. In this study, transgenic tobacco plant XD4V-26 carrying genes encoding a recombinant mouse AhR and the GUS reporter successfully detected residues of PCDD, PCDF, and Co-PCB congeners in the contaminated soils within 5 weeks. These results suggested that the assay using the plant seems to be useful for on-site assays of these residues. In contrast, cultivation of the transgenic plants on samples of the contaminated soils in the closed laboratory may provide some advantages such as public acceptance of the use of transgenic plants, and possible control of environmental conditions, including temperature, drought, and day length, all of which may influence on the stable GUS assay. Acknowledgments We thank Masayuki Hattori, Tomohito Arao, Shozo Endo, and Emiko Iizumi for technical assistance and useful discussions. This work was funded in part through the Bio-oriented Technology Research Advancement Institution (BRAIN) and a Grant-in-Aid for Scientific Research A from the Ministry of Education, Culture, Sports, Science and Technology of Japan (No. 17208029). Authors H. Inui and K. Gion contributed equally to this work. ==== Refs References [1] Van den Berg M. Birnbaum L.S. Denison M. De Vito M. Farland W. Feeley M. Fiedler H. Hakansson H. Hanberg A. Haws L. Rose M. Safe S. Schrenk D. Tohyama C. Tritscher A. Tuomisto J. Tysklind M. Walker N. Peterson R. The E. World Health Organization reevaluation of human and Mammalian toxic equivalency factors for dioxins and dioxin-like compounds Toxicol. Sci. 2006, 2005 93 2 223 241 [2] Anderson J.W. Hartwell S.I. Hameedi M.J. Regional comparisons of coastal sediment contamination detected by a biomarker (P450 HRGS; EPA Method 4425) Environ. Sci. Technol. 2005 39 1 17 23 15667070 [3] Windal I. Denison M.S. Birnbaum L.S. Van Wouwe N. Baeyens W. Goeyens L. Chemically activated luciferase gene expression (CALUX) cell bioassay analysis for the estimation of dioxin-like activity: critical parameters of the CALUX procedure that impact assay results Environ. Sci. Technol. 2005, 39 19 7357 7364 [4] Kodama S. Okada K. Akimoto K. Inui H. Ohkawa H. Novel recombinant aryl hydrocarbon receptors for bioassay of aryl hydrocarbon receptor ligands in transgenic tobacco plants Plant Biotechnol. J. 2009, 7 119 128 [5] Kodama S. Okada K. Inui H. Ohkawa H. Aryl hydrocarbon receptor (AhR)-mediated reporter gene expression systems in transgenic tobacco plants Planta 2007, 227 1 37 45 [6] Inui H. Wakai T. Gion K. Kim Y.S. Eun H. Differential uptake for dioxin-like compounds by zucchini subspecies Chemosphere 2008 73 1602 1607 18835616 [7] Kim Y.-S. Eun H. Katase T. Historical distribution of PCDDs, PCDFs and coplanar PCBs in sediment core of Ariake Bay Japan. Arch. Environ. Contam. Toxicol. 2008 54 3 395 405 [8] Heath-Pagliuso S. Rogers W.J. Tullis K. Seidel S.D. Cenijn P.H. Brouwer A. Denison M.S. Activation of the Ah receptor by tryptophan and tryptophan metabolites Biochemistry 1998 37 33 11508 11515 9708986 [9] Chen G. Bunce N.J. Interaction between halogenated aromatic compounds in the Ah receptor signal transduction pathway Environ. Toxicol. 2004, 19 5 480 489 [10] van der Plas S.A. Sundberg H. van den Berg H. Scheu G. Wester P. Jensen S. Bergman A. de Boer J. Koeman J.H. Brouwer A. Contribution of planar (0–1 ortho) and nonplanar (2–4 ortho) fractions of Aroclor 1260 to the induction of altered hepatic foci in female Sprague-Dawley rats Toxicol. Appl. Pharmacol. 2000, 169 3 255 268 [11] Jeong H.G. Kim J.Y. Effects of o p ’-DDT on the2,3,7,8-tetrachlorodibenzo-p-dioxin-inducible CYP1A1 expression in murine Hepa-1c1c7 cells Food Chem. Toxicol. 2002 40 11 1685 1692 12176094 [12] Long M. Laier P. Vinggaard A.M. Andersen H.R. Lynggaard J. Bonefeld-Jorgensen E.C. Effects of currently used pesticides in the AhR-CALUX assay: comparison between the human TV101L and the rat H4IIE cell line Toxicology 2003, 194 1–2 77 93 [13] Gion K. Inui H. Sasaki H. Utani Y. Kodama S. Ohkawa H. Assays of PCB congeners and organochlorine insecticides with the transgenic Agabidopsis and tobacco plants carrying recombinant guines pig AjR and GUS reporter genes J. Environ. Sci. Health B 2012 accepted [14] Hülster A. Müller J.F. Marschner H. Soil-plant transfer of polychlorinated dibenzo-p -dioxins and dibenzofurans to vegetables of the cucumber family(Cucurbitaceae ) Environ. Sci. Technol. 1994 28 1110 1115 22176237 [15] White J.C. Kottler B.D. Citrate-mediated increase in the uptake of weathered 2,2-bis (p -chlorophenyl) 1,1-dichloroethylene residues by plants Environ. Toxicol. Chem. 2002 21 3 550 556 11878468 [16] White J.C. Mattina M.I. Lee W.Y. Eitzer B.D. Iannucci-Berger W. Role of organic acids in enhancing the desorption and uptake of weathered p p ’-DDE by Cucurbita pepo Environ. Pollut. 2003 124 1 71 80 12683984 [17] Shimazu S. Ohta M. Inui H. Nanasato Y. Ashida H. Ohkawa H. Effects of biosurfactants on assays of PCB congeners in transgenic arabidopsis plants carrying a recombinant guinea pig AhR-mediated GUS reporter gene expression system J Environ Sci Health B 2010 45 8 773 779 20936563 [18] White J.C. Parrish Z.D. Gent M.P. Iannucci-Berger W. Eitzer B.D. Isleyen M. Mattina M.I. Soil amendments, plant age, and intercropping impact p p ’-DDE bioavailability to Cucurbita pepo J. Environ. Qual. 2006 35 4 992 1000 16738383	22428884	PMC3662081	NO-CC CODE	2021-01-04 22:45:14	yes	J Environ Sci Health B. 2012 Apr 19; 47(4):233-239
==== Front 960783520545Mol PsychiatryMol. PsychiatryMolecular psychiatry1359-41841476-55782347874510.1038/mp.2013.23nihpa441734ArticleGene Knockout of 5-Lipoxygenase Rescues Synaptic Dysfunction and Improves Memory in the Triple-Transgenic Model of Alzheimer’s Disease Giannopoulos Phillip F. 13Chu Jin 13Joshi Yash B. 13Sperow Margaret 2Li Jin-Luo 13Kirby Lynn G. 24Praticò Domenico 131 Center for Translational Medicine, Temple University School of Medicine, Philadelphia, PA 191402 Center for Substance Abuse Research, Temple University School of Medicine, Philadelphia, PA 191403 Department of Pharmacology, Temple University School of Medicine, Philadelphia, PA 191404 Department of Anatomy and Cell Biology, Temple University School of Medicine, Philadelphia, PA 19140Correspondence to: Domenico Praticò, MD, 940 Medical Education and Research Building, 3500 North Broad Street, Philadelphia, PA 19140, [email protected], Telephone: 215-707-9380, Fax: 215-707-98907 2 2013 12 3 2013 4 2014 01 10 2014 19 4 511 518 Users may view, print, copy, download and text and data- mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: http://www.nature.com/authors/editorial_policies/license.html#termsThe 5-Lipoxygenase (5LO) is upregulated in Alzheimer’s disease (AD), and in vivo modulates the amyloidotic phenotype of APP transgenic mice. However, no data are available on the effects that 5LO has on synaptic function, integrity and cognition. To address this issue we used a genetic and a pharmacologic approach by generating 3xTg mice deficient for 5LO, and administering 3xTg mice which a 5LO inhibitor. Compared with controls, we found that even before the development of overt neuropathology, both animals manifested significant memory improvement, rescue of their synaptic dysfunction and amelioration of synaptic integrity. In addition, later in life these mice had a significant reduction of Aβ and tau pathology. Our findings support a novel functional role for 5LO in regulating synaptic plasticity and memory. They establish this proetin as a pleiotropic contributor to the development of the full spectrum of the AD phenotype, making it a valid therapeutic target for the treatment of AD. ==== Body Introduction Alzheimer’s disease (AD) is a progressive neurodegenerative disorder and leading cause of dementia worldwide for which no effective treatments exist1,2. Memory loss is the most prominent clinical aspect of AD, and it typically manifests prior to the development of overt brain pathologies. While there is still debate on the actual contributors to the development of memory impairments, there is a consensus that alteration at the synaptic level, a phenomenon also known as synaptic dysfunction, is probably one of the most significant factors in the initial stages of memory loss3,4. In the last decade the development of transgenic mice has represented an invaluable tool for modeling diverse aspects of the AD phenotype. Although no model exactly and fully recapitulates it, the consensus is that the triple-transgenic mice, also known as 3xTg, have the advantage of presenting the most salient features of the human disease including synaptic dysfunction, memory impairments, Aβ and tau pathology 5. The 5-lipoxygenase (5LO) is a lipid-peroxidizing enzyme which inserts molecular oxygen into fatty acids leading to the biosynthesis of bioactive lipids such as leukotrienes6. The protein is widely expressed in the brain where its expression and activity increase in an age-dependent manner 7. Previous work showed that levels of 5LO are elevated in AD brains 8, and its genetic absence or pharmacological blockade reduced Aβ levels and deposition in a transgenic APP mouse model, Tg2576 9,10. More recently, we demonstrated that 5LO neuronal over-expression in the 3xTg exacerbated their neuro-pathologies 11. However, no data are currently available on the effect that 5LO genetic deficiency has on the AD-like synaptic phenotype, which includes synaptic function, synaptic integrity and cognition. To address this issue we used a genetic and a pharmacologic approach by generating 3xTg mice genetically deficient for 5LO (3xTg-5LOKO), and administering 3xTg mice which a selective 5LO inhibitor, zileuton 10. Compared with controls, we found that even before the development of overt neuropathology, 3xTg-5LOKO mice and 3xTg mice receiving zileuton manifested a significant improvement in cognition and memory, which was associated with a rescue of their synaptic dysfunctions and amelioration of synaptic integrity. In addition, later in life these mice had a significant reduction of their Aβ and tau pathology. Our findings support a novel role for 5LO at the synapse level whereby modulating synaptic plasticity and integrity as well as memory. Taken together, these new data establish the 5-LO as a key player in the development of the full spectrum of the AD-like phenotype and an important therapeutic target with true disease-modifying potential for the treatment of AD. Methods All animal procedures were approved by the Institutional Animal Care and Usage Committee, in accordance with the U.S. National Institutes of Health guidelines. The 3xTg mice harboring a mutant amyloid precursor protein (APP; KM670/671NL), a human mutant PS1 (M146V) knockin, and tau (P301L) transgenes; 3xTg wild type (WT), and mice genetically deficient for 5LO (5LOKO) used in the study were reported previously 5,12. All the animals were backcrossed 10 times on the same genetic background C57BL6/SJL. The 5LOKO mice were crossbred several times with 3xTg mice to obtain founder animals (3xTg/5LOKO), which were then crossed with each other and the animals from these crosses used for the studies. They were kept in a pathogen-free environment, on a 12hour light/dark cycle, and fed a normal chow and water ad libitum. Male and female mice were used throughout the studies. Animals underwent behavioral testing at two different age groups (6–8 months and 12–14 months). A separate group of five months old 3xTg mice were also randomized to receive zileuton (200 mg/L) or vehicle in their drinking water for a month. After this period they underwent to behavioral testing as described below then sacrificed for electrophysiology study. After sacrifice, mice were perfused with ice-cold 0.9% PBS containing EDTA (2mmol/L) pH7.4. Brain was removed, gently rinsed in cold 0.9% PBS and immediately dissected in two halves. One half was immediately stored at −80°C for biochemistry; the other half was fixed in 4% paraformaldehyde in PBS, pH7.4 for immunohistochemistry studies. The cortex and hippocampus were the two brain regions always used for biochemistry and immunohistochemistry studies, as also indicated in figure legends. Behavioral tests All the animals were handled for at least 3–4 days prior to testing. They were tested in random order and the experimenter conducting the tests was unaware of the genotype or treatment. Y-maze The Y-maze behavioral paradigm, a test widely used to assess working memory in rodents, was carried out as previously described 11. Briefly, each mouse was placed in the center of the Y-maze and allowed to explore freely through the maze during a 5-min session for assessment of spontaneous alternating behavior. The sequence and total number of arms entered were video recorded. Any entry into an arm was considered valid if all four paws entered the arm. An alternation was defined as three consecutive entries in three different arms (1,2,3 or 2,3,1, etc.). The percentage alternation score was calculated using the following formula: total alternation number/total number of entries-2)100. Testing was always performed in the same room and at the same time to ensure environmental consistency. Fear-conditioning Two weeks before sacrifice, fear conditioning experiments were performed following methods previously described 11, 13,14. Briefly, on day one animals are placed into the conditioning chamber for 2 min before the onset of a sound, which in its last 2 sec is paired with a foot shock. Mice are removed from the chamber 1 min after the shock, and on day two tested for contextual and cued fear conditioning, which is basically a 24hr memory retention test. Conditioning is measured by recording the freezing behavior as the complete absence of movement both during training as well as testing. The percentage time during which the mouse froze is calculated for analysis of cintexual ans cued fear memory assessments. ests were conducted in a conditioning chamber equipped with black methacrylate walls, a transparent front door, a speaker and grid floor (Start Fear System; Harvard Apparatus). Immunoblot Analyses Primary antibodies used in this paper are summarized in the Table. Proteins were extracted in EIA buffer containing 250mM Tris base, 750mM NaCl, 5% NP-40, 25mM EDTA, 2.5% Sodium Deoxycholate, 0.5% SDS and an EDTA-free protease and phosphatase inhibitors cocktail tablet (Roche Applied Science), sonicated, centrifuged at 13,000 rpm for 45 min at 4°C, and supernatants used for immunoblot analysis, as previously described 9,10. Total protein concentration was determined by using BCA Protein Assay Kit (Pierce, Rockford, IL). Samples were electrophoretically separated using 10% Bis–Tris gels or 3–8% Tris–acetate gel (Bio-Rad, Richmond, CA), according to the molecular weight of the target molecule, and then transferred onto nitrocellulose membranes (Bio-Rad). They were blocked with Odyssey blocking buffer for 1 hr; and then incubated with primary antibodies overnight at 4°C. After three washing cycles with T-TBS, membranes were incubated with IRDye 800CW or IRDye 680CW-labeled secondary antibodies (LI-COR Bioscience, NE) at 22°C for 1 hr. Signals were developed with Odyssey Infrared Imaging Systems (LI-COR Bioscience). Actin was always used as an internal loading control. Sarkosyl insolubility assay The assay for insoluble tau was performed as previously described 15. Briefly, ultracentrifugation and sarkosyl extraction (30 min in 1% sarkosyl) was used to obtain soluble and insoluble fractions of tau. Insoluble fractions were washed one time with 1% sarkosyl, then immunoblotted with HT-7 antibody (Table). Biochemical Analyses Mouse brain homogenates were sequentially extracted first in RIPA for the Aβ 1-40 and 1-42 soluble fractions, then in formic acid for the Aβ 1-40 and 1-42 insoluble fractions, and then assayed by a sensitive sandwich ELISA kits (WAKO Chem.), as previously described 10,14. Immunohistochemistry Primary antibodies used in this study are listed in Table. Immunostaining was performed as reported previously 9,13,14. Briefly, serial 6μm-thick coronal sections were mounted on 3-aminopropyl triethoxysilane-coated slides. Every eighth section from the habenular to the posterior commissure (8–10 sections per animal) was examined using unbiased stereological principles. The sections for testing Aβ were deparaffinized, hydrated, and pretreated with formic acid (88%) and subsequently with 3% H2O2 in methanol. The sections for testing total tau (HT7), phospho-tau (PHF-1, PHF-13, AT8, AT180, AT270), Synaptophysin (SYP), Postsynaptic Density Protein 95 (PSD95) and Microtubule associated protein-2 (MAP-2) were deparaffinized, hydrated, subsequently pretreated with 3% H2O2 in methanol, and then treated with citrate (10mM) or IHC-Tek Epitope Retrieval Solution (IHC World, Woodstock, MD) for antigen retrieval. Sections were blocked in 2% fetal bovine serum and then incubated with primary antibody overnight at 4°C. The following day, sections were incubated with biotinylated anti-mouse immunoglobulin G (Vector Laboratories, Burlingame, CA) and then developed by using the avidin-biotin complex method (Vector Laboratories) with 3,3′-diaminobenzidine as a chromogen. Light microscopic images were used to calculate the area occupied by Aβ immunoreactivity, and the cell densities of GFAP- and CD45-immunopositive reactions by using the software Image-Pro Plus for Windows version 5.0 (Media Cybernetics, Bethesda, MD). The threshold optical density that discriminated staining from background was determined and held constant for all quantifications. The area occupied by Aβ immunoreactivity was measured by the software and divided by the total area of interest to obtain the percentage area of immunoreactivity. Electrophysiology Six month old mice (n= # slices/# of animals): WT (n=23/8); 3xTg (n=21/7); 3xTg-5LOKO (n=20/6); 3xTg plus zileuton (n= 5/2) were sacrificed by rapid decapitation and brains placed into ice-cold artificial cerebral spinal fluid (ACSF) in which sucrose (248mM) was substituted for NaCl. Transverse hippocampal slices (400 μm thick) were cut using a Vibratome 3000 plus (Vibratome, Bannockburn, IL) and placed in ACSF (124 m MNaCl, 2.5 mM KCl, 2mM NaH2PO4, 2.5 mM CaCl2, 2 mM MgSO4, 10 mM dextrose, and 26 mM NaHCO3) at room temperature to recover for 1 hr bubbled with 95% O2/5% CO2. Slices were transferred to a recording chamber (Warner Instruments, Hamden, CT) and continuously perfused with ACSF at 1.5–2.0 ml/min flow, bubbled with 95% O2/5% CO2, and maintained by an in-line solution heater (TC-324; Warner Instruments) at 32–34 °C. We recorded field excitatory postsynaptic potentials (fEPSPs) from the CA1 stratum radiatum by using an extracellular glass pipette (3–5 MΩ) filled with ACSF. Schaffer collateral/commissural fibers in the stratum radiatum were stimulated with a bipolar tungsten electrode placed 200–300 μm from the recording pipette. Stimulation intensities were chosen to produce a fEPSP that was 1/3 of the maximum amplitude, based on an input/output curve using stimulations of 0–300 μA, in increments of 20μAs. Paired-pulse facilitation experiments were performed using a pair of stimuli of the same intensity delivered 20, 50, 100, 200 and 1000 ms apart. Baseline was recorded for 20 mins prior to tetanization with pulses every 30 seconds. LTP at CA3–CA1 synapses was induced by four trains of 100 Hz stimulation delivered in 20 second intervals. Recordings were made every 30 seconds for 3 hours following tetanization. The fEPSP rise/slope (mV/ms) between 30 and 90% was measured offline using Clampfit 10.3 (Molecular Devices, LLC) and normalized to the mean rise/slope of the baseline. Slices were eliminated if an unstable baseline was produced or if the normalized rise/slope dropped more than 20–50mV/ms in an approximately 10 min period. All the tests were always performed by an experimenter who was unaware of the different genotypes and treatment. Data Analysis One-way analysis of variance (ANOVA), unpaired Student’s t-test (two-sided) and Bonferroni multiple comparison tests were performed using Prism 5.0 (GraphPad Software, La Jolla, CA). All data are presented as mean +/− standard error of the mean. Significance was set at p <0.05. Results Genetic absence of 5LO ameliorates cognition To assess the effect of 5LO genetic absence on behavior, mice were initially tested in the Y-maze at two different ages: 6–8 and 12–14 months old. As shown in Figure 1A, initially we did not notice any differences among the four groups of mice considered in regard to their general activity as assessed by the total number of arm entries for each group at both ages (Fig. 1A). When we considered the number of alternations in the same test, we observed that 3xTg mice had a much lower number of alternations resulting in a significant lower percentage when compared with wild type and 5LOKO mice, suggesting that they have impairment in their working memory. However, compared with the 3xTg mice, the 3xTg-5LOKO mice had a greater number of alternations resulting in a significantly higher percentage at both ages, suggesting an improvement of their working memory (Fig. 1B). Next, mice underwent fear conditioning testing, which is a measure of a 24 hr retention memory for both the cued and the contextual form. No differences among the groups of mice were observed during the training session (not shown). When the four groups of mice were subjected to contextual fear conditioning they did not manifest any significant differences (Fig. 1C). In the cued phase of the conditioning we observed that 5LOKO and wild type mice exhibited similar levels of freezing at both ages. On the other hand, 3xTg mice had significant lower freezing percentages which were normalized in the 3xTg5LOKO mice (Fig. 1D). Genetic absence of 5LO decreases brain Aβ level and deposition Two weeks after completion of the last behavior tests (14 months of age), mice were sacrificed, brains harvested and assayed for Aβ levels and deposition. In comparing the two groups, we observed that 3xTg mice genetically deficient for 5LO displayed a significant decrease in the amount of RIPA-soluble and formic-acid soluble Aβ 1-40 and 1-42 (Fig. 2A). Similar results were obtained when brain tissues for 8-month old mice were assayed (Supplementary Fig. 1). Confirming the ELISA data, we found that absence of 5LO led also to a significant decrease of their brain Aβ immunoreactive areas (Fig. 2B). To explore for possible mechanisms responsible for this change, we assessed the steady-state levels of APP along with its cleavage products in the same samples. As shown in figure 2C, no differences were detected for total APP, the α-secretase (ADAM-10) and β-secretase (BACE-1) pathways between the two groups. By contrast, compared with controls, 3xTg-5LOKO mice had a significant decrease in the steady-state levels of the 4 components of the γ-secretase complex, PS1, nicastrin, Pen-2 and APH-1, which was associated with lower levels of C-terminal fragments (CTFs) (Fig. 2C, D). Genetic knockout of 5LO modulates tau metabolism We then examined the effect of 5LO knockout on tau metabolism. As shown in Figure 3, while we did not observe any change in levels of total soluble tau between the two groups, compared with the 3xTg, the 3xTg-5LOKO mice had a significant decrease in its phosphorylated forms at epitopes S396, as recognized by the specific antibody PHF-13, at S396/S404 as recognized by the specific antibody PHF-1, and S202/T205, as recognized by the specific antibody AT8 (Fig. 3A, B). By contrast, no changes were detected for other phosphorylation sites as recognized by the antibodies AT180 (T231/S235) and AT270 (T181). Additionally, compared with 3xTg we observed that 3xTg-5LOKO had a significant reduction in the levels of insoluble tau (Fig. 3C,D). In accordance with the western blot results, immunohistochemical staining showed decreased somatodendritic accumulations of the phosphorylated epitopes recognized by the antibodies PHF-1, PHF-13 and AT-8 in the 3xTg-5LOKO mice (ratios: PHF-13/tau=0.57; PHF-1/tau=0.64; AT8/tau=0.52) (Fig. 3E). To explore the molecular mechanism responsible for the hypophosphorylation of tau, next we assayed some of the kinases which are considered major regulators of tau post-translational modification. In comparing the two groups of mice, we did not observe any significant differences in the levels of total and phosphorylated GSK3-α and GSK3-β, JNK2, total and phosphorylated p38 and total and phosphorylated SAPK/JNK (Fig. 3F). However, we found that compared with controls, 3xTg-5LOKO mice had a statistically significant decrease in levels Cdk5 kinase along with its coactivators p35 and p25 (Fig. 3F, G). Absence of 5LO increases synaptic integrity Since changes in tau phosphorylation state have been implicated in alterations of synaptic integrity in AD, next we assessed this aspect of the 3xTg mice phenotype. Compared with the control group, 3xTg-5LOKO mice had a significant increase in the steady state levels of two main synaptic proteins: post-synaptic density protein 95 (PSD-95) and synaptophysin (Fig. 4A, B). A similar result was obtained also when the dendritic protein MAP2 was assayed (Fig. 4A, B). These results were further confirmed in brain sections of the same mice when they were assessed by immunohistochemical analyses (Fig. 4C). Finally, we observed that compared with brain homogenates from 3xTg, the ones from 3xTg-5LOKO mice had a significant decrease in GFAP and CD45 immunoreactivities, markers of astrocytes and microglia cells activation respectively (Fig. 4D, E). Genetic knockout of 5LO ameliorates synaptic deficits Since the absence of 5LO in 3xTg-AD mice yielded an improvement in memory at a very early stage of their AD-like phenotype (6 months; prior to plaque and tangle pathology), we then explored its effect on synaptic function at this age. To this end, first we investigated basal synaptic transmission by generating input/output (I/O) curves and measuring field-excitatory postsynaptic potentials (fEPSPs) elicited in CA1 by stimulation of the Schaffer collaterals at increasing strength of stimulations and intensities. As shown in Figure 5A, there were no differences observed in the I/O curves between any of the groups considered (WT, 3xTg, 3xTg-5LOKO). Next, we measured short-term plasticity by examining paired-pulse facilitation (PPF), which is due to an activity-dependent presynaptic modulation of transmitter release 16. Similar to the observation in the I/O curves, there were no differences in PPF between any of the groups analyzed (Fig. 5B). Finally, we investigated long-term potentiation (LTP) in the CA1 region of the hippocampus, which is thought to be a measure of neuronal plasticity and a major player in cognition17. In this test we found that, compared with WT, 3xTg mice had a significant reduction in LTP responses. However, the genetic absence of 5LO in the 3xTg completely restored the LTP responses to a level comparable to that of the WT mice (Fig. 5C–E). 5LO Pharmacologic blockade improves memory and rescues synaptic deficits To further confirm the involvement of the 5LO in the memory improvement and rescue of the pathological synaptic phenotype, 3xTg mice were randomized to receive zileuton, a selective and specific 5LO inhibitor in their drinking water for a month at a concentration we previously showed to completely block 5LO activation 10. At the end of this period, mice underwent memory assessment in the Y-maze as well as the fear conditioning paradigm. As shown in supplemental figure 2, first we observed that zileuton had no effect on the general motor activities of these mice. By contrast, we observed that compared with vehicle group, 3xTg mice receiving zileuton had a significant improvement in the number of alternations, suggesting an improvement of their working memory (Supplemental Fig. 2). In the fear conditioning paradigm, while we did not observe any differences between the two groups in the training phase, compared with their controls 3xTg mice receiving zileuton had a significant increase in the freezing percentage time in the cued phase, but no changes were detected in the contextual phase of the conditioning (Supplemental Fig. 2). Next, mice were sacrificed and their brains harvested for electrophysiology studies. First, we observed that 3xTg mice treated with zileuton did not differ from their controls in terms of basal synaptic transmission as measured by the field-excitatory postsynaptic potentials or short-term plasticity by examining paired-pulse facilitation (Fig. 5A,B). However, analysis of the LTP responses demonstrated that pharmacological blockade of 5LO was sufficient to restore the impairments noticed in the 3xTg mice back to levels indistinguishable from wild type animals (Fig. 5C–E). Discussion The data in the present paper unravel a new aspect of the neurobiology of 5LO by demonstrating its functional role in synaptic function and plasticity as well as memory. Together with the previous knowledge on this protein, they establish 5LO as a key player in the development of the full spectrum of the AD phenotype and an important therapeutic target with true disease-modifying potential for the treatment of AD. In recent years, there has been increasing evidence suggesting that alterations in synaptic integrity and function are the earliest phenotypic manifestations during the evolution of AD pathogenesis 18,19. Thus, synaptic loss, as reflected by changes in synaptic markers, is a constant feature of early stage AD pathology, and better correlates with clinical cognitive impairments than classical assessment of Aβ or tau brain lesions 20,21. However, the mechanisms involved in this phenomenon are still under investigation. Recent developments in gene-targeted and transgenic mice represent an invaluable tool for modeling diverse aspects of the AD phenotype, including synaptic dysfunction and pathology. Notably, the 3xTg mice exhibit deficits in synaptic plasticity, such as LTP which occurs at an early age prior to extracellular Aβ deposition and tau pathology 5. Previously, we showed that genetic manipulation or pharmacologic inhibition of the 5LO modulates the phenotype of the APP transgenic mice 9,10. However, this mouse model represents a limited version of AD since it manifests mainly AD-like brain amyloidosis and lacks the tau pathology. The availability of the 3xTg mice offered us the unique opportunity to better explore the biological relevance of this pathway in the context of the AD pathogenesis. In the current paper, first we showed that genetic absence of 5LO per se did not influence any of the memory tests performed in our study. Next, we observed that compared with their controls, the 3xTg-5LOKO mice did not manifest any difference in general motor activities. By contrast, they had a significant improvement in their memory performance already at 6 months of age, as demonstrated in the Y-maze paradigm, which by recording spontaneous alternation behavior, assesses working memory in rodents 11. Additionally, the same animals had an improvement in their learning memory ability, as assessed by the fear conditioning test. Thus, in this setting we observed that 3xTg mice performed significantly worse than the ones genetically deficient for the 5LO in the cue recall, but not in the contextual paradigm, suggesting a possible amygdala involvement. Consistent with these results, we observed that compared with 3xTg, the 3xTg-5LOKO mice had a significant increase in 3 distinct protein markers of synaptic integrity (i.e., synaptophysin, PSD95 and MAP2) suggesting an improvement of this important function for memory and learning secondary to the 5LO deficiency. In association to these changes, we observed a significant reduction in Aβ levels and deposition, which, confirming our previous report was secondary to an effect on the γ-secretase pathway 11. Besides the effect on the Aβ pathology, we also observed that 5LO genetic absence had an influence on tau metabolism. Thus, we observed that genetic absence of 5LO resulted in a significant decrease in phosphorylated tau epitopes as recognized by the immunoreactivity of the specific antibodies PHF-1, PHF13 and AT8, but not for the other two tested, AT180 and AT270, and confirmed the cdk-5 kinase pathway but not other kinases or phosphatases were involved 11. To corroborate the involvement of the 5LO in the improvement of the behavioral deficits we detected as early as at 6 months of age, we adopted a pharmacologic approach by administering a selective 5LO inhibitor to five-month old 3xTg mice for a month. The dosage selected was based on our previous published work where we showed that zileuton at this concentration significantly reduced 5LO activation up to 85% 10. Similar to the genetic study, we observed that 3xTg mice receiving the active drug had a significant amelioration of their working memory and conditioning learning. Because of the early improvements in memory deficits we noticed in both the 3xTg mice genetically deficient for 5LO and the ones administered with a 5LO inhibitor, next we assessed the effects that both conditions had on synaptic function by implementing an electrophysiological approach. First, we investigated basal synaptic transmission by measuring field-excitatory postsynaptic potentials elicited in CA1 by stimulation of the Schaffer collaterals. We found that there were no differences in this parameter amongst the four groups of mice, suggesting that synaptic transmission is not altered in any of the conditions implemented. Next, we measured short-term plasticity by examining paired-pulse facilitation, which is secondary to an activity-dependent presynaptic modulation of transmitter release 16. Similar to the observation for the basal synaptic transmission, there were no significant differences in paired-pulse facilitation among any of the groups investigated. These results suggest that there is not an increase or decrease in the probability of transmitter release in any of these groups of mice 5,22. Finally, we assessed the LTP response which is a type of plasticity that is thought to play a major role in learning and memory functions. As reported previously 5, there was a significant difference in LTP responses between the wild type and 3xTg mice with the latter showing deficits. However, the genetic absence of 5LO in the 3xTg mice was sufficient to restore their LTP responses to a level comparable to the ones measured in the wild type mice. The biological importance of this finding was further corroborated by the demonstration that 5LO pharmacologic blockade was also sufficient to rescue the abnormal synaptic phenotype of the 3xTg mice. Taken together these data unravel a new aspect of the neurobiology of 5LO pathway by demonstrating its functional role in synaptic function and plasticity as well as memory and learning. Interestingly, the above described 5LO-dependent beneficial effects on the LTP parameters are in line with previously reported enhancements of the GluR1/AMPA receptor phosphorylation by 5LO genetic absence or pharmacologic inhibition 23,24. Importantly, despite the fact that some of the 5LO biologic effects are mediated by an involvement of the γ-secretase pathway, no alteration of the Notch signaling has been reported 10. This observation makes any potential Notch-related side effect secondary to a chronic 5LO inhibition unlikely. Considering the anti-Aβ and tau effect of 5LO, our findings have important patho-physiological implications for AD since they establish this pathway as a major active contributor to all of the aspects of the disease phenotype. This new information by demonstrating the pleiotropic role of this protein in AD pathogenesis makes it not only a valid pharmacological target, since 5LO inhibitors are already FDA approved, but most importantly represents a unique therapeutic opportunity with a true disease-modifying potential for the treatment of AD. Supplementary Material 1 Supplementary Figure 1 Genetic absence of 5LO affects Aβ peptide levels of deposition in brains of 8month old 3xTg mice. (A) Radioimmunoprecipitation assay (RIPA)-soluble and formic acid (FA)-extractable Aβ1-40 and 1-42 levels in brain cortex of 3xTg mice and 3xTg-5LOKO mice at 8 months of age [n=8 (5M, 3F) for 3xTg and n=9 (4M, 5F) for 3xTg-5LOKO] (p<0.001). Values represent mean +/− standard error of the mean. 2 Supplementary Figure 2 Pharmacological blockade of 5LO improves memory in 3xTg mice. (A) Total number of arm entries for 3xTg and 3xTg+zileuton mice at 6 months of age (B) Percentage of alternations between the same groups of mice (p <0.001). (C) Contextual fear memory response in 3xTg and 3xTg+zileuton mice. (D) Cued recall fear memory response in the same groups of mice [n=4 (2M, 2F) for 3xTg, n=4 (2M, 2F) for 3xTg+zileuton (p<0.001). Values represent mean +/− standard error of the mean. This study was supported by US National Institute of Health (NIH) grants AG033568, NS071096 to D.P.; P30 DA 13429 and T32 DA 07237 to the Center for Substance Abuse Research. Additional support was provided by a grant from the Alzheimer Art Quilt Initiative to D.P. AUTHOR CONTRIBUTIONS P.F.G. and D.P. designed the study, developed the experimental design, performed data analyses, and wrote the paper. P.F.G., J.C., Y.B.J., J.L., M.S. performed the experiments. P.F.G., M.S. and L.G.K designed and performed electrophysiology experiments. All authors discussed the results and commented on the manuscript. CONFLICTS OF INTEREST The authors have no conflicting financial interest to disclose. Figure 1 Genetic absence of 5LO ameliorates behavioral deficits of 3xTg mice. (A) Total number of arm entries for WT, 5LOKO, 3xTg and 3xTg-5LOKO mice at 6–8 and 12–14 months of age. (B) Percentage of alternations between the same groups of mice (p <0.001). (C) Contextual fear memory response in WT, 5LOKO, 3xTg and 3xTg-5LOKO mice. (D) Cued recall fear memory response in the same groups of mice (6–8 months: n=8 (4M,4F) for WT, n=7 (4M, 3F) for 5LOKO, n=12 (6M, 6F) for 3xTg, n=13(7M, 6F) for 3xTg-5LOKO; 12–14 months: n=7(3M,4F) for WT, n= 7(4M, 3F) for 5LOKO, n=11 (6M,5F) for 3xTg, n=10 (4M, 6F) for 3xTg-5LOKO) (p<0.001). Values represent mean +/− standard error of the mean. Figure 2 Genetic absence of 5LO reduces Aβ peptide levels of deposition in brains of 3xTg mice. (A) Radioimmunoprecipitation assay (RIPA)-soluble and formic acid (FA)-extractable Aβ1-40 and 1-42 levels in cortex of 3xTg mice and 3xTg-5LOKO mice at 14 months of age [n=9 (5M, 4F)for 3xTg and n=10 (5M,5F) for 3xTg-5LOKO] (p<0.001). (B) Quantification of the area occupied with Aβ immunoreactivity in the brain cortices of the same group of mice (p<0.001). (C) Representative western blots of amyloid precursor protein (APP), ADAM-10, BACE-1, sAPPα, sAPPβ, CTFs, PS1, Nicastrin, APH-1 and Pen-2 in brain cortex homogenates from 14 month old 3xTg and 3xTg-5LOKO mice. (D) Densitometric analyses of the immunoreactivities to the antibodies shown in the panel C (p<0.01) [n=4 (2M, 2F) for 3xTg; n=4 (2M, 2F) for 3xTg-5LOKO]. Values represent mean +/− standard error of the mean. Figure 3 5LO modulates tau phosphorylation and metabolism in the brains of 3xTg mice. (A) Representative Western blot analyses for total tau (HT7) and phosphorylated tau at residues S202/T205 (AT8), T231/S235 (AT180), T181(AT270), S396(PHF-13) and S396/S404(PHF-1) in brain cortex homogenates of 3xTg and 3xTg-5LOKO mice at 14 months of age. (B) Densitometric analyses of the immunoreactivities to the antibodies shown in panel A (p <0.01). (C) Representative Western blot analysis of sarkosyl-soluble tau (HT7) in brain cortex homogenates from the same mice. (D) Densitometric analyses of the immunoreactivities shown in panel D (p <0.001). (E) Representative immunohistochemical stainings for AT8, PHF-13, PHF-1 positive areas in brain sections of 3xTg and 3xTg-5LOKO mice at 14 months of age. (F) Representative Western blot analyses for Cdk5, p35, p25, GSk3α, GSK3β, p-GSK3α, p-GSK3β, p38,p-p38, SAPK/JNK1, SAPK/JNK2, p-SAPK/JNK1 and p-SAPK/JNK2 protein levels in brain cortex homogenates from 3xTg and 3xTg-5LOKO mice 14 mo (G) Densitometric analyses of the immunoreactivities to the antibodies from the previous panel [3xTg, n=4 (2M,2F); 3xTg-5LOKO n=4 (2M,2F)] (p<0.001). Values represent mean +/− standard error of the mean. Figure 4 Genetic absence of 5LO ameliorates synaptic biomarkers and decreases neuroinflammation in 3xTg mice. (A) Representative Western blot analyses for synaptophysin (SYP), post-synaptic density protein 95 (PSD95) and MAP2 in brain cortex homogenates from wild type (WT), 3xTg and 3xTg-5LOKO mice. (B) Densitometric analyses of the immunoreactivities from panel A (p<0.001). (C) Representative immunohistochemical staining for SYP, PSD95 and MAP2 positive areas in brain sections of WT, 3xTg and 3xTg-5LOKO mice. (D) Representative Western blot analyses for GFAP and CD45 in brain cortex homogenates from 3xTg and 3xTg-5LOKO mice. (D) Densitometric analyses of the immunoreactivities to the antibodies from the previous panel (3xTg, n=4; 3xTg-5LOKO; n=4), (p<0.001). Values represent mean +/− standard error of the mean. Figure 5 Genetic absence or pharmacologic inhibition of 5LO rescues synaptic dysfunction in 3xTg mice. (A) Input/output (I/O) curves and representative fEPSPs at increasing stimulus strengths (0–300 μA) are shown for Wildtype, 3xTg, 3xTg/5LOKO and 3xTg+zileuton mice at 6 months of age. (B) Mean fEPSP slopes as a function of interpulse interval between the first and second fEPSPs evoked at CA3-CA1 synapses in slices from the same mice at 20, 50, 100, 200 and 1000 ms in the same animals. (C) fEPSP slopes were recorded for 3 hours and expressed as the percentage of pretetanus baseline in the same mice. (D) LTP magnitudes expressed as the percentages of baseline for 0–10 minutes post-tetanus [274.6% +/− 8.5% for WT (n=23 slices); 159.9% +/− 13.8% (n=19 slices) for 3xTg; 272.7% +/− 9.6% (n=20 slices) for 3xTg/5LOKO; 268.5% +/− 14.3% (n=5 slices) for 3xTg+zileuton)]. (E) For the same groups of mice, LTP magnitudes expressed as the percentages of baseline for 170–180 minutes post-tetanus (202.6% +/− 6.5% for WT; 121.0% +/− 3.4% for 3xTg; 197.1% +/− 11.5% for 3xTg/5LOKO; 156.9% +/− 6.9%; 199.5% +/− 7.4% for 3xTg+zileuton). (*p<0.0001). Values represent mean +/− standard error of the mean. Table 1 Antibodies used in this study Antibody Immunogen Host Application Source 4G8 aa 18-22 of human beta amyloid (VFFAE) Mouse IHC Covance APP aa 66-81 of APP {N-terminus} Mouse WB Millipore BACE-1 aa human BACE (CLRQQHDDFADDISLLK) Rabbit WB IBL ADAM10 aa 732-748 of human ADAM 10 Rabbit WB Millipore PS-1 aa arround valine 293 of human presenilin 1 Rabbit WB Cell Signaling Nicastrin aa carboxy-terminus of human Nicastrin Rabbit WB Cell Signaling APH-1 Synthetic peptide from hAPH-1a Rabbit WB Millipore Pen-2 aa N-terminal of human and mouse Pen-2 Rabbit WB Invitrogen sAPPα Synthetic peptide of the C-terminal part of Human sAPPα (DAEFRHDSGYEVHHQK) Rabbit WB Cell Signaling sAPPβ Synthetic peptide of the C-terminal part of human sAPPβ-sw (ISEVNL) Rabbit WB Cell Signaling CTFs a synthetic peptide [(C)KMQQNGYENPTYKFFEQMQN] corresponding to amino acids 751-770 of human precursor protein (APP), conjugated to KLH Rabbit WB Santa Cruz HT-7 aa 159-163 of human tau Mouse WB,IHC Pierce AT-8 Peptide containing phospho-S202/T205 Mouse WB,IHC Pierce AT-180 Peptide containing phospho-T231/S235 Mouse WB,IHC Pierce AT-270 Peptide containing phospho-T181 Mouse WB,IHC Pierce PHF-13 Peptide containing phospho-Ser396 Mouse WB,IHC Cell Signaling PHF-1 Peptide containing phospho-Ser396/S404 Mouse WB,IHC Dr. P. Davies GFAP aa spinal chord homogenate of bovine origin Mouse WB Santa Cruz CD45 Mouse thymus or spleen Rat WB BD Pharmingen SYP (H-8) aa 221-313 of SYP of human origin Mouse WB,IHC Santa Cruz PSD95 (7E3-1B8) Purified recombinant rat PSD-95 Mouse WB,IHC Thermo Scientific MAP2 Bovine brain microtubule protein Rabbit WB,IHC Millipore GSK3α/β aa 1-420 full length GSK-3β of Xenopus origin Mouse WB Millipore p-GSK3α/β aa around Ser21 of human GSK-3a. Rabbit WB Cell Signaling JNK2 aa of human JNK2 Rabbit WB Cell Signaling SAPK/JNK aa of recombinant human JNK2 fusion protein Rabbit WB Cell Signaling Phospho-SAPK/JNK aa Thr183/Tyr185 of human SAPK/JNK Mouse WB Cell Signaling p38 MAPK aa sequence of human p38MAPK Rabbit WB Cell Signaling p-p38 MAPK Aa resides surrounding Thr180/Tyr182 of human p38 MAPK Rabbit WB Cell Signaling Cdk5 aa C-terminus of Cdk5 of human origin Rabbit WB Santa Cruz P35/25 aa C-terminus of p35/25 of human origin Rabbit WB Santa Cruz Actin aa C-terminus of Actin of human origin Goat WB Santa Cruz ==== Refs 1 Selkoe DJ Alzheimer’s disease, genes, proteins, and therapy Physiol Rev 2001 81 741 766 11274343 2 Sze CI Troncoso JC Kawas C Mouton P Price DL Martin LJ Loss of the presynaptic vesicle protein synaptophysin in hippocampus correlates with cognitive decline in Alzheimer disease J Neuropathol Exp Neurol 1997 56 933 944 9258263 3 Sy M Inflammation induced by infection potentiates tau pathological features in transgenic mice Am J Pathol 2011 178 2811 2822 21531375 4 Terry RD Physical basis of cognitive alterations in Alzheimer’s disease synapse loss is the major correlate of cognitive impairment Ann Neurol 1991 30 572 580 1789684 5 Oddo S Triple transgenic model of Alzheimer’s disease with plaques and tangles: intracellular Abeta and synaptic dysfunction Neuron 2003 39 409 421 12895417 6 Radmark O Werz O Steinhilber D Samuelsson B 5-Lipoxygenase: regulation of expression and enzyme activity Trends Biochem Sci 2007 32 332 341 17576065 7 Chinnici CM Yao Y Praticò D The 5-lipoxygenase enzymatic pathway in the mouse brain: young versus old Neurobiol Aging 2007 28 1457 1462 16930777 8 Ikonomovic MD Abrahamson EE Uz T Manev H Dekosky ST Increased 5-lipooxygenase immunoreactivity in hippocampus of patients with Alzheimer’ diseases J Histochem Cytochem 2008 56 1065 1073 18678882 9 Firuzi O Zhuo J Chinnici CM Wisniewski T Praticò D 5-Lipoxygenase gene disruption reduces amyloid-β pathology in a mouse model of Alzheimer’s disease FASEB J 2008 22 1169 1178 17998412 10 Chu J Praticò D Pharmacological blockade of 5-lipoxygenase improves the amyloidotic phenotype of an Alzheimer’s disease transgenic mouse model Am J Pathol 2011 178 1762 1769 21435457 11 Chu J Giannopoulos PF Ceballos-Diaz C Golde TE Praticò D 5-Lipoxygenase gene transfer worsens memory, amyloid, and tau brain pathologies in a mouse model of Alzheimer disease Ann Neurol 2012 72 1762 1769 12 Goulet JL Snouwaert JN Latourt AM Coffman TM Koller BH Altered inflammatory responses in leukotriene-deficient mice Proc Natl Acad Sci 1994 91 12852 12856 7809134 13 Yang H Zhuo J Chu J Chinnici C Pratico D Amelioration of the Alzheimer’s disease phenotype by absence of 12/15-lipoxygenase Biol Psychiatry 2010 68 922 929 20570249 14 Zhuo JM Portugal GS Kruger WD Wang H Gould T Pratico D Diet-induced hyperhomocysteinemia increases amyloid-beta formation and deposition in a mouse model of Alzheimer’s disease Curr Alzheimer Res 2010 7 140 149 19939226 15 Andorfer C Hyperphosphorylation and aggregation of tau in mice expressing normal human tau isoforms J Neurochem 2003 86 582 590 12859672 16 Zucker RS Regehr WG Short-term synaptic plasticity Annu Rev Physiol 2002 64 355 405 11826273 17 Bliss TVP Collingridge GL A synaptic model of memory: long-term potentiation in the hippocampus Nature 1993 361 31 39 8421494 18 DeKosky ST Scheff SW Synapse loss in frontal cortex biopsies in Alzheimer’s disease correlation with cognitive severity Ann Neurol 1990 27 457 464 2360787 19 Scheff SW Scott SA DeKosky ST Quantitation of synaptic density in the septal nuclei of young and aged Fischer 344 rats Neurobiol Aging 1991 12 3 12 2002880 20 Dickson DW Crystal HA Bevona C Honer W Vincent I Davies P Correlations of synaptic and pathological markers with cognition of the elderly Neurobiol Aging 1995 16 285 304 7566338 21 Masliah E Altered expression of synaptic proteins occurs early during progression of Alzheimer’s disease Neurology 2001 56 127 129 11148253 22 Andersen P Morris R Amaral D Bliss T O’Keefe J The Hippocampus Book Oxford University Press Inc Oxford UK 2007 23 Imbesi M Zavoreo I Uz T Sharma RP Dimitrijevic N Manev H Manev R 5-Lipoxygenase inhibitor MK-886 increases GluR1 phosphorylation in neuronal cultures in vitro and in the mouse cortex in vivo Brain Res 2007 1147 148 153 2007 24 Chen H Manev H Effects of minocycline on cocaine sensitization and phosphorylation of GluR1 receptors in 5-lipoxygenase deficient mice Neuropharmacology 2011 60 1058 1063 20868701	23478745	PMC3688674	NO-CC CODE	2021-01-05 05:12:16	yes	Mol Psychiatry. 2014 Apr 12; 19(4):511-518
==== Front Int J Environ Res Public HealthInt J Environ Res Public Health101238455International Journal of Environmental Research and Public Health1661-78271660-4601Molecular Diversity Preservation International (MDPI) 19139533ijerph-05-00152ArticlesSanitary Conditions of Public Swimming Pools in Amman, Jordan Rabi Atallah 1Khade Yousef 1Alkafajei Ahmed 1Aqoulah Ashraf Abu 21 Department of Public Health, Faculty of Medicine, Jordan University of Science and Technology (JUST), Irbid, Jordan.2 Public Health Inspector, Health Department, Al-Rumtha, Jordan Correspondence to Dr. Atallah Rabi. E-mail: [email protected] 2008 30 9 2008 5 3 152 157 16 1 2008 20 5 2008 >© 2008 MDPI2008All rights reserved.This study was carried out in the summer of 2005 and investigated all of active public swimming pools (85 out of 93) in Amman, the capital of Jordan. The aim of this study was to find out if these swimming pools are in compliance with Jordanian Standards for Swimming Pools Water (JS 1562/2004). The pools were surveyed against the water microbial quality and other physicochemical parameters indicated in the standards. Two samples from each pool were collected for microbial analysis and pools monitoring were carried out during the afternoon of the weekends when the pools are most heavily used. The results indicated overall poor compliance with the standards. Compliance of the pools water to the microbial parameters was 56.5%, for residual chlorine 49.4%, for pH 87.7%, water temperature 48.8%, and bathing load 70.6%. The results also indicated that water microbial quality deteriorated with time. Multivariate analysis showed significant association of water contamination with time of sample collection, residual chlorine, water temperature and load of swimmers. The poor compliance was attributed to lack of proper disinfection, staff training, proper maintenance, and timely inspection. Swimming poolssanitary standardswater qualityJordanAmman ==== Body Introduction Swimming, one of the popular activities in Jordan, is a fun, active, and a healthy way to special leisure time especially in summer where ambient shade temperature exceeds 35°C (95°F) most of summer days. Therefore, swimming is a great way to relax and beat the summer heat. Pools are good recreational places where one can practice swimming as one of the popular sports. The importance of swimming pools in Jordan as a recreational tool acquires special importance. These pools are the only place for swimming for most of the Jordanians especially those living in Amman, the Jordanian capital, with a population of more than 2 million, or about 40% of the Jordanian population. The reason for that is because marine water is not easily accessible. The nearest normal sea water is the Red Sea which is more than 300 Km away from Amman. In spite of the importance and popularity of swimming pools in summer, they have been identified as posing a risk for infection by certain fast-growing environmental bacteria [1]. The most common disease caused by swimming pools is diarrhea. A person with diarrhea can easily contaminate the pool with fecal matter. Diarrhea is then spread when swimmers swallow this contaminated pool water. Moreover, swimming pools are used by a wide variety of people with different health conditions and are thus more susceptible to infection from opportunistic bacteria. Bathers contaminate the water with large amounts of microorganisms as a result of bathers’ secretions containing microorganisms which are shed from skin, mouth, nose and throat, urine, feces or by contaminated objects and clothes, making water a possible vehicle for the dissemination of diseases among swimmers. From the skin alone hundreds of millions of bacteria are rinsed during swimming [2, 3]. If left untreated, these pollutants will build up in water increasing the risk of infection to the swimmers. Microorganisms that are usually connected to swimming pools include Pseudomonas Aeruginosa, Legionellae, Cryptosporidium parvum, Giardia, Microsporidia, Dermatophytes and Keratinophilic fungi and Molluscum Contagiosum [4–11]. Swimming pools are well-documented transmission vehicle for infectious diseases throughout the world [2]. These diseases include: Bacterial diseases, such as, skin and eye infections, respiratory wound infections, gastroenteritis, folliculitis, external otitis, and corneal ulcer cheratitis (in wearers of contact lenses), swimming pool granuloma, cholera, paratyphoid fever, typhoid fever, hot-foot syndrome and haemolytic uraemic syndrome: Parasitic diseases, such as, chronic diarrhea, cryptosporidiosis, giardiasis, and dysentery: Fungal diseases, such as, a tinea pedis, superfacial mycoses, opportunistic mycoses, and hypersensitivity pneumonitis: Viral diseases, such as, a molluscum contagiosum infection, Hepatitis A, poliomyelitis, echovirus infection, and pharyngitis [2–6, 9–15]. Because of the health hazards of swimming pools, it is essential to monitor water quality in swimming pools to insure safety of water and complience with standards. The control of infection risks does require continuous water quality surveillance, assessment of the efficiency of treatment and disinfection processes, changes in chemical and physical characteristics of pools and evaluation of the behaviour of swimmers which may affect the water quality. Such water quality also depends on the total number of swimmers in the pools at any time, water temperature and environmental conditions. Ministry of Health carries out a water quality monitoring program which includes analysis of water samples and reporting on the swimming pool characteristics. It is beleived that such monitoring prgram does not reflect the quality of water because water samples are collected during the work days (Sunday to Thursday) and during working hours 8:00 pm– 2:00 am. Such timing occurs when the bathers, especially the young children who are brought by their working parents, at minimum. The highest number of bathers are expected to be on the weekends (Friday and Saturday), and Holidays. Therefore, it is believed that appropriate chemical and microbial water quality evaluation should be carried out on the weekend days to reflect the water quality and the consequent heath risk when peak number bathers are using the pools. This study was performed to ascertain the microbiological quality of swimming pool waters of Amman, in addition to some of the physical and chemical quality parameters and to find out if the swimming pools of Amman are in compliance with Jordanian Standards for Swimming Pools Water (JS 1562/2004), This study will provide baseline data about the quality of public swimming pools in Amman and identifies the risk factors associated with using these pools. It will suggest corrective measures to protect all swimmers from health hazards associated with swimming in unsanitary pools. Methods and Materials This study was conducted during the swimming season (June 18 – August 13, 2005). Amman has 93 swimming pools which constitute 65% of the total pools in Jordan. The study covered 85 swimming pools representing all operating swimming pools in Amman. The other 8 pools were not in operation at the time of the study. One hundred and seventy samples were collected from these pools. Two samples from each pool were collected at two different weekend days (85 in Saturday and 85 in Friday) and at different times of the day (111 at 12–3 PM, 32 at 3–4 PM, and 27 at 4–5 PM) from each swimming pool. Days and time of sampling were selected because the number of swimmers was at maximum. To insure the uniformity of water quality throughout the pool and to get a more representative sample, each water sample was collected at a depth of 50 cm of water surface and from four different points about one meter away from the pool edge. These samples were taken in 250 ml sterilized dark-colored glass bottles containing few drops of sodium thiosulphate to serve as inhibitor of chlorine action after sampling. The samples were transported refrigerated in ice box to the laboratory where they were analyzed, using multiple tube technique, for total coliform and fecal coliform test within 6 hours after collection. The sample was considered contaminated if it contained total coliform (TC) >1.1 MPN/100 ml, with or without fecal coliform, when tested by multi-tube method [19]. On the other hand, a separate sample was collected for on-site chemical analysis. The parameters measured included water temperature; pH, residual chlorine and transparency were measured. The microbial analysis for total coliform, and fecal coliform and chemical parameter measurement measurements were done according to the standard methods. 20 A pre-tested chick-list was prepared based on Jordanian Standard for Swimming Pools Water (JS 1562/2004) [18] and filled by direct observation, interview with swimming pool operator, field measurements in swimming pools area. The number of swimmers and swimming bathing load were noted. Data about pools and swimmers included: Type of pool, age and gender of pool users, taking shower before swimming, disinfection of feet by using disinfection liquid before stepping in the pool, cleanness of the pool water from floating material and availability of guidance board including health requirements. Swimming pool operators were interviewed for information about: recycling of the swimming pool water, treatment method of swimming pools water, method of disinfection and type of chlorine used. Data were analyzed using Statistical Package for Social Sciences (SPSS, version 11.5). Swimming pools, swimmers and sanitary parameters were described using frequencies and cross tabulation. Binary logistic regression was used to determine factors associated with TC > 1.1 MPN/100 ml. Comments about statistical significance refer to probability of less than 0.05. Results Characteristics of Swimming Pools The characteristics of the public swimming pools in Amman /Jordan that were open at the time of the study are depicted in Table 1. Sixty percent of swimming pools were outdoors. More than 60% of swimming pools were used by males and females of all ages, while one quarter of pools was used exclusively by males. About one-half of the swimming pools had water volume of more than 150 m3. Most of the pools (79%) were disinfected manually by chlorine powder. Almost all (98%) of swimming pools had wardrobes available for swimmers. Guidance board was available in 72% of pools. Adherence to bathing load was followed in 71% of pools. More than half (53.5%) of swimming pools had a number of swimmers, at the time of data collection, >20 persons. All swimming pools were free from floating material and were clean during visits. None of the surveyed public swimming pool was in full compliance with the Jordanian Standards for Swimming Pools Water (JS 1562/2004). Sanitary Parameters Table 2 shows the sanitary parameters of studied swimming pools. Less than half (48.8%) of the swimming pools met the water temperature standard (22 – 27°C). The majority (87.7%) of the analyzed samples revealed that pH value met the standard (7.2–7.8). Seventy four (43.5%) of the samples had TC > 1.1 MPN/100 ml and 94.7% of these samples were positive for fecal coliform. About half of the samples (49.4%) showed that free residual chlorine met the Jordanian standards (1–2 mg/l), while 51 (38.1%) of the analyzed samples had a concentration of free residual chlorine less than 1 ppm. This means that 38% of the swimming pools have unacceptable level of disinfection or do not comply with the standards limits. Moreover, 43.5% of the samples were unacceptable with respect to total coliform (> 1.1 MPN/100 ml). Factors Associated with Contamination of Swimming Pools by Total Coliform Out of 170 samples, 43.5% had a Total coliform >1.1 MPN/100 ml. Factors that were associated with contamination of swimming pools by TC in the multivariate analysis are shown in table 3. Compared with the samples collected at 12:00–3:00 p.m., samples collected at 3:00–4:00 p.m were more contaminated (OR= 2.91; 95% CI 1.06, 8.02) and samples collected at 4:00–5:00 p.m had higher probability of being contaminated that two types previous samples. (OR=3.50; 95% CI 1.07, 11.14) were more likely to have TC >1.1 MPN/100 ml. Total coliform was >1.1 MPN/100 ml in 51% of samples collected on Saturdays and in 35% of samples collected on Fridays without a statistically significant difference (p-value = 0.066). Contamination by total coliform was significantly associated with the total number of swimmers of more than 20 persons at the time of sampling (OR=3.15; 95% CI 1.41, 7.01). Free residual chlorine of less than 1 mg/l compared to that of 1–2 mg/l and water temperature of more than 27°C compared to water temperature of 22–27°C were significantly associated with increased odds (2.84) of contamination. However, pH value was not significantly associated with contamination. Discussion It is obvious from the results that none of the studied swimming pools completely comply with the Jordanian Standard for swimming pools especially for health related parameters, 43% of these pools decline severely from these standards. For free residual chlorine, more than half (50.6%) of the pools do not comply with the standards. The non complying pools have either higher (12.5%) or lower (38.1%) than the standards. Thus in the case of lower residual chlorine (< 1ppm) poor disinfection results are expected and they are actually reflected in the microbial contamination of the swimming pools. The assessment of some physical-chemical aspects, such as free available chlorine, pH and the swimming pool bathing could predict the quality of pool water. In fact, disinfection of swimming pools should control the organisms responsible for many diseases; however, some microbial contaminations may be relatively protected from disinfection by the filter material or structural features in the pool. Some researchers emphasize that the microbiological quality of swimming pools is best measured by using fecal coliform that indicates fecal contamination [20]. While others consider that the risk of infection is more associated with microorganisms derived from the skin, mouth, and upper respiratory tract of bathers rather than fecal contamination [21]. Some authors consider that microorganisms which indicate hygienic conditions (total coliform and heterotrophic bacteria) and fecal pollution are the best ones [22]. Nevertheless, there are doubts if any microorganism can reliably predict the health risks associated with swimming [14]. Prevalence of Bacterial Contamination The TC was >1.1 MPN/100 ml in 43.5% of water samples and FC was positive in 94.7% of samples which contained TC >1.1 MPN/100 ml. These figures were much higher than that reported in South America where total coliform and fecal coliform were 13.3% and 5.6% of swimming pools, respectively [14]. In Bologna, Italy, 34.2% of swimming pools did not conform to the recommended limits for microbiological characteristicsm [23]. The prevalence of contamination among the swimming pools in Amman is much higher than other countries. This can be explained by the finding that none of the swimmers take shower before swimming and most of the swimmers did not disinfect their feet by disinfection liquid before entering the pool. The prevalence of bacterial contamination in the swimming pools in this study is much higher than that reported by Ministry of Health (MOH) in the same period of the year (Jun – August). This finding may be due to the fact that MOH Employees collect sample during working days from 9:00 a.m. to 1:00 p.m. In addition, employees in MOH used to collect their samples from pool surface from any site in the pool (the employees collect the samples in this way because sample collection method is not mentioned in the Jordanian standard). This time of sample collection by MOH employees does not reflect the actual quality of swimming pools, because most of swimmers swim in the weekend after noon. Moreover, pools’ staff are not doing a proper control process either due to the lack of training of operators or the use of improper method of disinfection as using manual chlorination by un trained operator instead of using automatic disinfection that will produce fixed and continuous chlorine dose. However, the responsibility for controlling the quality of the swimming pools does not fall on ministry of health or pool staff alone. Swimmers need to be educated about the necessary hygienic behavior that is needed to prevent or reduce the spread of health hazards. Factors Associated with Bacterial Contamination Bacterial contamination was significantly associated with the time of sample collection where there was a trend of increase in the prevalence of bacterial contamination with late hours of the day. This may reflect a cumulative effect of swimmers as a source of contamination. The number of swimmers of more than 20 persons at the time of sample collection was associated with a high prevalence of contaminations, because none of the swimmers take shower before swimming and 97.5% of swimmers did not disinfect their feet before swimming. The finding is consistent with studies in South America [14]. The cutoff point (20 swimmers) was chosen because nearly half of pools contain more than 20 swimmers. Moreover, a significant association was found between water quality and the numbers of swimmers at this cut off point were water quality was acceptable in swimming pool with less than 20 swimmers. The pools with a temperature of more than 27°C temperature were more likely to be contaminated than pools with a temperature between 22–27°C. The temperature had direct influence on swimming pool water contamination by increasing microbial growth. The increase in temperature encourages the growth of bacteria. This finding is consistent with other studies [1, 6, 14]. Free residual chlorine had a significant negative effect on microbiological contamination. Free residual chlorine of less than 1mg/l was significantly associated with increased odds (8.29) of contamination. This indicates that chlorine concentration between 1–2 mg/l is a good operational index to control the quality of swimming pool water as shown by other studies [1, 6, 14]. Although free residual chlorine was available, some pools contained TC >1.1 MPN/100 ml; this may reflect recent contamination. On the other hand, there is no significant association between water pH value and microbial contamination. This finding may be explained by the small number of pools that had a pH < 7.2 (9%) and pH >7.8 (4%). In addition, although there was no significant association between day of sampling and contamination the results indicate that samples collected on Saturdays have increased odds (2.00) of fecal contamination when compared with Friday. This is an indication of ineffective water treatment procedures and the cumulative effect of contamination. It seems that water treatment does not cope with the increased load of bathers during the weekend days. Conclusions None of the surveyed public swimming pools was in full compliance with the Jordanian Standards for Swimming Pools Water 1562/2004. There was a significant association between pools contamination and: Time of sample collection (contamination was highest during the period from 4–5 p.m.). High number of swimmers (more than 20 swimmers in the pool). Water temperature (when it is more than 27°C). Free residual chlorine concentration (when it is less than1mg/l). Recommendations Using such swimming pools may result in health hazards for the swimmers. To avoid such hazards the following recommendations are suggested: Better and stricter inspection procedure by the ministry of health should be used. Inspection should be done during the weekends and holidays when the pools are most heavily used. Maintaining proper balance of swimming pool water chemistry especially water chlorination. Continuous monitoring for water quality indicators, (free residual chlorine and temperature) especially when the number of swimmers increases to more than 20 swimmers in the pool. Provide educational signs to advise bathers to: Wash their hands with soap and water after using the toilet. Take their children to bathroom before swimming. Refrain from swimming when they have diarrhea or infectious disease. Table 1: Characteristics of the Public Swimming Pools in Amman/ Jordan Variable Frequency n (%) Type of the pool Indoor 34 (40.0) Outdoor 51 (60.0) Users Males only 23 (27.1) Females only 03 (03.5) Males and females <18 years 07 (08.2) Males and females of all ages 52 (61.2) Volume of the pool (m3) <=150 41 (48.2) >150 44 (51.8) Method of disinfection Chlorination 80 (94.1) Hydrogen Peroxide (H2O2) 05 (05.9) Method of Chlorination* Gas (Auto) 03 (03.5) Powder (Manual) 67 (78.8) Powder (Auto) 06 (07.1) Liquid (Auto) 04 (04.7) Availability of wardrobe for each swimmer Yes 83 (97.6) No 02 (02.4) Availability guidance board Yes 61(71.8) No 24 (28.2) Adherence to bathing load Yes 60 (70.6) No 19 (22.4) Table 2: Public Swimming Pools Sanitary Parameters Parameter Frequency n (%) Water transparency Yes 157 (92.4) No 13 (07.6) Water temperature 22–27 °C (Required) 83 (48.8) >27 °C 87 (51.2) <22 °C 0 PH Value 7.2–7.8 (Required) 149 (87.7) < 7.2 15 (08.8) >7.8 06 (03.5) Free residual chlorine 1–2 mg/l (Required) 79 (49.4) < 1 mg/l 61 (38.1) > 2 mg/l 20 (12.5) Total coliform <1.1 MPN/100 ml (Required) 96 (56.5) >1.1 MPN/100 ml 74 (43.5) Fecal coliform* Negative 04 (05.3) Positive 71 (94.7) n = Numbers of samples. * Only in the samples that had TC >1.1 MPN/100 ml. Table 3: Multivariate analysis of factors associated with contamination by Total Coliform. Factors Total Coliform Total n (%) OR (95% CI) P - Value 170 74 (44) Hour of collection 12–3 PM 111 39 (35.1) 1 3–4 PM 32 17 (53.1) 2.91 (1.06, 8.02) 0.039 4–5 PM 27 18 (66.6) 3.50 (1.07,11.14) 0.038 Day of sampling Friday 85 30 (35.3) 1 Saturday 85 44 (51.8) 2.00 (2.12, 0.95) 0.066 Number of swimmers <=20 84 28 (33.3) 1 >20 86 46 (53.5) 3.15 (1.41, 7.01) 0.005 Temperature 22–27°C 83 24 (28.9) 1 >27 °C 87 50 (57.5) 2.84 (1.27, 6.31) 0.011 Free residual chlorine* 1–2 mg/l 79 20 (25.3) 1 <1 mg/l 61 45 (73.8) 8.29 (3.44,19.98) <0.0001 >2 mg/l 20 06 (30.0) 1.00 (0.27, 3.72) 0.997 pH value 7.2–7.8 149 63 (45.0) 1 <7.2 15 08 (53.3) 1.53 (0.30, 7.81) 0.606 >7.8 06 03 (50.0) 0.93 (0.13, 6.75) 0.941 OR: Adjusted odds ratio. ==== Refs Reference 1 Leoni E Legnani P Mucci MT Pirani R Prevalence of mycobacteria in a swimming pool environment Microbiology 1999 87 683 688 3 Borgmann-Strahsen R Comparative assessment of different biocides in swimming pool water International Biodeterioration & Biodegradation 2003 51 291 297 4 Sato MIZ Alves MN Stoppe NC Martinys MT Evaluation of Culture Media For Candida Albicans And Staphylococcus Aureus Recovery In Swimming Pool Pergamon 1995 29 10 2412 2416 5 Tate D Mawer S Newton A Outbreak of Pseudomonas aeruginosa folliculitis associated with a swimming pool inflatable Epidemiology & Infection 2003 130 187 192 12729186 6 Fiorillo L Zucker M Sawyer D Lin A The Pseudomonas Hot-Foot Syndrome The New England Journal of Medicine 2001 345 335 338 11484690 7 Leoni E Legnani P Buccisabattini MA Righi F Prevalence of legionella ssp. In swimming pool environment Pergamon 2001 35 15 3749 3753 8 Andrey E Floyd F Temothy A Elena N Andry T Ford F Serological Evidence of Cryptosporidium Infections in a Russian City and Evaluation of Risk Factors for Infections Ann Epidemiol 2004 14 129 136 15018886 9 Ekramul HM Virginia H Tord K Robert S Roy LY Risk of giardiasis in Aucklanders Int J Infect Dis. 2002 6 191 197 12718834 10 Fournier S Dubrou S Liguory O Gaussin F Santillana H Sarfati C Molina JM Derouin F Detection of microsporidia, cryptosporidia and giardia in swimming pools Immunology and Medical Microbiology 2002 33 209 213 12110483 11 Ali-Shtayeh MS Tayseer K Khaleel M Rana J Ecology of dermatophytes and other keratinophilic fungi in swimming pools and polluted and unpolluted streams Mycopathologia 2002 156 193 205 12749584 12 Khai Y Roberts C Roberts J Molluscum contagiosum, swimming and bathing Australasian Journal of Dermatology 1999 40 89 92 10333619 13 Parent L Salam M Appelbaum P Dossett J Disseminated Mycobacterium marinum infection and bacteremia in a child with severe combined immunodeficiency Clinical Infectious Diseases 1995 21 1325 1327 8589169 14 Von Reyn CF Pestel M Arbeit RD Clinical and epidemiologic implications of polyclonal infection due to Mycobacterium avium complex Research in Microbiology 1996 147 24 30 8761718 15 Martinys MT Sato MIZ Alves MN Stoppe NC Prado VM Sanchez PS Assessment Of Microbiological Quality For Swimming Pools In South America Pergamon 1995 29 10 2417 2420 16 Gonzalez-Mancebo E Gomez M Diez L Pulido Z Alfaya T Leon F Swimming-pool pneumonitis. European Journal of Allergy and Clinical Immunology 2000 55 782 783 17 Department of Statistics Statistical Year Book. Amman Jordan 2003 18 Ministry of Health, Department of Environment Health Amman Jordan 2004 19 Jordan institution for Standards and Metrology. Jordanian standard for swimming pool water. First edition. Hashemite Kingdom of Jordan. 1562/2004 20 American Public Health Association, American Water Works Association and Water Environment Federation Standard Methods for the Examination of Water and Wastewater 20th ed Washington 1998 21 Esterman A Roder DM Cameron AS Robenson BS Walters RP Lake JA Christy PE Determinants of the Microbiological Characteristic of South Australian Swimming Pools Appl. Environ. Microbial 1984 47 325 328 22 Mossel DA Microbiological markers for swimming-Associated infectious health hazard Am. J. Publ. 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==== Front 10094135421750Nat ImmunolNat. Immunol.Nature immunology1529-29081529-29162364450410.1038/ni.2607nihpa464426ArticleAkt and mTOR pathways differentially regulate the development of natural and inducible TH17 cells Kim Jiyeon S. 12Sklarz Tammarah 1Banks Lauren 12Gohil Mercy 1Waickman Adam T. 3Skuli Nicolas 1Krock Bryan L. 1Luo Chong T. 4Hu Weihong 5Pollizzi Kristin N. 3Li Ming O. 4Rathmell Jeffrey C. 5Birnbaum Morris J. 6Powell Jonathan D. 3Jordan Martha S. 7Koretzky Gary A. 181 Abramson Family Cancer Research Institute, University of Pennsylvania, Philadelphia, PA, USA2 University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA3 Sidney-Kimmel Comprehensive Cancer Research Center, Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, MA, USA4 Immunology Program, Memorial Sloan-Kettering Cancer Center, New York, NY, USA5 Department of Pharmacology and Cancer Biology, Department of Immunology, Sarah W. Stedman Nutrition and Metabolism Center, Duke University, Durham, NC, USA6 The Institute for Diabetes, Obesity and Metabolism, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA7 Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA, USA8 Department of Medicine, University of Pennsylvania, Philadelphia, PA, USACorrespondence should be addressed to: M.S.J ([email protected]) or G.A.K. ([email protected])2 5 2013 05 5 2013 6 2013 01 12 2013 14 6 611 618 Users may view, print, copy, download and text and data- mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: http://www.nature.com/authors/editorial_policies/license.html#termsNatural T helper 17 (nTH17) cells are a population of interleukin 17 (IL-17)-producing cells that acquire effector function in the thymus during development. Here we demonstrate that the serine/threonine kinase Akt plays a critical role in regulating nTH17 cell development. While Akt and the downstream mTORC1–ARNT–HIFα axis were required for inducible TH17 (iTH17) cell generation in the periphery, nTH17 cells developed independently of mTORC1. In contrast, mTORC2 and inhibition of Foxo proteins were critical for nTH17 cell development. Moreover, Akt controlled TH17 subsets through distinct isoforms, as deletion of Akt2, but not Akt1, led to defective iTH17 cell generation. These findings reveal novel mechanisms regulating nTH17 cell development and previously unknown roles of Akt and mTOR in shaping T cell subsets. ==== Body Interleukin-17 (IL-17) and the cells that produce this cytokine are important in mediating protection against extracellular pathogens1. Dysregulation of IL-17 has also been linked to autoimmunity and inflammatory disorders; hence, there is great interest to better define the cell types that produce IL-17 and to understand how its production is regulated. The best characterized source of IL-17 is T helper 17 (TH17) cells that arise from naïve CD4+ T cells in response to antigenic stimulation in the appropriate cytokine environment in the periphery, hereafter referred to as inducible TH17 (iTH17) cells. Recently, we and others identified another IL-17+ CD4+ T cell population that acquires the capability of producing IL-17 during development in the thymus2, 3. These natural TH17 (nTh17) cells are poised to produce cytokines upon stimulation without further differentiation in the periphery. While iTH17 and nTH17 cells share many features including expression of retinoid orphan receptor (ROR)γt, CD44 and CCR6 and production of IL-17 (IL-17A), IL-17F and IL-22, the signaling pathways directing their development are not well understood. Akt is a serine/threonine kinase that plays a central role in diverse processes including cell survival, proliferation, differentiation and metabolism. In T cells, Akt regulates development and is activated upon cytokine, costimulatory and antigen receptor engagement4. These extracellular signals activate phosphoinositol-3-kinase (PI(3)K) to generate phophatidylinositol -3′-phosphate (PIP3) to which Akt binds and thereby localizes to the plasma membrane, where it is phosphorylated at two key residues. Phosphatidylinositol-dependent kinase 1 (PDK1) phosphorylates Akt at threonine 308 (T308), while phosphorylation at serine 473 (S473) is mediated by mammalian target of rapamycin complex 2 (mTORC2). Akt phosphorylates an array of targets including glycogen synthase kinase 3 (GSK3), forkhead box protein O1 (Foxo1), Foxo3a and tuberous sclerosis complex 2 (TSC2), which leads to activation of the mTOR complex 1 (mTORC1). mTORC1 and mTORC2 are two distinct complexes that share a core catalytic subunit, mTOR5. mTORC1 consists of mTOR, Deptor, mLST8, PRAS40 and the scaffolding protein Raptor. Activation of mTORC1 promotes phosphorylation of downstream translational regulators, cell growth, and metabolism6. mTORC2 also contains Deptor and mLST8 but, unlike mTORC1, includes Protor, mSIN1 and Rictor. Disruption of mTORC2 specifically abolishes Akt phosphorylation at S473 but not at T308, resulting in loss of phosphorylation of Foxo proteins7, 8. Of note, loss of mTORC2 does not abrogate phosphorylation of all Akt substrates, as GSK3 and TSC2 are still phosphorylated in its absence. Both Akt and mTOR are essential for regulating the function and differentiation of CD4+ T cell subsets9. In vitro blockade of Akt signaling using Akt inhibitors results in robust induction of Foxp3 (ref. 10), a critical regulator of T regulatory (Treg) cells, whereas expression of constitutively active Akt inhibits Treg cell generation both in vitro from peripheral CD4+ T cells and in vivo among developing thymocytes11. Consistent with these findings, CD4+ T cells lacking mTOR fail to differentiate into TH1, TH2 or iTH17 cells and instead become Foxp3+ Treg cells12. Moreover, selective inhibition of mTORC1 results in defective TH1 and iTH17 cell differentiation leaving TH2 differentiation intact, while in the absence of mTORC2 activity, CD4+ T cells fail to differentiate into TH2 cells but retain their ability to become iTH17 cells13, 14. To date, however, neither the role of Akt or mTOR in the development of nTH17 cells had been studied. Using genetic and pharmacological modulation of Akt activity, we show that Akt is required for the development of both nTH17 and iTH17 cells. However, unlike iTH17 cells that require mTORC1- but not mTORC2-activity for their development, we found that nTH17 cells develop normally in the absence of mTORC1 activity but rely on mTORC2. In line with the role of Akt and mTORC2 in nTH17 cells, mice deficient in both Foxo1 and Foxo3a (inhibitory proteins whose function is blocked by Akt and mTORC2) showed greatly enhanced nTH17 cell development. In addition to distinct upstream activation, Akt isoform-specific activity also differentially contributes to nTH17 and iTH17 cell development. Deletion of Akt2 resulted in defective iTH17 cell differentiation but preservation of nTH17 cells. Collectively, our findings reveal critical roles of Akt isoforms and the two mTOR complexes in controlling the development of TH17 cell subsets. RESULTS Akt regulates the development of nTH17 and iTH17 cells To investigate the signaling pathways required for nTH17 cell development, we examined Akt phosphorylation in freshly isolated mouse nTH17 cells, without the addition of extracellular stimuli or presence of serum in the media. Flow cytometric analysis revealed constitutive phosphorylation+ of S473 in nTH17 cells but not in thymic Foxp3 natural Treg (nTreg) cells or “naïve” (CD44lo CCR6−) CD4 single positive (SP) thymocytes (Fig. 1a). In line with this finding, S6 kinase (S6K) and S6 were also phosphorylated in nTH17 cells but not in naïve CD4SP thymocytes (Fig. 1b), suggesting that Akt is constitutively activated in nTH17 cells. The selectivity of phospho-S6K and phospho-S6 staining was verified by the absence of staining in rapamycin-treated nTH17 cells. While nTH17 cells have been described in mice2, 3, it has been unclear whether a similar population exists in humans. In 18–19 week old human fetal thymic tissue, IL-17+ CD4SP TCRαβ+ cells were readily observed (Fig. 1c), constituting 1–2% of the CD4SP thymocyte population. Like their murine counterparts, human nTH17 cells expressed the transcription factor RORγt and the chemokine receptor CCR6 and did not co-express Foxp3 (Supplementary Fig. 1a,b). nTH17 cells were present in human umbilical cord blood, albeit at a lower frequency compared to the thymus (Supplementary Fig. 1c). Human thymic nTH17 cells showed constitutive phosphorylation of Akt (S473) (Fig. 1d). Given the constitutive phosphorylation of Akt in nTH17 cells, we hypothesized that Akt may regulate development of these cells. To test this, we utilized pharmacological and genetic approaches to modulate Akt activity. First, an allosteric Akt inhibitor, AKTi, which targets both Akt1 and Akt2 isoforms, was used in fetal thymic organ culture (FTOC) to assess the effect of Akt loss-of-function on nTH17 cell development. Since Akt is critical for early thymocyte development15, 16, fetal thymi were allowed to develop for 5 days before addition of the inhibitor to ensure normal transition from the CD4−CD8− double-negative (DN) to double-positive (DP) thymocyte stage. This strategy allowed us to interrogate the importance of Akt activity during the transition from DP to CD4SP stage. Although AKTi-1/2 treatment did affect overall thymic cellularity, primarily by reduction of DP cells, the development and frequency of CD4SP and CD8SP thymocytes were relatively preserved (Supplementary Fig. 2). Inhibition of Akt resulted in significant reduction of the nTH17 cell population compared to control FTOC (Fig. 1e, top). This reduction was not simply inhibition of cytokine production, since the population of RORγt+ CD44hi cells was also decreased, suggesting that Akt controls nTH17 cell development at the transcriptional level (Fig. 1e, middle). As expected given the known role of Akt as a negative regulator of Foxp3 expression11, in the same FTOC, Foxp3+ nTreg cells showed a reciprocal increase upon AKTi treatment (Fig. 1e, bottom). This effect was not specific to AKTi, as use of a pharmacologically distinct Akt inhibitor (MK-2206) resulted in the same effect on nTH17 and nTreg cells (Supplementary Figs. 2,3a). To complement the Akt loss-of-function studies, we investigated the effect of enhanced Akt activity on nTH17 cell development. For these studies, we used transgenic mice expressing a myristoylated form of Akt (myr-Akt) that causes its association with the plasma membrane, resulting in constitutive activation17. Myr-Akt mice showed greatly enhanced nTH17 cell development compared to controls assessed both by cytokine production and RORγt expression (Fig. 1f). These data suggest that activated Akt drives an increased number of developing thymocytes to adopt an nTH17 cell fate. These findings led us to investigate whether Akt also has a role in iTH17 cell generation. Akt regulates TH17-cytokine production in activated/memory human T cells18; however, the role of Akt in iTH17 cell development from naïve T cells has not been directly evaluated. Purified naïve CD4+ splenic T cells were activated for 18 h with anti-CD3 and anti-CD28 and further cultured for 36 h in the presence of iTH17-promoting cytokines with or without AKT inhibitor. Upon AKTi treatment, iTH17 cell differentiation was inhibited in a dose-dependent manner (Fig. 1g). A significant population of iTreg cells was generated under iTH17 cell-promoting conditions when AKTi was added, and this population increased in a dose-dependent manner, highlighting the reciprocal developmental relationship between iTH17 and iTreg cells. Similar results were observed with MK-2206 (Supplementary Fig. 3b). Taken together, these results show that activation of Akt is critical for both nTH17 and iTH17 cell development. nTH17 cell development is ARNT-HIFα-mTORC1 independent Akt phosphorylates a number of substrates including TSC2, leading to mTORC1 activation (Supplementary Fig. 4). Among mTORC1 target genes, recent studies have identified hypoxia-inducible factor (HIF)1α as a key transcriptional regulator of iTH17 development19, 20. Activity of HIF1α requires dimerization with aryl hydrocarbon receptor nuclear translocator (ARNT) (also known as HIF1β)21. Under in vitro iTH17 cell promoting conditions, ARNT-deficient CD4+ T cells, from Arntfl/fl Vav-cre (ARNT-cKO) mice, showed defective iTH17 cell differentiation and increased generation of iTreg cells (Fig. 2a), similar to results reported for HIF1α-deficient T cells20. The defect in iTH17 cell generation also occurs in vivo, as we observed markedly decreased iTH17 cells in the small intestine lamina propria (LP) of ARNT cKO mice (Fig. 2b). To our surprise, however, we found nTH17 cells were increased in thymi of ARNT cKO mice compared to wild-type controls (Fig. 2c). Like cells in wild-type mice, ARNT-deficient nTH17 cells also expressed both IL-17F and IL-22 (Supplementary Fig. 5). Thymic nTreg cells were not affected by ARNT deficiency (Fig. 2c), similar to what has been reported for Hif1αfl/fl CD4-cre mice19, 20. ARNT also regulates the activity of aryl hydrocarbon receptor (AhR), which is known to play an important role in iTH17 cell development22, 23. In contrast to the selectively high expression of AhR in iTH17 cells, nTH17 cells expressed lower amounts of AhR similar to mature naïve CD4+ T cells and other thymocyte populations (Fig. 2d). Thus, it appears likely that nTh17 cells develop independently of AhR. Our finding that ARNT is differentially involved in nTH17 versus iTH17 cell development led us to investigate the role of mTORC1 in nTH17 cells. mTORC1 is regulated by Rheb, a small GTPase, which is activated following phosphorylation and inhibition of the GTPase-activating protein TSC, composed of TSC1 and TSC2. Deletion of Rheb in T cells abrogates mTORC1 activation13. Consistent with the defective iTH17 cell differentiation previously reported in Rheb-deficient T cells13, we found that iTH17 cells were diminished in the small intestinal LP of Rhebfl/fl CD4-cre mice (RhebΔT) (Fig. 2e). In contrast, nTH17 cells were not decreased in the thymi of RhebΔT mice (Fig. 2f). Taken together, these results show that the mTORC1-ARNT-HIF1α pathway, while critical for iTH17 cells, is dispensable for nTH17 cell development and suggest differential roles of Akt signaling in nTH17 versus iTH17 cell development. nTh17 cell development is mTORC2 dependent Since mTORC2 is responsible for phosphorylating Akt on S473, the site constitutively phosphorylated in nTH17 cells, we investigated the role of this complex in nTH17 development. For these studies, we made use of mice with deletion of Rictor selectively in the T cell compartment (Rictorfl/fl CD4-cre; RictorΔT mice). Analysis of thymi from these mice revealed greatly defective nTH17 cell development (Fig. 3a). In contrast, in vivo iTH17 cell generation remained intact in RictorΔT mice as small intestinal LP iTH17 cells were present at normal numbers (Fig. 3b), consistent with the ability of Rictor-deficient T cells to differentiate into iTH17 cells in vitro13. Collectively, these data indicate that while Akt is critical for development of both nTH17 and iTH17 cells, these two cell subsets have opposing reliance on the functions of mTORC1 versus mTORC2. Foxo proteins negatively regulate nTH17 cell development mTORC2 and Akt have been shown to be responsible for phosphorylation of Foxo proteins in multiple cell types7, 8, including CD4+ T cells and thymocytes19,24. Given the requirement of both mTORC2 and Akt for nTH17 cell development, we investigated the role of Foxo proteins in the development of these cells. Since phosphorylation of Foxo proteins leads to their degradation, we hypothesized that Foxo proteins might be negative regulators of nTH17 cell development and that Foxo function might be diminished in nTH17 cells compared to other thymocyte populations. We tested this first by examining mRNA abundance of two transcriptional targets of Foxo, Klf2 and S1pr1. Using RT- PCR, we found that mRNA expression of both genes was diminished in nTH17 cells compared to CD44lo CCR6− CD4SP thymocytes (Fig. 4a). As expected since Foxo proteins bind to the Foxp3 locus and positively control Foxp3+ Treg cell differentiation25, 26, Klf2 and S1pr1 mRNA expression was relatively increased in nTreg cells (Fig. 4a). To directly test the function of Foxo proteins in nTH17 cell development, we utilized mice in which both Foxo1 and Foxo3a are deleted in the T cell compartment (Foxo1fl/fl Foxo3afl/fl CD4-cre referred to as Foxo1ΔT Foxo3ΔT mice). Since these mice develop an inflammatory phenotype as they age, we restricted our analysis to 3-week old animals25. Foxo1ΔT Foxo3ΔT mice exhibited enhanced nTH17 cell numbers in the thymus (Fig. 4b), suggesting that Foxo proteins negatively regulate nTH17 cell development. Although it is possible that peripheral cytokine dysregulation could alter the thymic environment, mice used in this study showed no overt signs of disease. Moreover, the previously reported defect in Treg development observed in 3-week old Foxo1ΔT Foxo3ΔT mice is cell- autonomous and occurs independently of peripheral T cell activation. Together with the cell intrinsic downregulation of Foxo targets, these data suggest Foxo proteins restrict nTH17 cell development. We also investigated the role of another key Akt substrate, GSK3, which is inhibited upon its phosphorylation. Using knock-in mice expressing an “uninhibitable” form of GSK3α/β, GSK3(S21A, S9A)27, we found intact nTH17 and iTH17 cell generation suggesting that inhibition of this kinase does not regulate either TH17 subset (Fig. 4c,d). iTH17 cell differentiation requires Akt2 An alternative mechanism by which Akt can regulate different signaling pathways is through the differential involvement of distinct Akt isoforms. In eukaryotes, three Akt isoforms exist (Akt1/PKBα, Akt2/PKBβ, and Akt3/PKBγ). Akt1 and Akt2 are the predominant isoforms in T cells, and deletion of both Akt1 and Akt2 results in defective thymocyte development15, 16. While all three isoforms share a high degree of structural similarity, a number of studies demonstrate isoform-specific functions30, 31. Therefore, we speculated that different Akt isoforms might mediate the selective involvement of downstream pathways in nTH17 and iTH17 cell development. To test this hypothesis, we utilized mice deficient in either Akt1 or Akt2. As reported previously15, 16, global thymic development was intact in these mice and we found that nTH17 cell development also proceeded normally in the absence of either Akt1 or Akt2 (Fig. 5a). In contrast, there was a unique requirement for Akt isoforms in iTH17 cell development. We observed a marked defect in iTH17 cells in the small intestinal LP of Akt2−/− mice, whereas iTH17 development in Akt1−/− mice was normal (Fig. 5b). These findings are not due to differential or predominant expression of Akt2 mRNA in iTH17 cells compared to other thymocyte and TH subsets (Supplementary Fig. 6). In addition, Akt1 and Akt2 are expressed similarly in resting and activated T cells and contribute equally to the pool of active Akt following stimulation with anti-CD3 and anti-CD28 at both early and late time points (Supplementary Fig. 7a,b). IL-6 is important for iTH17 cell differentiation and likely contributes to Akt activation in this context. However, IL-6 receptor expression was equivalent between wild-type, Akt1−/− and Akt2−/− CD4+ T cells (Supplementary Fig. 8a,b), and IL-6 stimulation of naïve CD4+ T cells did not differentially activate Akt1 versus Akt2 (Supplementary Fig. 8c,d). Thus, the ability of CD4+ T cells to mediate early IL-6 signaling events is not governed uniquely by Akt2. Cell-intrinsic regulation of iTH17 differentiation by Akt2 As Akt2−/− mice have significant metabolic abnormalities32, it was possible that their defective iTH17 cell development was a result of the abnormal physiological environment. To address this possibility, we first cultured purified naïve CD4+ T cells from wild-type and Akt2−/− mice under iTH17 cell promoting conditions in vitro. Akt2−/− T cells displayed defective iTH17 cell differentiation under optimal TH17-promoting conditions (Fig. 6a). This result was not due to a global defect in T helper cell differentiation, as TH1 and TH2 differentiation were unaffected (Supplementary Fig. 9a,b). Moreover, T cells from mice with restricted deletion of Akt2 to the T cell compartment (Akt2fl/fl CD4-cre; Akt2ΔT) exhibited similar defects in iTH17 differentiation in vitro (Supplementary Fig. 9c). Furthermore, iTreg cell differentiation of Akt2−/− T cells was enhanced compared to wild-type T cells when cultured under Treg-polarizing conditions (Fig. 6b). In light of our data and previous reports20, 21 indicating the importance of the ARNT-HIF1α-mTORC1 pathway in iTH17 development, we assessed the expression of HIF1α protein in in vitro-generated iTH17 cells. Detection of intracellular HIF1α protein expression by flow cytometry was verified by experiments using primary T cells from mice deficient in HIF1/2α and isotype control antibodies (Supplementary Fig. 10a,b). When cultured under TH17- promoting conditions, CD4+ T cells from Akt2ΔT mice showed less HIF1α expression compared to their wild-type counterparts (Fig. 6c). This defect was seen only under TH17 skewing conditions, as wild-type and Akt2 ΔT cells stimulated with TH1-promoting cytokines similarly upregulated HIF1α expression (Supplementary Fig. 10b). These data indicate that Akt2 is required for proper HIF1α expression during TH17 differentiation and provide a potential mechanism by which Akt2 regulates iTH17 and iTreg cell development. To further investigate the cell-intrinsic versus -extrinsic nature of the iTH17 cell defect in Akt2−/− mice, we created mixed bone marrow (BM) chimeras where wild-type (Thy1.1+) and Akt2−/−(Thy1.1−) BM progenitor cells were mixed at 1:1 ratio and transplanted into lethally irradiated wild-type (CD45.1+) hosts. T cell populations were assessed 8 weeks post-transplant. In the small intestine LP, iTH17 cells of Akt2−/− BM origin were greatly diminished compared to the cells of wild-type BM origin that had developed in the same host environment (Fig. 6d). Analysis of the mesenteric lymph nodes (MLN) also revealed a selective defect in iTH17 cell generation from the Akt2- deficient BM cells (Fig. 6e). Interestingly, in the intestine of these chimeras, Foxp3+ Treg cells of Akt2−/− BM origin were present at an increased frequency compared to those derived from the wild-type BM (Fig. 6f,g). Thymic nTH17 cells developed normally regardless whether they were of wild-type or Akt2−/− BM origin (Fig. 6h). Therefore, selective Akt2 deficiency leads to defective iTH17 cell generation in a cell-intrinsic manner. DISCUSSION Here we show that signaling through Akt is a shared and critical component for the development of nTH17 and iTH17 cells. This finding suggested that regulation of nTH17 and iTH17 populations might follow similar rules. However, we found substantial differences in Akt-, related signaling requirements for the development of these distinct subsets, as iTH17 cells require mTORC1 and the ARNT-HIFα pathway, whereas nTH17 cells do not. Moreover, mTORC2, an upstream activator of Akt, is essential for nTH17 cell development, yet dispensable for iTh17 cell differentiation. Additionally, we found differences in the requirement of Akt isoforms as iTH17 cells, but not nTH17 cells, are dependent on Akt2. mTORC1 and HIF1α are important for iTH17 cell differentiation19, 33. Thus, we were surprised to find that nTH17 cell generation was intact in RhebΔT mice and mice deficient in ARNT, a critical cofactor for HIF1α that associates with RORγt to drive IL17 gene expression19, 20. IL-17 production in the absence of ARNT indicates that IL17 transcription in nTH17 versus iTH17 cells is regulated in a fundamentally different manner, with possible implications for the function of these subsets in vivo. It is intriguing to speculate that ARNT/HIFα-independent IL-17 production allows nTH17 cells to serve as a basal and more consistent source of IL-17, one that optimally produces IL-17 regardless of O2 availability. Whether IL-17 production from nTH17 cells remains ARNT/HIFα-independent in peripheral tissues is under investigation. The notion of IL17 gene regulation being cell-type specific is consistent with a previous report showing that the NF-κB family members RelA and RelB are required for development of IL-17-producing γδ T cells and nTH17 cells yet dispensable for iTH17 cell differentiation34. Although the dependence of nTH17 cells on these NF-κB members was suggested to be downstream of lymphotoxin β receptor signaling, PKCθ phosphorylation and nuclear localization of NF-κB has also been shown to be dependent on mTORC214. Thus, it is possible that NF-κB activation by mTORC2 contributes to nTH17 cell development. We also demonstrated that Foxo proteins negatively regulate nTH17 cell development. Foxo proteins regulate a number of cellular responses and are silenced upon phosphorylation by Akt35. In many circumstances, Foxo phosphorylation by Akt is dependent upon mTORC2 mediated activation of Akt7, 8, 24. Given the requirements for mTORC2, Akt, and Foxo inhibition for proper nTH17 cell development, we speculate an mTORC2-Akt2-FoxO pathway is critical for nTH17 cell development. However, Foxo proteins can also be phosphorylated by other kinases, including serum glucocorticoid- regulated kinase 1 (SGK1)36. Therefore, it will be important to determine the role of these kinases in nTH17 cell development. Although we did not find a role for GSK3 in TH17 cell development, we note that a previous report showed enhanced in vitro iTH17 differentiation of GSK3(S21A,S9A) T cells28. While the nature of these discrepant results is unclear, the methodology for iTH17 induction in this prior report differs from our. Additionally, preliminary data from our laboratory have shown no difference in small intestinal LP iTH17 cell numbers in GSK3(S21A, S9A) compared to wild-type mice. Moreover, another recent report identified GSK3α as an upstream activator of Akt suggesting additional complexity in how this kinase may impact T cell subset development29. In addition to the differential reliance on mTOR complexes, we find distinct roles of Akt isoforms in nTH17 and iTH17 cell development. Analysis of mice deficient in Akt2 revealed reduced numbers of iTH17 cells in the small intestinal LP but normal numbers of nTH17 cells. Despite metabolic defects of Akt2−/− mice32, the iTH17 cell phenotype was determined to be cell-intrinsic, since we observed the same defects in mice in which Akt2 was deleted selectively in the CD4+ T cell compartment. Furthermore, in mixed BM chimeras generated with WT and Akt2−/− BM cells, only Akt2−/− derived T cells showed diminished IL-17 producing CD4+ T cells in the LP. This defect in iTH17 cell generation observed in Akt2−/− mice was recapitulated in vitro where the lack of IL-17+ cells correlated with reduced HIF1α expression. This finding is consistent with the role of HIF1α in iTH17 cell differentiation19, 20, and may provide a mechanistic explanation for the phenotype observed in Akt-deficient T cells. How Akt isoforms differentially activate downstream pathways has not been fully resolved. The three Akt isoforms are structurally similar, especially within their kinase domains and do not exhibit substrate specificity in vitro37. It does not appear that the differential reliance on Akt isoforms is due to preferential expression of one isoform over another38, as our analysis of the mRNA levels did not reveal predominant expression of Akt2 in iTH17 cells. In addition, Akt1 and Akt2 appear to be equivalently expressed and activated in CD4+ T cells upon stimulation with TCR or cytokines. A number of reports have shown requirements for specific Akt isoforms. In many of these cases, isoform-specific subcellular compartmentalization appeared to play a role in Akt isoform-specific functions39–42. In fact, in developing B cells where Akt2 plays an isoform-specific role in regulating IL-7R and RAG expression, differential subcellular localization of Akt1 and Akt2 was observed43. It remains to be determined whether Akt2 has a distinct subcellular localization pattern in T cells and, if so, whether that is responsible for its isoform- specific function in iTH17 cells. Lastly, in this report we also show that nTH17 cells are present in human fetal thymus and umbilical cord blood, with constitutive Akt phosphorylation. Human TH17 cells have been reported to develop from a precursor population, marked by CD161 expression44. Prior to differentiation, these CD161+ T cells express CCR6, RORγt, and IL-23R and are thus reminiscent of nTH17 cells. Therefore, it is likely that this previously defined precursor population contains nTH17 cells. Further characterization will be needed to determine the relationship between these two human TH17 populations. METHODS Mice Myr-Akt17, Rhebfl/fl CD4-cre and Rictorfl/fl CD4-cre13, Foxo1fl/fl Foxo3afl/fl CD4-cre25, Akt1−/− and Akt2−/−32, Akt2fl/fl 47, Hif1fl/fl 48, Hif2fl/fl 49, and Arntfl/fl50 mice were previously described. Rhebfl/fl mice were originally generated in M. Magnuson’s laboratory laboratory. Akt2fl/fl mice were mated to CD4-cre mice and Hif1fl/fl Hif2fl/fl and Arntfl/fl mice were mated to Vav-cre at the University of Pennsylvania. C57BL/6J and B6.PL-Thy1a/CyJ mice were purchased from Jackson laboratory. B6 CD45.1 mice were purchased from Taconic. Animals were housed at the University of Pennsylvania, and experiments were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee. Phospho-flow Cells were isolated into serum-free media (or incubated in serum-free media for at least 2 h) prior to staining. Following the indicated stimulation, phospho-proteins were fixed immediately using Phosflow Lyse/Fix buffer (BD) according to the manufacturer’s instructions. Surface stain was followed by permeabilization with Perm/Wash buffer (BD) and intracellular staining with antibodies including anti-pAkt(S473)-AF488 (BD, 56040), anti-pAkt(T308)-PE (BD, 558275), anti-pS6K (Cell Signaling; 9204), or anti-pS6 (Cell Signaling, 4856). The latter two stains were followed by secondary staining with anti-rabbit-IgG-AF488 (Invitrogen; A11034). Human lymphocyte samples Thymic tissue samples and lymphocytes from cord blood mononuclear cells were obtained from the Stem Cell and Xenotransplantation Core facility of the University of Pennsylvania in compliance with institutional review board (IRB) protocols. Fetal thymic organ culture Fetal thymic lobes were dissected from E15 mouse embryos and cultured on sponge- supported filter membranes (Gelfoam absorbable gelatin sponge, USP 7mm: Pfizer; Nuclepore track-etched membranes, 0.8 μm–13 mm round: Whatman) at an interphase between 5% CO2-humidified air and IMDM (10% FCS, 50 μM 2-mercaptoethanol, 2 mM L-glutamine/penicillin/streptomycin). Medium was changed after 3 days of culture. At day 5 of culture, Akt inhibitor, AKTi-1/2 (Akt inhibitor VIII, Calbiochem) or MK-2206 (ChemieTek), was added at indicated concentration and incubated for 2 additional days. T cell isolation and in vitro differentiation CD4+ T cells from spleens and lymph nodes of indicated mice were purified by negative selection and magnetic separation (Miltenyi Biotec) followed by sorting of naïve CD4+CD25−CD44loCD62Lhi population using the FACS Aria II (BD). Cells were activated by plate-bound anti-CD3 (1 μg/ml) and anti-CD28 (5 μg/ml) (both eBioscience, clones 2C11 and 37.51, respectively) in the presence of TGF-β(5 ng/ml), IL-6 (20 ng/ml), IL-23 (10 ng/ml), anti-IL-4 (10 μg/ml), anti-IFN-γ(10 μg/ml; BioXcell, BE0055) for iTH17 polarization; TGF-β(5 ng/ml) for iTreg polarization; IL-12 (50 U/ml), anti-IL-4 (10 μg/ml; eBioscience, 16704185) for TH1 polarization; and IL-4 (2000 U/ml), anti-IL- 12 (10 μg/ml; Biolegend, 505202), anti-IFNγ (10 μg/ml) for TH2 polarization. For Akt inhibitor treatment, cells activated with anti-CD3 plus anti-CD28 for 18 h were culture for additional 36 h in the presence of iTH17-polarizing condition containing indicated concentrations of inhibitor. Ex vivo stimulation Freshly isolated or cultured lymphocytes were stimulated for 5 h with 50 ng/ml phorbol-12-myristate-13-acetate (PMA) and 500 ng/ml ionomycin in the presence of 1 μg/ml brefeldin A. Cells were then assayed for cytokine production by intracellular flow staining. Isolation of lamina propria lymphocytes The small intestine was dissected, cleared from mesentery, fat and Peyer’s patches, washed in PBS, and cut into pieces. After incubation in RPMI 1640 with EDTA, epithelial cells were separated and the tissue was digested with Liberase TM and DNase I (both Roche) at 37 °C. LP lymphocytes were recovered after filtering the digested tissue through a 70 μm cell strainer and washed in media. Intracellular HIF1α staining Naïve CD4+CD25−CD44loCD62Lhi cells were sorted by flow cytometry and cultured under iTH17-polarizing (Fig. 6c) or indicated conditions (Supplementary Fig. 10). Following surface stain and fix/permeabilization using Foxp3 staining buffer (eBioscience), cells were incubated with anti-mouse HIF1α rabbit polyclonal antibody (Cayman, 10006421) at 1:100 dilution for 60 min or with an isotype control. Subsequent secondary staining was done using anti-rabbit-IgG-PE (Invitrogen, A105242). Immunoblot analysis Magnetically purified CD4+ T cells were rested in serum-free media for at least 2 h and stimulated in 0.1% BSA in PBS (with Ca2+ and Mg2+) with 5 μg/ml anti-CD3-biotin (eBioscience, 13003185), 5 μg/ml anti-CD28-biotin (BD, 553296), and soluble-SA (Molecular Probes, S888) at 37 °C for indicated time points or IL-6 (10 ng/ml) at 37 °C for 30 min. Then cells were collected and lysed, followed by immunoblot analysis. The following antibodies were used (Cell Signaling): anti-pAkt(T308) (9204S), anti-pAkt(S473) (9271S), anti-Akt (9272), anti-pFoxo1/3a (9464), anti-Foxo1 (2880), anti- pSTAT3 (9145P), anti-STAT3 (9139), and anti-β-actin (Sigma, A5441). Radiation bone marrow chimeras Recipient mice were irradiated with 950 rads and injected i.v. with a mixture of T cell- depleted (Magnetic bead depletion, Qiagen) BM from indicated donor mice. Recipients were reconstituted with 2×106 BM cells and maintained on sterile water with sulfamethoxazole/trimethoprim for 2–3 weeks. Chimeras were analyzed at 8 weeks post transplantation. Real-time PCR Total RNA was isolated from indicated cell populations using RNeasy Mini Kit (Qiagen), and cDNA was synthesized with the Super Script III First Strand Kit (Invitrogen). Real-Time PCR was performed with site-specific primers and probes (Applied Biosystems) with Fast Taq Master Mix (Applied Biosystems) on 7500 Fast Real-Time PCR system. For analysis, samples were normalized to β-actin abundance and then set relative to CD4SPCD44loCCR6− population (unless indicated otherwise) by the relative quantification method (ΔΔCT). List of primers and probes (Applied Biosystems): β-actin, Mm00607939_s1; Ahr, Mm00478932_m1; Klf2, Mm01244979_g1; Slpr1, Mm02619656_s1; Akt1, Mm01331626_m1; akt2, Mm02026778_g1. Flow cytometry The following antibodies were used for surface stain (BD unless noted): anti-CD3-PE-Cy5 or –PB (Biolegend, 100213), anti-CD4-PE-Cy7 (Biolegend, 100528) or –FITC (553055), anti-CD8-PETR (Invitrogen, MCD0817) or –APC-Cy7 (557654), anti-CD44-AF700 (Biolegend, 103026) or –PE (553134), anti-CD45.1-PE (553776), anti-CD45.2-FITC (eBioscience, 11045481) or –PE-Cy7 (560696), anti-CD62L-APC (561919), anti- Thy1.1-PE-Cy5 (eBioscience, 15090082) or –PE (eBioscience, 12090081), anti-TCRβ-APCe780 (eBioscience, 47596182), anti-TCRγδ-PE-Cy5 (eBioscience, 15571182), anti- NK1.1-PE-Cy7 (eBioscience, 25594181), anti-CCR6-PB (BioLegend, 129817). For intracellular cytokine or transcription factor expression, staining was performed using Foxp3 staining buffer (eBioscience) according to the manufacturer’s instructions. The following antibodies were used (eBioscience unless noted): anti-RORγt-PE (12698880), anti-Foxp3-FITC (11577380) or –APC (17577382), anti-IL-17A-AF660 (50717780) or –PE (12717781) or –FITC (11717780), anti-IL-17F-FITC (53747182), anti-IL-22-PE (12722780), anti-IFNγ-APC (BD, 554413), anti-IL-4-PE-Cy7 (25704241). Data were acquired using FACS LSR II (BD) and analyzed with FlowJo software (Tree Star). Statistical analysis P values were analyzed from Student’s t-test or one-way ANOVA followed by Dunnett’s or Bonferroni’s post-tests using Prism (GraphPad Software). Supplementary Material 1 We thank B. Stiles (University of Southern California) for tissue from Akt1−/− mice; the Stem Cell and Xenotransplantation Core facility of the University of Pennsylvania for assistance with obtaining previously collected and de-identified human fetal thymic tissue; B. Monks for invaluable technical assistance and animal husbandry; L. Dipilato for Akt inhibitors and helpful suggestions; S. Carty and T. Kambayashi for critical reading of the manuscript; J. Stadanlick for editorial assistance; and members of the Koretzky and Jordan labs for helpful discussions. This work was supported by grants from the National Institutes of Health R01 DK56886 (M.J.B), 5K01AR52802 (M.S.J), and R37GM053256 (G.A.K). Competing Financial Interests The authors declare no competing financial interests. Author Contributions J.S.K designed the research, did experiments and wrote the manuscript; T.S, L.B, and M.G did experiments; A.T.W, K.N.P, and J.D.P provided the Rhebfl/fl CD4-cre and Rictorfl/fl CD4-cre tissue; N.S. and B.L.K. provided the Arntfl/fl Vav-cre tissue; C.L and M.O.L provided the Foxo1ΔTFoxo3fl/fl CD4-cre tissue; W.H and J.C.R provided the myr-Akt tissue; M.J.B provided Akt1−/− and Akt2−/− mice and helpful suggestions; and M.S.J. and G.A.K. oversaw research and helped in the writing of the manuscript. Figure 1 Akt regulates development of both nTH17 and iTH17 cells. (a) Akt phosphorylation (p- Akt) at the S473 site was determined for the indicated wild-type (WT) thymocyte populations by phospho-flow staining. Naive CD4SP (CD4SP CD44lo CCR6−), nTreg (CD4SP Foxp3+) and nTH17 cells (CD4SP CD44hi CCR6+) were analyzed. (b) S6K phosphorylation (p-S6K) and S6 phosphorylation (p-S6) was assessed in the indicated thymocyte populations from WT mice by flow cytometry. The specificity of the phospho-flow staining was verified by treatment of thymocytes with rapamycin for 1 h at 37 °C prior to staining. (c) IL-17 is expressed in thymocytes from human fetal thymi upon ex vivo stimulation for 5 h with PMA/ionomycin and brefeldin A. Flow plots are gated on live lymphocytes (left) and CD4SP cells (middle and right). (d) Phospho-flow analysis of p-Akt at the S473 site is shown for the indicated thymocyte populations from human fetal thymi. (e) Expression of IL-17A, ROR-γt, CCR6, and Foxp3 is shown for thymocytes from day 7 of E15-initiated FTOC, cultured for the last 2 days in the presence of indicated concentrations of allosteric Akt inhibitor, AKTi. Cultures were stimulated with PMA/ionomycin prior to staining. Representative flow plots are gated on CD4SPTCRβ+TCRγδ− cells. Graphs show either the percent of IL-17A+ or Foxp3+ cells among CD4SP cells pooled from three independent experiments (n = 5 thymi per condition; mean ± SEM; P<0.0001, P=0.0124; P value from two-tailed Student’s t- test). (f) IL-17 production in thymocytes from WT and Myr-Akt mice following ex vivo stimulation was determined. Representative flow plots show staining on CD4SPTCRβ+TCRγδ− gated cells. Graphs are of pooled data from two independent experiments (n ≥ 5; mean ± SEM; P=0.013, P value from two-tailed Student’s t-test). (g) WT naïve (CD44lo CD62Lhi CD25−) CD4+ T cells were activated for 18 h with anti- CD3 plus anti-CD28, followed by 36 h of culture in iTH17-polarizing conditions in the presence of indicated concentrations of AKTi. IL-17 or Foxp3 expression was determined following restimulation with PMA/ionomycin. Representative flow plots are shown. The graph is of pooled data from n = 3 per condition (bars and error bars represent mean ±SEM. P ≤ 0.05, * P≤ 0.001 (one-way ANOVA followed by Dunnett’s post-test with 0μM as control group). Data are representative of at least three independent experiments (a–d,g). Figure 2 ARNT and mTORC1 regulate iTH17 but not nTH17 cell development. (a) CFSE dilution and expression of IL-17 and Foxp3 in WT and ARNT-cKO CD4+ T cells cultured with anti-CD3 plus anti-CD28 in iTH17-polarizing condition for 3 days. (b) IL-17 production in small intestinal lamina propria (LP) cells from WT and ARNT cKO mice following ex vivo stimulation. Representative flow plots are gated on CD4+CD3+TCRβ+ cells and the graph shows pooled data from two independent experiments (n = 3; mean ±SEM; P=0.002, P value from two-tailed Student’s t-test). (c) IL-17 and Foxp3 expression in thymocytes from WT and ARNT-cKO mice following ex vivo stimulation. Representative flow plots show CD4SPTCRβ+TCRγδ− gated cells, and graphs show pooled data from two independent experiments (n = 4; mean ±SEM; NS, not significant; P=0.010, P value from two-tailed Student’s t-test). (d) The relative quantity (RQ) of AhR mRNA transcripts in the indicated cell populations from in vitro differentiated (TH0 and TH17) or purified WT thymocytes (CD4SP: CD4+CD44loCCR6− and nTH17: CD4+CD44hiCCR6+), relative to β-actin, was determined by real-time PCR analysis. Data are from 3 independently sorted thymic or independently generated TH populations. All samples were run in triplicate; bars and error bars represent mean ± SEM. (e) IL-17 production in small intestinal LP cells from WT and RhebΔT mice following ex vivo stimulation. Representative flow plots are gated on CD4+CD3+TCRβ+ cells, and the graph shows pooled data from two independent experiments (n = 3; mean ±SEM; P=0.0100, P value from two-tailed Student’s t-test). (f) IL-17 production in thymocytes from WT and RhebΔT mice following stimulation. Representative flow plots are gated on CD4SPTCRβ+TCRγδ− cells, and the graph shows pooled data from three independent experiments (n = 5; mean ± SEM; NS, not significant; P value from two-tailed Student’s t-test). Figure 3 mTORC2 is required for nTH17 cell development. (a) IL-17 production in thymocytes from WT and RictorΔT mice following ex vivo stimulation. Representative flow plots are gated on D4SPTCRβ+TCRγδ− cells, and the graph shows pooled data from three experiments performed in parallel with analysis of RhebΔT mice (n = 5; mean ±SEM; P=0.0012, P value from two-tailed Student’s t-test). (b) IL-17 production in small intestinal LP cells from WT and RictorΔT mice following ex vivo stimulation. Representative flow plots are gated on CD4+CD3+TCRβ+ cells, and the graph shows pooled data from two experiments performed in parallel with analysis of RhebΔT mice (n = 3; mean ± SEM; NS, not significant; P value from two-tailed Student’s t-test). Figure 4 Foxo proteins regulate nTH17 cell development. (a) The relative quantity (RQ) of Klf2 and S1pr1 mRNA transcripts in the indicated purified thymocyte populations, relative to β-actin, were determined by real-time PCR. CD4SP: CD4+CD44loCCR6−; nTH17: CD4+CD44hiCCR6+; nTreg: CD4+Foxp3+ from Foxp3-GFP reporter mice. Data are from 3 independently sorted thymic populations. All samples were run in triplicate; bars and error bars represent mean ± SEM. P ≤ 0.05, P ≤ 0.01 (one-way ANOVA followed by Dunnett’s post-test with CD4SP as control group). IL-17 producing thymocytes from Foxo1+/+Foxo3+/+ and Foxo1ΔTFoxo3ΔT mice (b) or WT and GSK3(S21A,S9A) mice (c) following ex vivo stimulation. Representative flow plots are gated on CD4SP TCRβ+TCRγδ− cells and the graph shows pooled data from two independent experiments (n = 3; mean ± SEM; NS, not significant; P<0.0001, P value from two-tailed Student’s t-test). (d) Production of IL-17 from WT and GSK3(S21A,S9A) CD4+ T cells cultured with anti-CD3 plus anti-CD28 in iTH17-polarizing condition for 3 days followed by restimulation with PMA/ionomycin. Numbers represent mean ± SEM of IL-17+ cells from triplicate samples. Data are representative of three independent experiments. Figure 5 Isoform-specific deletion of Akt2 affects iTH17 cell differentiation. (a) IL-17 producing thymocytes from WT, Akt1−/−and Akt2−/− mice following ex vivo stimulation. Representative flow plots are gated on CD4SPTCRβ+TCRγδ− cells, and graphed data are pooled from three independent experiments (n = 5–8; mean ± SEM; ns, not significant from one-way ANOVA followed by Bonferroni’s post-test). (b) IL-17 producing small intestinal LP cells from WT, Akt1−/− and Akt2−/− mice following ex vivo stimulation. Representative flow plots are gated on CD4+CD3+TCRβ+ cells, and graphed data are pooled from three independent experiments (n = 3–4; mean ±SEM; NS, not significant; P≤0.001 from one-way ANOVA followed by Bonferroni’s post-test). Figure 6 Akt2 regulates iTH17 and iTreg cells in a cell-intrinsic manner. (a) CFSE dilution and production of IL-17 in WT and Akt2−/−CD4+ T cells cultured with anti-CD3 plus anti- CD28 in iTH17-polarizing condition for 3 days followed by restimulation with PMA/ionomycin. (b) CFSE dilution and expression of Foxp3 of WT and Akt2−/− CD4+ T cells cultured with anti-CD3 plus anti-CD28 in iTreg-polarizing condition for 3 days. (c) Expression of intracellular HIF1α in WT and Akt2ΔT CD4+ T cells cultured with anti- CD3 plus anti-CD28 in iTH17-polarizing condition for 3 days. Flow plots are gated on CD4+ T cells and numbers represent mean ± SEM of IL-17+ cells from triplicate samples. Data are representative of at least three independent experiments using Akt2−/− or Akt2ΔT mice (a–c). (d) IL-17 producing small intestinal (SI) LP cells from mixed BM chimeras following ex vivo stimulation. Representative flow plots are gated on CD4+CD3+TCRβ+ cells showing the percent of IL-17A+ cells among Thy1.1+ or Thy1.1− populations. The graph shows pooled data representing the proportion of IL-17+ iTh17 cells among CD4+ T cells of either WT (Thy1.1+)- or Akt2−/−(Thy1.1− CD45.2+)-origin from WT+Akt2−/− mixed BM chimeras into CD45.1+ hosts, P<0.0001. (e) IL-17 producing mesenteric lymph node (MLN) cells from mixed BM chimeras following ex vivo stimulation. Representative flow plots are gated on CD4+CD3+TCRβ+ cells showing the percent of IL-17+ cells among Thy1.1+ or Thy1.1− populations. Graph shows pooled data representing the proportion of IL-17+ cells among CD4+ T cells of either WT (Thy1.1+)-or Akt2−/−(Thy1.1− CD45.2+)-origin from WT+Akt2−/− mixed BM chimeras, P=0.0002. Foxp3 expression in SI LP cells (f) or MLN cells (g) from mixed BM chimeras. (f,g) Graphs show pooled data representing the proportion of Foxp3+ Treg cells among CD4+ T cells of either WT (Thy1.1+)- or Akt2−/−(CD45.2+)-origin from WT+Akt2−/− mixed BM chimeras, P=0.0241 in (f) and *P=0.0166 in (g). (h) IL-17 producing thymocytes from mixed BM chimeras following ex vivo stimulation. Representative flow plots are gated on CD4SPTCRβ+TCRγδ− thymocytes showing the percent of IL-17+ cells among Thy1.1+ or Thy1.1− populations. The graph shows pooled data representing the proportion of IL-17+ nTH17 cells among CD4SP cells of either WT (Thy1.1+)- or Akt2−/−(CD45.2+)-origin from WT+Akt2−/− mixed BM chimeras. NS, not significant. Mean ± SEM; P value from two- tailed Student’s t-test. 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==== Front J Biomed ResJ Biomed ResJBRJournal of Biomedical Research1674-8301Editorial Department of Journal of Biomedical Research jbr-27-04-31810.7555/JBR.27.20130001Research PaperPhysical and degradation properties of PLGA scaffolds fabricated by salt fusion technique Mekala Naveen Kumar a✉Baadhe Rama Raju aParcha Sreenivasa Rao aYalavarthy Prameela Devi ba Department of Biotechnology, National Institute of Technology, Warangal 506004, India;b Department of Zoology, Kakatiya University, Warangal 506009, India.✉ Corresponding author: Dr. Naveen Kumar Mekala, Deptartment of Biotechnology, National Institute of Technology, Warangal 506004, India. Tel/Fax.: +91-9581302090, E-mail: [email protected] authors reported no conflict of interests. 7 2013 25 6 2013 27 4 318 325 4 1 2013 6 2 2013 3 4 2013 © 2013 by the Journal of Biomedical Research. All rights reserved.2013Tissue engineering scaffolds require a controlled pore size and interconnected pore structures to support the host tissue growth. In the present study, three dimensional (3D) hybrid scaffolds of poly lactic acid (PLA) and poly glycolic acid (PGA) were fabricated using solvent casting/particulate leaching. In this case, partially fused NaCl particles were used as porogen (200-300µ) to improve the overall porosity (≥90%) and internal texture of scaffolds. Differential scanning calorimeter (DSC) analysis of these porous scaffolds revealed a gradual reduction in glass transition temperature (Tg) (from 48°C to 42.5°C) with increase in hydrophilic PGA content. The potential applications of these scaffolds as implants were further tested for their biocompatibility and biodegradability in four simulated body fluid (SBF) types in vitro. Whereas, simulated body fluid (SBF) Type1 with the optimal amount of HCO3− ions was found to be more appropriate and sensible for testing the bioactivity of scaffolds. Among three combinations of polymer scaffolds, sample B with a ratio of 75:25 of PLA: PGA showed greater stability in body fluids (pH 7.2) with an optimum degradation rate (9% to 12% approx). X-ray diffractogram also confirmed a thin layer of hydroxyapatite deposition over sample B with all SBF types in vitro. poly (lactic-co-glycolic acid) scaffoldssimulated body fluidsolvent immersionpolymer degradationhydroxyapatite ==== Body INTRODUCTION The core idea of tissue engineering is to allow the cells to repair and regenerate damaged tissues and organs by promoting cell growth and differentiation over the scaffolds[1],[2]. These scaffolds are biodegradable matrices designed to support cell proliferation, which finally provides a functional tissue[3]. Since scaffolds are temporary matrices, the degradation performance of the scaffolds must correspond to the regeneration rate of the affected tissues[4]. In this regard, the selection of scaffold material is very important to facilitate the cells to behave in the desired manner to generate tissues or organs of our requirement[5]. The materials used to synthesize biodegradable scaffolds for bone tissue engineering applications ranges between inorganic materials such as ceramics to synthetic polymers. Among them, synthetic polymers have the ability to tailor mechanical properties and degradation kinetics of scaffolds to suit various tissue engineering applications[6]. The intrinsic properties of the polymer materials play a strategic role in the morphology, texture and performances of the scaffold[7]. To be an artificial scaffold, the structure and the surface morphology of the scaffolds have to meet general requirements specific for the targeted tissue: i) three-dimensional architecture; ii) interconnected pores to ensure cell growth, diffusion of nutrients and metabolic waste; iii) suitable surface chemistry; iv) suitable mechanical properties; v) controllable biodegradation and bioresorbability[8],[9]. In the current study, three combinations of hybrid scaffolds were prepared by blending polylactic acid (PLA) and polyglycolic acid (PGA) at the ratios of 80:20, 75:25 and 70:30 and these polymers are known for their cell-based tissue engineering approaches. These polymers have been shown to be degraded mainly by hydrolysis of ester bonds into acidic monomers, which can be removed from the body by physiological metabolic pathways[10],[11] (Fig. 1). The process which we adopted for preparation of microporous biodegradable scaffolds was solvent casting/particle leaching, where we used non-dispersed sodium chloride (NaCl) as particulate porogen for improved pore interconnectivity[12]. In this case, pores were interconnected via fused salt particles prior to the synthesis of three-dimensional (3D) polymer scaffolds. Thus, dissolution of this fused porogen matrix leaves a highly interconnected pore structure in the polymer scaffolds[13]. Once the polymer scaffolds are made, there is an immediate need to test the scaffolds for both in vivo and in vitro in order to consider them for human applications[14],[15]. These studies include their physical, chemical and mechanical properties helpful for assessing their bioavailability[16]. In case of in vitro studies, scaffolds were exposed to a group of model solutions simulating the inorganic portions of blood plasma to study their surface interaction and changes. The composition of the most used simulated body fluids differs from that of human blood plasma by high content of Cl- and lower content of HCO3− ions. Considering the composition of bone like apatite, which contains carbonate ions, the test results could be influenced by this difference[17],[18]. In this study, we prepared four different simulated body fluids with varied concentrations of the above said ions and we monitored the influence of these simulated fluids on the physico-mechanical properties of polymer scaffolds. Fig. 1 Structure of PLGA molecule. The figure shows hydrolytic degradation to PLA and PGA monomers in the presence of physiological fluids. PLGA: poly (lactic co glycolic acid). MATERIALS AND METHODS Scaffold preparation PLA (2.9 kg/mol) and poly glycolic acid (PGA) (IV= 1.2 dL/g) were procured from Sigma (St. Louis, MO, USA). Porous polymer scaffolds were prepared by solvent casting/particulate leaching where we used NaCl salt as particulate porogen (200-300 µm). PLA and PGA with higher molecular weight/inherent viscosity were used in our studies to ensure that the scaffolds would hold adequate mechanical integrity despite their relatively high porosity (≥90%). Briefly, NaCl matrices were prepared by subjecting NaCl particles to 95% humidity for 12 hours prior to solvent casting. PLA and PGA were blended at the ratios 70:30, 75:25 and 80:20 and dissolved in Hexafluoro-2-propanol (Sigma). This molten polymer blends were poured in to non-dispersed NaCl matrices (or) scaffold before solvent evaporation. Then, these scaffolds were vacuum dried for 48 hours before NaCl particles were further leached out by immersing scaffolds in de-ionized water. Scaffold characterization Electron microscopy The transverse sections of NaCl scaffolds were imaged using scanning electron microscope (SEM) prior to solvent casting to monitor NaCl crystal fusion. In addition, transverse sections of polymer scaffolds after salt leaching were also imaged using SEM (Zeiss EVO® MA15). Determination of glass transition temperature (Tg) Glass transition temperature (Tg) of all three scaffold types was determined as per ASTM D7426 standard by differential scanning calorimeter (DSC) equipped with liquid nitrogen cooling system (Auto Q20, TA instruments). Ten mg of polymer samples were quantitatively transferred to sealed aluminum pans and subjected to cooling and heating cycles from 0°C to +200°C with cooling and heating rates of 5°C/min. During experiment, DSC cell was purged with dry nitrogen at 40 mL/min. The baseline correction was performed by recording a run with empty pans. Preparations of simulated body fluids We tested the polymer degradation as per ASTM F1635-04a standard with four simulated body fluid types by varying Cl− and HCO3−[19]. The composition of simulated body fluids is shown in Table 1. SBFs were prepared in polypropylene beakers by dissolving NaCl, NaHCO3, KCl, K2HPO4, MgCl2, 1M HCl, CaCl2, Na2SO4, Tris HCl in double distilled water and pH was adjusted to 7.4 and the fluids were further incubated at 37°C for 3 to 4 days and monitored for hydroxyapatite (HA) deposition. In vitro degradation of scaffolds by immersion method (ASTM F1635-04a) Polymer scaffolds were cut to uniform sizes and each sample was weighed before the immersion test[20]. Then, scaffolds were placed in separate polypropylene beakers and fully immersed in simulated body fluids (0.2 mL of SBF/mm3 of scaffold) and incubated for 21 days at 37°C. These polymer scaffolds were taken out at preferred time intervals (at 14 and 21 days) and rinsed with distilled water and dried further for studying the morphological changes and weight loss; simultaneously, we checked for pH shift in simulated body fluids due to polymer degradation. Analysis of the scaffold surface by X-ray diffraction The interaction of polymer scaffolds with body fluids was evaluated by studying surface modifications over the scaffolds by X-ray diffraction (XRD) analysis. Precisely, polymer scaffolds immersed in four different simulated body fluids for 21 days were further vacuum dried in order to test HA deposition over the scaffold surface using XRD (Shimadzu) with a 2 theta (2θ) angle between 10 to 80 degrees at a scan speed of 5°/min. Table 1 The composition of inorganic components required for the preparation of simulated body fluids (mmol/L) SBF1 SBF2 SBF3 SBF4 Na+ 142.0 142.0 142.0 142.0 K+ 2.0 2.0 2.0 2.0 Ca2+ 2.5 2.5 2.5 2.5 Mg2+ 1.0 1.0 1.0 1.0 Cl− 116 121.0 126.0 131.0 HCO3− 20.0 15.0 10.0 5.0 SO42− 1.0 1.0 1.0 1.0 HPO42− 1.0 1.0 1.0 1.0 RESULTS Scaffold preparation Highly porous (≥90%) hybrid poly (lactic co glycolic acid) (PLGA, scaffolds (3 mm thickness) were prepared by the solvent casting/particulate leaching method. These scaffolds were cut to uniform sizes for further characterization. For our convenience, scaffolds with 80:20, 75:25 and 70:30 of PLA: PGA are denoted as Sample A, Sample B and Sample C respectively. Scaffold characterization Electron microscopy Initial incubation of NaCl crystals in a humidifier (95%) resulted in fusion of salt crystals, creating interconnected matrices (Fig. 2A). This fused NaCl crystals prior to addition of molten polymer mixture increased pore interconnectivity, which improved the overall porosity of scaffolds (≥90%) (Fig. 2B). Determination of glass transition temperature (Tg) The DSC analysis of PLGA scaffolds revealed their amorphous nature identified by the presence of glass transition temperature (Tg) and by the absence of melting temperature (Tm). The PLGA scaffolds showed increased polymer degradation with increases in the PGA proportion in the scaffolds. We also noticed a gradual reduction in Tg of polymer scaffolds A, B and C to 48°C, 44.5°C and 42.5°C, respectively. Fig. 2 Scanning electronic microscopic images of fused salt crystals (A) and porous polymer scaffolds (B). In vitro degradation of scaffolds by the immersion method (ASTM F1635-04a) 1) Morphological variations with time After 14 days in simulated body fluids, the morphological changes were found to be irregular, with increase in pore size over the scaffold surface. After 21 days, regular and recognizable morphological changes were detected in PLGA scaffolds. We also compared the morphological variations with weight loss in PLGA scaffolds during degradation studies. 2) Weight loss Sample C had shown accelerated weight loss when compared to the other two samples; this may be due to higher PGA content in the scaffold, which is hydrophilic in nature. Though the percentage degradation was high in case of samples C, the degradation rate aws not even (approximately 11% to 22%). This uneven degradation property makes this combination inappropriate for in vivo application. Comparatively, sample B had a stable and optimum degradation rate (approximately 9% to 12%), whereas sample A exhibited a low degradation rate (approximately 4% to 8%) compared to the other two samples. These observations indicated that increase in the PLA content made the scaffolds more hydrophobic and denser, thus making them tougher to be degraded both in vivo and in vitro. 3) pH shift in the simulated body fluids over time By the end of 30 days of incubation, Fig. 3 shows the pH shift in the simulated body fluids over time at 37°C. Sample A showed a slight increase in pH from 7.4 to 7.6, which might be due to slower polymer degradation and continues release of (PO4)3- ions which acts as conjugate bases. Sample C, due to its accelerated degradation to lactic and glycolic acids; showed higher decline in pH from 7.4 to 6.7, making this sample vulnerable for in vivo applications whereas sample B was found to be stable with a shift in pH from 7.4 to 7.2. 4) Analysis of scaffold surface by XRD XRD analysis using the X-ray diffractometer revealed the presence of straight base line and semi-sharp peaks (Fig. 4), suggesting the semi crystalline nature of our PLGA scaffolds. The XRD patterns also clearly indicated the deposition of HA traces over the scaffold surface. Compared to other SBF types, the XRD spectrum of SBF1 showed better HA deposition whose carbonate content and phosphate contents are more similar to human blood plasma compared to other SBF solutions. Fig. 3 Time dependence on pH of simulated body fluids. A: 80:20 PLGA; B:75:25 PLGA; C: 70:30 PLGA. And 2, 3 and 4 represents four SBF types Fig. 4 XRD analysis of polymer scaffolds for HA deposition. A: XRD spectrum of Pure HA. B: XRD spectrum of Sample A. C: XRD spectrum of Sample B. D: XRD spectrum of Sample C. DISCUSSION Porous hybrid polymer scaffolds (PLGA) were prepared by the solvent casting/particle leaching method, where we fused NaCl particles by prolonged exposure to rich moist environment (95% humidity), resuling in enhanced pore interconnectivity in the PLGA scaffolds (Fig. 5A). In this study, fused NaCl particles resulted in the creation of holes on the walls of the scaffolds, which increased the comprehensive modulus of the polymer scaffolds[21],[22]. Improved pore interconnectivity is also helpful in a variety of tissue engineering applications, particularly those requiring close cell to cell contact[23]. Fig. 5B depicts the transverse section of PLGA scaffolds prepared using fused salt particles as porogen. Scanning electron micrographs illustrate the polymer scaffolds with highly porous and well interconnected network. The microstructures of the scaffold determine its interaction with the cells and molecular transport of nutrients and biological wastes from within the scaffold[24]. Exclusively, the pore size of the scaffolds determines the cell seeding efficiency into the scaffold; small pores prevent the cells from piercing into the scaffold, while very large pores prevent cell adhesion due to reduced area to colonize cells[25],[26]. Therefore, scaffold with an open and interconnected pore network and high degree of porosity (≈90%) is described as a perfect model to integrate with the host tissue[27]. PLGA scaffolds prepared by salt fusion also showed irregular pore sizes, ranging between few microns to 300 µm. This variation in porosity may be due to a phenomenon known as solid-liquid phase separation which is attributed to solvent crystallization[28]. When the temperature of the polymer solution is lower than the solvent freezing point (crystallization temperature), solvent crystallizes and the polymer phase is expelled as impurity. A continuous polymer-rich phase is formed by the aggregation of polymer fractions excluded from solvent crystals[29]. After solvent crystals have been sublimated, the scaffold is produced with a micro-porosity similar to the geometry of solvent crystals. In in vitro degradation studies, PLGA scaffolds were degraded by hydrolysis of their ester linkages[30]. The presence of methyl side chain in PLA makes it more hydrophobic and denser than PGA and hence lactide-rich PLGA copolymers are less hydrophilic, absorb less water and are subsequently degraded at a lower rate[31],[32]. Additionally, reduced molecular weight with increased PGA content influences the reduction in “Tg” of the PLGA scaffolds (from 48°C to 42.5°C, which was quite near the incubation temperature of 37°C)[33],[34]. All the above discussed features made sample B (PLA: PGA (75:25)) as a favorite in comparison with other scaffolds. Also, controlled degradation (9-12%) of sample B might provide the room for tissue growth both in vivo and in vitro as biodegradable or restorable material. Fig. 5 Micro structure of highly porous PLGA scaffolds. Whereas A depicts fused NaCl particles with contact points resulted in salt bridges between the particles; B: SEM images of the polymer scaffolds exhibit optimum porosity (≥90%). X-ray diffraction spectra of polymer scaffolds after interaction with different model solutions are explained in Figure 5. After 21 days of immersion in simulated boy fluids, a thin layer of HA deposition was observed over the scaffold surface. Comparatively, SBF1 showed characteristic peaks for HA with all three scaffold types. This fact indicates that SBF1 with carbonate content similar to the human blood plasma could be more suitable and sensitive for in vitro testing of bioactivity and the diffusive character of observed peaks might be the result of poor crystallinity of the precipitated product due to the relative short time of exposure in the simulated body fluids and/or thinner precipitated layer[35]. The slower apatite deposition with SBF1 in comparison with other body fluids could enable the more sensitive in vitro testing of bioactive materials. Moreover, during interaction with human blood plasma, the creation of carbonated hydroxyapatite could be awaited rather than pure HA precipitation. Therefore, the content of carbonate ions in the solutions can be important for the plausibility of in vitro test. In conclusion, in vitro degradation behaviors of PLGA scaffolds in three different formulations were tested systematically with four simulated body fluids for 30 days. Detailed quantitative studies on the physiological features of scaffolds in wet environment along with other material parameters were tested. During these studies, sample B was found to be more appropriate with better physiological characteristics for further in vivo studies. The composition of SBF1 also proved as a better source for further optimization studies. ==== Refs References 1 Tabata Y Significant role of cell scaffolding and DDS technology in tissue regeneration: tissue engineering strategies Int Congress Ser 2005 1284 257 65 2 Mekala NK Baadhe RR Parcha SR Prameela DY Osteoblast Differentiation of Umbilical Cord Blood-Derived Mesenchymal Stem Cells and Enhanced Cell Adhesion by Fibronectin Tissue Eng Regen Med 2012 9 259 64 3 Stevens B Yang Y Mohandas A Stucker B Nguyen KT A review of materials, fabrication methods, and strategies used to enhance bone regeneration in engineered bone tissues J Biomed Mater Res B 2007 85 573 82 4 Yeo A Rai B Sju E Cheong JJ Teoh SH The degradation profile of novel, bioresorbable PCLeTCP scaffolds: an in vitro and in vivo study J Biomed Mater Res B 2007 84 208 18 5 Flanagan TC Wilkins B Black A Jockenhoevel S Smith TJ Pandit AS A collagen-glycosaminoglycan co-culture model for heart valve tissue engineering applications Biomaterials 2006 27 2233 46 16313955 6 Willows A Fan Q Ismail F Vaz CM Tomlins PE Mikhalovska Li Assessment of tissue scaffold degradation using electrochemical techniques Acta Biomat 2008 4 686 96 7 Olah L Borbas L Properties of calcium carbonate-containing composite scaffolds Acta Bioeng Biomech 2008 10 1 61 6 18634355 8 Chung HJ Park TG Surface engineered and drug releasing pre-fabricated scaffolds for tissue engineering Adv Drug Deliv Rev 2007 59 249 62 17482310 9 Rezwan K Chen QZ Blaker JJ Boccaccini AR Biodegradable and bioactive porous polymer/inorganic composite scaffolds for bone tissue engineering Biomaterials 2006 27 3413 31 16504284 10 Hasirci V Berthiaume F Bondre SP Gresser JD Trantolo DJ Toner M Expression of liver-specific functions by rat hepatocytes seeded in treated poly (lactic-co-glycolic) acid biodegradable foams Tissue Eng 2001 7 385 94 11506728 11 Kuo YC Leou SN Effects of composition, solvent, and salt particles on the physicochemical properties of polyglycolide/poly(lactide-co-glycolide) scaffolds Biotechnol Prog 2006 22 1664 70 17137316 12 Po KY Hui S Vasiliy NG Katherine AF Three-dimensional interconnected microporous poly (dimethylsiloxane) microfluidic devices Lab on a Chip 2011 11 1541 4 21359315 13 William LM Robert GD Joel LK David JM Salt Fusion: An Approach to Improve Pore Interconnectivity within Tissue Engineering Scaffolds Tissue Eng 2002 8 43 52 11886653 14 Wang M Composite Scaffolds for Bone Tissue Engineering Am J Biochem Biotechnol 2006 2 80 4 15 Agarwal CM Athanasiou KA Technique to control pH in vicinity of biodegrading PLA-PGA implants J Biomed Mater Res 1997 38 105 14 9178737 16 Shinoka T Shum TD Ma PX Tanel RE Isogai N Langer R Creation of viable pulmonary artery autografts through tissue engineering J Thorac Cardiovasc Surg 1998 115 536 45 9535439 17 Ito A Maekawa K Tsutsumi S Ikazaki F Solubility product of OH-carbonated hydroxyapatite J Biomed Mater Res 1997 36 522 8 9294768 18 Helebrant A jonasova L sanda L The influence of simulated body fluid composition on carbonated hydroxyapatite formation Ceramics 2002 46 9 14 19 Kokubo T Kushitani H Sakka S Kitsugi T Yamamuro T Solutions able to reproduce in vivo surface-structure changes in bioactive glass-ceramic A-W J Biomed Mater Res 1990 24 721 34 2361964 20 Chandra R Renu R Biodegradable Polymers Prog Polym Sci 1998 23 1273 335 21 Kaufmann PM Heimrath S Kim BS Mooney DJ Highly porous polymer matrices as a three-dimensional culture system for hepatocytes Cell Transplant 1997 6 463 8 9331497 22 Mekala NK Baadhe RR Parcha SR Study on osteoblast like behavior of umbilical cord blood cells on various combinations of PLGA scaffolds prepared by salt fvusion Curr Stem Cell Res Ther 2013 8 253 9 23317433 23 Murphy WL Kohn DH Mooney DJ Growth of continuous bone-like mineral within porous poly(lactideco- glycolide) scaffolds in vitro J Biomed Mater Res 2000 50 50 8 10644963 24 Chen Y Zhou S Li Q Microstructure design of biodegradable scaffold and its effect on tissue regeneration Biomaterials 2011 32 5003 14 21529933 25 O Brien FJ Harley BA Yannas IV Gibson LJ The effect of pore size on cell adhesion in collagene GAG scaffolds Biomaterials 2005 26 433 41 15275817 26 Rossella D Claudia C Ida G Tiziana M Bice C Effect of porogen on the physico-chemical properties and degradation performance of PLGA scaffolds Polym Degrad Stabil 2010 95 694 701 27 Freyman TM Yannas IV Gibson LJ Cellular materials as porous scaffolds for tissue engineering Prog Mater Sci 2001 46 273 82 28 Zhang R Ma PX Poly(a-hydroxyl acids)/hydroxyapatite porous composites for bone-tissue engineering. 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Preparation and morphology J Biomed Mat Res A 1999 44 446 55 29 Wan Y Fang Y Wu H Cao X Porous polylactide/chitosan scaffolds for tissue engineering J Biomed Mat Res A 2006 80 776 89 30 Yoo JY Kim JM Seo KS Jeong YK Lee HB Khang G Characterization of degradation behavior for PLGA in various pH condition by simple liquid chromatography method Biomed Mater Eng 2005 15 279 88 16010036 31 Houchin ML Topp EM Physical properties of PLGA films during polymer degradation J Appl Polym Sci 2009 114 2848 54 32 Hirenkumar KM Steven JS Poly Lactic-co-Glycolic Acid (PLGA) as Biodegradable Controlled Drug Delivery Carrier Polymers 2011 3 1377 97 22577513 33 Kim K Yu M Zong X Chiu J Fang D Young SS Control of degradation rate and hydrophilicity in electrospun non-woven poly (d,l-lactide) nanofiber scaffolds for biomedical applications Biomaterials 2003 24 4977 85 14559011 34 Ramakrishna S Mayer J Wintermantel E Leong KW Biomedical applications of polymer-composite materials: a review Compos Sci Technol 2001 61 1189 224 35 Bigi A Boanini E Panzavolta S Roveri N Rubini K Bonelike apatite growth on hydroxyapatite-gelatin sponges from simulated body fluid Inc J Biomed Mater Res 2002 59 709 14	23885272	PMC3721041	NO-CC CODE	2021-01-04 22:58:57	yes	J Biomed Res. 2013 Jul 25; 27(4):318-325
==== Front Drug Des Devel TherDrug Des Devel TherDrug Design, Development and Therapy1177-8881Dove Medical Press 10.2147/DDDT.S39771dddt-7-645ReviewDrugs in development for treatment of patients with cancer-related anorexia and cachexia syndrome Mantovani Giovanni 1Madeddu Clelia 1Macciò Antonio 21 Department of Medical Oncology, University of Cagliari, Cagliari, Italy2 Department of Gynecologic Oncology, A. Businco Hospital, Regional Referral Center for Cancer Disease, Cagliari, ItalyCorrespondence: Giovanni Mantovani, Policlinico Universitario, SS 554, Km 4500, 09042 Monserrato, Cagliari, Italy, Tel +39 070 675 4250, Fax +39 070 675 4250, Email [email protected] 12 8 2013 7 645 656 © 2013 Mantovani et al, publisher and licensee Dove Medical Press Ltd2013This is an Open Access article which permits unrestricted noncommercial use, provided the original work is properly cited.Cancer-related anorexia and cachexia syndrome (CACS) is a complex multifactorial condition, with loss of lean body mass, chronic inflammation, severe metabolic derangements, reduced food intake, reduced physical activity, and poor quality of life as key symptoms. Cachexia recognizes different phases or stages, moving from precachexia through overt cachexia to advanced or refractory cachexia. The purpose of this review is to describe currently effective approaches for the treatment of cachexia, moving forward to drugs and treatments already shown to be effective but needing further clinical trials to confirm their efficacy. We then introduce novel promising investigational drugs and approaches which, based on a strong rationale from the most recent data on the molecular targets/pathways driving the pathophysiology of cachexia, need to be tested either in currently ongoing or appropriate future clinical trials to confirm their clinical potential. Although different drugs and treatments have been tested, we can speculate that a single therapy may not be completely successful. Indeed, considering the complex clinical picture and the multifactorial pathogenesis of CACS, we believe that its clinical management requires a multidisciplinary and multitargeted approach. In our opinion, appropriate treatment for cachexia should target the following conditions: inflammatory status, oxidative stress, nutritional disorders, muscle catabolism, immunosuppression, quality of life, and above all, fatigue. A comprehensive list of the most interesting and effective multitargeted treatments is reported and discussed, with the aim of suggesting the most promising with regard to clinical outcome. A critical issue is that of testing therapies at the earliest stages of cachexia, possibly at the precachexia stage, with the aim of preventing or delaying the development of overt cachexia and thereby obtaining the best possible clinical outcome for patients. Keywords proinflammatory cytokinesnutritional statusmetabolic derangementsquality of lifecachexia stagingmultimodal therapy ==== Body Introduction Cancer-related anorexia and cachexia syndrome (CACS) is a debilitating clinical condition that affects the course of several chronic diseases, including chronic heart failure, chronic obstructive pulmonary disease, chronic kidney disease, and especially cancer. During its progression, cancer induces changes in the host immune system and energy metabolism that affect the clinical status of the patient so profoundly that it can result in death.1 The following symptoms are associated with these events and involve various organs and systems: anorexia, nausea, weight loss (with a reduction in lean body mass and adipose tissue), increased energy metabolism (with changes in glucose, lipid, and protein metabolism), immunosuppression, and fatigue. All these symptoms ultimately result in the clinical picture of CACS, which, unless counteracted, has a negative impact on quality of life for patients.2 A recent consensus defined cachexia as “a complex metabolic syndrome associated with an underlying inflammatory disease and characterized by the loss of muscle with or without loss of fat mass”.3 The pathophysiology of cachexia is common, at least in part, in the different diseases, and represents the main background of cachexia symptoms. In this review, we focus on CACS, the mechanisms of which are shared by chronic illnesses. It is well established that proinflammatory cytokines, including interleukin (IL)-1, IL-6, and tumor necrosis factor (TNF)-α, which are produced by the activated immune system and by tumor cells, are involved in the pathophysiology of CACS and the associated metabolic changes.4 It may be hypothesized that the synthesis and release of proinflammatory cytokines may lead to an efficient antineoplastic effect during the initial phases of neoplastic disease. However, the inability of the immune system to counteract tumor growth ultimately results in chronic cytokine activity, with irreversible effects on cell metabolism, body composition, nutritional status, and immune system efficiency.5 In turn, proinflammatory cytokines promote the synthesis of acute-phase proteins, which contribute to the pathogenesis of altered energy metabolism.6 Proinflammatory cytokines, together with tumor-derived factors, such as proteolysis-inducing factors and the recently discovered myostatin,7 also play a central role in the pathogenesis of muscle wasting via activation of the ubiquitin-proteasome proteolytic pathway.8 A major clinical feature of CACS is loss of muscle mass, leading to fatigue, impairment of normal activity, and eventually death.9 Muscle wasting is the result of multiple alterations at both the molecular and metabolic levels, leading to a disturbance in the balance between protein degradation and protein synthesis, whereas loss of muscle mass is mainly related to enhanced use of muscle proteins as an energy source to supply the increased energy needs of patients with cachexia. Anorexia, which is also induced by proinflammatory cytokines,10,11 is often associated with CACS, leading to reduced food intake. However, anorexia alone cannot account for the complex alterations characterizing this syndrome, thus confirming that cachexia is not just a consequence of malnutrition, but that other events are involved in its pathogenesis.12 In this context, the finding that cancer patients in advanced stages of the disease show severe impairment of immune function, characterized by a cell-mediated immunity deficit, elevated serum levels of proinflammatory cytokines, and acute-phase proteins (fibrinogen and C-reactive protein), is very relevant13 and encompasses the chronic inflammation status typical of CACS.14 The exact time when these changes occur is difficult to establish, but they are probably due to an interaction between the tumor and host. The tumor and its continuous growth are responsible for increased energy expenditure and progressive weight loss.15 Moreover, tumor growth is accompanied by chronic activation of the immune system as it triggers a response to counteract the tumor. The immune response is also energetically costly (25%–30% of the basal metabolic rate, ie, 1750–2080 kJ/day).16 From the evidence discussed above, it is intuitive that the clinical management of CACS is complex and requires a multidisciplinary and multipharmacological approach.17,18 Appropriate treatment of CACS should include drugs that address the following conditions: inflammatory state, nutritional disorder, metabolic derangements, immunological defects, poor quality of life, and, in particular, fatigue. Accordingly, treatment for CACS should include as primary endpoints the following variables, which were recently identified as key in cachexia:19 an increase in lean body mass and functional activity (grip strength, physical activity measured by either the six-minute walk test, arm band device, or three-step treadmill test); a decrease in resting energy expenditure; and improvement of fatigue. Moreover, the following variables should be included as secondary end-points: increased appetite, improved quality of life assessed by EORTC-QLQ-C30, and a decrease in proinflammatory cytokines (IL-6, and TNF-α). In fact, only full knowledge of the pathophysiology of CACS will enable identification of the most appropriate drugs to counteract the constitutive symptoms. A comprehensive summary of potentially available drugs for CACS is shown in Table 1. Treatment of CACS Progestagens Progestagens were the first agents used for the treatment of CACS and are currently the only agents approved in Europe for its treatment. An extensive amount of literature is available on megestrol acetate and medroxyprogesterone acetate (MPA) for the treatment of patients with cancer.20 Megestrol acetate and MPA are equivalent in terms of effectiveness in the treatment of CACS. However, megestrol acetate has been more widely investigated for its effect on cachexia21 than MPA.20 The positive effects of megestrol acetate on weight and well-being have been observed at oral doses in the range of 160–1600 mg/day. However, because megestrol acetate may be associated with severe dose-related adverse effects, starting treatment at a low dose (160 mg/day) and titrating the dose upwards according to clinical response is recommended.22 MPA has been used at doses in the range of 300–4000 mg/day. In a placebo-controlled study by Simons et al,23 a significant improvement in appetite and body weight was achieved using an oral dose of MPA in the range of 500–1000 mg/day. Moreover, a systematic review of MPA for the treatment of CACS found that there were no significant differences between high and low doses.24 Therefore, an MPA dose of 500–1000 mg/day orally can be recommended in clinical practice. Both megestrol acetate and MPA may have adverse effects, including an increased risk of thromboembolic events, peripheral edema, breakthrough bleeding, hyperglycemia, hypertension, and Cushing’s syndrome. Recently, an oral suspension formulation of megestrol acetate was developed using nanocrystal technology. Preclinical pharmacokinetic data suggest that this formulation of megestrol acetate can produce a more rapid clinical response by rapidly increasing plasma megestrol acetate levels.25 The US Food and Drug Administration (FDA) approved the oral suspension for the treatment of acquired immune deficiency syndrome-related cachexia, and this drug is currently under evaluation for approval to treat CACS associated with other conditions. Corticosteroids Several randomized, placebo-controlled studies have shown that corticosteroids achieve a limited (up to one month) improvement in appetite, food intake, nausea, and feeling of well-being. However, none of these studies showed an increase in body weight.26–30 The rapid beneficial effect of corticosteroids on mood and behavior significantly improves quality of life. The mechanism of action of corticosteroids in CACS is not well understood, although inhibition of prostaglandin activity and suppression of IL-1 and TNF-α production and release are the most well recognized targets.22 The specific drug, dose, and route of administration of corticosteroids are not well established: however, low doses, equivalent to less than 1 mg/kg of prednisone, are recommended in clinical practice. Further, because of their well known adverse effects, short-term (no more than 1–2 months) or alternating use of these agents is recommended in the management of CACS. Anabolic agents Anabolic androgens are synthetic derivatives of testosterone, with a greater anabolic effect and less androgenic activity than testosterone. Studies on the use of these anabolic agents in cachectic patients have been limited largely to patients with chronic obstructive pulmonary disease and human immunodeficiency virus/acquired immune deficiency syndrome, in whom positive effects on body weight, lean body mass, and several functional parameters were observed. However, few studies have been carried out to date in patients with CACS. Recently, a prospective, randomized Phase III trial compared the effects of oxandrolone 10 mg twice daily and megestrol acetate 800 mg daily on weight, body composition, and quality of life in 155 adult patients with solid tumors and weight loss while receiving chemotherapy. This study showed that patients treated with oxandrolone experienced an increase in lean body mass, a reduction in fat mass, and fewer self-reported anorectic symptoms.31 The side effects of these agents include elevated transaminase levels (especially with nandrolone), decreased high-density lipoprotein levels, interactions with oral anticoagulants, oral hypoglycemics, and adrenal steroids, and hypogonadism (with decreased systemic testosterone levels). Oxandrolone is administered orally (at approved dose concentrations of 5–20 mg/day) and has a better safety profile and less potential for hepatic toxicity and virilizing effects than other androgens. This agent is well tolerated in women.32 Drugs with confirmed clinical results Nonsteroidal anti-inflammatory drugs COX-2 selective inhibitors The development of selective COX-2 inhibitors has resulted in safer modulation of cancer-associated inflammation, and these agents could help alleviate or control CACS. Moreover, the selective COX-2 inhibitors have shown potent inhibitory and preventive effects on tumor growth in animal models; therefore, their antineoplastic activity may contribute to their ability to counteract cachexia. In particular, use of celecoxib, a selective COX-2 inhibitor, has been investigated. Lai et al33 randomized 11 cachectic patients with head and neck or gastrointestinal cancer to receive celecoxib 200 mg twice daily or placebo for three weeks. The patients on celecoxib reported good compliance and no adverse events were observed. Patients on celecoxib also showed a nonsignificant increase in body weight (mean change +1.0 kg versus −1.3 kg in the placebo group) and a significant increase in quality of life. A recent nonrandomized, prospective Phase II study investigated celecoxib 300 mg/day for four months in 24 patients with advanced cancer.34 The results indicated a significant decrease in levels of the proinflammatory cytokine, TNF-α, and a significant increase in lean body mass. In addition, significant improvements were observed in quality of life, performance status, Glasgow Prognostic Score, and grip strength. Patient compliance was good and no severe toxicities were observed. On the basis of these results, celecoxib can be included as a component in the combined treatment approach to target the inflammatory environment of CACS. A randomized Phase II trial assessing the feasibility of recruitment and retention of patients with advanced non-small cell lung cancer (NSCLC) undertaking a 12-week multimodal intervention of celecoxib, oral nutritional supplements, and physical exercise is due for completion by December 2014.35 Thalidomide Thalidomide has complex immunomodulatory and anti-inflammatory properties. It downregulates the production of TNF-α and other proinflammatory cytokines, inhibits transcription factor nuclear factor (NF-kB), downregulates COX-2, and inhibits angiogenesis. Therefore, thalidomide is a novel and rational treatment approach for CACS. In a randomized, placebo-controlled trial, thalidomide was found to be well tolerated and effective in slowing weight loss and improving arm muscle mass and physical function in 33 patients with advanced pancreatic cancer and CACS.36 Recently, a meta-analysis was performed to assess whether thalidomide is an effective treatment for CACS,37 and the authors concluded that there is inadequate evidence to recommend the use of this drug in clinical practice. Further large, well conducted, randomized controlled trials are needed to assess properly the true benefits of thalidomide alone and in combination in CSCS. Lenalidomide (Revlimid™, Celgene Corporation, Summit, NJ, USA) is a derivative of thalidomide now approved by the FDA for the treatment of myelodysplastic syndromes. A randomized, multicenter Phase II trial is presently underway assessing the efficacy of lenalidomide in enhancing lean body mass and grip strength in patients with advanced cancer.35 Melatonin Del Fabbro et al38 performed a randomized, double-blind, 28-day trial of melatonin 20 mg versus placebo in patients with advanced lung or gastrointestinal cancer and a history of weight loss ≥5%. Assessments included weight, symptoms on the Edmonton Symptom Assessment Scale, and quality of life using the Functional Assessment of Anorexia/Cachexia Therapy questionnaire. After interim analysis of 48 patients, the study was closed because of futility. There were no significant differences between the treatment groups with regard to appetite or other symptoms, weight, Functional Assessment of Anorexia/Cachexia Therapy score, toxicity, or survival from baseline to day 28. Therefore, oral melatonin 20 mg at night did not improve appetite, weight, or quality of life compared with placebo. Investigational drugs with clinical effectiveness to be confirmed Ghrelin and ghrelin mimetics Ghrelin is a 28-amino acid peptide produced by the P/D1 cells of the stomach, and stimulates secretion of growth hormone (GH, through the GH secretagogue-1a [GHS-1a] receptor), promotes food intake (through the orexigenic neuropeptide Y system), and decreases sympathetic nerve activity. Based on animal and short-term human trials, the evidence for use of ghrelin and GHS-R agonists in the treatment of CACS seems promising. Synthetic human ghrelin has been shown to improve muscle wasting and functional capacity in patients with cardiopulmonary-associated cachexia.39 Single-dose intravenous administration of ghrelin to cancer patients with cachexia did not show univocal efficacy in increasing food intake. In a randomized placebo-controlled trial, RC-1291 (anamorelin, an orally active small molecule GHS-R agonist) was administered to 81 patients with a variety of cancers (predominantly lung cancer) over a 12-week period. RC-1291 improved total body mass and there was a trend towards increased lean body mass, but quality of life was unchanged.40 More recently, anamorelin was shown to increase body weight and anabolic hormone levels in healthy volunteers. This drug was also investigated as a treatment for CACS in 16 patients with different types of cancer, and achieved a significant increase in body weight and improvement in patient-reported symptoms, including appetite, compared with placebo.41 However, these were small Phase I and Phase II trials, so their results should be interpreted with caution. A randomized, double-blind, placebo-controlled Phase III trial is presently enrolling up to 477 patients with NSCLC and CACS to measure lean body mass and muscle strength. This trial, sponsored by Helsinn Therapeutics (Bridgewater, NJ, USA), started recruiting in 2011 and is expected to be completed by 2014 (see Table 2). A caveat to the use of ghrelin agonists for treating CACS is the potential for stimulating tumor growth. Ghrelin and its receptor are expressed in many tumor cells and may contribute to tumor progression. Although no clinical study has reported an increased tumor incidence with administration of ghrelin, the studies to date have been short-term only. Therefore, further randomized, controlled studies are warranted before the use of ghrelin can be translated into clinical practice. AEZS-130 is an oral peptidomimetic growth hormone secretagogue developed by Æterna Zentaris Inc (Quebec, Canada), and was shown to be well tolerated in healthy subjects.35 A proof-of-concept study in patients with cancer and cachexia was planned to start in 2011. Melanocortin antagonists Among the appetite stimulants, a promising approach is targeting of the melanocortin-4 receptor. Interesting results were observed in colon-26 tumor-bearing mice,42 and clinical studies of this agent are planned.35 Drugs targeting inflammatory cytokines The most effective anti-inflammatory drugs have been those targeting TNF-α and IL-6. A humanized monoclonal anti-IL-6 antibody, ALD518 (Alder Biopharmaceuticals Inc, Bothell, WA, USA), may also benefit patients with cancer-associated cachexia because its administration increases hemoglobin levels and prevents reduction in lean body mass in those with advanced NSCLC.43 Greater benefits may be conferred when TNF-α and IL-6 are targeted simultaneously. OHR Pharmaceutical Inc (New York, NY, USA) have developed the broad-spectrum peptide nucleic acid immune modulator drug, OHR/AVR118, which targets both TNF-α and IL-6 and maintains immune homeostasis. In a Phase II study, eight of 21 enrolled patients with advanced cancer completed the study, and showed an improvement in anorexia, dyspepsia, strength (assessed by grip strength), and depression.44 A Phase IIb trial is currently assessing the efficacy of OHR/AVR118 in improving appetite and enhancing body mass, lean mass, strength (assessed by grip strength), and quality of life in patients with recurrent or advanced cancer and was expected to be completed before the end of November 2011. A humanized anti-IL-6 antibody (BMS-945429) was shown to be safe and well tolerated during early clinical studies in patients with NSCLC, with treatment improving lung symptoms and reversing fatigue, with a trend towards a decrease in loss of lean body mass.45 These findings are consistent with the results of a Phase II trial that assessed selumetinib (an inhibitor of MAPK1 and IL-6 secretion) in 20 patients with cholangiocarcinoma.46 Overall, 84% of patients in this trial showed a mean muscle gain of 2.3 kg.46 Selective androgen receptor modulators Due to the lack of selectivity of anabolic androgens, a need for more selective anabolic agents has emerged, resulting in the development of nonsteroidal selective androgen receptor modulators (SARMs). These agents have the potential to elicit beneficial anabolic effects in a tissue-selective manner, while avoiding many of the side effects observed with steroidal agents. The first nonsteroidal SARM was reported in 1998, and many of the major pharmaceutical companies have disclosed the specific chemical structure of different SARMs. Currently, the agent furthest into clinical development is enobosarm (GTx Inc, Memphis, TN, USA) for the potential prevention and treatment of muscle wasting in patients with cancer. In a Phase IIb clinical trial (ClinicalTrials. gov, NCT00467844) in patients with CACS, treatment with enobosarm significantly improved lean body mass, physical performance, and quality of life compared with baseline. Currently, two Phase III trials (ClinicalTrials.gov, NCT01355484 and NCT01355497) are recruiting patients with NSCLC to assess the effects of enobosarm on muscle wasting. Further, enobosarm has potential in the treatment of other forms of muscle loss, including chronic sarcopenia; a Phase IIb trial in patients with chronic sarcopenia, a Phase II trial in patients with COPD and selective muscle loss, and a Phase II trial in burns patients with muscle wasting have been planned by GTx, but have not as yet started recruiment.9 Myostatin inhibitors Myostatin and activin are members of the transforming growth factor-beta (TGF-β) superfamily, and signal via the activin type IIB (ActRIIB) receptor to regulate skeletal muscle mass and function in a negative manner. They achieve this by several mechanisms, including inhibiting myogenesis and the Akt/mTOR pathway involved in muscle protein synthesis and increasing the expression of ubiquitin ligases to increase muscle proteolysis. Much research has focused on the therapeutic potential of inhibiting myostatin and more recently on treating CACS by inhibiting the ActRIIB receptor. PF-354 (Pfizer Global Research and Development, Groton, CT, USA), an inhibitory myostatin antibody, prevented muscle wasting and weakness in tumor-bearing mice,35 but the increases in muscle mass were not as great as those achieved using an ActRIIB decoy receptor (sActRIIB), indicating that greater hypertrophic effects could be achieved by simultaneous inhibition of multiple TGF-β ligands.47 Workers at Amgen Research (Thousand Oaks, CA, USA) showed that administration of sActRIIB not only prevented muscle wasting, but completely reversed prior weight loss and prolonged survival in C-26 tumor-bearing mice.47 A Phase II trial investigating whether AMG 745 (Amgen Research) can attenuate age-related muscle wasting was terminated prior to patient enrollment, and it is unknown whether Amgen Research will continue developing this compound. The ActRIIB decoy, ACE-031, is being developed by Acceleron Pharma Inc (Cambridge, MA, USA) and was shown to be well tolerated and to increase lean mass in healthy postmenopausal women. Further development of ACE-031 is planned. BYM338, a human antibody acting as a myostatin inhibitor, is being developed by Novartis Pharmaceuticals (Hanover, NJ, USA) to treat CACS. In August 2011, a multicenter, randomized double-blind placebo-controlled Phase II trial was initiated to investigate whether BYM338 can attenuate the loss of body mass in cachectic patients with stage IV NSCLC or stage III/IV pancreatic cancer. The estimated enrolment is 50 patients. The primary outcome is an increase in thigh muscle volume, and trial completion is expected in May 2013. The exact targets of BYM338 are not currently known (see Table 2). LY2495655 is another antimyostatin monoclonal antibody. A multi center, randomized, double-blind, placebocontrolled Phase II trial in patients with locally advanced or meta static pancreatic cancer will investigate two different doses of LY2495655 in combination with gemcitabine.48 Overall survival is the primary outcome of this study, with secondary endpoints including muscle mass and physical performance. β-adrenoceptor agonists The hypertrophic effects of β2-adrenoceptor agonists, such as formoterol, in cachectic tumor-bearing rodents are well established.49 APD209 (Acacia Pharma Ltd, Harston Mill, UK) is an oral fixed-dose combination of formoterol and megestrol, and a Phase IIa study investigating the effects of eight weeks of treatment in 13 patients with CACS was recently completed. Six of the seven patients who completed the study demonstrated improved muscle size and strength, and three patients had increased levels of daily physical activity. Few patients reported side effects, such as muscle tremor or tachycardia. Acacia Pharma is currently planning larger randomized studies of this agent. MT-102 (PsiOxus Therapeutics Ltd, Billericay, UK) is an anabolic/catabolic transforming agent with properties including nonspecific β1-adrenergic and β2-adrenergic receptor antagonism, intrinsic sympathomimetic activity, and 5-HT1a receptor antagonism. MT-102 increased food intake, body mass, fat and lean muscle mass, physical activity levels, and survival time in cachectic tumor-bearing rats.50 A multicenter, randomized, double-blind Phase II trial was initiated in April 2011 to investigate whether up to 16 weeks of treatment with MT-102 would improve the rate of change in body mass compared with placebo in at least 132 patients with stage III and IV NSCLC or colorectal cancer and CACS.51 The estimated study completion date was August 2012, and enrolled patients who completed the 16-week treatment period and still taking randomized, double-blind trial medication were offered the opportunity to join in a subsequent trial with a separate primary endpoint. Investigational new drugs registered at ClinTrials.gov The investigational new drugs registered at ClinicalTrials.gov for the treatment of CACS are shown in Table 2. Multimodal therapy To date, studies on CACS therapy using various single interventions have had limited success. The main features of cachexia, ie, progressive loss of muscle mass and function, are minimally influenced by the nutritional and pharmacological tools currently available. The lack of efficacy of monotherapy is due to the multifactorial pathogenesis of cachexia. Therefore, a combination of dietary, nutritional, and pharmacological approaches targeting the main factors contributing to cachexia may be able to normalize the metabolic milieu and thus reverse cachexia-related symptoms that impact quality of life for patients.18 Several studies in the last decade have investigated the combination of megestrol acetate with other drugs. The combination of megestrol acetate with tetrahydrocannabinol52 and with eicosapentaenoic acid53 did not provide any benefits compared with use of megestrol acetate alone. However, megestrol acetate with ibuprofen was more effective than either drug used alone.54 An interesting pilot study performed by Cerchietti et al55 demonstrated the efficacy of a combined approach in a homogeneous group of 15 patients with lung adenocarcinoma and evidence of systemic immune metabolic syndrome, which was defined by the authors as a distressing systemic syndrome characterized by weight loss, anorexia, fatigue, performance status ≤2, and an acute-phase protein response. The multitargeted approach consisted of MPA 500 mg twice daily and celecoxib 200 mg twice daily as well as oral food supplementation for six weeks. This combined treatment significantly improved the rate of change in body weight, nausea, early satiety, fatigue, appetite, and performance status. In a subsequent study, the same authors56 randomized 22 patients with advanced lung cancer and systemic immune metabolic syndrome to receive either fish oil 2 g three times daily plus placebo or fish oil 2 g times daily plus celecoxib 200 mg twice daily. All patients in both groups received oral food supplementation. After six weeks of treatment, patients in both arms showed a significantly increased appetite, improvement in fatigue, and lower C-reactive protein levels compared with baseline. Patients in the celecoxib group showed improved body weight and muscle strength compared with baseline and a significantly lower C-reactive protein level and greater muscle strength and body weight than patients who received placebo. Lundholm et al57 assessed whether a combined approach, including daily insulin plus anti-inflammatory treatment (indomethacin), rHuEPO, and specialized nutritional care (oral supplements plus home parenteral nutrition), attenuated the progression of cancer-related cachexia and improved metabolism and physical functioning in 138 unselected patients with advanced gastrointestinal cancer. The combined treatment significantly stimulated carbohydrate intake, decreased serum-free fatty acids, and increased whole body fat, whereas fat free lean tissue mass was unaffected. Moreover, the combined treatment improved metabolic efficiency during exercise, but did not increase maximum capacity during exertion and spontaneous physical activity. The safety and efficacy of a combined approach was also tested by Mantovani et al in controlled clinical studies of cachectic patients with advanced tumors at different anatomical sites. First, a Phase II study,58 carried out according to a Simon’s two-stage design in a population of 39 patients with advanced cancer and CACS, showed that a combined approach, which included antioxidants + L-carnitine + eicosapentaenoic acid supplementation + celecoxib + MPA, was both safe and effective in increasing body weight and lean body mass, decreasing proinflammatory cytokines, improving quality of life parameters, and ameliorating symptoms of fatigue. On the basis of these positive results, Mantovani et al carried out a randomized Phase III trial in 332 patients with CACS to establish the most effective and safest treatment for CACS with regard to the primary endpoints of increased lean body mass, decreased resting energy expenditure, and improvement of fatigue, and included several significant secondary endpoints, ie, improvement in appetite, improvement in quality of life, increase in grip strength, decrease in Glasgow Prognostic Score, and decrease in proinflammatory cytokine levels.59 All patients were given basic treatment with polyphenols plus antioxidant agents, ie, α-lipoic acid carbocysteine, and vitamins A, C, and E, all orally administered. The patients were then randomly assigned to one of five treatment arms: arm 1, MPA 500 mg/day or megestrol acetate 320 mg/day; arm 2, oral supplementation with eicosapentaenoic acid; arm 3, L-carnitine 4 g/day; arm 4, thalidomide 200 mg/day; or arm 5, a combination of the above. The treatment duration was four months. Analysis of variance showed a significant difference between the treatment arms, and post hoc analysis showed the superiority of the combination arm (arm 5) over the others for all primary endpoints. Subsequently, Mantovani et al60 carried out a randomized Phase III study to assess the efficacy of a combination including carnitine and celecoxib ± megestrol acetate for the treatment of CACS. Analysis of changes from baseline showed that lean body mass as well as physical performance increased significantly in both arms. No significant difference was found between the treatment arms, and treatment was well tolerated. These results suggest that this two-drug combination may be a feasible, effective, and safe approach for CACS in clinical practice. Macciò et al61 performed a randomized Phase III study in a large selected population of patients with advanced gynecological cancer to assess the safety and efficacy of a multitargeted approach including megestrol acetate, celecoxib, antioxidants (carboxycysteine and lipoic acid), and L-carnitine versus megestrol acetate alone as standard treatment for CACS. These drugs were selected on the basis of monotherapy studies published by Mantovani et al for their ability to target the inflammation, oxidative stress, and metabolic impairment, which are mainly involved in the pathogenesis of symptoms and impaired quality of life in patients with CACS.13,14,34,62 The combination treatment arm was found to be more effective than megestrol acetate alone in improving lean body mass, resting energy expenditure, fatigue, and global quality of life. Moreover, serum markers of inflammation (IL-6 and C-reactive protein) and oxidative stress decreased significantly in the combination arm, but did not change in the arm receiving megestrol acetate alone. Of note in this study, the gain in lean body mass and global improvement in quality of life and subjective symptoms, such as fatigue, which were observed in the combination therapy arm, were associated with a decrease in the inflammation-based Glasgow Prognostic Score. Therefore, the efficacy of the combined treatment in terms of modulation of the inflammatory response associated with improvement in the primary endpoints confirms our hypothesis that the main symptoms of CACS in patients with advanced cancer are driven by systemic inflammation. Moreover, the efficacy of the combination treatment was associated with a significant increase in leptin levels, which may reflect amelioration of metabolic and energy efficiency, as characterized by a reduced resting energy expenditure and an attenuated inflammatory response. These results suggest that monitoring of leptin levels during treatment for CACS can be a useful and relevant parameter of metabolic response. A multitargeted approach to CACS should be undertaken within the context of the “best supportive care”, which includes optimal management of symptoms and careful psychosocial counseling. Conclusion Although various treatments for CACS have been tested, from the results presented here, we can speculate that a single therapy may not be completely successful. Most trials with synthetic progestagens, although currently the only drugs approved for treatment of CACS in Europe, have not been shown to improve lean body mass and functional activity, nor have they been shown to improve global quality of life. Further, their significant adverse effects should be taken into account. Among the effective agents, corticosteroids may be useful for their rapid beneficial effects on mood and sense of well-being. However, because of their side effects, short-term and/or alternating administration is recommended. Among the drugs with confirmed clinical efficacy, COX-2 inhibitors and anabolic agents, such as oxandrolone, are well placed to achieve good results. Investigational drugs with potential clinical effectiveness yet to be demonstrated include ghrelin and ghrelin mimetics, SARMs, and drugs targeting inflammatory cytokines, such as OHR/AVR 118 and other anti IL-6 antibodies. Other drugs under investigation include the myostatin inhibitors and β-adrenoceptor agonists, such APD209 and MT-102. Considering the complex clinical picture and multifactorial pathogenesis of CACS, we believe that the clinical management of this condition requires a multidisciplinary and multitargeted approach. The recent randomized Phase III clinical trial of five different treatments59 should be considered as a template for future approaches. The clearly defined and appropriate endpoints used in that study should also be a reference for future trials, with primary endpoints including lean body mass, resting energy expenditure and fatigue, and secondary endpoints including muscle strength, anorexia, physical activity levels, quality of life, survival, and levels of proinflammatory cytokines.35 Finally, considering that cachexia is a progressive disease starting with precachexia, moving through different stages into overt clinical cachexia, and finally to advanced or refractory cachexia, and that not all patients will progress through the complete spectrum, it is critical to test therapies at the earliest stages of cachexia, possibly at the precachexia stage, with the aim of preventing or delaying the development of overt cachexia, to obtain the best possible clinical outcome for patients. Disclosure The authors report no conflicts of interest in this work. Table 1 Comprehensive summary of drugs potentially available for cachexia in cancer patients Ineffective treatments • Cyproheptadine • Hydrazine • Metoclopramide • Pentoxifylline Drugs commonly used • Progestagens ○ Megestrol acetate ○ Medroxyprogesterone acetate • Corticosteroids • Anabolic agents (oxandrolone) Drugs with a strong rationale that failed or showed equivocal results in clinical trials • Omega-3 fatty acids • Cannabinoids (dronabinol) • Bortezomib Drugs with confirmed clinical results • COX-2 inhibitors • Thalidomide • Melatonin • Insulin • Branch-chained amino acids Investigational drugs with clinical effectiveness to be confirmed • Ghrelin and ghrelin mimetics • Melanocortin antagonists • Drugs targeting inflammatory cytokines • Selective androgen receptor modulators • Myostatin inhibitors • β-adrenoceptor agonists Investigational new drugs registered at ClinicalTrials.gov Multimodal therapy Table 2 Investigational new drugs registered at ClinicalTrials.gov for the treatment of cachexia in cancer patients ClinicalTrials.gov identifier Title Purpose Intervention Phase Estimated enrolment Start date Completion NCT01387269 Safety and efficacy of anamorelin HCl in patients with NSCLC-C (ROMANA 1) Administration of anamorelin in patients with stage lll–IV NSCLC-C is expected to increase appetite, lean body mass, weight gain, and muscle strength. Anamorelin HCl Placebo III 477 July 2011 July 2013 NCT01387282 Safety and efficacy of anamorelin HCl in patients with NSCLC-C (ROMANA 2) Administration of anamorelin in patients with stage lll–IV NSCLC-C is expected to increase appetite, lean body mass, weight gain, and muscle strength. Anamorelin HCl Placebo III 477 July 2011 July 2013 NCT01206335 Open-label study with OHR/AVR118 in patients with advanced cancer and anorexia-cachexia To determine whether patients with advanced cancer who receive OHR/AVR 118 solution injected into the skin can achieve improvement in quality of life. Based on a previous study in patients with acquired immune deficiency syndrome, possible benefits may include improved appetite and strength, weight gain, improved mood, and decreased fatigue. OHR/AVR118 II 20 September 2010 February 2013 NCT01614990 Pilot clinical trial of repeated doses of macimorelin to assess safety and efficacy in patients with cancer-related cachexia To evaluate the safety and efficacy of repeated oral administration of macimorelin at different doses daily for 1 week in the treatment of cancer-related cachexia. Macimorelin Placebo II 26 May 2012 August 2013 NCT01767857 Study using MABpl to increase overall survival in patients with colorectal cancer and weight loss To determine if the true human monoclonal antibody MABp 1 can prolong life in patients with colorectal carcinoma who are losing weight. MABpl Megestrol acetate III 656 February 2013 December 2014 NCT01419145 Feasibility study of multimodal exercise/nutrition/anti-inflammatory treatment for cachexia (Pre-MENAC study) A multicenter, open, randomized Phase II study comparing a multimodal intervention (oral nutritional supplements, celecoxib, physical exercise) for cachexia versus standard cancer care. Multimodal intervention Standard care II 40 October 2011 October 2014 NCT01433263 Randomized, double-blind, placebo-controlled, multicenter study of BYM338 for treatment of cachexia in patients with stage IV NSCLC or stage lll/IV adenocarcinoma of the pancreas A safety and efficacy clinical study of the investigational medicinal product BYM338 for treatment of unintentional weight loss in patients with cancer of the lung or pancreas. BYM338 active drug Placebo II 50 August 2011 May 2013 NCT01501396 Treatment of CACS with mirtazapine and megestrol acetate To study the safety and efficacy of megestrol acetate ± mirtazapine in treating cancer patients with weight loss and loss of appetite. To date, no pharmacologic interventions have been approved by the FDA to treat CACS. Megestrol acetate has been shown to increase appetite in cancer patients. Adding mirtazapine may provide a more effective treatment and help improve quality of life. Megestrol acetate Arm Β (megestrol + mirtazapine) II 140 April 2012 April 2015 NCT00489593 Phase 1 dose-finding pilot study of the safety and tolerability of olanzapine in patients with advanced cancer and weight loss To find the highest tolerable dose of olanzapine that can be given to patients with advanced cancer who are experiencing weight loss. Researchers want to determine if olanzapine can help decrease weight loss in patients who are experiencing it because of cancer. How this drug affects performance status, cancer-related symptoms, and nutritional status in patients with advanced cancer will also be studied. Olanzapine I 57 October 26 April 2014 Study ongoing but not recruiting participants Abbreviations: CACS, cancer anorexia-cachexia syndrome; NSCLC-C, non-small cell lung cancer and cachexia; FDA, US Food and Drug Administration; HCl, hydrochloride. ==== Refs References 1 Delano MJ Moldawer LL The origins of cachexia in acute and chronic inflammatory diseases Nutr Clin Pract 2006 21 1 68 81 16439772 2 Vigano A Del Fabbro E Bruera E Borod M The cachexia clinic: from staging to managing nutritional and functional problems in advanced cancer patients Crit Rev Oncog 2012 17 3 293 303 22831160 3 Evans WJ Morley JE Argiles J Cachexia: a new definition Clin Nutr 2008 27 6 793 799 18718696 4 Argiles JM Busquets S Toledo M Lopez-Soriano FJ The role of cytokines in cancer cachexia Curr Opin Support Palliat Care 2009 3 4 263 268 19713854 5 McDonald N Cancer cachexia and targeting chronic inflammation: a unified approach to cancer treatment and palliative/supportive care J Support Oncol 2007 5 4 157 162 17500503 6 Oldenburg HS Rogy MA Lazarus DD Cachexia and the acute-phase protein response in inflammation are regulated by interleukin-6 Eur J Immunol 1993 23 8 1889 1894 8344351 7 Lokireddy S Wijesoma IW Bonala S Myostatin is a novel tumoral factor that induces cancer cachexia Biochem J 2012 446 1 23 36 22621320 8 Melstrom LG Melstrom KA Jr Ding XZ Adrian TE Mechanisms of skeletal muscle degradation and its therapy in cancer cachexia Histol Histopathol 2007 22 7 805 814 17455154 9 Dodson S Baracos VE Jatoi A Muscle wasting in cancer cachexia: clinical implications, diagnosis, and emerging treatment strategies Annu Rev Med 2012 62 265 279 20731602 10 Gautron L Laye S Neurobiology of inflammation-associated anorexia Front Neurosci 2009 3 59 20582290 11 Thaler JP Choi SJ Schwartz MW Wisse BE Hypothalamic inflammation and energy homeostasis: resolving the paradox Front Neuroendocrinol 2010 31 1 79 84 19822168 12 Tisdale MJ Mechanisms of cancer cachexia Physiol Rev 2009 89 2 381 410 19342610 13 Macciò A Lai P Santona MC Pagliara L Melis GB Mantovani G High serum levels of soluble IL-2 receptor, cytokines, and C reactive protein correlate with impairment of T cell response in patients with advanced epithelial ovarian cancer Gynecol Oncol 1998 69 3 248 252 9648596 14 Mantovani G Macciò A Lai P Massa E Ghiani M Santona MC Cytokine involvement in cancer anorexia/cachexia: role of megestrol acetate and medroxyprogesterone acetate on cytokine down-regulation and improvement of clinical symptoms Crit Rev Oncog 1998 9 2 99 106 9973244 15 Morrison SD Partition of energy expenditure between host and tumor Cancer Res 1971 31 2 98 107 5545274 16 Straub RH Cutolo M Buttgereit F Pongratz G Energy regulation and neuroendocrine-immune control in chronic inflammatory diseases J Intern Med 2010 267 6 543 560 20210843 17 Del Fabbro E More is better: a multimodality approach to cancer cachexia Oncologist 2010 15 2 119 121 20133501 18 Fearon KC Cancer cachexia: developing multimodal therapy for a multidimensional problem Eur J Cancer 2008 44 8 1124 1132 18375115 19 Fearon K Strasser F Anker SD Definition and classification of cancer cachexia: an international consensus Lancet Oncol 2011 12 5 489 495 21296615 20 Madeddu C Macciò A Panzone F Tanca FM Mantovani G Medroxyprogesterone acetate in the management of cancer cachexia Expert Opin Pharmacother 2009 10 8 1359 1366 19445562 21 Pascual Lopez A Roque i Figuls M Urrutia Cuchi G Systematic review of megestrol acetate in the treatment of anorexia-cachexia syndrome J Pain Symptom Manage 2004 27 4 360 369 15050664 22 Mantovani G Macciò A Massa E Madeddu C Managing cancer-related anorexia/cachexia Drugs 2001 61 4 499 514 11324680 23 Simons JP Aaronson NK Vansteenkiste JF Effects of medroxyprogesterone acetate on appetite, weight, and quality of life in advancedstage non-hormone-sensitive cancer: a placebo-controlled multicenter study J Clin Oncol 1996 14 4 1077 1084 8648360 24 Berenstein EG Ortiz Z Megestrol acetate for the treatment of anorexia-cachexia syndrome Cochrane Database Syst Rev 2005 2 CD004310 15846706 25 Femia RA Goyette RE The science of megestrol acetate delivery: potential to improve outcomes in cachexia BioDrugs 2005 19 3 179 187 15984902 26 Bruera E Roca E Cedaro L Carraro S Chacon R Action of oral methylprednisolone in terminal cancer patients: a prospective randomized double-blind study Cancer Treat Rep 1985 69 7–8 751 754 2410117 27 Willox JC Corr J Shaw J Richardson M Calman KC Drennan M Prednisolone as an appetite stimulant in patients with cancer Br Med J (Clin Res Ed) 1984 288 6410 27 28 Moertel CG Schutt AJ Reitemeier RJ Hahn RG Corticosteroid therapy of preterminal gastrointestinal cancer Cancer 1974 33 6 1607 1609 4135151 29 Della Cuna GR Pellegrini A Piazzi M Effect of methylprednisolone sodium succinate on quality of life in preterminal cancer patients: a placebo-controlled, multicenter study. 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==== Front Open Access J UrolOpen Access J UrolOpen Access Journal of Urology1179-1551Dove Medical Press 10.2147/OAJU.S13370oaju-2-177Original ResearchNeurotransmitter testing of the urine: a comprehensive analysis Hinz Marty 1Stein Alvin 2Trachte George 3Uncini Thomas 41 Clinical Research, NeuroResearch Clinics, Inc., Cape Coral, FL, USA2 Stein Orthopedic Associates, Plantation, FL, USA3 Department of Physiology and Pharmacology, University of Minnesota Medical School, MN, USA4 DBS Labs, Duluth, MI, USACorrespondence: Marty Hinz, 1008 Dolphin Dr, Cape Coral, FL 33904, USA, Tel +1 218 626 2220, Fax +1 218 626 1638, Email [email protected] 07 10 2010 2 177 183 © 2010 Hinz et al, publisher and licensee Dove Medical Press Ltd2010This is an Open Access article which permits unrestricted noncommercial use, provided the original work is properly cited.This paper analyzes the statistical correlation of urinary serotonin and dopamine data in subjects not suffering from monoamine-secreting tumors such as pheochromocytoma or carcinoid syndrome. Peer-reviewed literature and statistical analyses were searched and monoamine (serotonin and dopamine) assays defined in order to facilitate their proper interpretation. Many research findings in the literature are novel. Baseline assays completed with no monoamine precursors differ from baseline assays performed on a different day in the same subject. There is currently no scientific basis, value, or predictability in obtaining baseline monoamine assays. Urinary assays performed while taking precursors can demonstrate a lack of correlation or unexpected correlations such as inverse relationships. The only valid model for interpretation of urinary monoamine assays is the “three-phase model” which leads to predictability between monoamine assays and precursor administration in varied amounts. Purpose This paper reviews the basic science of urinary monoamine assays. Results of statistical analysis correlating baseline and nonbaseline assays are reported and provide valid methods for interpretation of urinary serotonin and dopamine results. Patients and methods Key scientific claims promoting the validity of the urinary neurotransmitter testing (UNT) model applications are discussed. Many of these claims were not supported by the scientific literature. Matched-pairs t-tests were performed on several groupings. Results of all statistical tests were compared with peer-reviewed literature. Results The statistical analysis failed to support the UNT model. Peer-reviewed literature search failed to verify scientific clams made in support of applications of the UNT model in many cases. Keywords serotonindopamineurinary neurotransmitter testing ==== Body Introduction Three applications have evolved with regard to urinary monoamine assays. The first is one of the older applications used in medicine. This is the use of monoamine assays for screening and diagnosing tumors that secrete serotonin or dopamine (herein referred to as the “tumor model”), such as pheochromocytoma (a catecholamine-secreting tumor) and carcinoid syndrome (a serotonin-secreting tumor).1,2 The validity of this type of monoamine testing application is well established in the scientific literature. The second application is the use of monoamine assays for renal organic cation transporter functional status determination (ie, the OCT model). Even though this model is relatively new, having been developed in 2003, this approach and the urinary serotonin and urinary dopamine applications developed according to this model are supported by the scientific literature, having been discussed and documented in several articles since February 2009.3–5 The basis for the OCT model requires two or more serial urinary serotonin and dopamine (ie, monoamine) assays while taking varied amino acid precursor daily dosing amounts. The results are then compared in order to determine the change in urinary serotonin and dopamine levels in response to changes in dosing. A urinary serotonin or dopamine value less than 80 or 475 μg of monoamine per g of creatinine, respectively, indicates a Phase II response. A urinary serotonin or dopamine value greater than 80 or 475 μg of monoamine per g of creatinine, respectively, is interpreted as being in Phase I or Phase III. Differentiation of Phase I from Phase III is as follows. If a direct correlation is found between amino acid dosing and urinary assay response, it is referred to as a Phase III response. An inverse correlation is referred to as a Phase I response.3–5 Unexpected results with matched-pairs t-test analysis revealed no significant difference when comparing baseline monoamine assays with assays performed while taking supplemental amino acid precursors in the same subject. Peer-reviewed scientific publications discussing urinary serotonin and urinary dopamine phase analysis according to the OCT model were first published in 20093,4 and 2010.5 These publications outlined the mechanics of the three-phase model in connection with urinary serotonin and urinary dopamine under a novel renal transporter model. This transporter model potentially describes the etiology of the three-phase response of monoamine assays during the administration of varied amino acid precursor daily dosing values.12 The third approach defining applications for the use of monoamine assays is the urinary neurotransmitter testing (UNT) model. This paper discusses the UNT model in depth because it is the only model of the three that lacks valid scientific literature discussing the model or supporting the monoamine assay applications that are being promoted. The goal of this writing is to assess monoamine assay applications statistically and define the validity of monoamine assays in the absence or presence of supplemental amino acid precursors. The premise of the UNT model is that baseline monoamine assays correlate with and are a good predictor of the peripheral and central nervous system neurotransmitter functional status. The basic assumption for this assertion is that serotonin and dopamine cross the blood–brain barrier6–8 and are then filtered at the glomerulus and enter the urine without further interaction with the kidneys.6,8 This argument is used on the basis of the UNT model to justify the conclusion that monoamine assays, in the presence and absence of serotonin and dopamine amino acid precursors, correlate with central nervous system and peripheral neurotransmitter functional status. It also asserts that baseline testing is the best approach to determine the neurotransmitter functional status of the central and peripheral nervous systems.7,8,10 Other conclusions made in support of utilizing monoamine assays under the urinary neurotransmitter testing model are as follows: Administration of amino acid precursors directly impacts urinary monoamine levels; therefore, the results of monoamine assays merely need to be interpreted as being either high or low values8,9,11 Baseline testing of urinary monoamines prior to starting supplemental amino acid precursors is required in order to define the amino acid precursor starting dose needed in treatment8–11 Baseline monoamine assays in the absence of supplemental amino acid precursors are required to diagnose and define the serotonin and dopamine imbalance in the central and peripheral nervous systems6,10 Baseline monoamine assays can serve as a reference point to gauge treatment effectiveness after amino acid precursors are started6,11 Baseline monoamine assays can be used to reduce the risk of side effects when amino acid precursor treatment is started.10 Materials and methods Statistical analysis was performed for each analyzed grouping considered. The statistical analysis involved the matched-pairs t-test. After initiation of supplemental amino acid precursor administration or a change in daily dosing levels was maintained constant, a minimum period of seven days without missing one or more doses was required for data to be considered valid. This time period allows the amino acid precursors and the urinary monoamines to achieve equilibrium in order to ensure that valid urinary serotonin and urinary dopamine test results are obtained. A P value ≤0.05 was considered statistically significant. JMP software (SAS Institute, Cary, NC) was used to perform the statistical analysis. Processing, management, and assay of the urine samples collected for this study were as follows. Urine samples were collected six hours prior to bedtime, with 4 PM being the most frequent collection time point. The samples were stabilized in 6 N HCl to preserve urinary dopamine and urinary serotonin. The urine samples were collected after a minimum of one week, during which time the patient was taking a specific daily dose of amino acid precursors of serotonin and dopamine. Samples were shipped to DBS Laboratories. Urinary dopamine and serotonin were assayed utilizing commercially available radioimmunoassay kits (3 CAT RIA IB88501 and IB89527; Immuno Biological Laboratories, Inc., Minneapolis, MN). The DBS laboratory is accredited as a high complexity laboratory by Clinical Laboratory Improvement Amendments to perform these assays. Results Two approaches to analyze the validity of the UNT model were undertaken. The first approach was a literature search intended to test claims made in support of applications for monoamine assays under the UNT model. After an exhaustive search, no indepth valid peer-reviewed studies were found documenting the UNT model. In most cases, the claims justifying use of urinary serotonin and urinary dopamine assays according to the UNT model were contrary to the identified scientific literature. The second approach was the statistical analysis of baseline monoamine assays in the presence or absence of supplemental amino acid precursors in order to assess the UNT model critically. Five significant divergences from the UNT model from the existing scientific literature were identified. Specifically, divergences were noted from the established science, ie, serotonin and dopamine do not cross the blood–brain barrier3,5,12,13 and peripheral serotonin and dopamine are filtered at the glomerulus and then enter the proximal tubules.5 They are then actively transported into the proximal convoluted renal tubule cells where they are essentially completely metabolized.5 Due to the high efficiency of this metabolic process, significant amounts of serotonin and dopamine filtered at the glomerulus do not reach the urine in patients not suffering from a tumor secreting serotonin or dopamine.3,5 From a practical standpoint, urinary serotonin and urinary dopamine represent serotonin and dopamine that have not previously been in the central or peripheral nervous system.3,5 The literature notes that urinary serotonin and urinary dopamine are monoamines that are newly synthesized from serotonin and dopamine amino acid precursors by the kidneys in the proximal convoluted renal tubule cells.3,5 These newly synthesized serotonin and dopamine molecules are then either transported out of the proximal convoluted renal tubule cells across the basolateral membrane and then into the peripheral system via the renal vein or across the apical membrane and then into the urine.3–5,14,15 It is noted that there are many other renal interactions that exist between synthesis of serotonin and dopamine transported across the basolateral membrane and the apical membrane prior to arriving at the final destination of the renal vein or urine, respectively. These interactions appear small in comparison with the effects of the basolateral monoamine transporter and the apical monoamine transporter under the three-phase model.5 There is also no correlation between urinary serotonin and dopamine levels and the serotonin or dopamine levels within the central and peripheral nervous systems.3–5 The renal interaction of urinary serotonin, urinary dopamine, and their amino acid precursors is counterintuitive. It is expected that when serotonin and/or dopamine amino acid precursors are administered, levels of the associated urinary serotonin or urinary dopamine will increase or decrease with increases or decreases in the amino acid precursor daily dosing levels, ie, a direct relationship. The literature reveals that this is not the predominant response. Outcomes are not intuitive because the process is complex, and there is no simple, dominant, direct relationship between serotonin and dopamine amino acid dosing and monoamine assays. Instead, a complex interaction is found, giving rise to the three-phase model, as we have previously proposed.3–5 Furthermore, there is no significant statistical difference between baseline monoamine levels in the urine and those resulting from administration of monoamine precursors. Given that support for this is not found in the literature, the following statistical analysis is put forth. The data for the following analysis was obtained from the DBS Laboratories monoamine assay database. The database was assembled according to the criteria discussed in the Materials and methods section. By definition, the laboratory baseline reference range for a given assay is calculated by taking all baseline data generated for that assay, then defining the group of values that are within two SDs from the mean. This grouping size represents approximately 95% of the initial group data generated. In the following reports of statistical analysis, when use of the reference range values is referred to, the following values were used. A laboratory promoting the UNT model has defined the urinary serotonin reference range as 150–300 μg of serotonin per g of creatinine. The same laboratory defined the urinary dopamine reference range as 150–300 μg of dopamine per g of creatinine.9 Urinary serotonin at baseline versus while taking 5-hydroxytryptophan Matched-pairs groupings were queried from the database as follows. Two urinary serotonin samples from the same subject were obtained, one sample while taking no supplemental amino acid precursors and the other sample while taking 5-hydroxytryptophan (5-HTP), and these were match-paired together. A group of these matched-pairs samples were then defined for analysis, revealing a group of n = 167. The serotonin reference range values as reported above were used to query the baseline urinary matched-pairs serotonin group of n = 167 further, revealing a group of n = 103. The group taking 5-HTP was then queried from the group of n = 103 using the parameter 5-HTP < 301 mg per day, to give a final matched-pairs group of n = 78 for analysis. The final matched-pairs group was then analyzed using a t-test, and a P value of 0.0809 was found indicating lack of a significant statistical difference between baseline urinary serotonin levels and serotonin levels when taking less than 301 mg of 5-HTP per day. Urinary dopamine at baseline versus while taking levodopa Matched-pairs groups were queried from the database as follows. Two samples from each subject, one sample taking no supplemental amino acid precursors and the other sample taking levodopa, were paired together. This revealed a group of n = 617. The baseline assay portion of the entire matched-pairs group was queried with the dopamine reference range values reported earlier, to give a population size of n = 230. The group taking levodopa was then queried to find only subjects taking less than 361 mg of levodopa per day, leading to a final population size of n = 166. This matched-pairs group was then analyzed using a matched-pairs t-test, and a P value of 0.0742 was found indicating no significant statistical difference between baseline dopamine assays and dopamine assays performed while taking less then 361 mg of levodopa per day. Baseline serotonin assays from different days in the same subject Data were analyzed in the following manner, with the following numbers reported in μg of serotonin per g of creatinine. From a matched-pairs group of n = 146, the mean (SD) for both baseline serotonin urinary assay groups was determined. For Group 1, the mean serotonin value was found to be 239.0 (±2282.8). For Group 2 (baseline testing performed on a different day after the first assay) the mean serotonin value was found to be 273.2 (±8214.51). All data greater than the value found in calculating the sum of two SDs plus the mean were removed from consideration, revealing a group of n = 134. The matched-pairs grouping was then analyzed using the matched-pairs t-test. The baseline urinary serotonin assay grouping analysis revealed a P value of 0.0080. These findings indicate that baseline urinary levels do differ in a statistically significant manner when baseline assays are performed on different days for the same subject and are not uniform or reproducible from day to day. Baseline dopamine assays from different days in the same subject Data were analyzed in the following manner, with numbers reported in μg of dopamine per g of creatinine. From a matched-pairs group of n = 146, the mean SD for both baseline serotonin urinary assay groups was determined. For Group 1, the mean dopamine value was found to be 144.0 (±286.9). For Group 2 (baseline testing performed on a different day after the first assay), the mean dopamine value was found to be 198.6 (±484.8). All data greater than the value found in calculating the sum of two SD plus the mean were removed from consideration, revealing a group of n = 138. The matched-pairs grouping was then analyzed using the matched-pairs t-test. The baseline urinary serotonin assay grouping analysis revealed a P value of 0.0049. These findings indicate that baseline urinary dopamine levels do differ in a statistically significant manner when baseline assays are performed on different days in the same subject, and are not uniform or reproducible from day to day. Discussion The focus of this research is the applications of urinary serotonin and dopamine assays, whereby three distinctly different application models of monoamine assays are being promoted. The basis of the tumor model is screening for a monoamine-secreting tumor. This methodology is well founded. The OCT model is a relatively new application of monoamine assays, but its validity is supported by the literature.3–5 The third application model for monoamine assays, the urinary neurotransmitter testing model, has no indepth, valid peer-reviewed scientific literature to support its use. The UNT model distinguishes itself from the two other approaches by requiring use of baseline urinary monoamine assays, and advocates a direct relationship between urinary serotonin and urinary dopamine when the serotonin and dopamine amino acid precursor daily dosing levels are varied. The following is a consolidation of the findings and scientific concepts discussed in this paper with the claims and approach for use of monoamine assay applications under the UNT model. Significant challenges to the urinary neurotransmitter testing model include the widely recognized finding that serotonin and dopamine do not cross the blood–brain barrier.16–19 In support of applications for urinary serotonin and urinary dopamine assays, the UNT model claims that serotonin and dopamine do cross the blood–brain barrier.6–8 This assertion is widely known to be untrue.16–19 No significant amount of serotonin and dopamine filtered at the glomerulus reaches the urine. Serotonin and dopamine found in the urine are newly synthesized in the kidneys, and their levels are a function of the interaction between the basolateral monoamine transporters and the apical monoamine transporters of the proximal convoluted renal tubule cells.19 The UNT model claims that serotonin and dopamine are merely filtered at the glomerulus, and then enter the urine without further renal interactions.6 This assertion is not supported by review of the relevant science. Urinary serotonin and urinary dopamine found in the urine have no correlation with brain or peripheral serotonin and dopamine levels. Significant levels of urinary serotonin and urinary dopamine molecules assayed in the urine have never been shown in the brain or peripheral nervous system.3,5 The UNT model, based on assertions that serotonin and dopamine cross the blood–brain barrier and are then simply filtered at the glomerulus and enter the urine, claims that urinary monoamine assays represent the functional neurotransmitter status of the central nervous system, peripheral nervous system, and urine.1 This assertion again is not supported by the relevant science. There is no consistent direct relationship between serotonin and dopamine amino acid precursor daily dosing levels and the amount of serotonin and dopamine that appears in the urine on monoamine assays.3–5 The peer-reviewed literature notes that there is no relationship between administration of the serotonin precursor, 5-HTP, in varied doses and subsequent urinary serotonin levels.4 The literature also notes that there is a correlation between administration of L-tyrosine and urinary dopamine levels, but this is an inverse relationship,4 and not the direct relationship predicted by the UNT model.6,7 The UNT model advocates that there is a dominant direct correlation between amino acid doses and urinary serotonin and urinary dopamine found on assay.6,7 This leads to the assertion under the UNT model that simply determining whether the urinary serotonin and urinary dopamine levels found on assay are high or low is the focal point of proper monoamine assay interpretation.6,7 This assertion is not supported on review of the science involved. Statistical analysis of baseline monoamine assays reveals that these assays do not predict the response to precursor therapy. They differ significantly with subsequent baseline assays undertaken on different days from the same subject, and no significant difference exists with assays performed when amino acid precursors are taken. These findings are contrary to the assertions of the UNT model.6–8,11 The UNT model claims that baseline monoamine assays obtained prior to ingestion of supplemental amino acid precursors can identify neurotransmitter imbalance in the central nervous system, peripheral nervous system, and urine.6–8 Due to the statistical difference in baseline monoamine assays in the same subject from day to day, an unlimited number of different neurotransmitter imbalances might theoretically be diagnosed with serial assays performed on many different days from the same subject. There is a statistical difference between baseline urinary serotonin and urinary dopamine assays in subjects not harboring a monoamine-secreting tumor. The assertion that baseline monoamine assays can diagnose central nervous system, peripheral nervous system, and urinary neurotransmitter dysfunction is not supported on review of the scientific literature. The UNT model also claims that baseline assays of urinary serotonin and urinary dopamine are required prior to starting serotonin and/or dopamine amino acid precursors to assist in selecting the optimal daily serotonin and dopamine amino acid precursor doses.8–10 Using any laboratory criteria to diagnose serotonin and dopamine imbalance prior to selecting the starting point of amino acid dosing gives results that differ statistically from day to day and are not reproducible. The assertion on the part of the UNT model that baseline monoamine assays are needed to determine a starting point for serotonin and dopamine amino acid precursor treatment is not supported. The UNT model claims that baseline assays are required to minimize side effects when treatment with amino acid precursors is started. The results of baseline assays obtained from the same subject on different days vary statistically, and are not reproducible relative to the first baseline assay obtained. The ability to minimize side effects claimed on the basis of the UNT model is not supported by the reported science. The UNT model incorrectly asserts that baseline monoamine assays can serve as a reference point during treatment to gauge effectiveness of treatment when serotonin and dopamine amino acid precursors are started.8,10 As noted already, there is a significant statistical difference between values found with baseline monoamine assays and baseline assays performed on a different day in the same subject, leading to a host of different reference points being generated when baseline assays are obtained on multiple days. The baseline assays cannot be used as a reference point to measure treatment progress or indicate results of treatment. The only valid correlation that exists between monoamine assays performed with and without administration of amino acid precursors in subjects not suffering from a monoamine-secreting tumor is the three-phase model described in the literature. When the three-phase model is applied correctly to urinary serotonin and urinary dopamine assay results, it leads to a predictable course of outcomes with urinary serotonin and urinary dopamine assay interpretation. The three-phase model is based on the interaction between the newly synthesized serotonin and dopamine by the kidneys with the basolateral monoamine (serotonin and dopamine) transporters and the apical monoamine (serotonin and dopamine) transporters of the proximal convoluted renal tubule cells of the kidneys, leading to the serotonin and dopamine that is found in the urine on assay.3–5 Conclusion The application and interpretation of baseline monoamine assays according to the urinary neurotransmitter testing model is not a valid approach because there is a significant statistical difference between baseline monoamine assays and monoamine assays obtained on a different day from the same subject and no significant statistical difference in subsequent monoamine assays performed while taking amino acid precursors. The UNT model has no ability to diagnose central or peripheral nervous system serotonin and dopamine imbalance using baseline monoamine assays in subjects not suffering from monoamine-secreting tumors. Urinary serotonin and urinary dopamine assays are not assays of serotonin and dopamine that have been in the central nervous system. Serotonin and dopamine do not cross the blood–brain barrier. Significant amounts of urinary serotonin and urinary dopamine found on assay have not been in the brain or in the peripheral system. Urinary serotonin and urinary dopamine are filtered at the glomerulus and are then metabolized in the kidneys, with no significant amounts of serotonin or dopamine filtered at the glomerulus being found in the urine. Levels of urinary serotonin and urinary dopamine found on assay are newly synthesized in the kidneys, and are a function of the interaction between the basolateral monoamine transporters and apical monoamine transporters of the proximal convoluted renal tubule cells. A simple direct relationship between the daily dosing levels of amino acid precursors and monoamine assays does not exist in most cases. Due to complex renal physiologic interactions between serotonin and dopamine newly synthesized by the kidneys, a complex relationship is observed that is defined by the three-phase model described in the already published peer-reviewed literature. The goal of this paper is to spark interest, research, awareness, and scrutiny of the topics discussed. A laboratory assay is only valid if properly interpreted. Correct interpretation of monoamine assays while taking amino acid precursors is complex, and not a direct linear relationship as predicted by the UNT model. Disclosure TU and MH are director and owner of DBS Laboratories, Duluth, Minnesota respectively. AS and GT have no conflicts of interest to report in this work. ==== Refs References 1 Oates JA Sjoerdsma A A unique syndrome associated with secretion of 5-hydroxytryptophan by metastatic gastric carcinoids Am J Med 1962 32 333 342 14480945 2 Szakacs JE Cannon AL Noreprinephrine myocarditis Am J Clin Pathol 1958 30 425 434 13594914 3 Hinz M Depression Kohlstadt I Food and Nutrients in Disease Management CRC Press 2009 4 Trachte G Uncini T Hinz M Both stimulatory and inhibitory effects of dietary 5-hydroxytryptophan and tyrosine are found on urinary excretion of serotonin and dopamine in a large human population Neuropsychiatr Dis Treat 2009 5 227 235 19557117 5 Hinz M Stein A Uncini T The dual gate lumen model of renal monoamine transport Neuropsychiatr Dis Treat 2010 6 387 392 20856602 6 Alts J Alts D Bull M Urinary Neurotransmitter Testing: Myths and Misconceptions Osceola, WI NeuroScience, Inc 2007 7 Watkins R Validity of urinary neurotransmitter testing with clinical applications of CSM (Communication System Management) model Asheville, NC Sanesco International 2009 Available at: http://www.neurolaboratory.net/lab/neurolab%20pdf%20files/2009%20Urinary%20NT%20White%20Paper.pdf Accessed 2010 Aug 4 8 Theirl S Clinical relevance of neurotransmitter testing The Original Internist 12 2009 Available at: http://www.clintpublication.com/documents/Dec_OI_2009.pdf Accessed 2010 Aug 4 9 Sanesco Neurolab baseline sample report Available at: http://sanesco.net/images/files/resourcelibrary/baseline_sample_report.pdf Accessed 2010 Jul 2 10 Neuroscience Assessing nutritional imbalances Available at: https://www.neurorelief.com/index.php?option=com_content&task=view&id=131&Itemid=48 Accessed 2010 Jul 2 11 Kellermann G Bull M Ailts J Understanding diurnal variation Technical Bulletin Issue 4 Osceola, WI NeuroScience, Inc 2004 1 9 Available at: https://www.neurorelief.com/index.php?option=com_content&task=view&id=224&Itemid=48 Accessed 2010 Jul 2 12 Carley C Radulovacki M Role of peripheral serotonin in the regulation of central sleep apneas in rats Chest 1999 115 1397 1401 10334159 13 Volkow N Fowler JS Gatley J PET evaluation of the dopamine system of the human brain J Nucl Med 1996 37 1242 1256 8965206 14 Wang Y Berndt T Gross T Peterson M So M Know F Effect of inhibition of MAO and COMT on intrarenal dopamine and serotonin and on renal function Am J Physiol Regul Integr Comp Physiol 2001 280 R248 R254 11124158 15 Vieira-Coelho MA Soares-Da-Silva P Apical and basal uptake of L-dopa and L-5-HTP and their corresponding amines, dopamine and 5-HT, in OK cells Am J Physiol 1997 272 5 Pt 2 F632 F639 9176374 16 Pyle AC Argyropoulos SV Nutt DJ The role of serotonin in panic: Evidence from tryptophan depletion studies Acta Neuropsychiatr 2004 16 79 84 17 Verde G Oppizzi G Colussi G Effect of dopamine infusion on plasma levels of growth hormone in normal subjects and in agromegalic patients Clin Endocrinol (Oxf) 1976 5 419 423 971548 18 Gozzi A Ceolin L Schwarz A A multimodality investigation of cerebral hemodynamics and autoregulation in pharmacological MRI Magn Reson Imaging 2007 25 826 833 17451905 19 Ziegler MG Aung M Kennedy B Sources of human urinary epinephrine Kidney Int 1997 51 324 327 8995750	24198626	PMC3818889	NO-CC CODE	2021-01-04 23:22:24	yes	Open Access J Urol. 2010 Oct 7; 2:177-183
==== Front Open Access J UrolOpen Access J UrolOpen Access Journal of Urology1179-1551Dove Medical Press 10.2147/OAJU.S16637oaju-3-019Original ResearchUrinary neurotransmitter testing: considerations of spot baseline norepinephrine and epinephrine Hinz Marty 1Stein Alvin 2Uncini Thomas 31 Clinical Research, NeuroResearch Clinics Inc., Cape Coral, FL, USA2 Stein Orthopedic Associates, Plantation, FL, USA3 DBS Laboratories, Duluth, MN, USACorrespondence: Marty Hinz, 1008 Dolphin Dr, Cape Coral, FL 33904, USA, Tel +1 218 626 2220, Fax +1 218 626 1638, Email [email protected] 04 2 2011 3 19 24 © 2011 Hinz et al, publisher and licensee Dove Medical Press Ltd2011This is an Open Access article which permits unrestricted noncommercial use, provided the original work is properly cited.Background The purpose of this paper is to present the results of statistical analysis of spot baseline urinary norepinephrine and epinephrine assays in correlation with spot baseline urinary serotonin and dopamine findings previously published by the authors. Our research indicates a need for physicians and decision-makers to understand the lack of validity of this type of spot baseline monoamine testing when using it in the decision-making process for neurotransmitter deficiency disorders. Methods Matched-pairs t-tests were performed for a group of subjects for whom spot baseline urinary norepinephrine and epinephrine assays were performed on samples collected on different days then paired by subject. Results The reported laboratory test results for urinary serotonin, dopamine, norepinephrine, and epinephrine, obtained on different days from the same subjects, differed significantly and were not reproducible. Conclusion Spot baseline monoamine assays, in subjects not suffering from a monoamine-secreting tumor, such as pheochromocytoma or carcinoid syndrome, are of no value in decision-making due to this day-to-day variability and lack of reproducibility. While there have been attempts to integrate spot baseline urinary monoamine assays into treatment of peripheral or central neurotransmitter-associated disease states, diagnosis of neurotransmitter imbalances, and biomarker applications, significant differences in day-to-day reproducibility make this impossible given the known science as it exists today. Keywords neurotransmitter testingepinephrinenorepinephrinedopamineserotonin ==== Body Introduction A previously published paper by the authors of this paper discussed the reproducibility of spot baseline urinary serotonin and dopamine assays.1 This companion paper discusses the reproducibility of spot baseline urinary norepinephrine and epinephrine assays, and explores the feasibility and validity of using spot urinary norepinephrine or epinephrine assays in subjects not suffering from a monoamine-secreting tumor as a basis for decision-making. The paper then correlates the novel spot baseline norepinephrine and epinephrine findings reported here with our earlier reports relating to spot baseline urinary serotonin and dopamine. Urinary neurotransmitter testing samples can be generated in several ways. “Spot urine” is a single urine sample obtained at a specific time.1 A 24-hour urine sample is a collection of all urine excreted in a defined time period, and is used when the total daily excretion of a substance by the kidneys into the urine is to be studied. One application of the 24-hour urine test is in the diagnosis of monoamine-secreting tumors. Collection of a 24-hour urine sample is burdensome, and requires the subject to carry sample collection materials during all daily activities.2,14 Urinary monoamines exist in two states, ie, “the endogenous state”, found when no amino acid precursors of the monoamines are being administered, and “the competitive inhibition state”, found when significant amounts of both serotonin and dopamine amino acid precursors are being administered simultaneously. Obtaining urine samples in the endogenous state is known as “baseline testing”.3 The focus of this paper is spot urine measurements obtained in the endogenous state, which is also known as “baseline urinary neurotrans-mitter testing”. Spot baseline urinary neurotransmitter testing samples obtained in the endogenous state are of no value in patients not suffering from a monoamine-secreting tumor, such as pheochromocytoma or carcinoid syndrome, due to a lack of reproducibility of the testing involved.1 Previously published peer-reviewed literature has established the validity and utility of OCT interpretation of monoamine assays in the competitive inhibition state when performed under proper conditions.1,3–5 Materials and methods Results of statistical analysis of spot baseline urinary neurotransmitter testing of serotonin and dopamine assays have been discussed and published previously by the authors of this paper.1 Novel statistical results of spot baseline urinary neurotransmitter testing of norepinephrine and epinephrine assays from a database accumulated by two of the authors of this paper are reported here. Urinary norepinephrine and epinephrine samples obtained on different days from the same subject were statistically analyzed using a matchedpairs t-test. A P value ≤0.05 was considered to reveal a significant difference between groupings. JMP (SAS Institute, Cary, NC) software was used to perform the statistical analysis. Processing, management, and assay of the urine samples collected for this study were as follows. Urine samples were collected six hours prior to bedtime, with 4 pm being the most frequent collection time point. The samples were stabilized in 6 N HCl to preserve urinary dopamine and urinary serotonin. Samples were shipped to DBS Laboratories, Duluth, MN. Urinary norepinephrine and dopamine were assayed utilizing commercially available radioimmunoassay kits (3 CAT RIA IB88501 and IB89527; Immuno Biological Laboratories Inc, Minneapolis, MN). DBS Laboratories is accredited as a high complexity laboratory by Clinical Laboratory Improvement Amendments to perform these assays. Results In order for laboratory testing to be valid it needs to be reproducible. The following is a discussion of the statistical reproducibility of spot baseline urinary neurotransmitter testing of norepinephrine and epinephrine performed on a group of subjects in whom two urine samples were obtained on different days. The matched-pairs t-test was used to evaluate these spot baseline samples. To complete the serotonin and catecholamine discussion, previously published data by the authors relating to spot baseline urinary neurotransmitter testing of serotonin and dopamine is included, because norepinephrine and epinephrine production and balance are related to balanced levels of serotonin and dopamine. Spot baseline norepinephrine matched-pairs t-test The following norepinephrine data are novel. The laboratory values are reported in μg of norepinephrine per g of creatinine. From a matched-pairs group of n = 54, the mean and standard deviation (SD) for both spot baseline norepinephrine urinary assay groups was determined. For Group 1, the mean norepinephrine value was found to be 64.66 (±148.98). For Group 2 (spot baseline norepinephrine testing performed on a different day after the first assay), the mean norepinephrine value was found to be 42.01 (±173.39). All data greater than the value found in calculating the sum of two SDs plus the mean were removed from consideration, revealing a group of n = 44. This matched-pair values group was then analyzed using the matched-pairs t-test, revealing a P value of 0.0399. These findings indicate that spot baseline urinary norepinephrine levels do differ in a statistically significant manner when spot baseline assays are performed on different days from the same subject. This supports the assertion that spot urinary norepinephrine values are not uniform or reproducible from day to day. The epinephrine group (n = 44) comprised 21 females aged 48.22 (±13.34) years and 23 males aged 46.31 (±14.63) years. Spot baseline epinephrine matched-pairs t-test The following epinephrine data are also novel. The laboratory values are reported in μg of epinephrine per g of creatinine. From a matched-pairs group of n = 135, the mean and the SD for both spot baseline epinephrine urinary assay groups was determined. For Group 1, the mean epinephrine value was found to be 6.55 (±5.5). For Group 2 (spot baseline testing performed on a different day after the first assay), the mean epinephrine value was found to be 10.4 (±14.12). All data greater than the value found in calculating the sum of two SDs plus the mean were removed from consideration, leaving a group of n = 122. This matched-pair values group was then analyzed using the matched pairs t-test, revealing a P value of <0.0001. These findings indicate that spot baseline urinary epinephrine levels do differ in a statistically significant manner when spot baseline assays are performed on different days from the same subject. This supports the assertion that spot urinary epinephrine values are not uniform or reproducible from day to day. The epinephrine group (n = 122) comprised 63 females aged 59.09 (±11.87) years and 59 males aged 45.89 (±18.72) years. Spot baseline serotonin matched-pairs t-test A 2010 peer-reviewed paper by the authors presented results of a novel spot serotonin matched-pairs t-test (n = 134). Spot baseline–baseline grouping of urinary serotonin samples obtained on different days from the same patient revealed a P value of 0.0080. This indicates that spot baseline urinary serotonin levels differ in a statistically significant manner when they are performed on different days from the same subject. This supports the assertion that spot urinary serotonin values are not uniform or reproducible from day to day.1 Spot baseline dopamine matched-pairs t-test A 2010 peer-reviewed paper by the authors of this paper presented results of a novel spot dopamine matched-pairs t-test (n = 138). Spot baseline–baseline grouping of urinary dopamine samples obtained on different days from the same patient revealed a P value of 0.0049. This indicates that spot baseline urinary dopamine levels differ in a statistically significant manner when they are performed on different days from the same subject. This supports the assertion that spot urinary dopamine values are not uniform or reproducible from day to day.1 Results of the four matched-pairs t-tests shown in Table 1 reveal that there are significant differences between spot baseline urinary neurotransmitter testing performed on different days from the same subject for all four monoamines under scrutiny. Simply asserting that testing differs significantly and is not reproducible from day to day in the same subject may not have the impact of reviewing the data used for the statistical analysis. The data in the accompanying tables illustrate that the urinary neurotransmitter testing results are not reproducible from day to day, and that spot baseline urinary neurotransmitter testing is not a valid foundation for medical decision-making. Tables 2–5 contain the paired results of 160 spot baseline urinary neurotransmitter tests. All values are reported in μg of monoamine per g of creatinine. The urine samples analyzed were collected approximately six hours prior to bedtime, with 4 pm being the most common time of collection. A review of all samples collected at other times of the day revealed results that were similar to the aforementioned findings. Spot baseline urinary monoamine samples differed significantly from day to day in the same subject, regardless of the time collected, and were not reproducible. Discussion In the scientific world, there are two highly polarized views regarding the validity of spot baseline urinary neurotransmitter testing. One view advocates that baseline urinary neurotransmitter testing has no value in patients not suffering from a monoamine-secreting tumor.1,3–5 The other view advocates that it is very beneficial, and that it has numerous applications in medical decision-making, including diagnostic, therapeutic, and biomarker applications.6–12 The purpose of this writing is to educate medical practitioners regarding the selection of laboratory testing for neurotransmitter diseases so that they do not use invalid testing methods. The science supporting the view of the authors is as follows. It is a well-known fact that norepinephrine, epinephrine, serotonin, and dopamine do not cross the blood–brain barrier. These monoamines are filtered at the glomerulus and are then metabolized by the kidneys. Significant amounts of these monoamines filtered at the glomerulus do not reach the final urine. Monoamines found in the urine of patients not suffering from a monoamine-secreting tumor are primarily synthesized by structures in the kidneys.1,3–5,13 Spot baseline testing lacks reproducibility and is of no value in patients not suffering from a monoamine-secreting tumor.1 Those who claim that spot baseline urinary neurotransmitter testing is valid assert that monoamines cross the blood-brain barrier, are filtered at the glomerulus, and simply excreted into the urine without further renal involvement. They conclude that spot baseline urinary neurotransmitter testing is a valid assay for peripheral and central nervous system neurotransmitter levels.6–12 Spot baseline urinary neurotransmitter testing of norepinephrine, epinephrine, serotonin, and dopamine is not reproducible from day to day in the same subject; therefore, this type of testing is not valid. An infinite number of assays performed on an infinite number of days would generate an infinite number of differing test results.1 The following are true, based on the statistics put forth in this paper and the lack of reproducibility as demonstrated in this writing: Spot urinary neurotransmitter testing is not a reliable assay for peripheral or central nervous system function; the majority of serotonin and catecholamine molecules found in the urine of patients not suffering from a monoamine-secreting tumor have never been in the peripheral or central nervous system, having been synthesized by renal structures Spot urinary neurotransmitter testing does not correlate with monoamine neurotransmitter-related disease states in patients not suffering from a monoamine-secreting tumor Spot urinary neurotransmitter testing, due to lack of reproducibility, cannot assist the health care practitioner in making informed decisions regarding the choice of amino acids, or the dosing value for intervention with a disease state associated with monoamine neurotransmitters Spot urinary neurotransmitter testing, due to lack of reproducibility, does not have a place in clinical practice for identifying biomarkers of peripheral or central nervous system function and disease states Spot urinary neurotransmitter testing cannot determine monoamine imbalances that exist in subjects because the results are not reproducible Spot baseline monoamine assays cannot serve as a predictor of expected efficacy once amino acid precursors are started due to lack of reproducibility. There is evidence that urinary monoamines, such as norepinephrine reported on 24-hour urine samples, may be elevated in a specific group of patients with depression.15 However, these are group findings, and do not necessarily translate to individual testing validity on spot testing due to the lack of reproducibility of the test from day to day in the same subject. Conclusion This research underscores the fallacy of the attempt to use spot baseline urinary neurotransmitter testing as a potential biomarker in the treatment of patients with presumed monoamine neurotransmitter-related diseases who are not suffering from a monoamine-secreting tumor. Levels of urinary norepinephrine, epinephrine, serotonin, and dopamine, found in the urine on spot baseline testing, differ significantly from day to day in the same subject. Results are not reproducible, so spot baseline urinary neurotransmitter testing in the endogenous state in subjects not suffering from a monoamine-secreting tumor is of no clinical value. Health care practitioners need to understand this difference when selecting a form of testing for their patients. It is hoped that this writing will spark interest and scrutiny of the topic, leading to advancement of the relevant science. Disclosure The authors report no conflicts of interest in this work. Table 1 Matched-pairs t-test values. A P value <0.05 indicates that a significant difference between the test 1 grouping and test 2 grouping exists on different days in the same individual. Spot baseline monoamine assays are not uniform and reproducible from day to day in the same subject, and therefore the testing is not reproducible or valid n P value Norepinephrine 44 0.0399 Epinephrine 122 <0.0001 Serotonin 134 0.0080 Dopamine 138 0.0049 Tables 2a, b Serial spot baseline–baseline norepinephrine assays from the same subject. Some of the norepinephrine data used to determine the norepinephrine matched-pairs t-test values found in Table 1. Comparison of norepinephrine 1 with norepinephrine 2 from the same subject (by row) illustrates the lack of test reproducibility. The number of days column is the number of days between urinary sample collection dates a) Sort: High-low by NE-1 b) Sort: High-low by NE-2 Days (n) NE-1 NE-2 Days (n) NE-1 NE-2 217 595.42 270.20 272 145.46 861.92 58 479.59 8.50 225 7.67 581.60 28 416.86 132.37 32 386.01 540.17 41 399.75 49.38 79 151.44 482.38 32 386.01 540.17 217 595.42 270.20 19 381.86 10.62 29 232.14 261.01 42 357.80 61.73 189 132.09 233.98 41 301.00 203.70 41 301.00 203.70 50 268.04 31.36 28 0.97 195.92 29 232.14 261.01 64 214.24 186.00 Abbreviation: NE, norepinephrine. Tables 3a, b Serial spot baseline–baseline epinephrine assays from the same subject, including epinephrine data used to determine the epinephrine matched-pairs t-test values found in Table 1. Comparison of EPI-1 with EPI-2 from the same subject (by row) illustrates lack of test reproducibility. The number of days column is the number of days between urinary sample collection dates a) Sort: High-low by EPI-1 b) Sort: High-low by EPI-2 Days (n) EPI-1 EPI-2 Days (n) EPI-1 EPI-2 43 36.06 3.90 77 8.98 29.09 22 24.83 9.58 272 13.09 16.37 364 22.81 11.99 104 8.43 15.34 27 21.37 3.22 35 8.62 15.01 46 20.44 14.76 42 14.80 14.99 49 18.80 6.69 46 20.43 14.76 380 18.59 13.83 98 6.39 14.10 185 16.49 4.87 380 18.59 13.83 42 14.80 8.43 225 6.16 13.42 41 12.86 6.57 22 24.83 13.02 Abbreviation: EPI, epinephrine. Tables 4a, b Serial spot baseline-baseline serotonin assays from the same subject, including some of the serotonin data used to determine the serotonin matched-pairs t-test values found in Table 1, from a previously published paper by the authors.1 Comparison of serotonin 1 with serotonin 2 from the same subject (by row) vividly illustrates lack of testing reproducibility. The number of days column is the number of days between urinary sample collection dates a) Sort: High-low by Serotonin 1 b) Sort: High-low by Serotonin 2 Days Serotonin 1 Serotonin 2 Days Serotonin 1 Serotonin 2 42 9885.64 179.65 272 307.07 6004.24 28 5178.39 415.45 79 1159.95 5194.81 32 3309.76 1191.05 41 2451.00 4049.95 41 2451.00 4049.95 41 96.77 3655.97 98 2157.10 368.47 103 9885.65 3246.75 42 1569.16 432.35 217 828.22 2275.38 79 1159.95 5194.81 204 276.97 2183.79 29 1005.58 851.43 47 227.30 2000.00 217 828.22 2275.38 383 60.32 1996.24 19 763.47 31.14 32 3309.76 1191.05 Tables 5a, b Serial spot baseline–baseline dopamine assays from the same subject, including dopamine data from a previous study used to determine the dopamine matched-pairs t-test values found in Table 1.1 Comparison of dopamine 1 with dopamine 2 from the same subject (by row) illustrates the lack of test reproducibility. The number of days column is the number of days between urinary sample collection dates a) Sort: High-low by dopamine 1 b) Sort: High-low by dopamine 2 Days (n) Dopamine 1 Dopamine 2 Days (n) Dopamine 1 Dopamine 2 46 7854.32 1884.93 41 1129.58 2891.23 41 1129.58 2891.23 98 300.37 2623.79 204 1034.63 71.76 6 138.81 2504.14 28 785.00 181 103 164.50 2109.03 77 652.35 1288.47 46 7854.32 1884.93 27 498.23 68.80 28 785.00 1806.00 58 419.82 88.41 77 652.35 1288.48 168 405.20 180.51 314 197.72 1220.54 28 387.64 169.78 47 785.00 853.00 29 372.51 208.49 383 289.88 430.71 ==== Refs References 1 Hinz M Stein A Trachte G Uncini T Neurotransmitter testing of the urine, a comprehensive analysis Open Access Journal of Urology 2010 2 177 183 2 Smythe G Edwards G Graham P Lazarus L Biochemical diagnosis of pheochromocytoma by simultaneous measurement of urinary excretion of epinephrine and norepinephrine Clin Chem 1992 38 486 492 1568311 3 Hinz M Stein A Uncini T The dual-gate lumen model of renal monoamine transport Neuropsychiatr Dis Treat 2010 6 387 392 20856602 4 Hinz M Depression Kohlstadt I Food and Nutrients in Disease Management Boca Raton, FL CRC Press 2009 5 Hinz M Stein A Uncini T Amino acid responsive Crohn’s disease Clinical and Experimental Gastroenterology 2010 3 171 177 21694863 6 Alts J Alts D Bull M Urinary Neurotransmitter Testing: Myths and Misconceptions Osceola, WI NeuroScience Inc 2007 7 Watkins R Validity of urinary neurotransmitter testing with clinical applications of CSM (Communication System Management) model Asheville, NC Sanesco International 2009 Available at: http://www.neurolaboratory.net/lab/neurolab%20pdf.%20files/2009%20Urinary%20NT%20White%20Paper.pdf Accessed November 24, 2010 8 Theirl S Clinical relevance of neurotransmitter testing The Original Internist 12 2009 Available at: http://www.clintpublication.com/documents/Dec_OI_2009.pdf Accessed November 24, 2010 9 Sanesco Neurolab baseline sample report Available at: http://sanesco.net/images/files/resourcelibrary/baseline_sample_report.pdf Accessed November 24, 2010 10 Neuroscience Assessing nutritional imbalances Available at: https://www.neurorelief.com/index.php?option=com_content&task=view&id=131&Itemid=48 Accessed November 24, 2010 11 Kellermann G Bull M Ailts J Understanding diurnal variation Technical Bulletin Issue 4 Osceola, WI NeuroScience Inc 1 9 2004 Available at: https://www.neurorelief.com/index.php?option=com_content&task=view&id=224&Itemid=48 Accessed November 24, 2010 12 Marc D Ailts J Ailts-Campeau D Neurotransmitters excreted in the urine as biomarkers of nervous system activity: validity and clinical applicability Neurosci Biobehav Rev 2011 35 635 644 20696183 13 Trachte G Uncini T Hinz M Both stimulatory and inhibitory effects of dietary 5-hydroxytryptophan and tyrosine are found on urinary excretion of serotonin and dopamine in a large human population Neuropsychiatr Dis Treat 2009 5 227 235 19557117 14 Hughes J Watkins L Blumenthal J Kuhn C Sherwood A Depression and anxiety symptoms are related to increased 24-hour urinary norepinephrine excretion among healthy middle-aged women J Psychosom Res 2004 57 353 358 15518669	24198631	PMC3818932	NO-CC CODE	2021-01-04 23:22:24	yes	Open Access J Urol. 2011 Feb 4; 3:19-24
==== Front J Cell Mol MedJ. Cell. Mol. MedjcmmJournal of Cellular and Molecular Medicine1582-18381582-4934Blackwell Publishing Ltd Oxford, UK 1776084010.1111/j.1582-4934.2007.00067.xArticlesCaveolin and MAP kinase interaction in angiotensin II preconditioning of the myocardium Das Manika Das Samarjit Das Dipak K Cardiovascular Research Center, University of Connecticut School of Medicine, Farmington, USACorrespondence: Dr Dipak K. DAS Cardiovascular Research Center University of Connecticut School of Medicine Farmington, CT 06-030-1110 USA Tel.: (860)679-3687 Fax.: (860)679-4606 E-mail.: [email protected] 2007 24 6 2007 11 4 788 797 28 2 2007 17 5 2007 Abstract Angiotensin II (Ang II) has been found to exert preconditioning (PC)–like effect in mammalian hearts. The present investigation reported for the first time a unique mitogen activated protein (MAP) kinase signalling in Ang II PC of the heart involving lipid rafts, which generated a survival signal by differentially associating MAP kinases with caveolin. A group of rat hearts was treated with Ang II in the absence or presence of NADPH oxidase inhibitor, apocynin or a cell permeable reactive oxygen species (ROS) scavenger, N-acetyl-cysteine (NAC). Ang II pre-treatment improved post-ischaemic ventricular recovery, myocardial infraction and decreased the number of cardiomyocyte apoptosis indicating PC effect of Ang II. Both apocynin and NAC abolished the PC ability of Ang II. In Ang II treated heart, there was a decreased association of p38MAPKβ & extracellular-signal regulated kinase (ERK) 1/ 2 (anti-death signalling component) with caveolin while there was an increased association of p38MAPKα & Jun N-terminal kinase (JNK) (death signalling component) indicating reduced amount of death signal components and increased amount of anti-death signalling components being available to the Ang II treated heart to generate a survival signal, which was reversed with NAC or apocynin. The survival signal was also demonstrated by increased phosphorylation of serine/threonine-protein kinase B (AKT) and enhanced induction of expression of Bcl-2 during Ang II PC and its reversal with NAC & apocynin treated heart. heartischaemia/reperfusioncaveolinMAP kinasesangiotensin IIROS ==== Body Introduction A large number of studies have demonstrated the role of angiotensin II (Ang II) in cardiac preconditioning (PC) against ischaemia-reperfusion injury [1–5]. Generally, Ang II is a detrimental factor for the heart and its inhibition with angiotensin converting enzyme (ACE) inhibitor provides cardio protection [6]. There is no clear explanation for such paradoxical behaviors of Ang II. A number of different mechanisms of action have been put forward to explain the cardio protective ability of Ang II. Almost a decade ago, Ang II receptor stimulation was found to pre-condition rabbit myocardium [7]. Evidence is rapidly accumulating to support that Ang II can stimulate NADPH oxidase dependent superoxide generation by increasing activity of NADPH oxidase subunits (p22phox and gp91phos) [8]. A previous study showed the phosphorylaion of p47phox by Ang II with concomitant induction of reactive oxygen species (ROS) formation via NADPH oxidase [9]. The mitogen activated protein (MAP) kinases have been shown to play a crucial role in ischaemic PC [6, 10 and 11]. Several MAP kinases including ERK (1/2), JNK and p38MAPK are main targets of ROS signalling. Unlike ERK (1/2), which is activated by growth signal via Ras dependent signal trans-duction pathway, the activation of JNK and p38MAPK are potentiated by diverse stresses and proinflamatory cytokines [8]. To date, four members of the p38MAPK family have been identified: p38α, p38β, p38γ, p38δ[12]. Although these isoforms share functional similarities, difference exist in their upstream and downstream kinase specificity, sug-gesting that they may have non-overlapping functions [13]. In general, p38MAPKα is linked to death signal while p38MAPKβ linked with the survival signal [14]. Recent investigation from our laboratory showed that differential interaction and/or translocation of p38MAPKα/β with caveolin 1/3 play an important role in generation of survival signal during PC [15]. Since MAP kinases play a crucial role in ischaemic PC, we reasoned that Ang II might modulate MAP kinase signalling through its interaction and/or translocation with caveolin. Our results determined that Ang II indeed pre-conditioned the ischaemic heart by differentially regulating MAP kinase interaction with lipid raft thereby converting the death signal into a survival signal. Materials and methods Chemicals: Ang II and N-acetyl-cysteine (NAC) were obtained from Sigma (St Louis, MO, USA). Apocynin was obtained from Calbiochem, CA, USA. Antibodies against caveolin-1, caveolin-3, p38MAPKβ, eNOS and Bcl-2 were purchased from Santa Cruz Biotechnology, CA, Santa Cruz, USA. Antibodies against p38MAPKα, JNK, ERK, AKT, phospho-AKT were purchased from Cell Signalling technology (Danvers, MA, USA). Experimental protocol The study used isolated working rat hearts subjected to ischaemia/reperfusion protocol. Isolated rat hearts were randomly divided into seven groups: perfused with KHB buffer only for 15 min (group I); the hearts were perfused with Krebs-Henseleit biocarbonate buffer (KHB) buffer for 15 min in the presence of Ang II [100 nM] (group II); NAC [1 uM] (group III); Apocyanin [2mg/Kg, i.p. 4 hrs before experiment] (Group IV); Ang II + NAC (group V) and Ang II + apocynin (Group VI). All hearts were then subjected to 30 min global ischaemia followed by 2 hrs reperfusion with KHB buffer. Control hearts (group VII) after perfusion with KHB buffer for 15 min, were not exposed to ischaemia and reperfusion. They were only subjected to 2 hrs and 30 min continuous perfusion. Isolated working rat heart preparation Male Sprague Dawley rats of 250 gm body weight were used for this study. All animals received humane care in compliance with the ‘Principles of Laboratory Animal Care’ formulated by the National Society for Medical Research and the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Sciences and published by the National Institute of Health (NIH Publication No. 85–23, revised 1996). The rats were anaesthetized with sodium pentobarbital (80 mg/kg b/w, ip.) (Abbott Laboratories, North Chicago, IL, USA) and anticoagulated with heparin sodium (500 IU/kg b.w, i.p) (Elkin-Sinn Inc., Cherry Hill, NJ, USA) injection. After ensuring sufficient depth of anaesthesia, the hearts excised and then the isolated hearts were perfused in Langendorff mode (cannulation viaaorta) against at constant perfusion pressure of 100 cm of water (10 kPa) for stabilization period [16]. Any heart that showed any cardiac disturbance (ventricle arrhythmia, and fibrillation) during the entire experiment was excluded from this study. After 5 min perfusion in Langendroff mode the hearts were converted in working mode (i.e. left ventricular preparation) and perfused by working mode according to the protocol as described earlier. To ascertain the normal function of the heart, the heart rate, left ventricular developed pressure (the difference between the maximum systolic and diastolic pressure), left ventricular end-diastolic pressure and the first derivative of developed pressure were recorded by Gould p23XL transducer (Gould Instrument System Inc., Valley View, OH, USA). The signal was amplified by using Gould 6600 series signal conditioner (Gould Instrument System Inc.) and monitored on Cordat II real-time acquisition system (Triton technologies, San Diego, CA, USA) [17]. The aortic flow was measured by flow meter. The coronary flow was measured by time-collection of the coronary effluent dripping from the heart. Measurements of the infarct size After ischaemia/ reperfusion (I/R) procedure, the heart was infused with 10% solution of the Triphenyl tetrazolium (TTC) in phosphate buffer through the aortic cannula for 20 min [18]. The left ventricle was removed and sliced into 1-mm thickness of cross-sectional pieces and weighted. Each slice was scanned with computer-assisted scanner (Scanjet). The infarct zone remained unstained while the non-infract part of myocardium was stained red by TTC. These were measured by using of computerized software (Scion Image); areas were multiplied by the weight of each section, and summed up to obtain the total infarct zone. The infarct size was expressed as the ratio of the infarct zone to the non-infract zone. TUNEL Assay for assessment of apoptotic cell death Immunohistochemical detection of apoptotic cells was carried out by TUNEL assay using ApopTag in situ apoptosis detection kit (Intergen Company, Purchase, NY, USA) [19]. Tissue sections were prepared from left ventricular tissue of the heart. Cells were counted at 100X magnification and at least four fields per sample. The sections were incubated again with mouse monoclonal antibody recognizing cardiac myosin heavy chain to specifically recognize apoptotic cardiomyocytes. The fluorescence staining was viewed with a confocal laser microscope. The number of apoptotic cells was counted and expressed as a percent of total myocytes population. Isolation of caveolin-rich membrane fractions The hearts were homogenized in sodium carbonate buffer containing protease inhibitor cocktail, pH 11.0 using a Polytron homogenizer [three 10s bursts][Brinkman Instruments, Westbury, NY, USA]. The homogenate was sonicated [three 20s bursts], and adjusted to 45% sucrose by the addition of 2 ml of 90% sucrose prepared in MBS [25 mM Mes, pH 6.5, 0.15 M NaCl] and placed at the bottom of an ultracentrifuge tube as described previously [20]. A 35% discontinuous sucrose gradient was formed above [4 ml of 5% sucrose/4 ml of 35% sucrose–both in MBS containing 250 mM sodium carbonate] and centrifuged at 39,000 rpm for 16–20 hrs in an SW41 rotor [Beckman Instruments, Palo Alto, CA, USA]. From the top of each gradient, 1 ml gradient fractions were collected to yield a total of 12 fractions as described elsewhere. Caveolin migrates mainly in fractions 5 and 6 of these sucrose density gradients [20]. Immunoprecipitation with caveolin-1 and caveolin-3 Only caveolin rich fractions (fraction 5 & 6) were used for immunoprecipitation. Immunoprecipitation was performed with Protein-A sepharose CL-B4 (Pharmacia Biotech Inc.) using a polyclonal antibody against caveolin-1 or caveolin-3 [Santa Cruz Biotechnology] Incubation conditions were maintained as instructed by the supplier. Western blot analysis was then performed with antibodies against P38MAPKα P38MAPKβ, JNK, ERK, eNOS, AKT and Phospho-AKT and Bcl-2 according to established Method [21]. Statistical analysis The values for number of apoptotic cardiomyocytes and infarct sizes as well as the functional parameters were all expressed as the Mean ± Standard Error of Mean (SEM) for at least six animals per group. The Western blot analyses were performed with at least three animals per group. The statistical analysis was performed by analysis of variance followed by Bonferroni's correction for any differences between the mean values of all groups. Differences between data were analysed for significance by performing a Student's t-test. The results were considered significant if P < 0.05. Results Effects of NAC and apocynin on Ang II pre-conditioning As expected, pre-perfusion of the hearts with Ang II triggered the PC effect as evidenced by its ability to enhance post-ischaemic left ventricular function. As shown in Table I, Ang II group displayed better ventricular recovery as reperfusion progressed, and left ventricular developed pressure (LVDP), dp/dt, and aortic flow was significantly improved (P < 0.05) in the Ang II group compared to those of I/R group. This cardio protective ability of Ang II was abolished when Ang II treatment was done in presence of NAC or apocynin. There were no significant differences in coronary flow and heart rate among the groups at any time point. The groups of heart that received only NAC showed significant effects on the post-ischaemic ventricular recovery, but apocynin showed only better recovery (non-significant). However, both compounds abolished the PC effects of Ang II. The values of LVDP, dp/dt and aortic flow were significantly lower in the treated groups compared to Ang II group (Table 1). 1 Effect of NAC and Apocyanin on Ang II pre-conditioning. Isolated hearts were treated as mentioned in the methods section. The hearts were then subjected to 30 min ischaemia followed by 2 hrs reperfusion. Measurements were taken at the baseline and at the end of reperfusion. Results are expressed as mean ± SEM. P < 0.05 versus I/R, †P < 0.05 versus Ang II. LVDP = Left Ventricular Developed Pressure, dp/dt = Max. First Derivative of Developed Pressure Control I/R Ang II NAC Apocynin Ang II + NAC Ang II + apocynin Heart Rate (beats/min) % change from baseline 369 ± 17.3 414 ± 24.3 443 ± 12.5 421 ± 30.3 437 ± 26.8 422 ± 16.7 453 ± 19.2 12%↑ 33%↑ 20%↑ 28%↑ 30%↑ 34%↑ 32%↑ Aortic Flow (ml/min) % change from baseline 22.6 ± 4.1 4.2 ± 1.4 12.5 ± 2.7 * 7.9 ± 2.1 * 5.8 ± 0.5 4.7 ± 1.2† 3.7 ± 0.7† 30%↓ 94%↓ 83%↓ 87%↓ 90%↓ 93%↓ 94%↓ Coronary Flow (ml/min) % change from baseline 29.7 ± 1.9 21.5 ± 1.2 20.2 ± 1.7 22.4 ± 1.1 20.2 ± 0.9 21.9 ± 0.9 20.7 ± 1.4 12%↓ 27%↓ 25%↓ 20%↓ 25%↓ 21%↓ 21%↓ LVDP (mm Hg) % change from baseline 105 ± 5.8 42.5 ± 7.6 81 ± 3.4 * 70 ± 7.2 * 50.4 ± 2.7 69.8 ± 2.8† 58.6 ± 4.6† 16%↓ 66%↓ 36%↓ 47%↓ 50%↓ 46%↓ 56%↓ dp/dt (mm Hg/ Sec) % change from baseline 2772 ± 90 849 ± 70 1139 ± 93 * 988 ± 67 897 ± 78.4 908 ± 66.5† 885 ± 83† 40%↓ 75%↓ 60%↓ 68%↓ 69%↓ 71%↓ 69%↓ Effects of NAC and apocynin on infarct size lowering ability of Ang II Figure 1A shows the effects of Ang II PC on the myocardial infarct size. The infarct size was about 37.6% for the I/R heart and Ang II reduced the infarct size by 31.2%. NAC and apocynin alone significantly reduced myocardial infarct size, but when used in conjunction with Ang II; they abolished infarct size lowering ability of Ang II. 1 Effect of Ang II, NAC & apocynin on the infract size of the heart and cardiomyocyte apoptosis. A:Infract size, B:car-diacmyocyte apoptosis.The results are mean ± SEM of six animal per group P < 0.05 versus I/R, †P < 0.05 versus Ang II. Effects of Ang II, NAC and apocynin on cardiomyocyte apoptosis Ang II PC significantly lowered the number of apoptotic cardiomyocytes (Fig. 1B). The number of apoptotic cardiomyocytes was 24.7% in the I/R heart that had been subjected to 30 min ischaemia and 2 hr reper-fusion as compared to only 12.2% in the Ang II treated hearts. NAC and apocynin also significantly reduced cardiomyocyte apoptosis compared to control. When used in conjunction with Ang II, NAC and apocynin significantly increased the number of apoptotic cell compared to Ang II alone. Effect of Ang II, NAC and apocyanin on differential interaction of caveolin & MAP kinase Caveolin rich fractions (5 th and 6 th fraction of sucrose density gradient) of all seven groups of heart were immuno-precipitated with caveolin-1 and caveolin-3. The examination of the existence of p38MAPKα/β in the caveolin immunoprecipitated sample by Western blot revealed that high amount of p38MAPKα bind with caveolin-1 whereas interaction of caveolin-3 with p38MAPKα was very negligible. In contrast, high amount of p38MAPKβ bind with Caveolin-3 whereas its binding with caveolin-1 was very negligible. Earlier investigation of our laboratory showed the similar results [15]. JNK & ERK1/2 also bind with caveolin-1 whereas interaction with caveolin-3 is very negligible. In I/R heart, there was increased association of p38MAPKβ & ERK1/2 (anti-death signalling component) with caveolin-3 and caveolin-1, respectively, while there was reduced association of p38MAPKα & JNK (death signalling component) with caveolin-1 indicating increased amount of death signalling component were available to I/R heart to generate a death signal. In contrast, in Ang II pre-conditioned heart, there was decreased association of anti-death signalling component (p38MAPKβ, ERK 1/2) with caveolin-3 and caveolin-1, respectively, while there was an increased association of death signalling components (p38MAPKα, JNK) with caveolin-1 indicating reduced amount of death signalling components and increased amount of anti-death signalling components were available to the Ang II treated heart to generate a survival signal. When Ang II pre–conditioned hearts were treated with NAC and apocynin, caveolin–MAPKs interaction showed a similar pattern like I/R heart making heart more abundance to death signalling component to induce death signal and suppress survival signal (Fig. 2). 2 Differential interaction of p38MAPK, JNK, ERK 1/2 and eNOS with caveolin-1 and caveolin-3 during ischaemia reperfusion, Ang II precondition in presence or absence of NAC or apocynin.The results are mean ± SEM of six animal per group P < 0.05 versus I/R, †P < 0.05 versus Ang II. In order to reconfirm our immunoprecipitation data, we tested all the 12 fraction (sucrose density gradient) of the Ang II pre-conditioned heart and found that p38MAPKβ and ERK was present in non-caveolin fraction and p38MAPKα and JNK was present in caveolin-rich zone. From these results it can be concluded that during Ang II PC, p38MAPKα and JNK (death signalling component) remain bound with caveolin and making them non-available to induce death signal. But at the same time p38MAPKβ and ERK (anti-death signalling component) remain in non-caveolin fraction and making the heart more abundance to them to induce survival signal (Fig. 3). 3 Distribution of p38MAPKα, p38MAPKβ, ERK and JNK in different fraction of the Ang II preconditioned heart. Nitric oxide (NO) derived from vascular endothelium has many physiological effects related to the protection of the ischaemic-reperfused heart [22]. Similar to p38MAPKβ, interaction of eNOS with caveolin-3 was very high compared to its binding with caveolin-1. In Ang II treated heart, a negligible amount of eNOS bound with caveolin-3 (Fig. 2). Whereas more eNOS bound with caveolin-3 in I/R heart and NAC & apocynin treated heart, indicating more eNOS was available in Ang II pre-conditioned heart to induce cardio protection. Effect of ANG II, NAC and apocynin on the expression of AKT, pAKT and Bcl-2 Bcl-2 and AKT are known to transmit survival signal. We did not find these proteins in the caveolin immunoprecipitated samples. The expression of these proteins was found in the cytosolic and crude fraction in of the heart. ANG II heart showed the upregulation of Bcl-2 compared to the control and I/R heart. NAC and apocynin abolished such increase in Bcl-2 protein (Fig. 4). Phospho-AKT was only present in the Ang II treated heart but not in the control, I/R, NAC and apocynin treated hearts. 4 Effect of Ang II, NAC & apocyanin on the induction of expression of Bcl-2, AKT and Phospho-AKT. The results are mean ± SEM of six animal per group *P < 0.05 versus I/R, †P < 0.05 versus Ang II. Discussion The results of the present study clearly demonstrated that Ang II could exert precondition-like cardioprotection confirming previous reports [1–6]. Ang II PC was associated with improved post-ischaemic ventricular recovery, reduced myocardial infarct size and decreased cardiomyocyte apoptosis [23]. Such PC effects of Ang II were almost completely abolished by ROS scavenger, NAC and NADPH oxidase inhibitor, apocynin as evidenced by depressed ventricular function and increased infarct size and cardiomyocyte apoptosis. Although NAC and apocynin block the cardio protection offered by Ang II, cadioprotective abilities of NAC and apocynin were also demonstrated when they were treated alone (without Ang II). The dual nature of NAC and apocynin can be explained by the dual nature of ROS. ROS are central to cardiac ischaemic and reperfusion injury. They contribute to myocardial stunning, infarction and apoptosis, and possibly to the genesis of arrhythmias. Multiple laboratory studies and clinical trials have evaluated the use of scavengers of ROS to protect the heart from the effects of ischaemia and reperfusion. A possible role for oxygen radicals in PC and/or cardio protection was suggested by variety of laboratories. Administration of superoxide dismutase (SOD), an enzyme that removes superoxide anions, during the reperfusion period following the initial PC (short period of ischaemia), could prevent the phenomenon [24]. The authors hypothesized that myocardial reperfusion after the first, short ischaemic episode results in the generation of low amounts of oxygen free radicals, not sufficient to causes cell necrosis, but which could modify cellular activities and thus induce cardio protection. In another study, treatment with N-acetylcysteine negated the beneficial effect of PC on post-ischaemic recovery of contractile function [25]. Similarly, the beneficial effect of PC on reperfusion-induced arrhythmias was prevented by SOD administration during the PC [26]. Several mechanisms might explain the effects of oxygen radicals. Recently, it has become appreciated that exposure of cells to mild oxidative stress or low dose of oxygen radicals can reversibly modify several cellular activities, in the absence of cell damage, but secondary to changes in the activity of various enzymes and other cellular components [27, 28, 29]. Among others, low dose of ROS can modify some of the cellular activities that have been implicated in vivo as mediators of the signalling cascade of cardio protective phenomenon. Earlier investigation from our laboratory showed that Ang II increased ROS activities, which were reduced by either NAC or apocynin. Most interestingly, Ang II induced the expression of two NADPH subunits, p22phox and gp91phox, which were completely inhibited with apocynin, and partially with NAC [8]. These results tend to suggest that Ang II PC be triggered by redox cycling of ROS, which are generated by both NADPH oxidase-dependent and independent pathway. [30, 23]. In another related study using renal homogenate, Ang II could stimulate NADPH oxidase dependent O2 generation [10]. NAD/NADPH oxidase is considered as the major source of ROS in VSMCs responsible for redox signalling [9]. ROS generated by Ang II could precondition the myocardium through the redoxregulated cell survival signalling pathways [11]. It is well established that ROS have either direct or indirect effect on MAPKs, under the family of serinethreonine kinase. MAPKs are considered as one of the major mediators of signal transduction in the stress activation pathway. Several recent studies have demonstrated that Ang II strongly phosphorylate ERK 1/2 [31–34]. In another study, Ang II induced robust phosphorylation of p38MAPK, ERK and JNK which was blocked by inhibition of NADPH oxidase, tyrosine kinase or ROS scavenger [35]. In another study using cardiac microvascular endothelial cells, Ang II activated ERK (1/2), but not p38MAPK in redox sensitive manner [36]. In smooth muscle cells, Ang II activated ERK 1/2 and p38 MAPK by a redox regulated mechanism [37]. The inhibition of JNK and p38MAPK activation was shown by using an antioxidant in the rat aortic smooth muscle cells, but interestingly the antioxidant did not change the activation of ERK (1/2) [38]. Lipid rafts are specialized membrane domain enriched with certain lipids, cholesterol and proteins. Caveolae, small plasma membrane invaginations that are coated with cholesterol binding protein, caveolin, are a subset of lipid raft. These lipid rafts serve as platforms for organizing and integrating signal transduction process. Recent investigation of our laboratory showed that p38MAPK translocation to caveolin during reperfusion and interacts differentially with caveolin-1 and caveolin-3. This differential interaction of p38MAPKα and p38MAPKβ with caveolin-1 and caveolin-3, respectively, function as a switch for conversion of I/R induced death signal to PC induced survival signal [13]. From the present results, it can be concluded that caveolin and MAPKs interaction play a very important role in generation of survival signal in Ang II preconditioned heart. In I/R heart anti-death signalling component (p38MAPKβ, ERK 1/2) bound with caveolin and binding of the death signalling component (p38MAPKα, JNK) with caveolin is less—exposing the heart to more abundance death signalling components thereby generating death signal. In contrast, in Ang II preconditioned heart binding of anti-death signalling component with caveolin is much less compared to the I/R heart and exposing the heart to more abundance anti-death signalling component, generating a survival signal. It appears that differential translocation and/or interaction of MAPKs with caveolin functions as a switch for the conversion of I/R induced death signal into Ang II PC induced survival signal. NO derived from vascular endothelial has many physiological effects related to the protection of the ischaemic-reperfused heart [30]. Here, in the present investigation, only a negligible amount eNOS bound with caveolin-3 in Ang II pre-conditioned heart, whereas more eNOS bound with caveolin-3 in I/R heart indicating generation of survival signal by free eNOS in Ang II pre-conditioned heart. The survival signal was further confirmed by increased phosphorylation of AKT and enhanced induction of expression of Bcl-2 during Ang II pre-condition and its reversal with I/R, NAC & apocynin treated heart. This result indicated that caveolin play a unique role in Ang II PC of the heart by interacting with different MAPKs and eNOS. In another ward, caveolin control the generation of survival signals in Ang II pre-conditioned heart by controlling the availability of survival signalling components to induce cardio protection. This study was supported by NIH HL 22559, HL33889 and HL 34360. ==== Refs References 1 Ferreira AJ Santos RA Almeida AP Angiotensin: cardio protective effect in myocardial ischemia/reperfusion Hypertension 2001 38 665 8 11566952 2 Sharma A Singh M Possible mechanism of cardio protective effect of angiotensin preconditioning in isolated rat heart Eur J Pharmacol 2000 406 85 92 11011038 3 Sharma A Singh M Effect of ethylisopropyl amiloride, a Na + -H + exchange inhibitor, on cardio protective effect of ischemic and angiotensin preconditioning Mol Cell Biochem 2000 214 31 8 11195787 4 Nakano A Miura T Ura N Suzuki K Shimamoto K Role of angiotensin II type I receptor in preconditioning against infarction Coronary Artery Dis 1997 8 343 50 5 Diaz RJ Wilson GJ Selective blockade of AT1 angiotensin II receptors abolishes ischemic preconditioning in isolated rabbit hearts J Mol Cell Cardiol 1997 29 129 39 9040028 6 Das DK Maulik N Engleman RM Redox regulation of Angiotensin II signaling in the heart J. 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==== Front 10126486032902Obesity (Silver Spring)Obesity (Silver Spring)Obesity (Silver Spring, Md.)1930-73811930-739X2383979110.1002/oby.20559nihpa500203ArticleIncreased HO-1 Levels Ameliorate Fatty Liver Development Through a Reduction of Heme and Recruitment of FGF21 Hinds Terry D. Jr.+Sodhi Komal 1Meadows Charles 1Fedorova Larisa +Puri Nitin +Kim Dong Hyun 1Peterson Stephen J. 2Shapiro Joseph 1Abraham Nader G. 13Kappas Attallah 31 Department of Medicine, Joan C. Edwards School of Medicine, Marshall University, Huntington, WV, 257552 Department of Medicine, New York Methodist-Weill Cornell Medical College, New York, NY 100653 The Rockefeller University, New York, NY 10065Corresponding author: Dr. Attallah Kappas, Professor, The Rockefeller University, 1230 York Avenue, New York, New York, NY 10065 Tel: 212 327-8494; or Nader G. Abraham, Ph.D., Dr.H.C. Marshall University Joan C. Edwards School of Medicine, Huntington WV 25701, [email protected]* contributed equally to this work * Present Address: Department of Physiology & Pharmacology, University of Toledo College of Medicine, Toledo OH 43614 2 8 2013 02 12 2013 3 2014 01 9 2014 22 3 705 712 Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:http://www.nature.com/authors/editorial_policies/license.html#termsObjective Obese leptin deficient (ob/ob) mice are a model of adiposity that displays increased levels of fat, glucose and liver lipids. Our hypothesis is that HO-1 overexpression ameliorates fatty liver development. Design and Methods Obese mice were administered cobalt protoporphyrin (CoPP) and stannic mesoporphyrin (SnMP) for 6 weeks. Heme, HO-1, HO activity, PGC1α, FGF21, glycogen content and lipogenesis were assessed. Results CoPP administration increased hepatic HO-1 protein levels and HO activity, decreased hepatic heme, body weight gain, glucose levels and resulted in decreased steatosis. Increased levels of HO-1 produced a decrease in lipid droplet size, FAS levels involving recruitment of FGF21, PPARα and Glut 1. These beneficial effects were reversed by inhibition of HO activity. Conclusion Increased levels of HO-1 and HO activity reduced the levels of obesity by reducing hepatic heme and lipid accumulation. These changes were manifested by decreases in cellular heme, increases in FGF21, glycogen content and fatty liver. The beneficial effect of HO-1 induction results from an increase in PPARα and FGF21 levels and a decrease in PGC1α, levels they were reversed by SnMP. Low levels of HO-1 and HO activity are responsible for fatty liver. non-alcoholic fatty liver disease (NAFLD)non-alcoholic steatohepatitis (NASH)FGF21Heme, PGC-1α, heme oxygenasePPARαobesity ==== Body Introduction Nonalcoholic fatty liver (NAFLD) occurs in a setting of high fat diets, insulin resistance, obesity and dyslipidemia (1, 2). NAFLD is a spectrum of diseases that range from fatty infiltration of the liver all the way to Nonalcoholic Steatohepatitis, NASH. Individuals with NAFLD have an increased risk of developing metabolic syndrome (3), the most severe form can progress to liver failure. If the liver contains fat levels that are in excess of 5 to 10%, fatty liver (hepatic steatosis) occurs. Low levels of antioxidants, excess heme, cytochrome P450-heme and mitochondrial dysfunction are factors that increase oxidative stress and ROS (reviewed in (4)). Increased HO-1 expression enables the cell to resist heme-mediated cell injury (reviewed in (4)). The well-documented protective role of HO-1 against the development of diabetes and the metabolic syndrome has been ascribed to several mechanisms that include a reduction in the levels of cellular heme-dependent proteins that increase oxidative stress and increased formation of the antioxidant, bilirubin (5). Excess heme-iron has also been implicated in liver damage (reviewed in (4)) and increased lipid accumulation generated in adipose tissues (6, 7). Heme iron-mediated oxidative stress magnifies the adverse effects of obesity by inducing inflammation in liver leading to NAFLD and NASH and systemic antioxidant treatment has yielded positive results in small studies (8). Probucol treatments improved NASH through the lowering of lipid levels and an increase in antioxidants level (9). However, broad systemic antioxidant treatment remains a “shotgun” approach. Targeting of HO-1 to adipose tissue and the vascular system (5, 10) has yielded positive effects on adipose dysfunction and insulin resistance (reviewed in (4)). HO-1 overexpression increased cellular antioxidant capabilities by increasing ferritin, CO and bilirubin (11, 12) leading to an increase in insulin sensitivity (12). HO-1 inducers, such as cobalt-protoporphyrin (CoPP), decrease heme levels (13) and increase phosphorylation of AKT in animal models of experimental diabetes (reviewed in (4)). Thus, increasing HO activity results in the reversal of oxidative stress and a decreases in liver damage (reviewed in (4)). Several transcription factors regulate liver metabolism and food intake including. peroxisome proliferator activated receptors-gamma coactivator-1(PGC-1α) and fibroblast growth factor 21(FGF21)(2, 14). PGC-1α regulates FGE21, and cellular heme biosynthesis (15). FGF21 regulates hepatic carbohydrate and glycogen content and is PGC-1α expression dependent (16). Thus, cellular heme modulates the levels of PGC-1α, lipid metabolism and adipogenesis (14, 17). Increases in hepatic heme content inhibit PGC-1α leading to suppression of FGF21 (18). Glycogen levels in liver are maintained by PPARα and increased FGF21 (19). FGF21 increases the phosphorylation of AMPK and AKT resulting in an increase in insulin sensitivity and the lowering of blood glucose and fatty acid levels (20, 21). The objective of this study was to examine the consequences of HO-1 induction and increased HO activity on the hepatic heme-HO system, FGF21, glycogen and hepatic fat content in obese mice. CoPP increased HO-1 expression and HO activity, attenuated the development of fatty liver, decreased lipid droplet size, PGC1α and increased FGF21 levels. Inhibition of HO activity increased lipid droplet size, FAS and decreased FGF21 and PPARα, thus substantiating a significant role of HO-1 (reviewed in (4)) against heme mediated adiposity and fatty liver. Pharmacological agents that increase HO-1 levels and gene targeting of HO-1 offer a promising therapeutic target for NAFLD and suggest the existence of a significant link between the heme-HO system and the extent and severity of heme-dependent fatty liver. Methods and Procedures Animal Treatment All experiments were performed following a New York Medical College IACUC approved protocol in accordance with the NIH Guide for the Care and Use of Laboratory Animals. Procedures for animal treatments have been previously published (12). In brief, male obese mice (B6v-Lep obese/J) were purchased from Harlan (Chicago, IL) at the age of 8 weeks. Age- and sex-matched lean mice (B6.V, lean; Harlan) were used as controls. Mice were fed a normal chow diet and had free access to water. Glucose monitoring was performed using an automated analyzer (Life scan, Milpitas, CA). Cobalt protoporphyrin (CoPP), an inducer of HO-1 expression, was administered intraperitoneally (i.p.) once a week (5 mg/kg) for 6 weeks to obese mice. Stannous mesoporphyrin (SnMP), an inhibitor of HO activity, was administered i.p. three times a week (20 mg/kg) for 6 weeks. Metalloporphyrins were dissolved in 10 mmol/l Tris base, and the pH was adjusted to 7.8 with 0.1 N HCl. A Tris/HCl solution free of metalloporphyrins was used to inject control animals. The animals were divided into four groups: 1) lean, 2) obese 3) obese CoPP and 4) obese CoPP+SnMP. Food intake did not change in mice treated with the various treatments. At the time of sacrifice the body weight of all mice was measured. After a 6-hour fast, mice were anesthetized with sodium pentobarbital (65 mg/kg, i.p.) and blood was obtained from a tail vein for glucose measurement using a glucometer. Blood samples were collected in K3EDTA tubes at sacrifice and the plasma was separated. Samples were flash frozen in liquid nitrogen and maintained at -80°C until needed. Other methodological details are provided in the online Data Supplement of Obesity. Statistical analyses Statistical significance between experimental groups was determined by ANOVA with Tukey-Kramer post-hoc analysis. The data are presented as means ± SEM and the null hypothesis was rejected at p<0.05. Results Effect of CoPP on Obese and Diabetic Phenotypes A significant (p<0.05) decrease in the expression of HO-1 protein in livers of obese compared to lean mice was observed (Figure 1A). CoPP, and CoPP plus tin mesoporphyrin (SnMP), resulted in a significant (p<0.05) increase of HO-1 protein levels in obese mice when compared to obese mice administered vehicle. In addition, decreased HO-1 expression in liver of obese mice resulted in a significant increase in cellular heme (p<0.05) compared to the lean control (Figure 1B). Treatment with CoPP increased HO-1 protein and HO activity and reduced heme levels. Administration of SnMP to obese mice treated with CoPP reversed the effect of CoPP and increased hepatic heme content (Figure 1B). CoPP increased HO-1 protein levels in obese mice several fold compared to untreated obese mice. As expected obese mice treated with both CoPP and SnMP resulted in a further increase in HO-1 protein levels. CoPP increased HO activity to 1.35 ± 0.17 nmol bilirubin/mg/hr. vs. 0.392 ± 0.029 nmol bilirubin/mg/hr., in untreated obese mice (n=6, p<0.05). Administration of SnMP to CoPP-treated mice resulted in significantly lower hepatic HO activity, 0.032± 0.005 nmol bilirubin/mg/hr., (n=6, P< .05) when compared to CoPP-treated obese mice. Obese mice weight gain was significantly (p<0.05) more compared to lean control (Figure 1C). Treatment with CoPP prevented body weight gain in obese mice resulting in a level that was comparable to age matched lean mice. SnMP, administered to CoPP treated mice, prevented weight loss and resulted in an increase in body weight in obese mice. Compared to lean animals, blood glucose was significantly (p<0.05) increased in obese mice. However, obese mice treated with CoPP showed significantly (p<0.05) lower glucose levels compared to obese control (Figure 1D). SnMP ablated the beneficial effects of CoPP on glucose levels (Figure 1D). Effect of HO Activity on Lipid Accumulation in Liver As shown in Figure 2A, obese mice have significantly (p<0.05) more lipid accumulation and larger lipid droplets (Figure 2B) in liver compared to age matched lean animals. Oil red O staining of liver from lean and obese mice showed that CoPP decreased both lipid accumulation and droplet size (Figure 2B). The decrease in lipid accumulation and lipid droplet size in mice treated with CoPP was reversed by co-administration of SnMP, Figure 2A, and 2B. SnMP abrogated the beneficial effects of CoPP on both oil red staining and lipid droplet size. Furthermore, obese mice have significantly (p<0.05) higher FAS expression compared to lean (Figure 2C). CoPP-reduced FAS expression to below that of both obese and lean mice. SnMP administration resulted in significantly (p<0.05) higher FAS expression compared to lean controls (Figure 2C). Effect of CoPP on glycogen content in liver In comparison to lean animals, glycogen content was significantly (p<0.05) decreased in the liver of obese mice (Figure 3A). CoPP administered to obese mice, resulted in significantly (p<0.05) higher glycogen levels in liver. SnMP significantly (p<0.05) lowered glycogen content compared to both lean and obese control animals (Figure 3A). Glycogen synthase 2 (GS2) mRNA expression was suppressed in obese mice and increased by CoPP (Figure 3C). SnMP significantly (p<0.05) reduced GS2 mRNA expression to levels below both lean and obese animals treated with CoPP (Figure 3C). Obese mice had reduced phosphorylation of AKT in liver when compared to lean animals (Figure 3B). CoPP restored the phosphorylation of AKT to levels comparable to lean control while SnMP reversed the beneficial effects of CoPP on AKT phosphorylation. Effect of CoPP on PPARα expression PPARα expression is decreased 54% (mean, 0.36 ± 0.04 n=5, p<0.05) in obese mice compared to lean control (Figure 4A). CoPP significantly (p<0.05) increased PPARα expression to levels that were comparable to lean control. In contrast SnMP significantly (p<0.05) decreased the perturbations in PPARα expression to levels lower than those of obese mice. In parallel to PPARα expression, phosphorylation of AMPK was significantly (p<0.05) decreased in obese mice as well as in mice treated with SnMP (Figure 4B). CoPP decreased heme levels, and restored AMPK phosphorylation in obese mice to levels comparable to those of lean control animals. The liver specific gene carnitine palmitoyltransferase 1A (CPT1A) was decreased 39% (0.58 ± 0.08 n=5, p<0.05) in obese mice when compared to age matched lean animals. Treatment of obese mice with CoPP resulted in a 487% increase in CPT1A expression (4.65 ± 0.76 n=5, p<0.05) over that of lean control. However, co-administration of SnMP to CoPP treated mice reversed the increase in the levels of CPT1A by 62.37% (0.35 ± 0.04 n=5, p<0.05) (Figure 4C). Effect of CoPP on FGF21 expression in liver We analyzed FGF21 expression in the liver of both lean and obese mice. In comparison to lean, obese mice displayed an increase of 340% (3.69 ± 0.70, p<0.05) in FGF21 mRNA expression (Figure 5A). CoPP increased FGF21 expression by 868% in obese (9.39 ± 0.61, p<0.05) when compared to lean animals and 255% greater than obese controls. SnMP prevented the CoPP-mediated increase in FGF21 expression resulting in levels similar to those of the obese control (Figure 5A). We examined the glucose lowering effect of increased HO activity to elucidate if it was the result of an increase in the levels of the epigenetic molecules of FGF21 signaling. Glut1 and PGC1α expression were decreased in obese mice, 53% (0.48 ± 0.06) and 39% (0.66 ± 0.08) respectively when compared to age-matched lean animals. CoPP increased Glut1 expression 570% (mean, 5.85 ± 0.99) and PGC1α 639.21% (mean, 6.94 ± 0.91) in obese mice when compared to lean controls. SnMP decreased Glut1 expression 82% (0.18 ± 0.02, p<0.05) compared to lean animals and 63% to obese controls; PGC1α expression was reduced to 86% (0.15 ± 0.01, p<0.05) when compared to lean controls and 77% of obese controls (Figures 5B and C). Discussion In this report, we demonstrate that increased HO-1 expression and HO activity lowers hepatic heme content, improves glycogen content and FGF21 expression and attenuates hepatic steatosis. In addition, the upregulation of HO-1 expression decreases both hepatic FAS levels and lipid droplet accumulation. Inhibition of HO-1 expression results in the opposite effect. These beneficial effects of HO-1 overexpression are reflected by a reduction the content of heme. PPARα and FGF21 tightly regulate hepatic lipid accumulation and glycogen content. HO activity was lower in untreated obese mice when compared with age matched controls. CoPP increased HO-1 protein and HO activity to levels significantly greater than those seen in age-matched vehicle treated lean animals. Several key findings in this report offer potential mechanisms by which increased HO-1 expression resulted in decreased levels of steatosis. Firstly, cellular heme content in obese mice is increased. Upregulation of HO-1 decreased heme content and increased FGF21 levels in obese mice. Others have shown that intracellular heme mimics the effect of PGC-1α on FGF21 recruitment, i.e. PGC-1α negatively regulates hepatic levels of FGF21 (2) and the heme modulating REV-ERb (alpha) axis. Heme, a ligand of the nuclear receptors REV-ERBα and B (14, 15), decreases FGF21 levels. It is noteworthy that heme levels increased during adipogenesis (14) while HO-1 protein levels and HO activity decreased (12) and that induction of HO-1 decreased adipogenesis, total fat and body weight gain in both obese mice and mice fed a high fat diet (5). Another potential mechanism by which HO-1 upregulation decreases fat content may be related to increased levels of CO and bilirubin which contribute to vascular cytoprotection (10, 22). Thus an increase in heme degradation resulted in increased antioxidant levels with a resultant increase in PPARα and FGF21 expression. PPARα knockout (KO) mice have steatosis (21, 23), due to decreased FGF21 expression. FGF21 is an important mediator of glycogen storage in liver, most likely through the FGF21 directed phosphorylation of AKT. An increase in HO activity in obese animals resulted in a reduction in oxidative stress, inflammation and lipid accumulation in bone marrow (12). Increases in HO-1 expression and HO activity decreased the mitochondrial release of ROS via an increase in superoxide dismutase, catalase and the mitochondrial signaling pathway (13). A decrease in both human and rodent HO-1 levels caused severe oxidative stress due a concomitant increase in cellular heme iron levels ((24, 25), (reviewed in (4)). Heme, a potent pro-oxidant (26, 27), is critical for the synthesis of NADPH oxidase and ROS (28, 29). Thus, a significant relationship exists between HO-1 expression and steatosis in obese mice. The present data confirm that glucose deprivation increases HO-1 protein levels which are reversed by the addition of glucose (30, 31). The suppressive effect of high glucose on HO-1 levels despite increased levels of ROS is well documented. Elevated glucose levels decrease HO-1 expression (13, 32), analogous to what is observed in humans and rodents with obesity (33). A decrease in HO-1 levels appears to magnify the heme-mediated activation of NADPH oxidase and O-2 generation in diabetes and in obesity (reviewed in (4)). Thus, HO-1 levels control the levels of bilirubin which have potent anti-inflammatory properties both in vitro and in vivo ((34), (reviewed in (4)). In humans, elevated bilirubin levels have been reported in vascular dysfunction and in patients with Gilbert's syndrome (35). In contrast, a decrease in human HO-1 levels and bilirubin results in elevated heme levels and premature aging (24, 25). Another potential mechanism by which CoPP-mediated upregulation of HO-1 and HO activity decreases hepatic lipids is through elevated levels of FGF21. A decrease in oxidative stress results in the sensitization of the FGF21 signaling cascade. Thus, FGF21 may be considered a therapeutic target for HO-1 in reducing NAFLD. In contrast, inhibition of HO activity decreased the expression of FGF21 target genes confirming previous reports suggest that obesity is a FGF21 resistant state (19). Thus increased heme levels, as a result of reduced HO-1 expression and HO activity, inhibits FGF21 signaling and reduces the attenuation of lipid accumulation. HO-1 has been implicated as a major regulator of several epigenetic signaling pathways by limiting the amount of heme and/or CO, which strongly binds to heme proteins CYP450, 4A10, 2E,1 prevents NAFLD fibrosis and decreases iNOS or COX-2 (36) resulting in the suppression of inflammatory cytokines (4). In contrast, the epigenetic signaling of FGF21 was increased by increased HO activity. Thus, activation of the HO-1-PPARα-FGF21 axis decreases hepatic lipid accumulation but increases hepatic glycogen content. The HO-1/PPARα activity relationship is an important mediator of blood glucose levels. Increased HO activity improved insulin and glucose sensitivity in Zucker diabetic fat (ZDF) rats (6). These actions reflect an increased phosphorylation of AMPK and AKT, and suggest the existence of a symbiotic relationship between HO and AMPK/AKT. Induction of the HO-1-PPARα axis reduced blood glucose, through increased phosphorylation of AMPK. AMPK plays an important role in lipid metabolism and adipogenesis (37). Phosphorylation of AMPK is elevated in the presence of increased PPARα activity (38). In agreement with increased PPARα expression, CPT1A expression was reduced in obese mice and greatly enhanced by the induction of HO-1, indicative that increased levels of HO-1 expression and HO activity are paralleled by an increase in PPARα activity. The HO modulated increase in PPARα expression is well documented (39). In summary, administration of CoPP to obese mice increased HO-1 levels and HO activity and decreased cellular heme levels. The reduction in hepatic heme content by administration of CoPP resulted in the recruitment of FGF21, PPARα and the abrogation of lipid accumulation in liver of obese mice. Activation of the FGF21 signaling cascade resulted in enhanced glycogen content and AMPK-AKT phosphorylation, as well as, reduced body weight and blood glucose levels. These results clearly imply that the activation of FGF21 level is dependent on HO-1 induction and the reduction in hepatic heme levels (diagrammed in Figure 6). These studies suggest a new functional role for the HO-1 mediated recruitment of PPARα-FGF21. This potential role, as a signaling paradigm, may prove crucial in the control of obesity and the metabolic syndrome related NAFLD, predicted to become the leading cause for liver transplantation in the next decade. This is both an economic burden and a major impact on quality of life. As the epidemic of obesity continues to loom, this problem will only increase. All authors had full access to the data and take responsibility for its integrity. All authors have read and agree with the manuscript as written. This work was supported by National Institutes of Health grants HL55601 and HL34300 (NGA) and gifts from the Renfield Foundation to The Rockefeller University (AK). Conflict Of Interest/Disclosure: The authors declared no conflict of interest. Figure 1 A) Western blot and densitometry analyses of liver HO-1 and actin; B) measurement of Heme levels in liver; C) body weight; and D) blood glucose in lean, obese control, obese treated with CoPP, and obese treated with CoPP and SnMP. Values represent means ± SEM of five independent treatments. , P < 0.05 vs. lean or #, P < 0.05 vs. obese control. Figure 2 A) Oil Red O staining of lipids in liver and quantitative analysis of lean (1), obese control (2), obese treated with CoPP (3), and obese treated with CoPP and SnMP(4), magnifications: 40× (n=3). A representative section for each group is shown; B) lipid droplet size from Oil Red O stained livers; and C) Western blot and densitometry analyses of liver fatty acid synthase (FAS) and actin in lean, obese control, obese treated with CoPP, and obese treated with CoPP and SnMP. Values represent means ± SEM of five independent treatments. , P < 0.05 vs. lean or #, P < 0.05 vs. obese control. Figure 3 A) Periodic acid Schiff staining of glycogen in liver of lean (1), obese control (2), obese treated with CoPP (3), and obese treated with CoPP and SnMP(4), magnifications: 40× (n=3). A representative section for each group is shown; B) Western blot and densitometry analyses of AKT phosphorylation (pAKT) and total AKT (AKT) (n=4); and C) Real-time PCR of glycogen synthase 2 (GS2) expression in lean, obese control, obese treated with CoPP, and obese treated with CoPP and SnMP. Values represent means ± SEM of five independent treatments. , P < 0.05 vs. lean or #, P < 0.05 vs. obese control. Figure 4 Western blot and densitometry analyses of A) PPARα and actin (n=4); and B) AMPK phosphorylation (pAMPK) and total AMPK (AMPK) (n=4) in lean, obese control, obese treated with CoPP, and obese treated with CoPP and SnMP. C) Real-time PCR of CPT1A expression (n=4-5) in lean, obese control, obese treated with CoPP, and obese treated with CoPP and SnMP. Values represent means ± SEM of five independent treatments. , P < 0.05 vs. lean or #, P < 0.05 vs. obese control. Figure 5 A) Real-time PCR of FGF21; B) Glut1; and C) PGC1α expression in lean, obese control, obese treated with CoPP, and obese treated with CoPP and SnMP. Values represent means ± SEM of five independent treatments. *, P < 0.05 vs. lean or #, P < 0.05 vs. obese control. Figure 6 Schematic diagram of potential mechanisms underlying HO-1-mediated improvement of hepatic steatosis in NAFLD. Fatty liver is accompanied by decreases in HO-1 protein and HO activity, increased heme content and derangement of cell signaling including the increase in PGC1α expression and the decrease in FGF21 levels. Upregulation of HO-1 protein and HO activity by pharmacological agents leads to an increased in heme degradation and the generation of CO and bilirubin. CO and/or bilirubin enhance antioxidant mechanisms, thereby decreasing lipid droplets along an elevation of PPARα expression. Induction of PPARα leads to an increase in FGF21. Increase in HO activity enhances the phosphorylation of AKT and AMPK, resulting in increased Glut1 expression and the lowering of blood glucose levels. Thus, activation of the HO-1 protein and HO- activity along with an increase in PPARα-FGF21 module results in higher glycogen content in the liver and a decrease of liver steatosis. What is already known about this subject Excessive heme causes an increase in ROS, lipid peroxidation and inflammatory heme dependent enzymes e.g. iNOS, COX-2-derived PGE2. Induction of HO-1 increases insulin sensitivity and reduces body fat content (visceral and subcutaneous fat). What this study adds Increased hepatic HO activity reduces PGC-1α, reverses FGF21 decrease and is associated with a reduction in blood glucose and body weight gain in obese mice. Increased HO activity decreases hepatic heme content and enhances glycogen levels. HO-1 induction reduced steatosis and adiposity. ==== Refs 1 Krahenbuhl L Lang C Ludes S Seiler C Schafer M Zimmermann A Reduced hepatic glycogen stores in patients with liver cirrhosis Liver Int 2003 23 101 109 12654132 2 Estall JL Ruas JL Choi CS Laznik D Badman M Maratos-Flier E PGC-1alpha negatively regulates hepatic FGF21 expression by modulating the heme/Rev-Erb(alpha) axis Proc Natl Acad Sci U S A 2009 106 22510 22515 20018698 3 Adams LA Waters OR Knuiman MW Elliott RR Olynyk JK NAFLD as a risk factor for the development of diabetes and the metabolic syndrome: an eleven-year follow-up study Am J Gastroenterol 2009 104 861 867 19293782 4 Abraham NG Kappas A Pharmacological and clinical aspects of heme oxygenase Pharmacol Rev 2008 60 79 127 18323402 5 Cao J Peterson SJ Sodhi K Vanella L Barbagallo I Rodella LF Heme oxygenase gene targeting to adipocytes attenuates adiposity and vascular dysfunction in mice fed a high-fat diet Hypertension 2012 60 467 475 22753217 6 Nicolai A Li M 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==== Front 04104626011NatureNatureNature0028-08361476-46872430504810.1038/nature12785ems55279ArticleHmga2 functions as a competing endogenous RNA to promote lung cancer progression Kumar Madhu S. 1Armenteros-Monterroso Elena 1East Philip 2Chakravorty Probir 2Matthews Nik 3Winslow Monte M. 4Downward Julian 151 Signal Transduction Laboratory, Cancer Research UK London Research Institute, 44 Lincoln’s Inn Fields, London WC2A 3LY, UK. 2 Bioinformatics and Biostatistics Group, Cancer Research UK London Research Institute, 44 Lincoln’s Inn Fields, London WC2A 3LY, UK. 3 Advanced Sequencing Facility, Cancer Research UK London Research Institute, 44 Lincoln’s Inn Fields, London WC2A 3LY, UK. 4 Department of Genetics, Department of Pathology, the Stanford Cancer Institute, Stanford University School of Medicine, Stanford CA 94305, USA. 5 Lung Cancer Group, Division of Cancer Biology, The Institute of Cancer Research, 237 Fulham Road, London SW3 6JB, UK. Correspondence and requests for materials should be addressed to J.D. ([email protected]).Author Contributions. M.S.K. and J.D. designed the study. M.S.K. and E.A.M. performed the experiments described. M.S.K., P.E., and P.C. conducted bioinformatics analyses. N.M. performed the next-generation sequencing studies. M.M.W. provided necessary reagents and conceptual advice. M.S.K. and J.D. wrote the manuscript. 21 10 2013 04 12 2013 9 1 2014 09 7 2014 505 7482 10.1038/nature12785Users may view, print, copy, download and text and data- mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: http://www.nature.com/authors/editorial_policies/license.html#termsNon-small cell lung cancer (NSCLC) is the most prevalent histological cancer subtype worldwide1. As the majority of patients present with invasive, metastatic disease2, it is vital to understand the basis for lung cancer progression. Hmga2 is highly expressed in metastatic lung adenocarcinoma where it contributes to cancer progression and metastasis3-6. Here we show that Hmga2 promotes lung cancer progression by operating as a competing endogenous RNA (ceRNA)7-11 for the let-7 microRNA (miRNA) family. Hmga2 can promote the transformation of lung cancer cells independent of protein-coding function but dependent upon the presence of let-7 sites; this occurs without changes in the levels of let-7 isoforms, suggesting that Hmga2 affects let-7 activity by altering miRNA targeting. These effects are further observed in vivo, where Hmga2 ceRNA activity drives lung cancer growth, invasion and dissemination. Integrated analysis of miRNA target prediction algorithms and metastatic lung cancer gene expression data reveals the TGF-β co-receptor Tgfbr312 as a putative target of Hmga2 ceRNA function. Tgfbr3 expression is regulated by the Hmga2 ceRNA via differential recruitment to Argonaute-2 (Ago2), and TGF-β signalling driven by Tgfbr3 is largely necessary for Hmga2 to promote lung cancer progression. Finally, analysis of NSCLC patient gene expression data reveals that HMGA2 and TGFBR3 are co-ordinately regulated in NSCLC patient material, a vital corollary to ceRNA function. Taken together, these results suggest that Hmga2 promotes lung carcinogenesis as both a protein-coding gene and a non-coding RNA; such dual-function regulation of gene expression networks reflects a novel means by which oncogenes promote disease progression. Cancer Research UK : A3570 \|\| CRUK_ ==== Body The competing endogenous RNA hypothesis posits that specific RNAs can function as sinks for pools of active miRNAs, functionally liberating other transcripts targeted by that set of miRNAs10. Downregulation of the transcription factor Nkx2.1 promotes lung adenocarcinoma progression partially via derepression of Hmga26, a non-histone chromosomal high mobility group protein. Intriguingly, Hmga2 has been described as a prototypic let-7 target transcript, with seven conserved sites in its 3′ untranslated region (3′ UTR)13. Reduction of Hmga2 by RNA interference, which would deplete both Hmga2 protein and transcript, greatly reduces metastatic ability. Thus, it is possible that the transcript could operate independent of the protein in lung cancer progression. To determine if Hmga2 can operate as a ceRNA for the let-7 family, we generated an allelic series of Hmga2 expression constructs (Fig. 1a). In this series, we expressed the wild-type full-length Hmga2 cDNA (wt); Hmga2 with mutation of all seven predicted let-7 binding sites13 (m7); Hmga2 with mutation of the single in-frame start codon (ATG wt); or Hmga2 with mutation of both the start codon and the let-7 binding sites (ATG m7). We then examined these constructs in two lung cancer cell lines generated from the KrasLSL-G12D;Trp53flox/flox mouse model: a cell line derived from a non-metastatic lung tumour which expresses very low levels of Hmga2 (368T1); and a cell line derived from a lymph node metastasis which expresses high levels of Hmga2 (482N1) 6. Using two antibodies that recognize either the N-terminus or the second AT-hook of the protein (M. Narita, personal communication), we found that the Hmga2 wt and m7 constructs efficiently express full-length Hmga2 protein (m7 over-expresses Hmga2 due to loss of let-7 mediated suppression) while the Hmga2 ATG wt and ATG m7 constructs do not (Fig. 1b). Importantly, we observe similar levels of Hmga2 transcript expressed in the allelic series (in the case of the 482N1 cell line, the allelic series was mutated to abrogate binding to a short hairpin RNA [shRNA] against Hmga2) (Fig. 1c). Moreover, expression of the allelic series has no effect on the expression of various let-7 family members (Extended Data Fig. 1a). Taken together, this Hmga2 allelic series allows us to compare specifically the roles of Hmga2 protein and transcript function on lung cell transformation. We therefore compared the ability of the Hmga2 allelic series to promote anchorage-independent growth of the lung cancer cell lines. We observed a striking promotion of soft agar growth by both Hmga2 wt and ATG wt in the 368T1 and 482N1 cells (Figs. 1d and e); more modest growth was observed with Hmga2 m7, despite elevated protein expression relative to Hmga2 wt, and no growth was provided by Hmga2 ATG m7. This effect can be observed further in two additional human lung cancer cells (H1299 and SK-MES-1), as suppression of soft agar growth by HMGA2 depletion can be rescued robustly by Hmga2 wt and ATG wt but more modestly by Hmga2 m7 (Extended Data Fig. 1b-e). Importantly, exogenous expression of let-7 reversed the ability of the Hmga2 ceRNA to promote anchorage-independent growth, suggesting that let-7 regulates this effect (Extended Data Fig. 2b). To demonstrate that the effect of the Hmga2 ceRNA is driven by let-7 sites in the 3′ untranslated region (3′UTR), we expressed only the wild type or let-7 site mutated 3′UTRs in 368T1 cells and examined the consequences on anchorage-independent growth. Strikingly, expression of the wild type but not let-7 mutant 3′UTR was sufficient to promote soft agar growth in 368T1 cells (Extended Data Fig. 2c). Beyond direct Hmga2 depletion, the Hmga2 wt and Hmga2 ATG wt constructs substantially rescued anchorage-independent growth in 482N1 cells stably over-expressing Nkx2.1, which we have previously shown to suppress lung cancer progression6 (Extended Data Fig. 2d). Notably, this effect is not due to a general proliferative benefit of Hmga2 wt and ATG wt cells, as BrdU incorporation in adherent conditions was comparable across the allelic series (Extended Data Fig. 3a). However, when lung cancer cells are placed in suspension, Hmga2 depletion suppressed proliferation and this proliferation was rescued substantially by Hmga2 wt and ATG wt and more marginally by Hmga2 m7 (Extended Data Fig. 3b); in contrast, the rate of apoptosis in the allelic series was not affected by growth in suspension (data not shown). Taken together, these results suggest that the Hmga2 transcript functions in a largely protein coding-independent but let-7 site-dependent manner to promote lung cancer cell transformation in vitro. To examine the effect of Hmga2 ceRNA activity on lung cancer cell dissemination in vivo, we intravenously transplanted 482N1 cells expressing either the control shRNA and expression construct or the shRNA targeting Hmga2 plus the Hmga2 allelic series into syngeneic mice. As seen in Figure 2a, micro-CT analysis revealed a substantial rescue of lung tumour formation with expression of either the Hmga2 wt or ATG wt constructs (with more modest effects with the Hmga2 m7 construct). Histopathological analysis and quantification of surface lesions confirmed the effects of Hmga2 ceRNA activity on in vivo lung tumourigenesis (Figs. 2b and c). Moreover, the control shRNA, the shHmga2 plus Hmga2 wt and the shHmga2 plus Hmga2 ATG wt transplants generate a highly metastatic disease, with lesions disseminating to both local and distant lymph nodes, kidney, and the abdominal and thoracic cavities (data not shown). We also examined the effect of Hmga2 ceRNA function on survival in the transplant system. We observed a dramatic reduction in survival in animals transplanted with the Hmga2 wt and ATG wt cells, similar to that seen with transplant of the control shRNA cells (Fig. 2d); we noted a more modest reduction in survival with transplant of the Hmga2 m7 cells. In total, these findings indicate that Hmga2 competing RNA activity dramatically promotes in vivo lung cancer formation. To elucidate the mechanism of Hmga2 ceRNA function on lung carcinogenesis, we analysed the set of genes differentially expressed between metastatic and non-metastatic KrasG12D;Trp53flox/floxlung cancer cells6 and compared them to the list of predicted let-7 target genes based upon the miRNA target prediction algorithm TargetScan14 (Supplementary Table 5). Kras was not a candidate in this analysis, in spite of previous description of Kras as an important let-7 target15. Moreover, the Hmga2 allelic series had no impact on either expression of K-Ras protein or activity of downstream K-Ras signalling pathways (Extended Data Fig. 3c). In contrast, we observed several known Hmga2 transcriptional targets, including components of the Igf2bp family16, validating this approach. To elucidate more broadly which transcripts are Hmga2 ceRNA targets, we initially examined whether let-7 sites are enriched among transcripts induced by the Hmga2 ceRNA through RNA-seq of the 482N1 allelic series combined with Sylamer analysis, which detects miRNA seed sites as nucleotide strings enriched within the 3′UTRs of transcripts17. We first compared control to Hmga2 knockdown cells and observed a specific enrichment of let-7 sites lost with Hmga2 depletion (Extended Data Fig. 4a). We then determined if this was specific to ceRNA activity by determining let-7 site enrichment upon re-expression of either the Hmga2 wt or ATG wt constructs in Hmga2 knockdown cells. In both conditions, let-7 sites were enriched among the up-regulated transcripts (Extended Data Fig. 4b and c) Importantly, let-7 sites were not enriched with re-expression of either Hmga2 m7 or ATG m7 in the Hmga2-depleted background (Extended Data Fig. 4d and e). Moreover, analysis of fragments per kilobase of exon per million fragments mapped (FPKM) in the RNA-seq results from control 482N1 cells showed Hmga2 was among the most highly expressed predicted let-7 target transcripts, suggesting that Hmga2 constitutes a physiologically germane fraction of the let-7 target milieu (Supplementary Table 6). Taken together, these results indicate that the Hmga2 ceRNA broadly regulates let-7 targets. To more specifically assess Hmga2 ceRNA targets, we examined which transcripts were suppressed in response to Hmga2 depletion; 13 out of 34 predicted targets were suppressed by Hmga2 knockdown (Extended Data Fig. 5a). To delineate which of these were Hmga2 transcriptional targets versus ceRNA targets, we re-expressed either Hmga2 wt or ATG wt in knockdown cells. As seen in Extended Data Figure 5b, 6 out of 13 transcripts were rescued by both Hmga2 wt and ATG wt, suggesting they are putative ceRNA targets; the remaining targets were rescued only by Hmga2 wt, suggesting they are targets of Hmga2 transcription factor function. These Hmga2 ceRNA targets were strikingly enriched in let-7 regulated transcripts, as their repression by Hmga2 loss could be reversed with the use of a ‘tough decoy’ let-7 sponge transcript, designed to be an efficient and long-term suppressor of miRNA function (Extended Data Fig. 5c)18. Conversely, over-expression of let-7 suppressed these transcripts, although Hmga2 transcriptional targets were also affected due to depletion of Hmga2 (Extended Data Fig. 5d). Taken together, these studies outline a collection of putative target transcripts regulated by Hmga2 ceRNA function. Among these six Hmga2 ceRNA target transcripts, we found the TGF-β co-receptor Tgfbr312 to be both up-regulated in metastatic lung cancer cells and a putative let-7 target. Furthermore, several Hmga2 ceRNA targets have been described as targets of TGF-β signalling19. Thus, we examined whether Hmga2 exerts ceRNA function through enhanced TGF-β signalling via Tgfbr3. In line with this, we found that in both 368T1 and 482N1 cells, Hmga2 wt and ATG wt promote the expression of Tgfbr3 protein (Fig. 3a). This Tgfbr3 up-regulation also occurs to a lesser degree at the mRNA level, as has been described previously for miRNA targets20 (Fig. 3b). Moreover, exogenous expression of let-7 reversed the ability of the Hmga2 ceRNA to up-regulate Tgfbr3, suggesting this effect is controlled by let-7 (Extended Data Fig. 2a). An important consideration in ceRNA/target analysis is the absolute levels of Hmga2, Tgfbr3, and let-7 transcripts in cells, so we determined the copies per cell of these factors (Extended Data Fig. 4f). We observed that Hmga2 and Tgfbr3 had similar levels of transcript, as one might expect for two factors that can titrate expression of one another; similar results were observed in FPKM analysis of these transcripts in control 482N1 cells by RNA-seq (Supplementary Table 6). Furthermore, total let-7 family expression was within an order of magnitude of Hmga2 and Tgfbr3. As this pool of let-7 regulates the entire target set, one could expect miRNA occupancy to be a limiting factor, allowing for the contribution of a ceRNA-like Hmga2. Taken together, these results suggest that Hmga2 could regulate Tgfbr3 expression as a let-7 ceRNA. In line with these observations of Hmga2 promoting Tgfbr3 expression, Hmga2 wt and ATG wt activated TGF-β signalling via phosphorylation of Smad2 (Fig. 3a). This effect was let-7 dependent, as exogenous let-7 suppressed Smad2 phosphorylation (Extended Data Fig. 2a). That the TGF-β pathway is active in the absence of exogenous ligand is likely due to low but detectable levels of TGF-β in serum during cell culture21. We further examined whether Hmga2 ceRNA function affects TGF-β pathway activation by two methods. First, we found that a TGF-β reporter (CAGA12)22 was potently induced by Hmga2 wt and ATG wt (Extended Data Fig. 5e). Second, analysis of TGF-β target transcript levels revealed specific expression of these genes with the Hmga2 wt and ATG wt constructs (Extended Data Fig. 5f). Notably, we observed little activation of the TGF-β pathway by Hmga2 m7, in spite of previous reports of Hmga2 functioning as a Smad2/3/4 co-activator in the epithelial-mesenchymal transition (EMT)23; this is likely due to the lack of upstream activation of the pathway. In line with this, the Hmga2 ceRNA does not induce an EMT in either 368T1 or 482N1 cells (Extended Data Fig. 6a). Overall, these results indicate that the Hmga2 ceRNA induces expression of Tgfbr3 and potentiates TGF-β signalling. To determine whether the effects of Hmga2 on Tgfbr3 occur via let-7 mediated de-repression, we first examined the effect of Hmga2 ceRNA function on a reporter containing the Tgfbr3 3′ UTR. In both 368T1 and 482N1 cells, Hmga2 wt and ATG wt induced expression of luciferase under the control of the wild type Tgfbr3 3′ UTR, but not if the let-7 site was mutated (Fig. 3c). Furthermore, we found that the effect of the Hmga2 ceRNA was broadly miRNA dependent, as Hmga2 wt and ATG wt induced the reporter expression in Dicer1-intact sarcoma cells, but not in a Dicer1-null derivative cell line24 (Extended Data Fig. 6b). To assess directly whether Hmga2 induces Tgfbr3 via competition away from Ago2, we performed RNA immunoprecipitation (RIP) on Ago2 in lung cancer cells expressing the Hmga2 allelic series. As seen in Figure 3d, we found that Hmga2 wt and ATG wt were as comparably recruited to Ago2 as endogenous Hmga2 while Hmga2 m7 and ATG m7 were not. Moreover, Hmga2 wt and ATG wt cells had a substantial decrease in Tgfbr3 recruitment to Ago2. Of note, these effects on Ago2 occupancy by Tgfbr3 are not caused by a change in let-7 activity, as various let-7 family members were comparably loaded on Ago2 across the Hmga2 allelic series (Extended Data Fig. 7). Thus, these results demonstrate Hmga2, via its let-7 binding sites, displaces Tgfbr3 from miRNA-mediated repression by RISC. In total, these results suggest the Hmga2 ceRNA directly functions by blocking recruitment of Tgfbr3 to the Ago2-based miRNA repression complex. To examine whether Hmga2 ceRNA activity through Tgfbr3 is functionally relevant, we used shRNAs to deplete Tgfbr3 in 482N1 cells and 368T1 cells expressing either Hmga2 wt or ATG wt. At both the mRNA and protein level, multiple shRNAs reduced Tgfbr3 expression (Figs. 3e and f). Moreover, knockdown of Tgfbr3 led to substantial suppression of TGF-β signalling, as evinced by loss of Smad2 phosphorylation, CAGA12 reporter activity and expression of TGF-β target genes (Fig. 3f and Extended Data Fig. 8a and b). We then assessed the functional effect of Tgfbr3 loss on Hmga2 ceRNA-driven soft agar colony formation. In all cells, Tgfbr3 knockdown strikingly suppressed anchorage-independent growth, though not to the same extent as Hmga2 depletion in 482N1 cells (Fig. 3g). This occurred without generally affecting proliferation, as measured by BrdU incorporation (Extended Data Fig. 8c). We further functionally analysed the broader set of six Hmga2 ceRNA targets by individually depleting them by siRNA (Extended Data Fig. 8d). When we compared their effects on anchorage-independent growth, both Hmga2 and Tgfbr3 loss strongly suppressed growth, Hmga1 depletion modestly reduced colony formation, while the remaining targets had little effect (Extended Data Fig. 8e). It should be noted that these other targets include extracellular factors like Angptl2 and Col1a2 that might promote lung cancer progression in vivo in a non-cell autonomous manner, and could thus still be relevant to Hmga2 ceRNA activity in lung cancer progression. This considered, these results still indicate that Tgfbr3, though certainly not the only relevant Hmga2 ceRNA target, is an important effector of Hmga2 ceRNA function in lung cancer cell transformation. To determine if this effect of Tgfbr3 is driven through potentiation of TGF-β signalling, we inhibited the TGF-β pathway with the TGF-β receptor kinase inhibitor SB-431542 (SB)25. In 368T1 and 482N1 cells, SB treatment led to a substantial inhibition of Smad2 phosphorylation (Extended Data Fig. 9a). In addition, SB treatment of 482N1 and 368T1 Hmga2 wt and ATG wt cells markedly suppressed CAGA12 reporter activity and expression of TGF-β target genes (Extended Data Fig. 9b and c). We then examined whether SB could inhibit Hmga2 ceRNA driven soft agar colony formation and observed a striking reduction in anchorage-independent growth (Extended Data Fig. 9d). This impaired colony formation was not due to general proliferative arrest, as SB treated cells had a similar rate of BrdU incorporation (Extended Data Fig. 9e). Strikingly, many Hmga2 ceRNA targets are in fact TGF-β target genes, as their expression is suppressed by SB treatment and induced by exogenous addition of TGF-β (Extended Data Fig. 9f and g). Thus, it is possible that Hmga2 could function in a feed forward loop in which it regulates TGF-β target genes directly through ceRNA function and indirectly through TGF-β signalling via Tgfbr3. In sum, these results indicate that TGF-β signalling via Tgfbr3 is an important pathway downstream of Hmga2 ceRNA function. Based upon the above findings, we wanted to explore if HMGA2 functions as a ceRNA for TGFBR3 (the human orthologues of Hmga2 and Tgfbr3, respectively) in NSCLC patients. An important corollary of the ceRNA hypothesis is the coordinate regulation of a competing RNA and its targets, such that up-regulation of the ceRNA should lead to higher expression of the target RNA and vice versa10. To assess this, we used NSCLC gene expression data generated by the Cancer Genome Atlas (TCGA) and sorted the patient cohort into the top and bottom quartiles of HMGA2 expression (HMGA2 high and low, respectively) (Fig. 4a). As seen in Figure 4b, we observed significantly higher levels of TGFBR3 transcript in HMGA2 high versus low patient samples. To address the converse relationship, we sorted the TCGA data set into top and bottom quartiles of TGFBR3 expression (TGFBR3 high and low, respectively) (Fig. 4c). When we compared HMGA2 transcript levels between the groups, we found HMGA2 to be significantly over-expressed in TGFBR3 high versus low patient samples (Fig. 4d). To extend and validate these findings, we performed similar gene expression analyses of HMGA2 and TGFBR3 in an independent lung adenocarcinoma patient gene expression cohort, the Director’s Challenge data set26. Similar to the findings with the TCGA cohort, we observed HMGA2 and TGFBR3 to be co-ordinately expressed in the Director’s Challenge data set (Extended Data Fig. 10a-d). While we focused specifically on high and low expressors of HMGA2 and TGFBR3, where ceRNA activity is more likely to occur10, HMGA2 and TGBR3 expression was broadly correlated across both data sets (Extended Data Fig. 10e and f). As these two data sets constitute two of the largest collections of NSCLC gene expression data available, we believe these findings are in line with HMGA2 functioning as a ceRNA for TGFBR3 in NSCLC patients. It is possible that the co-expression of HMGA2 and TGFBR3 could correspond to additional tumour characteristics these data sets do not include; future studies in independent data sets would be needed to assess this issue. In total, our results suggest a model by which Hmga2 promotes lung cancer progression by competing for let-7 occupancy with other targets, including Tgfbr3, leading to the up-regulation of those targets (Fig. 4e). Importantly, this occurs without changes in the levels of let-7 family microRNAs, reflecting specific competition for microRNA binding among targets. Here we have outlined a novel gene expression pathway in which a protein-coding gene, Hmga2, operates largely independently of its protein-coding function to promote lung cancer progression as a competing endogenous RNA. While much of this ceRNA activity is driven by over-expression of TGF-β signalling via Tgfbr3, there are likely additional Hmga2 ceRNA targets to be found in future studies. Moreover, HMGA2 is over-expressed in many other cancer types27, making it possible that HMGA2 functions as a ceRNA in cancer sites beyond lung. Taken more broadly, these findings raise the possibility that many protein-coding genes differentially expressed in cancer might contribute to tumourigenesis through this distinct mode of regulatory gene expression. Moreover, these results raise issues with the validation of candidates in RNA interference screens. The “gold standard” assay for validating an siRNA target is expression of an siRNA-resistant form of the coding sequence28; however, such an approach overlooks the possibility that depletion of both the full-length RNA and protein might contribute to a given phenotype, requiring complementation by the full-length transcript. Such dual-function ceRNA/protein activities necessitate a deeper exploration of the coding genome in biological systems. Online-only Methods Cell lines 368T1 and 482N1 cells were generated previously6. KPD F/- and −/− sarcoma cells were a kind gift of Phillip A. Sharp24. Cancer Research UK Cell Services provided HEK293, Phoenix ampho, NCI-H1299, and SK-MES-1 cells. All cell lines were cultured in DMEM medium with 10% FBS, 10 mM glutamine and 1% penicillin and streptomycin at 37°C in a 5% CO2/95% air incubator. Drug and reagent treatment For Western, luciferase, qRT-PCR, and BrdU analysis, 368T1 cells were co-treated with 2 ng/mL TGF-β and either DMSO or 2.5 μM SB-431542 (“SB”) and 482N1 cells were treated with either DMSO or 2.5 μM SB-431542 for 24 hours. For soft agar analysis, cells were fed with media containing either DMSO or SB. For Western analyses, 368T1 and 482N1 cells were plated (2.5 × 105 cells) into 6-well plates overnight. Cells were then treated with DMSO, 1 μM AZD6244 or 1 μM PIK-75 for two hours and lysates were prepared. Lentivirus and retrovirus production Short hairpin RNA (shRNA) lentiviruses (which are listed in Supplementary Table 1) were generated by co-transfection of HEK293 cells with shRNA vector and packaging vectors pCMV-VSVG and pCMV-8.2. MSCV-based retroviruses were generated by transfection of Phoenix ampho cells with retroviral vectors. 48 and 72 hours after transfection, virus particles in the medium were harvested, filtered, and transduced onto cells. Stable cell line generation Expression vectors were initially linearized overnight using BglII. Linearised DNA was then transfected into cells in 24-well plates, with media replaced six hours later. Cells were then replated into 6-well plates in the presence of 500 μg/mL G418 for selection over two weeks. For lentiviral and retroviral infection, cells were infected in 6-well plates and subsequently split into 10 cm dishes in the presence of 2.5 μg/mL puromycin for selection over 72 hours. Western blotting Western blotting was performed by standard methods. Antibody details are provided in Supplementary Table 2. qRT-PCR RNA was isolated using RNA-Bee using manufacturers’ instructions. Both standard and miRNA-specific qRT-PCR was performed using QuantiTect SYBR Green primers as per manufacturers’ instructions (QIAGEN). Primer details are provided in Supplementary Table 3. Luciferase assays CAGA12 Cells were seeded into 96-well plates in triplicate and co-transfected with pRL-TK (Promega) and either control pGL3 or the TGF-β reporter pCAGA12-GL322. 24 hours later, luciferase activities were determined using the Dual-Luciferase Assay System as per manufacturers’ instructions (Promega). Tgfbr3 3′ UTR Cells were seeded into 96-well plates in triplicate and co-transfected with pGL3 (Promega) and either the control pRL-CXCR430 or the wild type/mutant fragment of the Tgfbr3 3′ UTR. Luciferase activities were determined as above. BrdU analysis Cells were plated in triplicate into six-well plates overnight (2.5 × 105 cells per well). They were then treated with 10 μM BrdU for one hour, trypsinized, washed in PBS and stained for BrdU/DAPI analysis using standard methods by the Cancer Research UK Flow Cytometry Facility. siRNA/miRNA transfection Cells were transfected with siRNA/miRNA as described previously29. Cells were subsequently plated into soft agar assays as above or harvested for RNA/protein as above. polyHEMA plating analysis 6-well plates were either mock coated or coated with (polyHEMA) as previously described31. Cells were subsequently plated into these 6-wells overnight and BrdU analysis was performed as described above. Histopathological analysis After sacrifice by CO2 asphyxiation, lungs were collected from animals and fixed overnight in 10% formalin. They were subsequently placed in 70% ethanol and submitted for embedding, sectioning, and hematoxylin and eosin (H&E) staining. In vitro transcription TOPO cloned fragments of Hmga2 and Tgfbr3 were in vitro transcribed using the SP6 MAXIscript kit as per manufacturers’ instructions (Ambion). RNA was subsequently purified using the RNEasy cleanup kit as per manufacturers’ instructios (Qiagen). Transcript copy number analysis For determination of transcript copy number, RNA was prepared from 2 × 106 482N1 cells using RNA Bee as described above. qRT-PCR was performed for Hmga2, Tgfbr3, and let-7a/c/e/f/i as described above. CT values were compared to a two-fold dilution series of either in vitro transcribed Hmga2/Tgfbr3 or synthetic let-7a/c/e/f/i miRNA mimics (Qiagen). Moles of transcript and copies of transcript per cell were then calculated using standard stoichiometric methods. Confidence intervals were calculated using a t-statistic. RNA library production and next-generation sequencing (RNA-seq) Total RNA was generated from the 482N1 Hmga2 allelic series (with biological triplicates) as described above. The Total RNA samples were initially quality controlled (QC) using the 6000 Nano RNA Chip on the BioAnalyser 2100 (Agilent, Santa Clara, CA, USA) to ensure RNA integrity and estimate concentration before starting the procedure. If the samples passed the initial QC the Total RNA samples were subjected to poly-A selection using Sera-Mag oligo (dT) beads (Thermo Fisher Scientific Inc.), and the bound poly-A RNA species utilized for downstream library prep using the Illumina mRNA kit RS-122-2101 (TruSeq Stranded mRNA Sample Prep. Illumina San Diego, CA, USA). The standard PCR cycles suggested in the protocol were also altered to match the concentration of the total RNA from the initial QC. After production of the mRNA libraries a final QC was performed on a DNA 1000 chip using the BioAnalyser 2100 (Agilent, Santa Clara, CA, USA). If the mRNA libraries passed the QC they were ready for flow cell cluster formation on a cBot and then 100 b.p. Paired End (PE) sequencing by synthesis on the HiSeq 2500 was performed. RNA-seq data processing and analyses We aligned 100 b.p. paired-end sequencing reads to the mouse genome (mm9, UCSC) using Tophat2/Bowtie232 allowing for 5 mismatches, a mate-inner-distribution of −40 and a mate-standard-deviation of 50. These library fragment metrics were derived from an alignment of 5e5 sampled reads to the mouse transcriptome. We identified read-pair mappings to gene structures derived from RefSeq (RefGene table, UCSC) using the summarizeOverlaps function with mode IntersectStrict (GenomicRanges, Bioconductor). This generated a mean fragments per sample value of 33.5e6. Using these raw counts we identified genes expressed across the sample groups. We removed genes with a count <= 10 across all samples prior to statistical analysis. The differential analysis was carried out using edgeR33 applying TMM library normalisation and a 0.0005 false discovery rate (fdr) to select expressed transcripts. In addition, we calculated gene level fragments per kilobase of exon per million fragments mapped (FPKM) values for the same gene set using Cufflinks34. Sylamer analysis A list of Ensembl gene IDs was generated from the set of transcripts expressed in the samples described above. This set of transcripts was then placed in rank order by their differential expression between two groups and analysed using the Sylamer algorithm via the SylArray platform17. A Sylamer plot was then generated for all 8mer miRNA strings whose target sites were enriched significantly (p<0.001 by hypergeometric testing). Statistical testing Unless otherwise specified, statistical significance was assessed by the Student’s t-test. Vector cloning Hmga2 pCDNA3.1-Hmga2 wt and Hmga2 m7 (“wt” and “m7”) were acquired from Addgene (Plasmid nos. 14789 and 14792)13. In order to generate pCDNA3.1-Hmga2 ATG wt and pCDNA3.1-Hmga2 ATG m7 (“ATG wt” and “ATG m7”), the wt and m7 constructs were mutated by site-directed mutagenesis as per manufacturers’ instructions (Stratagene) using primers 5′-GGTAGCGGCGGCGGGAGGCAGGCTGAGCGCACGCGGTGAGG-3′ and 5′-CCTCACCGCGTGCGCTCAGCCTGCCTCCCGCCGCCGCTACC-3′. For generation of 482N1 shHmga2 stable cell lines, “wt”, “m7”, “ATG wt” and “ATG m7” the shRNA binding site was mutated using primers 5′-CCATTTCTGCAAGCTAAGTATGTTTGCAGGAGCCCCGGCTCCGGACGCGTAACTG CATCCAACTTTCTCC-3′ and 5′-GGAGAAAGTTGGATGCAGTTACGCGTCCGGAGCCGGGGCTCCTGCAAACATACT TAGCTTGCAGAAATGG-3′. pRL-Tgfbr3 A fragment containing the Tgfbr3 3′UTR was PCR amplified from 482N1 cDNA using primers 5′-TAAGAACTCGAGCTGCGTGTGTTCTCCGCAG-3′ and 5′-TAACAAGCGGCCGCCTGTCAGTTTAATGAACGAACC-3′. Both the PCR product and pRL-CXCR4 were digested with XhoI and NotI and ligated together to generate pRL-Tgfbr3. The let-7 mutant was generated by site-directed mutagenesis as above using primers 5′-GCAGGCGCGTGCCTGTCTTTTTGTACTGTAACGGGCTCATGGTTTGAATGATGAG CGTACTGCTGGTTG-3′ and 5′-CAACCAGCAGTACGCTCATCATTCAAACCATGAGCCCGTTACAGTACAAAAAGA CAGGCACGCGCCTGC-3′. Hmga2-TOPO/Tgfbr3-TOPO A fragment containing the Hmga2 cDNA sequence recognized by the respective qRT-PCR primers was PCR amplified from 482N1 cDNA using primers 5′-CTACATCCCGTCTCCCGAAAGGTGCTGG-3′ and 5′-GGATCCGGTAGAAATTGAATGTCGGCGCCCCCTAATC-3′. A fragment containing the Tgfbr3 cDNA sequence recognized by the respective qRT-PCR primers was PCR amplified from 482N1 cDNA using primers 5′-CTCGAGCAGAAGAAGTGCAAGGGGGCGTGAATATCG-3′ and 5′-CCATGTTGAAGGTAGCATTTCCATCGAGCTGGTCCTGGAAG-3′. PCR products were subsequently TOPO cloned as per manufacturers’ instructions (Invitrogen) and used as substrates for in vitro transcription. Hmga2 wt/m7 3′UTR A fragment containing the Hmga2 3′UTR was PCR amplified from the Hmga2 wt or Hmga2 m7 pcDNA3.1 constructs used for transfection. Products were amplified using primers 5′-GCAGAATTCGGGGCGCCGACATTCAATTTC-3′ and 5′-TAAGCGGCCGCGCCCACAGAGGCTGTTATGTTTATTG-3′. PCR products were subsequently TOPO cloned as per manufacturers’ instructions (Invitrogen). TOPO clone inserts and pcDNA3.1-Hmga2 wt were EcoRI digested and ligated together to generate pcDNA3.1-Hmga2 wt 3′UTR and pcDNA3.1-Hmga2 m7 3′UTR. Supplementary Material Supplementary Tables 1-6 Acknowledgements We thank the laboratory of T. Jacks for providing the 482N1 and 368T1 lung cancer cell lines and the laboratories of P.A. Sharp and P. Chambon for providing the KPD sarcoma cell lines. We thank the LRI FACS Facility for the BrdU analyses and the LRI Biological Resources Unit for assistance with the animal studies. We thank M.S. Ebert and E. De Bruin for critical review of the manuscript. M.S.K. is a Long-Term Fellow of the Human Frontier Science Program and the European Molecular Biology Organization. This work was funded by Cancer Research UK and by the European Commission’s Seventh Framework Programme (FP7/2007-2013) under the grant agreement Lungtarget (project n° 259770). Author Information: Reprints and permissions information is available at www.nature.com/reprints. The authors declare no competing financial interests. Figure 1 Hmga2 promotes lung cancer cell transformation in a protein-coding independent but let-7 site dependent manner a, Diagram of Hmga2 allelic series: expression constructs containing the entire Hmga2 cDNA (“wt”); the cDNA with all seven let-7 sites in the 3′ UTR mutated (“m7”); the cDNA with the start codon mutated (“ATG wt”); and the cDNA with both the start codon and let-7 sites mutated (“ATG m7”). b, Hmga2 wt and m7 induce Hmga2 expression in non-metastatic lung cancer cells (368T1) and restores expression cells in metastatic lung cancer cells (482N1) depleted for endogenous Hmga2 (shHmga2). Two distinct HMGA2 antibodies are used: one recognizes the N-terminus of HMGA2 (HMGA2-CST) and the other recognizes the central AT-hook region of HMGA2 (HMGA2-Narita). c, Hmga2 RNA is comparably expressed by the wt, m7, ATG wt, and ATG m7 in both 368T1 and 482N1 cells. Hmga2 expression is normalized to Gapdh. 368T1 values are normalized to empty and 482N1 values are normalized to shluc empty. Values are technical triplicates, have been performed independently three times, and represent mean +/− standard deviation (s.d.) with propagated error. d, Hmga2 wt and ATG wt promote substantial anchorage-independent growth in both 368T1 and 482N1 cells. Values are technical triplicates, have been performed independently three times, and represent mean +/− s.d. e, Representative images of soft agar colonies. Magnification is 10X. *: p<0.0005; : p<0.005; : p<0.05. Figure 2 Hmga2 ceRNA activity enhances lung cancer progression in vivo a, Hmga2 wt and ATG wt restore lung tumour growth in response to endogenous Hmga2 knockdown. B6129SF1/Tac males were intravenously injected with 482N1 cells expressing either a control shRNA and empty vector (shluc empty) or shHmga2 with the Hmga2 allelic series. Three weeks afterwards, animals were scanned by micro-CT and representative transverse images are shown. The heart is demarcated (labelled ‘H’) and white arrows identify lung tumours. b, Representative histological images of lungs transplanted with 482N1 cells from the series described in a. Magnification is 1X. c, Lung surface tumour counts were taken from animals transplanted with 482N1 cells from the series described in a (n=3 animals per group). Values are technical triplicates represent mean +/− s.e.m. d, Hmga2 wt and ATG wt substantially reduce survival of animals transplanted with 482N1 cells expressing the shRNA targeting Hmga2. Animals were intravenously transplanted with cells as in a. Animals were subsequently aged for survival and a Kaplan-Meier analysis was performed (n=9 animals per group). Median survival was 34 days for shluc empty/shHmga2 wt transplants; 37 days for shHmga2 ATG wt transplants; 43 days for shHmga2 m7 transplants; and 50 days for shHmga2 empty/ATG m7 transplants. Statistical significance was assessed by log-rank tests compared to shHmga2 empty. *: p<0.00005; : p<0.0005; : p<0.005; : p<0.05; n.s.: not significant. Figure 3 Hmga2 ceRNA activity enhances TGF-β signalling through over-expression of Tgfbr3 a, Hmga2 wt and ATG wt substantially induce both Tgfbr3 protein expression and phosphorylation of Smad2 (pSmad2) in both 368T1 and 482N1 cells. b, Hmga2 wt and ATG wt significantly promote expression of Tgfbr3 mRNA in both 368T1 and 482N1 cells. Tgfbr3 expression is normalized to Gapdh. 368T1 values are normalized to empty and 482N1 values are normalized to shluc empty. Values are technical triplicates, have been performed independently three times, and represent mean +/− s.d. with propagated error. c, Hmga2 wt and ATG wt specifically induce expression of a luciferase Tgfbr3 3′ UTR reporter in a let-7 site-dependent manner in both 368T1 and 482N1 cells. Cells were transfected with Renilla constructs of the control siCXCR4 multimer30 and either the Tgfbr3 wild type or let-7 mutant 3′ UTR reporter. Values are normalized to co-transfected pGL3 plasmid. 368T1 values are normalized to empty and 482N1 values are normalized to shluc empty. Values are technical triplicates, have been performed independently three times, and represent mean +/− s.d. with propagated error. d, Hmga2 wt and ATG wt displace Tgfbr3 from Argonaute-2 (Ago2) based RNA-induced silencing complexes. Lysates from 368T1 and 482N1 cells of the Hmga2 allelic series underwent either control immunoprecipitation (IgG) or immunoprecipitation for Ago2. RNA was purified and qRT-PCR was performed for Hmga2 and Tgfbr3 on both the immunoprecipitated and input RNAs. Values are depicted as the percentage of input RNA, are technical triplicates, have been performed independently twice, and represent mean +/− s.d. e, multiple shRNAs elicit substantial knockdown of Tgfbr3 mRNA in both 368T1 and 482N1 cells. 482N1 cells were infected with control shRNA (shluc) or a set of shRNAs targeting Tgfbr3 (shTgfbr3.1-3.5), while 368T1 wt and ATG wt cells were infected with shluc or shTgfbr3.1, 3.2, 3.4, and 3.5. RNA was purified and qRT-PCR was performed. Tgfbr3 expression is normalized to Gapdh and 368T1 wt and ATG wt and 482N1 values are normalized to shluc. Values are technical triplicates, have been performed independently three times, and represent mean +/− s.d. with propagated error. f, multiple shRNAs induce knockdown of Tgbr3 and suppress TGF-β pathway activity in 368T1 and 482N1 cells. Cells were infected with shRNAs as in e and Western analysis was performed for Tgfbr3, pSmad2 and total Smad2 (Smad2). g, Tgfbr3 depletion reduces anchorage-independent growth of 368T1 wt and ATG wt and 482N1 cells. Cells were infected with the listed shRNAs and plated for anchorage-independent growth and colonies were counted as above. Values are technical triplicates, have been performed independently three times, and represent mean +/− s.d. *: p<0.0005; : p<0.005; : p<0.05. Figure 4 HMGA2 and TGFBR3 are reciprocally and co-ordinately upregulated in NSCLC patients a, The Cancer Genome Atlas (TCGA) NSCLC gene expression data set was sorted on HMGA2 expression. The top and bottom quartiles (HMGA2 low and high, respectively) were selected (45 patients per group) and HMGA2 expression was compared by box and whisker plot. b, The TCGA data set was sorted into top and bottom quartiles of HMGA2 expression as in a, and TGFBR3 expression was compared by box and whisker plot. c, The TCGA data set was sorted into top and bottom quartiles of TGFBR3 expression (TGFBR3 low and high, respectively) as in a, and TGFBR3 expression was compared by box and whisker plot. d, The TCGA data set was sorted into top and bottom quartiles of TGFBR3 expression as in c, and HMGA2 expression was compared by box and whisker plot. In all box and whisker plots, values are presented on a log2 scale. Significance was assessed by the Mann-Whitney test with a Bonferroni correction for multiple hypothesis testing. **: p<0.0005. e, Model for Hmga2 acting as a competing endogenous RNA for Tgfbr3. In nonmetastatic NSCLC, Hmga2 expression is low, leading to suppressed Tgfbr3 expression by let-7. In metastatic NSCLC, Hmga2 expression is elevated, titrating away let-7 from Tgfbr3 and allowing for its over-expression. 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==== Front AIDS CareAIDS CarecaicAIDS Care0954-01211360-0451Taylor & Francis 10.1080/09540121.2013.819404Research ArticleComplex care needs of patients with late-stage HIV disease: A retrospective study Halman Mark abcCarusone Soo Chan deStranks Sarah afSchaefer-McDaniel Nicole dStewart Ann cda Department of Psychiatry, St Michael's Hospital, Toronto, Canadab Centre for Research on Inner City Health, The Keenan Research Centre in the Li Ka Shing Knowledge Institute of St. Michael's Hospital, Toronto, Canadac Faculty of Medicine, University of Toronto, Toronto, Canadad Casey House, Toronto, Canadae Department of Clinical Epidemiology & Biostatistics, McMaster University, Hamilton, Canadaf School of Medicine, Queen's University, Belfast, UK Corresponding author. Email: [email protected] 7 2013 3 2014 26 3 320 325 15 11 2012 20 6 2013 © 2013 The Author(s). Published by Taylor & Francis2013This is an open access article distributed under the Supplemental Terms and Conditions for iOpenAccess articles published in Taylor & Francis journals, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.This retrospective chart review provides a profile of an emerging population of vulnerable HIV patients with complex comorbidities. Data were abstracted from all 83 patients admitted in 2008 to Casey House, a community-based hospital dedicated to supportive and palliative care for persons with HIV in Toronto, Canada. We describe patient characteristics, including medical and psychiatric conditions, and use a Venn diagram and case study to illustrate the frequency and reality of co-occurring conditions that contribute to the complexity of patients' health and health care needs. The mean age at admission was 49.2 years (SD = 10.5). Sixty-seven patients (80.7%) were male. Patients experienced a mean of 5.9 medical comorbidities (SD = 2.3) and 1.9 psychiatric disorders (lifetime Axis I diagnoses). Forty patients (48.2%) experienced cognitive impairment including HIV-associated dementia. Patients were on a mean of 11.5 (SD = 5.3) medications at admission; 74.7% were on antiretroviral medications with 55.0% reporting full adherence. Current alcohol and drug use was common with 50.6% reporting active use at admission. Our Venn diagram illustrates the breadth of complexity in the clients with 8.4% of clients living in unstable housing with three or more medical comorbidities and two or more psychiatric diagnoses. Comprehensive HIV program planning should include interventions that can flexibly adapt to meet the multidimensional and complex needs of this segment of patients. Researchers, policy-makers, and clinicians need to have greater awareness of overlapping medical, psychiatric and psychosocial comorbidities. Inclusion of the needs of these most vulnerable patients in the development of evidence-based guidelines is an important step for effectively treating, preventing, and planning for the future of HIV/AIDS care. HIV/AIDScomorbiditiesvulnerable populationshomelessnessmental healthHIV-associated neurocognitive disorders ==== Body Introduction Highly Active Antiretroviral Therapy (HAART) has changed the face of the AIDS epidemic (Palella et al., 1998; Puhan et al., 2010). Many now thrive; however, a subset of people living with HIV struggle and contend with high levels of chronic medical, psychiatric and psychosocial morbidity (Rubin, Colen, & Link, 2009; Walley et al., 2008). Patients, such as Abigail (Appendix 1) require ongoing complex care, though this aspect of HIV disease remains scarcely researched and underreported in the literature. Current clinical guidelines are most strongly informed by large randomized controlled trials. Methods used in these trials aim to maximize internal validity, resulting in the underrepresentation of complex patients (Fortin et al., 2006). It is important that clinicians, policy-makers, and researchers understand the breadth and complexity of issues that some people with HIV/AIDS are facing, so that effective resource planning can address the needs of the entire HIV population. HIV medical clinics and AIDS Service Organizations must evolve their services to meet the needs of all patients with HIV disease, including the most marginalized and vulnerable, who may not maximally benefit from traditional models of care. In this paper, we add to the literature by describing the complex and overlapping medical, psychiatric, and psychosocial care needs of some of the most vulnerable patients living with HIV disease. We conducted data abstraction through retrospective chart review of all patients admitted to Casey House, a community-based HIV/AIDS hospital, during the course of one year. Casey House began as an HIV/AIDS hospice focused on end of life care in 1988 and evolved over time into a community-based hospital with sub-acute inpatient care and community outreach programs that provide home care, case management, and service coordination with acute care hospitals and partner agencies in Toronto. Methods Research setting The research focused specifically on the patients admitted to the 13-bed inpatient sub-acute HIV/AIDS hospital. Individuals with HIV may be admitted for sub-acute, palliative or respite care, provided by an inter-professional team including physicians, nurses, rehabilitation therapists, and social workers. Research methods and data analysis We employed an in-depth retrospective chart review to collect data on patient demographics, self-reported substance use, medical and psychiatric history. Cognitive impairment was diagnosed according to the nosology for HIV-Associated Neurocognitive Disorders (Antinori et al., 2007). Data were analyzed in SPSS version 17.0. Data are from all 83 patients admitted to Casey House between 1 January and 31 December 2008. Sixty-seven patients were admitted once, 16 patients were admitted on multiple occasions. Only first admission data are reported. Ethical approval was obtained through the St. Michael's Hospital Research Ethics Board. Results Participant characteristics Patient demographics and medical and psychiatric history are summarized in Tables 1 and 2. The majority of patients was male (n = 67; 80.7%) and patients’ mean age was 49.2 years (SD = 10.5). About half of the patients identified as homosexual (n = 44; 61.1%). Eleven patients (13.6%) reported living on the street or in a shelter at the time of admission and five patients (6.1%) were under-housed, staying with friends or family. Table 1. Patient demographics (n = 83). Frequency Percentage Mean age (years) 49.2 SD = 10.5 Gender (n = 83) Male 67 80.7 Female 16 19.3 Marital status (n = 82) Single, never married 46 56.1 Married, common law 16 19.5 Divorced, separated 16 19.5 Widowed 4 4.9 Sexual orientation (n = 72) Homosexual 44 61.1 Heterosexual 24 33.3 Bisexual 4 5.6 Ethnicity (n = 77) White 56 72.7 Black 8 10.4 Aboriginal 8 10.4 Asian 5 6.5 Citizen status (n = 75) Canadian citizen 65 86.7 Permanent resident 9 12.0 Refugee claimant 1 1.3 Income source* (n = 79) Disability and/or national pension plan 70 88.6 Employment 2 2.5 Family support 2 2.5 No reported income 3 3.8 Other 6 7.6 Housing (n = 81) Renting own dwelling 44 54.3 Supported housing (renting) 17 21.0 Unstable housing (shelter, streets, staying with family or friends) 16 19.8 Homeowner 2 2.5 Nursing home 2 2.5 Note: Patients could report more than one income source. Table 2. Patient medical and psychiatric history (n = 83). Frequency Percentage Medical History Average number of years living with HIV 14.8 SD = 7.5 CD4 + at admission (n = 79) CD4 + < 200 46 58.2 CD4+ 200–500 23 29.1 CD4+ > 500 10 12.7 Viral load recorded on chart (n = 53) Viral load detectable 32 60.4 Viral load undetectable 21 39.6 Presence of anemia 26 31.3 No family physician 9 10.8 Medical co-morbidities (n = 83) Mean number of medical co-morbidities 5.9 SD = 2.3 AIDS defining opportunistic condition 44 53.0 Respiratory disease/condition 36 43.4 Liver disease 24 28.9 Non-AIDS defining malignancies 14 16.9 Cardiac disease 16 19.3 Kidney disease 8 9.6 AIDS defining malignancies 5 6.0 Medications (n = 83) Mean number of all meds at admission 11.5 SD = 5.3 On HAART 62 74.7 Mean CPE 2010 Rank of HAART regime (n = 60) 9.7 SD = 3.0 Self-reported full adherence to ART (n = 60) 33 55.0 Lifetime history of psychiatric disorder (n = 83) Mean number of Axis I diagnoses 1.9 SD = 1.1 Drug misuse disorder 52 62.7 Cognitive disorders includingdementia 40 48.2 Depressive disorder 32 38.6 Anxiety disorder 12 14.5 Bipolar disorder 6 7.2 Schizophrenia disorder 4 4.8 PTSD 3 3.6 Adjustment disorder 1 1.2 Other psychiatric disorder 6 7.2 Psychiatric medications (n = 63) Mean number of psych meds at admission 1.9 SD = 1.1 Hypnotics 40 63.5 Antidepressants 28 44.4 Antipsychotics 23 36.5 Psychostimulants 3 4.8 Mood stabilizers 1 1.6 Substance misuse (n = 83) Alcohol 11 13.3 Any substance use (other than alcohol) 36 43.4 Cocaine 20 24.1 Marijuana 14 16.9 Crystal meth 7 8.4 Other drugs 5 6.0 Hospital stay Twenty-two patients (26.5%) were admitted as a planned 14-day respite stay, while the remaining patients entered through a general admission (mean stay = 46.9 days (SD = 50.2)). Patients could have multiple reasons for admission (summarized in Table 3): the most common being for supportive care with a medical focus (86.7%) that is, failure to thrive and recovery from acute medical illness, and HAART adherence support (20.5%). Table 3. Reason for admission (n = 83). General admission (n = 61) Respite admission (n = 22) Reason for admission Frequency Percentage Frequency* Percentage Supportive care/medical focus 51 83.6 21 95.5 ART adherence support 11 18.0 6 27.3 End of life care 9 14.8 0 n/a Supportive care/psychosocial focus 7 11.5 5 22.7 Caregiver relief 1 1.6 3 13.6 Note: Patients could be admitted for more than one reason. Sixteen of the patients admitted in 2008 died at Casey House (19.3%). The remaining patients were discharged to various locations including home (n = 34; 41.0%), hospital (n = 13; 15.7%), supportive housing (n = 13; 15.7%), community shelters (n = 4; 4.8%), and nursing homes (n = 2; 2.4%). Medical history Patients had been living with HIV for an average of 14.8 years (SD = 7.5). Forty-six patients (58.2%) had CD4 counts below 200 cells/ml. Nine patients (10.8%) did not have a primary care physician. At admission, patients were taking an average of 11.5 medications (SD = 5.3). Seventy-five percent were on HAART (n = 62); just over half of these patients reported being adherent (55.0%). On average, patients experienced 5.9 (SD = 2.3) medical co-morbidities, the most common being AIDS-defining opportunistic infections (n = 44; 53.0%), respiratory disease (n = 36; 43.4%) and liver disease (n = 24; 28.9%). Psychiatric history, neurocognitive status, and substance use Psychiatric comorbidity was significant with a group mean of 1.9 lifetime Axis I diagnoses (SD = 1.1). The most common psychiatric disorders were substance misuse (n = 52; 62.7%), cognitive disorders including dementia (n = 39; 47.0%), and depressive disorders (n = 32; 38.6%). Of the 40 individuals with cognitive impairment, 18 (45.0%) had mild neurocognitive impairment, and 22 (55.0%) had dementia. Upon admission, 63 (75.9%) patients were on psychotropic medications. Eight (9.6%) had previously attempted suicide and 10 (12.0%) had a bipolar or psychotic disorder. At admission, 11 (13.3%) reported using alcohol and 36 (43.4%) reported using other substances, most commonly cocaine (n = 20; 24.1%) and marijuana (n = 14; 16.9%). Patient complexity We used a Venn diagram to demonstrate the coexistence of complicating conditions (Figure 1). We defined three variables of complexity: medical complexity, psychiatric complexity, and housing instability. Medical complexity is defined as having three or more medical comorbidities. Psychiatric complexity is defined as having two or more lifetime Axis I diagnoses (which includes substance misuse and cognitive disorders). Housing instability was defined as living on the street, in a shelter or with family or friends. One patient (1.2%) did not have any of these complexities. Seventy-seven patients (92.8%) had two or more Axis I diagnoses, 28 patients (33.7%) had three or more medical comorbidities, and 16 patients (19.3%) had unstable housing. Seven patients (8.4%) experienced all three complexity variables. Figure 1. Patient complexity Venn diagram. This Venn diagram demonstrates the complex interaction of psychiatric history, medical morbidity and unstable housing in 83 patients. Only 1.2% (n = 1) did not have any of the complexity variables. Note: Psychiatric diagnoses include substance misuse disorder and HIV-associated neurocognitive impairment. Discussion This chart review provides a profile of issues experienced by a segment of the evolving HIV-positive population with high care needs in Toronto. These patients are too unwell to manage independently and require a community-based flexible alternative to acute care hospitalizations. Appendix 1 provides a case example of the context and care provided. Today, treatment with antiretroviral therapies enables the majority of the HIV-positive population in developed countries to live longer, healthier lives. However, we highlight a vulnerable population that is unable to optimally benefit from existing therapies. Nineteen percent of our patients died during their stay illuminating the fact that people with HIV disease continue to contend with early mortality. In addition to HIV disease and considerable psychosocial challenges, patients had multiple medical comorbidities, were taking, on average, more than 11 medications, and almost half had cognitive impairment. The co-occurrence of medical, psychiatric and psychosocial complexities in Casey House patients was significant, as illustrated in our Venn diagram. Our findings are analogous to those reported from an HIV-infected veterans’ cohort. Kilbourne et al. (2001) demonstrated significant overlap of conditions when examining depressive symptoms, at-risk alcohol or illicit drug use, and two or more general comorbidities. These results emphasize the need to both, identify coexisting conditions and to improve our understanding of how they influence standard treatment protocols. As Parekh et al. (2011) recommended, this includes improving the external validity of clinical trials and incorporating the issue of multi-morbidity in clinical guidelines. Our results also highlight the need for improved coordination of medical and psychiatric care, as well as an integration of psychosocial, recovery oriented addictions and harm reduction services, to enable all patients to benefit from the advances in HIV/AIDS medicine. Our report shares many findings with a study examining medical and psychiatric comorbidities in HIV-positive patients cared for between 1995 and 1998, at an AIDS designated long-term care facility in New Haven, USA (Selwyn et al., 2000). In their sample of patients with late stage HIV disease, they also found high rates of medical illness, HIV dementia (32%) and psychiatric illness (44%). As these authors noted, with the effectiveness of HAART, people are living longer with HIV and a growing number experience morbidity, marginalization, and disability. Our findings, from 10 years later in the HIV epidemic, echo this notion and demonstrate the need for interventions that can flexibly support these patients. Limitations and future directions Our study provides some of the missing context to the HIV/AIDS literature, focusing on complex patients in Toronto, Canada. We acknowledge that this study has important limitations. Our ability to explore clinical outcomes is limited by issues associated with chart reviews including the lack of standardized diagnoses. The use of self-reports for determining alcohol and substance use likely resulted in an underestimation of use. The complexity and often disjointed care in multiple centers, common to this population, provided additional barriers to obtaining both health and care variables. However, the importance of this study is strengthened by the inclusion of all patients seen over a 12-month period at a community-based HIV/AIDS hospital. Although stigma remains a barrier for some to seek treatment, this research design allowed us to capture marginalized and medically complex individuals, who are often not represented in prospective studies. Planning for the future, both HIV care and research should include a holistic view of individuals with HIV, addressing their medical, psychiatric, and social needs and the various interactions between them. We believe that we need to plan for the future informed by health promotion and determinants of health models for improving the lives of those living with HIV. In order to reduce health disparities for all patients with HIV disease, comorbidities, such as psychiatric illness and addictions, and social issues, such as housing insecurity must be addressed. Practical suggestions should include key components of continuity of care that have been found to be helpful in related fields involving complex clients and vulnerable populations, such as: attending to service coordination; facilitating transitions in care; involving the meaningful voice of patients living with HIV and multiple comorbidities, and; developing care plans that are reasonable, feasible and appropriately meet individuals where they are situated (LHINC, 2011). Furthermore, we must take the effort to include complex patients in research so that we can make evidence-based decisions. Guidelines based solely on randomized controlled trials of healthier patients with HIV/AIDS may fail to address the complex care needs of the most vulnerable patients. Policy-makers must also strive to make just allocation of resources so that needs of patients such as Abigail may be met. Acknowledgments We would like to acknowledge the Krembil Foundation for supporting research at Casey House and Tim Guimond for providing valuable suggestions for this manuscript. Appendix 1: Casey House case study Abigail (name changed for reasons of confidentiality) is a 46-year-old HIV-positive Aboriginal woman with a history of schizophrenia, depression, and crack dependence. She is homeless. Following an episode of crack use she is found on the railings of a highway overpass and endorses a desire to die. Her weight is 95 pounds. She is weak with unsteady gait, poor color, hair loss and many missing teeth. She has a CD4 count of 23 and a viral load of 220,000. She has normocytic anemia, chronic Hepatitis B, and Hepatitis C. Her behavior is grossly disorganized, and she has persistent auditory hallucinations. Her personal history is marked by repeated exposures to trauma and psychosocial deprivation, including repeated sexual assaults, both as a child and as an adult, multiple arrests for prostitution and related charges, and a third grade level of education. She is brought to an acute care hospital but refuses to stay. Following counseling with her community case worker she agrees to go to a community facility for the care and support of HIV-positive clients, known as Casey House. Following her admission to Casey House, Abigail is stabilized on IM (long-acting) risperidone and her HIV disease is treated with a combination of tenofovir/emticitibine/lopinavir/ritonavir. She decreases her crack use, gains 45 pounds and her CD4 count rises to 145. She sees a dentist and gets an upper plate. She begins to form stable working relationships with the care team, is able to establish stable housing and begins to contemplate treatment for her addictions. She remains supported through outpatient case management and has required readmissions to Casey House during periods of medical and/or psychiatric decline. ==== Refs References Antinori A. Arendt G. Becker J. T. Brew B. J. Byrd D. A. Cherner M. Wojna V. E. Updated research nosology for HIV-associated neurocognitive disorders Neurology 2007 69 1789 1799 10.1212/01.WNL.0000287431.88658.8b 17914061 Fortin M. Dionne J. Pinho G. Gignac J. Almirall J. Lapointe L. Randomized controlled trials: Do they have external validity for patients with multiple comorbidities? Annals of Family Medicine 2006 4 2 104 108 10.1370/afm.516 16569712 Kilbourne A. M. Justice A. C. Rabeneck L. Rodriguez-Barradas M. Weissman S. VACS 3 Project Team General medical and psychiatric comorbidity among HIV-infected veterans in the post-HAART era Journal of Clinical Epidemiology 2001 54 Suppl. 1 S22 28 10.1016/S0895-4356(01)00443-7 11750206 Local Health Integration Network Collaboration Mental Health and Addictions Working Group Through the door 2011 Ontario Local Health Integration Network Retrieved from http://www.ofcmhap.on.ca/sites/ofcmhap.on.ca/files/LHINC%20Mental%20Health%20and%20Addictions%20Working%20Group%20Report%20-%20Through%20the%20Door.pdf Palella F. J. Jr. Delaney K. M. Moorman A. C. Loveless M. O. Fuhrer J. Satten G. A. Holmberg S. D. Declining morbidity and mortality among patients with advanced human immunodeficiency virus infection. HIV Outpatient Study Investigators The New England Journal of Medicine 1998 338 13 853 860 10.1056/NEJM199803263381301 9516219 Parekh A. K. Goodman R. A. Gordon C. Koh H. K. HHS Interagency Workgroup on Multiple Chronic Conditions Managing multiple chronic conditions: a strategic framework for improving health outcomes and quality of life Public Health Reports 2011 126 4 460 471 Retrieved from http://www.publichealthreports.org/issueopen.cfm?articleID=2684 21800741 Puhan M. A. Van Natta M. L. Palella F. J. Addessi A. Meinert C. Ocular Complications of AIDS Research Group Excess mortality in patients with AIDS in the era of highly active antiretroviral therapy: Temporal changes and risk factors Clinical Infectious Diseases, 2010 51 8 947 956 10.1086/656415 20825306 Rubin M. S. Colen C. G. Link B. G. Examination of inequalities in HIV/AIDS mortality in the United States from a fundamental cause perspective American Journal of Public Health 2009 100 6 1053 1059 10.2105/AJPH.2009.170241 20403885 Selwyn P. A. Goulet J. L. Molde S. Constantino J. Fennie K. P. Wetherill P. Kennedy C. HIV as a chronic disease: implications for long-term care at an AIDS-dedicated skilled nursing facility Journal of Urban Health: Bulletin of the New York Academy of Medicine 2000 44 2 187 203 10.1007/BF02390530 10856000 Walley A. Y. Cheng D. M. Libman H. Nunes D. Horsburgh C. R. Jr Saitz R. Samet J. H. Recent drug use, homelessness and increased short-term mortality in HIV-infected persons with alcohol problems AIDS 2008 22 3 416 420 10.1097/QAD.0b013e3282f423f8	23869624	PMC3919151	NO-CC CODE	2021-01-04 23:50:39	yes	AIDS Care. 2014 Mar 22; 26(3):320-325
==== Front 04104626011NatureNatureNature0028-08361476-46872408921010.1038/nature12594nihpa517922ArticleOdour receptors and neurons for detection of DEET and new insect repellents Kain Pinky 1Boyle Sean Michael 2Tharadra Sana Khalid 1Guda Tom 1Pham Christine 3Dahanukar Anupama 1234Ray Anandasankar 12341 Department of Entomology2 Genetics, Genomics and Bioinformatics Program3 Neuroscience Program4 Institute of Integrative Genome Biology, University of California, Riverside, CA 92521Corresponding Author: Anandasankar Ray, Department of Entomology, University of California Riverside, 3401 Watkins Drive, Riverside, CA 92521, USA, Phone: +1(951) 827-5998, Fax:+1(951)827-3086, [email protected]* These authors contributed equally 5 2 2014 02 10 2013 24 10 2013 24 4 2014 502 7472 507 512 Users may view, print, copy, download and text and data- mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: http://www.nature.com/authors/editorial_policies/license.html#termsSummary There are major impediments to finding improved DEET alternatives because the receptors causing olfactory repellency are unknown, and new chemistries require exorbitant costs to determine safety for human use. Here we identify DEET-sensitive neurons in a pit-like structure in the Drosophila antenna called the sacculus. They express a highly conserved receptor Ir40a and flies in which these neurons are silenced or Ir40a is knocked down lose avoidance to DEET. We use cheminformatics to screen >400,000 compounds and identify >100 natural compounds as candidate repellents. We test several and find that most activate Ir40a+ neurons and are repellents for Drosophila. These compounds are strong repellents in mosquitoes as well. The candidates contain chemicals that do not dissolve plastic, are affordable, smell mildly like grapes, with three being considered safe for human consumption. Our findings pave the way to discover new generations of repellents that will help fight deadly insect-borne diseases worldwide. ==== Body Blood-feeding insects transmit deadly diseases such as malaria, dengue, lymphatic filariasis, and West Nile fever, to hundreds of millions of people, causing untold suffering and more than a million deaths every year. Insect repellents can be very effective in reducing disease transmission by blocking contact between blood-seeking insects and humans. N,N-Diethyl-m-toluamide (DEET) has remained the primary insect repellent used for more than 60 years. However, DEET has little impact on disease control in endemic regions due to high costs and inconvenience of continuous application on skin at high concentrations. DEET also dissolves some plastics, synthetic fabrics, and painted surfaces1. Additionally, DEET inhibits mammalian acetylcholinesterase2. Instances of DEET resistance have also been reported in flies3, and mosquitoes4,5. However, major barriers in developing improved repellents are the estimated cost for identification6 and subsequent cost of safety analyses for new chemistries. A significant challenge in finding improved DEET substitutes is that the target receptors through which it causes repellency in insects are unknown. Recent studies have put forth multiple models of DEET action. Pure DEET causes inhibition7,8 or mild electrophysiological modification of neural responses to weakly-activating odours in Drosophila antennal olfactory neurons9, but whether these effects contribute to repellency is unknown. Mosquitoes can also directly detect DEET10 and mutations in the Orco co-receptor gene in A. aegypti cause reduction in repellency11. Some DEET-sensitive olfactory neurons have been identified in C. quinquefasciatus10 and A. aegypti5, but it is not yet known whether they are responsible for repellency or which odour receptors they express. A broadly tuned larval odour receptor responds to DEET12,13, however its role in avoidance in larval or adult mosquitoes has not been demonstrated. Not only can more than one pathway contribute to olfactory repellency, analyses are further confounded by the observation that DEET also activates bitter taste neurons that mediate contact-avoidance in Drosophila14,15. DEET detected by neurons of the sacculus In order to identify the elusive DEET-sensing neurons of the olfactory system in an unbiased manner, we used the NFAT-based system to report DEET-evoked neural activity via expression of green fluorescent protein (GFP) in Drosophila melanogaster16 (Fig. 1a). Exposure to 10% DEET showed an increase in expression of GFP in neurons that innervate sensilla within the sacculus, a pit-like structure in the antenna (Fig. 1b, c, Supplementary Fig.1a, Supplementary Video 1). The dendrites of GFP+ neurons primarily innervated the most distal chamber (I) of the sacculus (Fig. 1c, Supplementary Fig. 1b). Previous studies of DEET overlooked the sacculus, since it is intractable to traditional electrophysiology methods. Contrary to expectations from a previous report17 we were unable to find DEET-activated reporter expression in ORNs of the maxillary palps (Fig. 1b). We therefore performed single-sensillum electrophysiology analyses and found that the previously reported Or42a+ pb1A neurons responded poorly to DEET, but strongly to hexane that was used as solvent in the previous study (Supplementary Fig. 2a, b). ORNs innervating the sacculus do not express Or genes, but instead members of the ancient Ir (Ionotropic Receptor) gene family18-21. In the antennal lobes robust DEET-dependent GFP was detected in the characteristic “column” glomerulus (Fig.1d, Supplementary Fig. 3a), which is innervated by axons of Ir40a-expressing neurons of the sacculus18. Faint GFP was also observed in the Or67d+ DA1 glomerulus, which is likely caused by exposure to male pheromone CVA in the assay, since the CVA-responsive at1 neuron did not respond to DEET (Supplementary Fig. 2c). The DC4 glomerulus, which is innervated by other sacculus ORNs that express Ir64a19, showed a very faint signal as well (Supplementary Fig. 3a). The simplest interpretation of these results is that Ir40a+ sacculus ORNs innervating chamber I, and projecting to the column glomerulus, may represent a major olfactory detection pathway for DEET. Consistent with previous electrophysiological analysis14,15, we found DEET-dependent GFP expression in gustatory neurons of the labellum (Fig. 1e). In addition we observed DEET-dependent GFP in neurons innervating the Labral Sense Organ (LSO) of the pharynx (Fig. 1e). The DEET activity mapped to neurons marked by Gr33a and Gr89a, which are bitter-sensing deterrent neurons (Supplementary Fig. 3b). Axonal projections of DEET-sensitive gustatory neurons in the sub-oesophageal ganglion (SOG) revealed arborization patterns similar to those of taste neurons originating in the labellum and the pharynx (Fig. 1e, Supplementary Fig. 3b). In order to directly test physiological responses of the sacculus Ir40a+ ORNs to DEET we performed in vivo calcium imaging in flies expressing GCaMP3 using Ir40a-Gal418,22,23. Ir40a neurons show robust activation in response to a puff of DEET delivered from an atomizer but not to control DMSO (Fig. 2a,b). Moreover, DEET response is dependent on Ir40a (Fig. 2c). In order to test whether the Ir40a+ ORNs are required for DEET repellency we blocked synaptic transmission in these neurons using Ir40a-Gal4 to express the active form of tetanus toxin (TNTG)24. We employed a trap lured by 10% apple cider vinegar (ACV) in which a DEET-treated filter paper was placed inside the trap. Avoidance was significantly decreased in Ir40a-TNTG flies as compared to various controls, including a non-functional version of the tetanus toxin (IMPTV), suggesting that Ir40a+ neurons are required for DEET repellency (Fig. 2d). All genotypes exhibited attraction to 10% ACV in 2-choice trap assays (Supplementary Fig. 4a). Ir40a is necessary for DEET avoidance To test directly whether Ir40a is required for olfactory avoidance to DEET we examined the behaviour of flies in which Ir40a was knocked down pan-neuronally using an elav-Gal4 driver to express UAS-Ir40a-RNAi. In 2-choice trap assays (Fig. 3a), we found a significant loss of DEET avoidance in Ir40a-RNAi flies as compared to control flies (Fig. 3b). Similar results were obtained when Ir40a-RNAi was executed selectively in Ir40a+ ORNs using two independent UAS-Ir40a-RNAi transgenes (Fig. 3c). Not only was avoidance completely abolished, Ir40a knockdown flies in fact showed a mild attraction to the DEET trap. Attraction to ACV was unaffected (Supplementary Fig. 4b,c). We next wanted to rule out the possibility of a developmental role for Ir40a. We therefore suppressed expression of Ir40a-RNAi during development using a temperature-sensitive Gal80ts transgene (Fig. 3d). Flies were raised at the permissive temperature (18°C) until just before adult eclosion, at which point they were left at 18°C (RNAi Off) or shifted to the Gal80ts restrictive temperature 29°C (RNAi On). Behavioural assays performed four days after the temperature shift showed that post-developmental Ir40a-RNAi was sufficient to abolish DEET avoidance when RNAi was induced in Ir40a+ ORNs (Knockdown, Fig. 3e). Moreover, DEET avoidance was completely restored when flies were returned to the Gal80ts permissive temperature (Recovery, Fig. 3e). Attraction to ACV was unaffected (Supplementary Fig. 4d). Taken together, these experiments demonstrate that Ir40a is required in adult Ir40a+ sacculus ORNs for olfactory avoidance of DEET. In silico prediction of new repellents Identification of DEET receptors and neurons offer a powerful system to screen for improved repellents. However, volatile chemical space that can be exploited to find DEET substitutes is vast and therefore poses unfeasible requirements in terms of cost and time to screen. The receptor structure is unavailable for screening and the most effective repellents may require detection by both olfactory and gustatory pathways. To circumvent these limitations we developed a high-throughput chemical informatics screen. Previous studies using such structure-activity approaches have given encouraging results25. We identified structural features shared by DEET and other known repellents and used them to screen a vast library of compounds in silico for the presence of these features. We assembled a training set of known repellents that included: the two commercially approved repellents DEET and picaridin; 34 N-acyl piperidines25 that were identified by structural relatedness to picaridin; natural repellents eucalyptol, linalool, alpha-thujone, and beta-thujone10,26,27; and a structurally diverse panel of other odours as negatives28,29. We focused on a descriptor-based computational approach and using a Sequential-Forward-Selection method30 we incrementally identified a unique subset of 18 descriptors that were highly correlated with repellency (correlation of 0.912) (Fig. 4a, Supplementary Table 1). The repellents clustered together if the optimized descriptor subset was used to calculate Euclidean distances amongst odorants of the training set (Fig. 4b). The optimized descriptor set was utilized to train a Support Vector Machine (SVM), which is a well-known supervised learning approach31, to predict compounds that shared optimized structural features with known repellents (Fig. 4a). A 5-fold cross-validation on the training set of repellents was performed and a mean Receiver-Operating-Characteristic (ROC) analysis curve generated. The Area-Under-Curve was determined to be high (0.994) indicating that the in-silico approach was extremely effective at predicting repellents from compounds that were excluded from the training set (Fig. 4c). We next used the 18-optimized-descriptor and SVM method to screen in silico a large virtual chemical library consisting of >440,000 volatile-like chemicals. Inspection of the top 1,000 predicted repellents (0.23% of hits) revealed a diverse group of chemicals that retain some structural features of the known repellents (Fig. 4d,e). We computed partition coefficient (logP) values of the 1,000 compounds to exclude those predicted to be lipophilic (logP >4.5) and therefore more likely to pass through the skin barrier in topical applications32 (Fig. 4e). In addition, we computed predicted vapour pressures of these chemicals, since volatility may be a useful predictor of spatial volume of repellency (Fig. 4e). Although the in-silico screen was feasible, a more significant challenge lies in identifying safe and effective DEET substitutes that can be rapidly approved for human use. To identify such compounds, we applied our in-silico screen to an assembled natural odour library consisting of >3,000 chemicals identified as originating from plants, insects, or vertebrate species, and compounds already approved for human use as fragrances, cosmetics, or flavours (Supplemental Materials). While many of the top 200 hits share structural features with known repellents from the training set, they also represent structurally diverse chemicals, allowing targeted exploration of previously untested chemical space (Fig. 4f). For example, several anthranilates and pyrazines were identified, although such compounds were largely missing from the training set. Ir40a+ cells activated by repellents We selected 4 compounds from the list, methyl N,N-dimethyl anthranilate (MDA), ethyl anthranilate (EA), butyl anthranilate (BA), and 2,3-dimethyl-5-isobutyl pyrazine (DIP), of which the first 3 have a mild grape-like aroma, have excellent safety profiles, have been thoroughly tested and approved for human consumption/oral inhalation by the FDA, World Health Organization and European Food Safety Authority, and have been listed in the “generally recognized as safe” (GRAS) list by the Flavour and Extract Manufacturer's Association (Fig. 4g, Supplementary Table 2). The fourth, a pyrazine, is an ant trail pheromone33. The anthranilate and pyrazine classes also contain a large diversity of chemicals found in nature and therefore present attractive repositories of structural substitutes. For all 4 chemicals we found robust activation of sacculus ORNs (Fig. 5a, Supplementary Video 2) that innervate the Ir40a+ “column” glomerulus (Fig. 5b, as shown for BA). They also activated gustatory neurons that project to similar areas of the SOG as DEET (Fig. 5b, as shown for BA). GCaMP3 imaging in Ir40a+ neurons showed robust responses to these chemicals, while several other classes of common odorants did not (Fig. 5c, Supplementary Fig. 5). These results demonstrate that the computationally predicted chemicals activate the same chemosensory pathways as DEET and are therefore ideal candidates for new repellents. In order to test the effect of these compounds on behaviour we used a 2-choice trap assay in which flies can sense a DEET-treated filter paper positioned at the entrance of a trap via both olfactory and gustatory systems3,17 (Fig. 5d). All 4 compounds had strong dose-dependent repellent effects on D. melanogaster (Fig. 5d). Measurements were taken at 24 hours and 48 hours after the start of the assay, and were found to be consistent. Six additional predicted repellents were tested in a similar manner, at least four of which elicited strong repellency similar to DEET (Supplementary Fig. 6). To confirm the role of Ir40a+ neurons in mediating avoidance to these new repellents, we examined behavioural avoidance of flies in which synaptic activity of Ir40a+ neurons was silenced using TNTG as before. We found that avoidance of chemical treated traps was substantially decreased in Ir40a-TNTG flies as compared to control flies (Fig. 5d), showing that Ir40a+ neurons are required for repellency to the four chemicals. Mosquitoes avoid predicted repellents To test the effects of the identified chemicals on mosquito behaviour, we adapted a hand-in-glove assay that allows quantitative analysis of chemical repellency on mosquitoes attracted to a human arm (described in Supplementary Methods) (Fig. 6a, Supplementary Fig. 7). Female A. aegypti mosquitoes showed strong avoidance behaviour to DEET, irrespective of whether or not they could directly contact DEET (Fig. 6b). However, for sporadic landings the average time spent on the net before escape while not significant (p=0.203 for 10% DEET and p=0.06 for 1% DEET, Student's t-test) was reduced when direct contact with DEET was permitted, particularly at the lower concentrations (Fig. 6c). While it is difficult to asses from these experiments the direct contribution of the gustatory system alone, it demonstrates that mosquitoes can avoid DEET strongly at close range, even without making direct contact with it. In order to test whether the 4 newly identified Drosophila repellents were also olfactory repellents to mosquitoes, we performed behaviour trials using the non-contact version of the assay. Notably, we found that all 4 compounds applied at 10% concentration demonstrated substantial repellency (Fig. 6d). The fraction of mosquitoes present on the net throughout the duration of the assay (Fig. 6d), as well as the cumulative number of mosquitoes present on the net were substantially decreased in the presence of the test compounds (Fig. 6e). For the mosquitoes that did land on the repellent treatment, the escape index, as measured by the frequency of take off, was substantially higher as compared to those landing on controls (Supplementary Fig. 8,9). One of the major disadvantages of DEET is its property of solubilizing plastics and synthetic materials1, which impacts its usefulness. We tested the ability of the 4 repellents to dissolve a 3 × 3 mm square of vinyl. While the vinyl completely disappeared in DEET within 6 hrs, there was no significant difference in the weight of the vinyl squares immersed in the 4 DEET substitutes after 6-hrs or 30-hrs (Fig. 6f). Discussion The unbiased strategy to use a genetic-reporter of neural activity was instrumental in identifying DEET-sensitive Ir40a+ neurons. These reside in the pit-like sacculus that could protect neurons from harsh chemicals. Both olfactory and gustatory systems are activated by DEET, with additional modes of detection in the antenna being mediated by Orco11 and a yet to be identified tuning Or gene (Fig. 6h). Additionally, DEET has been reported to have a mild enhancing or suppressing effect on the activity of various Or-expressing neurons of antennal basiconics in Drosophila, although a causal relationship between this effect and repellency has not been established9. DEET also has a solvent effect that slows down volatile odour release, potentially also from skin10. Thus, multiple pathways and mechanisms are likely to participate in overall repellency. Ir40a can account for the widespread effect of DEET olfactory repellency since it is highly conserved in species that show strong avoidance to it including Drosophila, mosquitoes, head lice34, and tribolium35, but not in the honey bee36. Ir40a orthologs are conserved across multiple insects and arthropods, with several regions of amino acid similarity across the length of the protein (Supplementary Fig. 10). This degree of conservation may better explain the repellent effects of DEET across several arthropod species compared to Or pathways that are not as well conserved. The Ir40a pathway therefore has major implications in the development of safe and affordable strategies to control several types of insects and arthropods that vector diseases of animals and plants or are plant pests. The chemical informatics enabled us to identify a number of affordable and safe potential repellents that are good candidates for regulatory approval for human use (Fig. 6g). This screen identified ∼1000 compounds and >100 additional natural compounds, many approved for use in human food and cosmetics, which may lead to other effective repellents. The repellency strategy may also have promise for use in combination with other behaviour control strategies, such as masking of CO2-mediated attraction behaviour or population control by trapping as a part of an integrated pull-mask-push strategy37,38. Moreover, these DEET substitutes may be of value in controlling DEET-resistant strains as well. Since several of the new repellents are affordable, activate both the olfactory and bitter gustatory neurons, are approved for human consumption and are strong repellents for fruit flies, they may also have major implications for control of agricultural pest insects that cause enormous crop loss. Novel repellents that are safe and affordable can be used to limit insect-human contact in disease-endemic areas of the world and to provide an important line of defence against deadly vector-borne diseases. Methods Summary Physiological experiments NFAT-based neural tracing16 and GCaMP3-based Calcium imaging22,23 were performed as previously described with some modifications (see Online Methods). Single-unit recordings from olfactory sensilla were performed as described previously37. Behavioural experiments For olfactory trap assays 20 Drosophila were released in cylindrical arenas containing Eppendorf tube traps (Fig. 2d and Fig.3a) with 10% apple cider vinegar as a lure. Repellents were presented on filter papers placed near the trap openings in a manner that did not allow physical contact with the fly prior to its entering the trap. Trap assays to measure repellency when both olfactory and gustatory inputs were possible were performed as described previously3. Mosquito arm-in-cage avoidance assays were performed with 40 mated A. aegypti females held in a cage and presented a human arm that was inserted in a glove containing a window covered with a double-layer of netting. Test compounds were applied to the nettings. Attraction towards the arm was measured using video recordings and analysts were blind to treatments. Chemical informatics Optimized molecular descriptors were selected from 3,224 Dragon descriptors based on their ability to increase the correlation between descriptor values and repellency. The repellency-optimized descriptor set was utilized to first train a Support Vector Machine to predict repellents and then applied to predict new repellents from large compound libraries. Insects Fly lines were obtained from the Bloomington Drosophila Stock Center for TNT and GCaMP3 experiments, the Vienna Drosophila RNAi Center for Ir40a-RNAi, Jing Wang (UC San Diego, CA) for NFAT tracing, and Richard Benton (University of Lausanne, Switzerland) for Ir40a-Gal4. Flies were grown on standard cornmeal-dextrose media, at 25°C unless otherwise noted and mosquitoes at 27°C and 70% RH. Full Methods and associated references are available in the online version of the paper in Supplementary Information. Methods Fly stocks Wild type flies were w1118 backcrossed to Canton-S for 5 generations. UAS-GCaMP3 (BL#32236), UAS-TNTG (BL#28838), UAS-IMPTV (BL#28840) and Tub-PGal80ts (BL#7017) were obtained from the Bloomington Drosophila Stock Center. The following stocks were generously provided: LexAop-CD8-GFP-2A-CD8-GFP; UAS-mLexA-VP16-NFAT, LexAop-CD2-GFP by Jing Wang (UC San Diego, CA), Ir40a-Gal4 by Richard Benton (University of Lausanne, Switzerland), and elav-Gal4 by Liqun Luo (Stanford, CA). UAS-Ir40a RNAi (1) (v101725) and UAS-Ir40a RNAi (2) (v3960) RNAi lines were obtained from the Vienna Drosophila RNAi Center. Ir40a RNAi is predicted to have no off-targets. Fly stocks were grown on standard cornmeal-dextrose media, at 25°C unless otherwise noted. Flies of appropriate genotypes for behaviour experiments were randomly sorted from populations before performing behavioural or electrophysiological experiments. NFAT Based neural tracing Late dark Drosophila pupae ready to emerge (∼95-97 hrs) of genotype elav-Gal4/LexAop-CD8-GFP-2A-CD8- GFP; UAS-mLexA-VP16- NFAT, LexAop-CD2-GFP/+ 16 were collected on moist filter paper strips in culture vials which contained 2 Kimwipes soaked in 5ml of water in as much of an odour free environment. A 100μl of odour at indicated concentration, dissolved in acetone, was spread on a filter strip (∼1cm × 3 cm), dried for 1 minute and placed, in a vial with 10-15 pupae. The exposure was given for 24 hrs and the filter paper strip with odour was replaced at ∼12-14hrs with fresh odour. Calcium Imaging using GCaMP3 DEET, dimethyl sulfoxide (DMSO), hexane and candidate compounds were purchased from Sigma-Aldrich or the emolecules database (http://www.emolecules.com) from Enamine, Vitas M Labs or Chembridge and were of highest purity available. Approximately 10-12 days old flies raised at 29°C (to improve Gal4 expression) were anesthetized and secured by their wings on the double-sided sticky tape (ventral side up) on a petri dish (BD Falcon, 50 × 9 mm). The fly proboscis, head and body was immobilized by sticky tape as shown (Fig. S11). One antenna was stably held down using a glass electrode on thin layer of 70% glycerol that enhanced imaging of fluorescence. The antenna was orientated with the arista and sacculus pointing upwards accessible to odours. Odorants were delivered using 5ml plastic syringes containing 2 Whatman filter paper strips (2×3 cm). A fine mist of DEET at indicated concentrations in DMSO was sprayed into the syringe using an atomizer. Fresh atomized odour syringes were prepared immediately before odour delivery. For DEET substitutes (BA, EA, MDA and DIP) a 100μl of 50% dilution in DMSO was applied to the filter paper directly and for other odorants a 100μl of (10-2) solution in paraffin or water for apple cider vinegar (ACV) was applied directly on the filter paper. The odour puff (∼ 2 sec) was delivered using the syringe over the antenna manually. For imaging odour-evoked activity from the antenna using GCaMP3 a Leica SP5 inverted confocal microscope was utilized. A filter block with 488 nm excitation filter and 500-535 nm emission filter was used and images were acquired at 3.3 frames per second with a resolution of 330×330 pixels using a 10× objective. The settings were optimized to capture odour-induced responses of GCaMP3 with high spatial and temporal resolution while limiting reporter bleaching. Data analysis for calcium imaging was performed using the Leica SP5 LAS AF software (in Quantify mode) to obtain the heat map images and fluorescence intensity changes. The % ΔF/F was calculated separately for each selected cell body by taking the mean intensity value of all frames for 5 seconds prior to the odour puff (Fpre) and taking the mean intensity value of all frames for 5 seconds around the peak responses (Fpost) after the end of the ∼2 second of stimulus delivery period. Similarly, the mean intensity values were taken for a background area in the vicinity of the cells. % ΔF/F was calculated according to the formula below: %ΔF/F=[Fpost–Fbackground(post)]−[Fpre−Fbackground(pre)][Fpre−Fbackground(pre)]×100 Immunohistochemistry After 24hrs exposure to either odour or solvent (control), flies were anaesthetized on ice and the tissue dissected in chilled 1XPBS and fixed for 30 minutes in 4% PFA (0.3% Triton X- 100) at room temperature. After washes with PBST (PBS with 0.3% Triton X-100) brains were blocked using PBST with 5% bovine serum albumin (BSA). Rabbit anti-GFP (1:1000, invitrogen) and anti-nc82 (1:10 Developmental Studies Hybridoma Bank) were used as primary antibodies and samples were incubated for 3 nights at 4 degrees. Alexa Fluor 488 anti-rabbit immunoglobulin G (IgG) (Invitrogen; 1:200) and Alexa Flour 546 anti-mouse IgG (Invitrogen; 1:200) were used as secondary antibodies, respectively followed by over night incubation at 4 degrees. Images were acquired with a Zeiss or Leica SP5 confocal microscope and images processing was done using ImageJ and Photoshop software. Data analysis was performed offline, and the investigator was blind to the treatment while counting GFP+ antennal neurons in the confocal micrographs. Temperature sensitive Gal80ts experiment For the two-choice behaviour assay in Figure 3 and supplementary Figure S4, flies (10 males and 10 females) with genotypes Ir40a-Gal4/+; UAS-Ir40aRNAi(2)/Gal80ts flies were grown throughout at 18°C (permissive temperature) where Gal80 is active and RNAi is off. Such flies were treated as control. In parallel, flies of the same genotype were shifted to 29°C (non-permissive temperature) from 18°C as late black pupae for 4 days to activate Gal4 and switch on RNAi. These flies were used as knockdown flies. A subset of flies that were shifted to 29°C was shifted back to 18°C for 4 additional days to turn off the RNAi and these were used as recovery flies. Electrophysiology Flies used were 4-7 days old and raised on cornmeal food at 25°C. Extracellular recording were made by inserting a glass electrode into the base of a palp sensillum as done previously37,39. Odorants were diluted in hexane or DMSO, at indicated concentrations (made fresh for every stimulus). For DEET stimulation, 10 μl of diluted odorant was applied to a filter paper strip, the hexane solvent was evaporated for 30 seconds (as in a previous study17) or for 5 minutes, and placed into a glass pasture pipette cartridge, and each cartridge was only used once. The evaporation of hexane from filter paper strip was much slower upon mixing with DEET and lingering dampness of the filter paper could be observed visually as well. Behaviour testing Drosophila olfactory avoidance assay for DEET For each trial flies that were to 3-6 day old flies (10 males and 10 females) were starved for 18 hours. Trap Assay Flies were transferred to a cylindrical 38.1 mm D × 84.1 mm H chamber containing a trap fashioned from an upturned 1.5 ml microcentrifuge tube with 2 mm removed from the tapered end. A pipette tip (1000 μl) was cut 2.5 cm from narrow end and 0.5 cm from top and inserted into the bottom of the inverted microcentrifuge tube. A 15mm × 16 mm #1 Whatmann filter paper was inserted in between the pipette tip and tip of microcentrifuge tube in a manner that entering flies cannot make physical contact with it. A 25 μl sample of test compound was applied to filter paper and 125 μl of 10% ACV is applied to the upturned lid of the microcentrifuge tube as attractant. Trials were run for 24 hours, and numbers of flies entering trap counted (Fig. 2d). 2-choice trap assay For 2-choice test two 10% ACV (125 μl) lured traps as described above were placed in the cylinder, one with 50 μl solvent (DMSO) and another with 50 μl the test odorant at 50% applied to the filter paper (Fig. 3). The more volatile DIP was tested at a lower concentration of 25%. For positive control tests in Supplementary Fig. 4, 125 μl of 10% ACV in test traps and 125 μl of water in control traps is added in the upturned microcentrifuge tube lid. Both traps contained filter papers as before with 50 μl solvent (DMSO). All trials were run for 24 hours, positions randomized, and counted. Only trials with >35% participation was considered. Preference Index = (number of flies in treated trap-number in control trap)/(number of flies in treated + control traps). Drosophila olfactory and gustatory avoidance assay for DEET Repellency was tested in Fig. 5d and Supplementary Fig. 6 using a Drosophila melanogaster 2-choice trap assay as described previously3,17 with minor modifications. Briefly, traps were made with two 1.5 ml microcentrifuge tubes (USA Scientific) and 200 μl pipette tips (USA Scientific), each cap contained standard cornmeal medium. T-shape piece of filter paper (Whatman #1) was impregnated with 5 ul of acetone (control) or 5 μl of 10%, 1%, 0.10% test odour, diluted in acetone. Traps were placed within a petri dish (100 × 15mm, Fisher) containing 10ml of 1% agarose to provide moisture. Ten wild-type Canton-S flies 4-7days old were used per trial, which lasted 48 hours by which time point nearly all flies in the assays had made a choice. For the 24 hour time point data was considered only if >35% of flies had made a choice, at 48 hours the majority of flies had made choices. Preference Index= (number of flies in treated trap-number in control trap)/(number of flies in treated + control traps). Mosquito arm-in-cage avoidance assay for DEET Repellency was tested in mated and starved A. aegypti females using an arm-in-cage assay. A. aegypti mosquitoes (eggs obtained from Benzon Research Inc.) were maintained at ∼27 °C and 70% RH on 14h: 10h L: D cycle. Behavioural tests were done with 40 mated, non-blood fed, ∼24 hour starved, 4-10 day old females in 30cm × 30cm × 30cm cages with a glass top to allow for video recording (Fig. 6a, Supplementary Fig. 7). The experimental protocol was reviewed and approved by the Institutional Review Board (IRB) Compliance Analyst at UCR and determined not to require additional Human Research Review Board approval. Each test compound solution (500μl) of 10% concentration in acetone solvent was applied evenly to a white rectangular 7cm × 6cm polyester netting (mesh size 26 × 22 holes per square inch) in a glass petri-dish and suspended in the air for 30 minutes to allow solvent evaporation. The more volatile 2,3-dimethyl-5-isobutyl pyrazine was dissolved in paraffin oil. Acetone or paraffin oil (500μl) served as control. A nitrile glove (Sol-vex) was modified as described in Supplementary Fig. 7 such that a 5.8cm × 5cm window was present for skin odour exposure. A set of magnetic window frames were designed to secure the treated net ∼1.5 mm above skin, and a second untreated netting ∼4.5 mm above the treated net in a manner so that mosquitoes were attracted to skin emanations in the open window but unable to contact treated nets with tarsi, or contact and pierce skin. Additionally the test compound had minimal contact with skin. A clean set of glove and magnets were used for every trial. Care was taken that experimenter did not use cosmetics, soap etc on arms. For each trial the arm was first inserted for 5 min and the number landing or escaping test window recorded on video for 5-min period. Solvent controls were always tested prior to treatment. It was determined first that a solvent treated arm when offered to the same cage with a gap of 5 minutes slightly more mosquitoes were attracted the second time around, therefore providing a more rigorous assay for the repellents. No cage was tested more than once within 1 hour of a testing session and not more than twice on any single day. Videos were analysed blind and the numbers of mosquitoes present for a 5-sec continuous duration were counted every minute. Mosquitoes reliably started accumulating in controls at the 2 min point, and data from this time point was considered for analysis. Percentage present = average number of mosquitoes on window for 5 seconds at a given time-point across trials. All values were normalized to percentage of the highest value for the comparison, which was assigned a 100 percent present. Percentage repellency = [1 - (mean cumulative number of mosquitoes on the window of treatment for 5 seconds at time points 2,3,4,5 min/mean cumulative number of mosquitoes that remained on window of solvent treatment for 5 seconds at time points 2,3,4,5 min)] × 100. Escape Index = (Average Number of mosquitoes in treatment that landed yet left the mesh during a five second window over the following time points: 2 minutes, 3 minutes, 4 minutes, 5 minutes)/(Average Number of mosquitoes that landed yet left the mesh during a five second window over the same time points in (treatment + control)) Each time point has N=5 trials, 40 mosquitoes per trial, Except for EA, where N=4. Chemical Informatics Calculation of descriptors A single energy-minimized 3-Dimensional structure was predicted for each compound using of the Omega2 software package40. The commercially available software package Dragon (3,224 individual descriptors) from Talete was used to calculate molecular descriptors41. Descriptor values were normalized across compounds to standard scores by subtracting the mean value for each descriptor type and dividing by the standard deviation. Molecular descriptors that did not show variation across compounds were removed. Classification of repellent compounds For our analysis, compounds from different studies were approximated into a single metric of “protection duration” as a rough indicator of repellency. The non-repellent diversifying training set of odours were assigned protection times of zero, while the approved repellents DEET and Picaridin were assigned the highest value since we made the assumption that these would have structural properties important for regulatory approval. Compounds were clustered using Euclidean distance and hierarchical clustering based on differences in repellency values, and a set of 5 compounds with the highest activity that clustered together was classified as “training repellents”. Determination of optimized descriptor subsets A compound-by-compound repellency distance matrix was calculated from repellency data. A separate compound-by-compound descriptor distance matrix was calculated using the 3,224 descriptor values calculated by the Dragon software package. Using a Sequential Forward Selection (SFS) approach, all descriptors are individually compared and selected for their ability to increase the correlation between descriptor values and repellency. The descriptor that correlates best is retained and each further iteration adds an additional descriptor to improve the correlation values. This process is continued until additional descriptors fail to improve the correlation value from the previous step. This process results in unique descriptor set that is optimized for repellency. Support Vector Machine predictions from Odour compound libraries This repellency-optimized descriptor set was utilized to train a Support Vector Machine (SVM) using regression and a radial basis function kernel available in the R package e1071, which integrates libsvm 42,43. Optimal gamma and cost values were determined using the Tune.SVM function. The resulting trained SVM was then applied to predict activity for compounds from two libraries in silico, a natural compound library of ∼3200 volatiles and a >440,000 compounds library. For the natural compound library we assembled a subset of 3,197 volatile compounds from defined origins including plants, humans, insects44, food flavours and a fragrance collection45 including fruit and floral volatiles46-53. For the larger library we assembled a subset of >440,000 small molecules from the eMolecules database54 that have properties of volatile odourants. (MW <325 and atoms: C, O, N, H, S). We performed a 5-fold cross-validations by dividing the dataset randomly into 5 equal sized partitions. Four of the partitions were applied to train the SVM and the remaining partition, which was not used for training, was used to test predictive ability. This process is repeated five times, each trial excluding a different subset of compounds as the training set and assigning the remainder as the test set. The whole process is repeated 20 times to improve consistency. A receiver operating characteristics (ROC) analysis is then used to analyze the performance of our computational repellency prediction. The overall predictive ability was calculated as a single receiver operating characteristic (ROC) curve for all 20 independent validations. Calculation of LogP and vapor pressure values SMILES structures of the predicted repellent odours were used with EPI Suite (http://www.epa.gov/oppt/exposure/pubs/episuite.htm) to calculate predicted LogP and Vapour Pressure values. Vinyl solubility test One 3 × 3 mm square of 4 gauge vinyl was submerged in 1mL of each test compound in a glass container and stirred at a constant rate on a shaker and checked every 30 minutes until the vinyl square in DEET was completely dissolved (6 Hrs). The vinyl pieces in each of the other compounds was removed, rinsed in ethanol and weighed. The process was repeated at 30 Hrs (24 Hrs after the vinyl square completely disappeared in DEET). Statistical analyses For behaviour experiments with preference index, arcsine-transformed data were analyzed. Tests used are indicated in the figure legends and they are Students t-test,1-way ANOVA and Tukey's post hoc analysis. Statistical tests for each experimental category and sample trails sizes were selected on the basis of previously published studies using similar assays, which are cited throughout the manuscript. For all graphs, error bars indicate S.E.M. Supplementary Material 2 3 4 5 6 We thank Anindya Ganguly and David Carter for help with calcium imaging; Iliano Coutinho-Abreu for Ir40a sequences, Zev Wisotsky for help with gustatory experiments; Dyan MacWilliam for help with olfactory experiments; Jing Wang for sharing the NFAT transgenic fly line, and Richard Benton for sharing the Ir40a-Gal4 fly line. This work was partly funded by a Whitehall Foundation grant to A.D, R21NS074332 (NINDS) to A.D and A.R, and an R56AI099778 (NIAID) and R01AI087785 (NIAID) to A.R. The granting agencies had no role in experimental design or analysis. Author Contributions S.M.B planned and performed the chemical informatics and solubility experiment, and helped design the behaviour experiments. P.K. planned and performed the NFAT imaging, Ca2+ imaging and Drosophila behaviour experiments. S.K.T. performed Ca2+ imaging, electrophysiology and some behaviour experiments. T.G. performed the arm-in-cage experiments. C.P. performed behaviour analysis. S.M.B, P.K. and S.K.T helped prepare drafts of the manuscript and figures. A.D. planned experiments and helped write the manuscript. A.R. planned experiments, managed the project, and wrote the manuscript. Author Information P.K., S.M.B., C.P. and A.R. are listed as inventors in pending patent applications filed by the University of California Riverside. Readers are welcome to comment on the online version of this article at http://www.nature.com/nature. Figure 1 DEET is detected by Ir40a+ sacculus neurons a, Schematic of the NFAT (CaLexA)-based method to label neurons activated by DEET. b, Confocal micrographs of olfactory organs from flies stimulated with 10% DEET or solvent (acetone). c, Quantification of GFP+ antennae (Top) and mean numbers of GFP+ cells in chamber I. n=35 (blank), n=30 (solvent), n=20 (10%DEET), n=20 (100%DEET). P < 0.0001,1-way ANOVA with Tukey's post hoc test. d, GFP+ axonal termini in antennal lobes of flies treated as indicated. e,f Expression of GFP+ in the labellum, labral sense organ (LSO), the sub-esophageal ganglion (SOG). Anti-GFP (green) and anti-nc82 (red). For SOG, dorsal is top. Figure 2 Ir40a neurons detect DEET and are required for repellency a, Images of calcium activity in Ir40a-Gal4/+;UAS-GCaMP3/+ neurons color-coded as indicated (right). Measurements taken from areas in dashed circles: cells (white), background (red). b, Mean fluorescence intensities for 6 different cells. Red arrowhead indicates onset of ∼2-sec puff of DEET. c, Mean percentage change in fluorescence intensity after application of ∼2-sec indicated stimulus; genotypes were Ir40aGal4/+;UAS-GCaMP3/+ (control) and Ir40aGal4/Ir40aGal4;UAS-GCaMP3/UAS-Ir40aRNAi(2) (Ir40a-RNAi). n=10-13. P < 0.01, Student's t-test. d, Schematic (left) and results (right) for DEET-treated trap assays for indicated genotypes. n=6 trials, 20 flies/trial for each genotype. Letters indicate statistical significance, P ≤ 0.008, 1-way ANOVA with Tukey's post hoc analysis. Error bars=S.E.M. Figure 3 Ir40a is required for DEET avoidance a, Set-up for behavioural 2-choice assay. b,c Mean preference index of indicated genotypes for DEET in 2-choice assays using b, elav-Gal4, and c, Ir40a-Gal4. n=6 trails (20 flies/trial) except elav-Gal4/+;Ir40aRNAi(2) n=10 trials and RNAi experiments with Ir40a-Gal4 n=12 trials each. d, Genotype and schematic for post-developmental knockdown and recovery of Ir40a. e, Mean DEET preference index of flies derived from indicated treatments in 2-choice assays. n=6 trials for all conditions, with 20 flies/trial. For b-e, P < 0.001, 1-way ANOVA with Tukey's post hoc analysis. Error bars=S.E.M. Figure 4 Chemical informatics prediction of new repellents a, Cheminformatics discovery pipeline to identify novel DEET-like repellents. b, Hierarchical cluster analysis of 201 training set odorants using optimized descriptors to calculate distances in chemical space. c, Receiver-operating-characteristic curve (ROC) representing computational validation of repellent predictive ability from 20 independent 5-fold cross validations. AUC=Area under the curve. d, DEET, Picaridin, and two unapproved repellents25. e, Representative predicted repellents from >400,000 odorant library (Left) and computationally determined values for 1000 top-ranked predicted repellents (Right). f, Representative predicted repellents from >3,000 natural odour library (Left) and computationally determined values for 150 top-ranked predicted repellents (Right). Colour arrowheads indicate values for DEET and selected odours shown in g. Figure 5 Predicted repellents activate Ir40a neurons and are strong repellents for Drosophila a, Images of antenna of elav-Gal4/LexAop-CD8-GFP-2A-CD8-GFP; UAS-mLexA-VP16- NFAT, LexAop-CD2-GFP/+ flies exposed to indicated stimuli for 24 hrs. b, BA-activated GFP+ neurons in indicated tissues. c, Mean changes in fluorescence intensity in Ir40aGal4/+;UAS-GCaMP3/+ cells after ∼2-sec application of indicated odorants. n=9-17. d, Mean responses of flies to predicted repellents in 2-choice olfactory and gustatory trap assays measured at 24 and 48 hrs. n=3-10 trials (24 hours) and 7-10 (48 hrs); 10 flies/trial, trials with <40% participation were excluded. e, Quantification of flies of indicated genotypes entering repellent-treated traps. n=6 trials for each genotype, ∼20 flies for each trial. P < 0.001, 1-way ANOVA with Tukey's post hoc test. For c-e, error bars=S.E.M. Figure 6 A new class of mosquito repellents with desirable safety profiles a, Arm-in-cage assay to measure repellency in mosquitoes. b, Mean percentage of female A. aegypti present for >5 sec on top net (Left=10% DEET, Right=solvent). Solvent controls performed separately (dark gray). c, Average time on net for each landing event in b. d, Mean percentage of female A. aegypti present for >5 sec on top net in non-contact assay. e, Cumulative repellency summed across minutes 2-5 of indicated non-contact treatment (10%) in comparison to appropriate solvent control. 40 mosquitoes/trial, n=5 trials/treatment for b,c,d, and e. f, Mean weight of vinyl pieces following submersion in indicated compounds for indicated amount of time. n=3, *P < 10-5, Student's t-test. 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==== Front J Cell Mol MedJ. Cell. Mol. MedjcmmJournal of Cellular and Molecular Medicine1582-18381582-4934John Wiley & Sons, Ltd Chichester, UK 1712559310.1111/j.1582-4934.2006.tb00533.xOriginal ArticleDifferential proteomic profiling to study the mechanism of cardiac pharmacological preconditioning by resveratrol Bezstarosti Karel bDas Samarjit aLamers Jos M J bDas Dipak K aa Cardiovascular Research Center, University of Connecticut School of MedicineFarmington, CT, USAb Department of Biochemistry, Cardiovascular Research School COEUR, Erasmus Medical CenterRotterdam, The Netherlands Correspondence to: Dipak K. DAS, Ph.D., FAHA Cardiovascular Research Center, University of Connecticut, School of Medicine, Farmington, CT 06030-1110, USA. Tel.: (860) 679-3687; Fax: (860) 679-4606 E-mail: [email protected] 2006 09 10 2008 10 4 896 907 25 9 2006 01 11 2006 Recent studies demonstrated that resveratrol, a grape-derived polyphenolic phytoalexin, provides pharmacological preconditioning of the heart through a NO-dependent mechanism. To further explore the molecular mechanisms involved in resveratrol-mediated cardioprotection, we monitored the effects of resveratrol treatment after ischemia-reperfusion on the protein profile by implementation of proteomic analysis. Two groups of rats were studied; one group of animals was fed resveratrol for 7 days, while the other group was given vehicle only. The rats were sacrificed for the isolated working heart preparation and for isolation of cytoplasmic fraction from left ventricle homogenates to carry out the proteomic as well as immunoblot at baseline and at the end of 30 min ischemia/2-h perfusion. The results demonstrate significant cardiopro-tection with resveratrol evidenced by improved ventricular recovery and reduced infarct size and cardiomyocyte apopto-sis. The left ventricular cytoplasmic fractions were separated by two-dimensional electrophoresis (2-DE). Differentially regulated proteins were detected with quantitative computer analysis of the Coomassie blue stained 2-DE images and identified by MALDI-TOF (MS) and nanoLC-ESI-Q-TOF mass spectrometry (MS/MS). Five redox-regulated and precondi-tioning-related proteins were identified that were all upregulated by resveratrol: MAPKK, two different aB-crystallin species, HSP 27 and PE binding protein. Another HSP27 species and aldose reductase were downregulated and peroxire-doxin-2 remained constant. The results of the immunoblot analysis of phosphorylated MAPKK, -HSP27 and -aB-crys-tallin and PE binding protein were consistent with the proteomic findings, but not with peroxiredoxin-2. The proteomic analysis showed also downregulation of some proteins in the mitochondrial respiratory chain and matrix and the myofila-ment regulating protein MLC kinase-2. The results of the present study demonstrate that proteomic profiling enables the identification of resveratrol induced preconditioning-associated proteins which reflects not only changes in their expression level but also isoforms, post-translational modifications and regulating binding or activating partner proteins. ischemia/reperfusionheartresveratrolproteomicsαB-crystallinhsp27phosphatidylethanolamine binding protein ==== Refs References 1 Bradamante S Piccinini F Barenghi L Bertelli AA De Jonge R Beemster P De Jong JW Does resveratrol induce pharmacological preconditioning Int J Tissue React 2000 2 1 4 10937348 2 Hung LM Chen JK Huang SS Lee RS Su MJ Cardioprotective effect of resveratrol, a natural antioxidant derived from grapes Cardiovasc Res 2000 47 549 55 10963727 3 Hattori R Otani H Maulik N Das DK Pharmacological preconditioning with resveratrol: role of nitric oxide Am J Physiol Heart Circ Physiol 2002 282 H1988 95 12003802 4 Kopp P Resveratrol, a phytoestrogen found in red wine. A possible explanation for the conundrum of the ’French paradox’ Eur J Endocrinol 1998 138 619 20 9678525 5 Das DK Maulik N Resveratrol in cardioprotection: a therapeutic promise of alternative medicine Mol Interv 2006 6 36 47 16507749 6 Imamura G Bertelli AA Bertelli A Otani H Maulik N Das DK Pharmacological preconditioning with resveratrol: an insight with iNOS knockout mice Am J Physiol Heart Circ Physiol 2002 282 H1996 2003 12003803 7 Das S Alagappan VK Bagchi D Sharma HS Maulik N Das DK Coordinated induction of iNOS-VEGF-KDR-eNOS after resveratrol consumption: a potential mechanism for resveratrol preconditioning of the heart Vascul Pharmacol 2005 42 281 9 15905131 8 Hung LM Su MJ Chen JK Resveratrol protects myocardial ischemia-reperfusion injury through both NO-dependent and NO-independent mechanisms Free Radic Biol Med 2004 36 774 81 14990356 9 Das S Tosaki A Bagchi D Maulik N Das DK Resveratrol-mediated activation of cAMP response element-binding protein through adenosine A3 receptor by Akt-dependent and -independent pathways J Pharmacol Exp Ther 2005 314 762 9 15879002 10 Das S Cordis GA Maulik N Das DK Pharmacological preconditioning with resveratrol: role of CREB-dependent Bcl-2 signaling via adenosine A3 receptor activation Am J Physiol Heart Circ Physiol 2005 288 H328 35 15345477 11 Faber MJ Agnetti G Bezstarosti K Lankhuizen IM Dalinghaus M Guarnieri C Caldarera CM Helbing WA Lamers JM Recent developments in proteomics: implications for the study of cardiac hypertrophy and failure Cell Biochem Biophys 2006 44 11 29 16456231 12 Faber MJ Dalinghaus M Lankhuizen IM Bezstarosti K Dekkers DH Duncker DJ Helbing WA Lamers JM Proteomic changes in the pressure overloaded right ventricle after 6 weeks in young rats: correlations with the degree of hypertrophy Proteomics 2005 5 2519 30 15912512 13 McGregor E Dunn MJ Proteomics of the heart: unraveling disease Circ Res 2006 98 309 21 16484627 14 Van Eyk JE Proteomics: unraveling the complexity of heart disease and striving to change cardiology Curr Opin Mol Ther 2001 3 546 53 11804269 15 Engelman DT Watanabe M Engelman RM Rousou JA Kisin E Kagan VE Maulik N Das DK Cardiovasc Res 1995 29 133 140 7895231 16 Sato M Cordis GA Maulik N Das DK SAPKs regulation of ischemic preconditioning Am J Physiol Heart Circ Physiol 2000 279 H901 7 10993748 17 Maulik N Sasaki H Addya S Das DK Regulation of cardiomyocyte apoptosis by redox-sensitive transcription factors FEBS Lett 2000 485 7 12 11086156 18 Wang X Li N Liu B Sun H Chen T Li H Qiu J Zhang L Wan T Cao X A novel human phosphatidylethanolamine-binding protein resists tumor necrosis factor alpha-induced apoptosis by inhibiting mitogen-activated protein kinase pathway activation and phos-phatidylethanolamine externalization J Biol Chem 2004 279 45855 64 15302887 19 Das DK Redox regulation of cardiomyocyte survival and death Antioxid Redox Signal 2001 3 23 37 11291596 20 Ray PS Martin JL Swanson EA Otani H Dillmann WH Das DK Transgene overexpression of alphaB crystallin confers simultaneous protection against cardiomyocyte apoptosis and necrosis during myocardial ischemia and reperfusion FASEB J 2001 15 393 402 11156955 21 Uchiyama T Engelman RM Maulik N Das DK Role of Akt signaling in mitochondrial survival pathway triggered by hypoxic preconditioning Circulation 2004 109 3042 9 15184284 22 Arrell DK Neverova I Fraser H Marban E Van Eyk JE Proteomic analysis of pharmacologically preconditioned cardiomyocytes reveals novel phosphorylation of myosin light chain 1 Circ Res 2001 89 480 7 11557734 23 Arrell DK Neverova I Van Eyk JE Cardiovascular pro-teomics: evolution and potential Circ Res 2001 88 763 73 11325867 24 Lam L Lind J Semsarian C Application of proteomics in cardiovascular medicine Int J Cardiol 2006 108 12 9 16466817 25 Das S Tosaki A Bagchi D Maulik N Das DK Potentiation of a survival signal in the ischemic heart by resveratrol through p38 mitogen-activated protein kinase/mitogen- and stress-activated protein kinase 1/cAMP response element-binding protein signaling J Pharmacol Exp Ther 2006 317 980 8 16525036 26 Kutuk O Poli G Basaga H Resveratrol protects against 4-hydroxynonenal-induced apoptosis by blocking JNK and c-JUN/AP-1 signaling Toxicol Sci 2006 90 120 32 16322078 27 Maulik N Watanabe M Zu YL Huang CK Cordis GA Schley JA Das DK Ischemic preconditioning triggers the activation of MAP kinases and MAPKAP kinase 2 in rat hearts FEBS Lett 1996 396 233 7 8914993 28 Pataki T Bak I Kovacs P Bagchi D Das DK Tosaki A Grape seed proanthocyanidins improved cardiac recovery during reperfusion after ischemia in isolated rat hearts Am J Clin Nutr 2002 75 894 9 11976164 29 Yeung K Seitz T Li S Janosch P McFerran B Kaiser C Fee F Katsanakis KD Rose DW Mischak H Sedivy JM Kolch W Suppression of Raf-1 kinase activity and MAP kinase signalling by RKIP Nature 1999 401 173 7 10490027 30 Ping P Identification of novel signaling complexes by functional proteomics Circ Res 2003 93 595 603 14525921 31 Vondriska TM Ping P Multiprotein signaling complexes and regulation of cardiac phenotype J Mol Cell Cardiol 2003 35 1027 33 12967625 32 Edmondson RD Vondriska TM Biederman KJ Zhang J Jones RC Zheng Y Allen DL Xiu JX Cardwell EM Pisano MR Ping P Protein kinase C epsilon signaling complexes include metabolism- and transcription/translation-related proteins: complimentary	17125593	PMC3933086	NO-CC CODE	2021-01-05 03:50:49	yes	J Cell Mol Med. 2006 Oct 9; 10(4):896-907
==== Front 980967121092Nat NeurosciNat. Neurosci.Nature neuroscience1097-62561546-17262416265510.1038/nn.3560nihpa560026ArticleTGF-β Signaling Regulates Neuronal C1q Expression and Developmental Synaptic Refinement Bialas Allison R. 12Stevens Beth 121 Department of Neurology, F.M. Kirby Neurobiology Center, Boston Children's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA2 Program in Neuroscience, Harvard Medical School, Boston, Massachusetts 02115, USAAll correspondence should be addressed to B.S. [email protected] 3 2014 27 10 2013 12 2013 01 6 2014 16 12 1773 1782 Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:http://www.nature.com/authors/editorial_policies/license.html#termsImmune molecules, including complement proteins C1q and C3, have emerged as critical mediators of synaptic refinement and plasticity. Complement localizes to synapses and refines the developing visual system via C3-dependent microglial phagocytosis of synapses. Retinal ganglion cells (RGCs) express C1q, the initiating protein of the classical complement cascade, during retinogeniculate refinement; however, the signals controlling C1q expression and function remain elusive. Previous work implicated an astrocyte-derived factor in regulating neuronal C1q expression. Here we identify retinal TGF-β as a key regulator of neuronal C1q expression and synaptic pruning in the developing visual system. Mice lacking TGF-β receptor II (TGFβRII) in retinal neurons have reduced C1q expression in RGCs, reduced synaptic localization of complement, and phenocopy refinement defects observed in complement-deficient mice, including reduced eye specific segregation and microglial engulfment of RGC inputs. These data implicate TGF-β in regulating neuronal C1q expression to initiate complement- and microglia-mediated synaptic pruning. ==== Body Increasing evidence implicates immune molecules in synapse development and refinement. Several molecules best known for their functions in the immune system, including MHC class I1, neuronal pentraxins2, and complement3, mediate synaptic remodeling in the developing mouse brain, yet surprisingly little is known about the signals regulating the expression and function of these immune molecules at developing synapses. Classical complement cascade proteins are components of the innate immune system that mediate developmental synaptic pruning, a process critical for the establishment of precise synaptic circuits. Complement proteins, C1q and C3, are expressed in the postnatal brain and localize to subsets of synapses during synaptic remodeling in the mouse retinogeniculate system3--a classic model for studying developmental synapse elimination. Early in postnatal development, retinal ganglion cells (RGCs) form transient functional synaptic connections with relay neurons in the dorsal lateral geniculate nucleus (dLGN). Prior to eye opening (~P14), many of these transient retinogeniculate synapses are eliminated while the remaining synaptic arbors are elaborated and strengthened4–6. C1q−/− and C3−/− mice exhibit sustained defects in synaptic refinement and elimination as shown by the failure to segregate into eye-specific territories and the retention of multi-innervated dLGN relay neurons3. However, the signals regulating complement-mediated pruning during development remain poorly understood. As the initiator of the classical complement cascade, C1q is a critical point of regulation in this pathway. C1q, a large secreted protein composed of C1qA, C1qB, and C1qC peptide chains, is the recognition domain of the initiating protein, C1, in the classical complement cascade. In the immune system, binding of C1q to apoptotic cell membranes or pathogens triggers a proteolytic cascade of downstream complement proteins, resulting in C3 opsonization and phagocytosis by macrophages that express complement receptors. The function of complement proteins in the brain appears analogous to their immune system function: clearance of cellular material that has been “tagged” for elimination. Consistent with the well-ascribed role of complement proteins as opsonins or “eat me” signals, C1q and C3 localize to retinogeniculate synapses, and presynaptic terminals of retinal ganglion cells are similarly eliminated by phagocytic microglia expressing complement receptors. Genetic deletion of C1q, C3 or the microglia-specific complement receptor, CR3 (CD11b) results in sustained defects in eye-specific segregation7, suggesting that these molecules function in a common pathway to refine synaptic circuits3,7. Importantly, microglial engulfment of retinogeniculate inputs occurs during narrow window of postnatal development (P5–P8) coincident with retinal C1q expression3, suggesting that complement-dependent synaptic pruning may be initiated by C1q in the developing brain. Remarkably little is known about the signals controlling C1q expression and function in the brain. In contrast to microglia, which express C1q continuously throughout development, C1q expression in RGCs is developmentally restricted to the early postnatal period when RGC axons undergo synaptic pruning3. Given the developmental expression of C1q in RGC neurons, we hypothesize RGC-derived C1q plays a key role in refinement of RGC synapses onto their target neurons in the dLGN. In the current study, we sought to identify the signals regulating C1q expression in RGCs and to determine whether retinal C1q is required to initiate downstream complement-dependent synaptic refinement and microglia-mediated synaptic pruning in the dLGN. A screen investigating how astrocytes influence neuronal gene expression first identified the C1q genes among the few genes that were highly upregulated in developing RGCs in response to astrocytes3. The presence of immature astrocytes in the retina corresponds to C1q expression in RGCs in vivo, suggesting that the astrocyte-derived factor that upregulates C1q in RGC cultures may also regulate postnatal C1q expression in RGCs in vivo. Each of the three C1q genes (c1qa, c1qb, and c1qc) were found to be highly upregulated in purified RGCs upon exposure to a feeder layer of astrocytes3, implicating a secreted factor; however, the identity of the astrocyte-derived signal(s) that regulate C1q expression has remained unknown. In the immune system, expression of complement and other immune genes can be modulated by rapid cytokine signaling pathways that regulate the inflammatory response. In the developing brain, astrocytes are a major source of cytokines, several of which potently regulate synapse development and function8–11. In the current study, we identified transforming growth factor beta (TGF-β) as the factor secreted by astrocytes necessary and sufficient for C1q expression in purified RGCs. TGF-β ligands are expressed in the retina during the refinement period and TGF-β receptors (TGFβRII) are developmentally expressed in the postnatal RGCs in vivo. Blocking TGF-β signaling in retinal neurons significantly reduced C1q expression in postnatal RGCs and decreased synaptic localization of complement in the dLGN in vivo. Moreover, specific disruption of TGFβRII in retinal neurons inhibited complement-and microglia-mediated synaptic pruning in the dLGN, suggesting that TGF-β-dependent regulation of neuronal C1q in the retina regulates downstream complement-dependent synapse elimination in the dLGN. Taken together, our data reveal a novel role for the TGF-β cytokine signaling pathway in regulating C1q expression in neurons and in initiating complement-dependent synaptic refinement in the developing CNS. Results C1q upregulation by a secreted factor is rapid and direct Previous findings suggested that an astrocyte-derived factor triggers neuronal C1q expression3. Consistent with previous findings, we found that purified RGC neurons significantly upregulated the C1q genes (c1qa, c1qb, and c1qc) upon chronic exposure (6 days) to cortical astrocytes grown on tissue culture inserts suspended above cultured RGCs (Fig. 1a). To determine if C1q upregulation in RGCs was dependent on indirect, bidirectional signaling between astrocytes and neurons, we measured C1q mRNA levels in purified postnatal (P8) RGC cultures (DIV 4) treated with conditioned medium (ACM) collected from cortical astrocytes. Treating RGCs with ACM or astrocyte inserts resulted in a comparable 10–20 fold upregulation of C1q in RGCs (Fig. 1a), suggesting that an astrocyte-secreted factor directly upregulates neuronal C1q expression. Moreover, ACM collected from purified postnatal (P8) retinal astrocyte cultures12 stimulated a similar upregulation compared to purified or standard cortical astrocytes (Fig. 1a), suggesting that the astrocyte-derived factor that upregulates C1q in RGC cultures may also regulate C1q expression in RGCs in vivo. Our results support a direct astrocyte-to-neuron signaling pathway in C1q upregulation; however, chronic exposure to astrocytes promotes many developmental changes in neurons, including robust increases in synapse number and neuronal activity9,13. To determine the timecourse for C1q upregulation, we measured C1q mRNA levels by quantitative PCR (qPCR) following ACM treatment (15 min. – 3 days). Surprisingly, RGCs upregulated C1q after only 15 minutes of ACM treatment, further supporting that ACM directly triggers C1q upregulation (Fig. 1b). Moreover, RGCs rapidly upregulated C1r and C1s, the enzymes that associate with C1q to initiate the complement cascade (Supplemental Fig. 1a). This rapid upregulation occurred only in neurons and not in microglia or astrocytes treated with ACM for 15 minutes, suggesting differential regulatory mechanisms in neurons and glia (Fig. 1c). C1q upregulation was blocked by actinomycin, a transcriptional inhibitor, confirming that this effect was a result of transcription (Supplemental Fig. 1b). Furthermore, treating cells with boiled ACM prevented C1q upregulation, implicating a protein in ACM in neuronal C1q upregulation (Supplemental Fig. 1c). In addition to the upregulation of C1q mRNA, we observed a corresponding increase in C1q protein levels as measured by immunocytochemistry for C1q (Rabbit anti-C1qA14) in purified RGC cultures treated with ACM (6 hours) (Fig. 1e,f) and by western blot analysis of media samples from ACM-treated RGC cultures (1 week) (Fig. 1d). Media samples of ACM alone or from RGCs treated with ACM acutely showed undetectable levels of C1q by western blot (data not shown), indicating that C1q produced by RGCs accumulates in the media over time in response to ACM. Thus, we used this purified culture system as a robust assay to screen for secreted signals that rapidly upregulate and sustain C1q expression in RGCs. TGF-β is necessary and sufficient for C1q upregulation To identify the astrocyte-derived factor responsible for C1q upregulation, we screened several candidate molecules for the ability to upregulate C1q in purified RGC neuronal cultures. Cytokines, potent modulators of immune and neural function, were the main class of molecule tested since astrocytes are a major source of cytokines in the brain and several cytokines elicit systemic increases in C1q in the bloodstream. We first measured the levels of several candidate cytokines in ACM by ELISA (MSD mouse 7-plex inflammatory cytokine assay and individual ELISAs for TGF-β1, 2, and 3) (Fig. 2a). Several cytokines were enriched in ACM including CXCL1, IL-12, and transforming growth factor beta (TGF-β) 1, 2, and 3. To determine which cytokines were required for astrocyte-dependent C1q upregulation in RGC neurons, we immunodepleted individual cytokines from ACM using specific antibodies. Two of the cytokines most enriched in ACM, CXCL1 and IL-12, showed no effect on C1q expression when added to cultures as recombinant protein (Supplemental Fig. 1d,e), thus immunodepletion of these cytokines was not performed. Although TNFα and IL-6 induced modest C1q upregulation when added directly to RGC cultures (Supplemental Fig. 1f), immunodepletion of these cytokines using neutralizing antibodies (R&D systems) did not affect ACM-induced C1q upregulation (Fig. 2b). In contrast, specific immunodepletion of TGF-β from ACM using an anti-pan TGF-β neutralizing antibody (1D11, R&D Systems) prevented ACM-induced C1q upregulation after 15 min. treatment as measured by qPCR (Fig. 2b). We verified TGF-β immunodepletion from ACM by ELISA (Fig. 2c). Together these data showed that TGF-β is necessary to upregulate neuronal C1q in purified RGC cultures. To determine which TGF-β isoform was upregulating C1q, we specifically depleted individual TGF-β isoforms from ACM using antibodies specific to TGF-β1, 2, or 3 (R&D Systems). Interestingly, only ACM depleted of TGF-β3 failed to upregulate C1q in RGCs, suggesting that this isoform may be key in regulating neuronal C1q (Fig. 2d). Furthermore, we generated concentration-response curves (50ng/ml-25pg/ml) in RGC cultures for each of the TGF-β isoforms and measured C1q expression by qPCR. TGF-β3 most effectively upregulated C1q in RGCs at concentrations similar to those measured in ACM, whereas TGF-β2 modestly upregulated C1q at high concentrations (Fig. 2e). In addition, glycine elution to release bound TGF-β from anti-pan or anti-TGF-β3 neutralizing antibodies produced an eluate which could upregulate C1q to the same extent as ACM when added to RGC cultures (Fig. 2f). We also found that retinal astrocytes show enrichment for TGF-β3 compared with RGCs, cortical astrocytes, and microglia (Supplemental Fig. 1g). However, treating retinal astrocyte or microglial cultures with recombinant TGF-β3 (50pg/ml, 15 min.) failed to induce C1q upregulation (Fig. 2g), consistent with our findings with ACM treatment (Fig. 1c). Taken together, these results demonstrate that, of the cytokines tested, TGF-β a major component in ACM responsible for C1q upregulation in RGCs, but not in microglia or astrocytes (Fig. 2g). Furthermore, our findings suggest that TGF-β3 may be the key isoform regulating retinal C1q expression (Fig. 2d,f). We next addressed whether TGF-β receptor signaling in RGCs is required for C1q upregulation in RGCs. In the canonical TGF-β signaling pathway, TGF-β1, 2, or 3 binds to TGFβRII, which then phosphorylates TGFβRI. Once activated, TGFβRI phosphorylates a Smad transcription factor to alter gene expression. A third receptor, TGFβRIII, can also associate with TGFβRII and alters ligand affinity15. Purified RGCs express all of the components required for TGF-β signaling (Fig. 2h,i). Furthermore, treating RGCs with ACM stimulated rapid (15–30 min.) nuclear accumulation of phosphorylated Smads (pSmad 2/3), as measured by immunocytochemistry for phospho-Smad (Fig. 2h), consistent with the timing of C1q upregulation. In addition, transcripts for TGFβRII, TGFβRIII, and TGFβRI were present in purified RGCs (Fig. 2i). These data indicate that RGCs express functional TGF-β receptors that can be activated with a timecourse consistent with C1q upregulation. To determine whether the TGF-β receptors, I and II, were responsible for this upregulation, we pre-incubated RGC cultures with specific TGFβRI and II inhibitors prior to acute treatment with either TGF-β3 (50 pg/ml) or ACM. Adding TGFβRII neutralizing antibodies (R&D Systems) or a TGFβRI-specific inhibitor16 (SB431542, Tocris, 30µM) blocked both TGF-β-induced and ACM-induced C1q upregulation (Fig. 2j). Thus, signaling through TGFβRI and II is required for ACM-induced C1q upregulation in purified RGCs. TGF-β regulates developmental C1q expression in the retina C1q is developmentally regulated in the postnatal retina, showing peak expression around P5 and sharply decreasing by P10–P153, corresponding to the synaptic refinement period in the retinogeniculate system and the presence of immature astrocytes throughout the developing CNS (Supplemental Fig. 2a). To determine if TGF-β signaling regulates developmental C1q expression in RGCs in vivo, we examined the expression of TGF-β signaling components during the refinement period. Expression of all three isoforms of TGF-β was detected by RT-PCR at P5 and in the mature retina (Fig. 3a). In addition, TGFβRII protein was detected in retinal lysates by western blot (Fig. 3b). Of the three ages examined, the level of TGFβRII protein was highest at P5 and sharply decreased by P15 and P28 (Fig. 3b,c), coinciding with C1q expression in the postnatal retina (Supplemental Fig. 2a). Consistent with western blot analysis, immunohistochemistry for TGFβRII showed TGFβRII localization to the RGC and inner plexiform layers (IPL) of the retina at P5 and TGFβRII levels decreased by P15 (Fig. 3d). Co-staining with anti-calretinin (Millipore), a marker of a subset of RGCs, confirmed TGFβRII localization to RGCs (Supplemental Fig. 2b). Taken together, these data show that TGF-β ligands and receptors are present at the right time in the retina to regulate C1q expression. To determine if TGF-β signaling regulates C1q expression in vivo during retinogeniculate refinement, we used genetic and pharmacological approaches to block TGF-β signaling and then measured C1q expression in vivo. Retina-specific TGFβRII−/− mice were generated by crossing floxed TGFβRII mice with CHX10-Cre mice that express Cre in all retinal neurons at E13.517,18. TGFβRII knockout was confirmed by immunostaining retinas (Fig. 3d) and performing RT-PCR using mRNA from WT and transgenic P5 whole brain (Supplemental Fig. 2c) and purified RGCs acutely isolated from P5 WT and transgenic mice (Fig. 3e). TIEG, a known TGF-β-dependent gene, also showed reduced expression in P5 acutely isolated RGCs (Supplemental Fig. 2d). Moreover, while total Smad2 levels were not significantly different between WT and retinal TGFβRII−/− mice (Fig. 3f), pSmad 2/3 levels, corresponding to active TGF-β signaling, were reduced in Brn3a-positive RGCs (Fig. 3g), further indicating that the TGFβRII retina-specific knockout was achieved. Since TGF-β signaling has many roles in development, we measured cell numbers, axon density, and axon caliber and found no significant difference from WT retinas (Supplemental Fig. 2e–h). Given that there were no gross abnormalities in retinal development, we used this mouse to analyze the role of TGF-β signaling in C1q regulation and synaptic refinement. To determine if retinal TGF-β signaling is required for developmental C1q expression in RGCs, we assayed C1q expression levels in retinas and RGCs from retinal TGFβRII−/− mice relative to WT littermates, and from mice receiving intraocular injection of anti-TGF-β relative to vehicle treated littermates. In situ hybridization for c1qb revealed a decrease in signal for c1qb in the RGC layer in retinal TGFβRII−/− retinas (P5) compared to WT littermates (Fig. 4a; anti-TGF-β: Supplemental Fig. 3a). To verify reduction in C1q expression was in RGCs and not other cell types in the RGC layer, we acutely isolated RGCs and microglia from P5 retinas using established immunopanning techniques12,19. Acutely isolated RGCs from the retinal TGFβRII−/− showed a significant reduction in C1q expression by qPCR while acutely isolated retinal microglia showed no change in C1q levels (Fig. 4b), as expected given that microglia do not express the retinal neuron-specific gene, chx10. Similarly, RGCs acutely isolated from P5 mice receiving intraocular injection of anti-TGF-β (injected at P3) also showed significantly reduced C1q expression, while microglia again showed no change in C1q (Supplemental Fig. 3b). We verified cell purity after immunopanning using qPCR for the neuron-specific gene, nse, and the microglia-specific gene, iba1 (Supplemental Fig. 3c). Moreover, immunohistochemistry for C1q at P5 in retinal TGFβRII−/− (Fig. 4c,d) and anti-TGF-β injected mice (Supplemental Fig. 3d,e) also showed a 40–50% reduction in C1q immunostaining in the IPL and RGC layers of the retina, but showed no change in microglial staining. Taken together, these data show that TGF-β signaling is required for postnatal C1q expression in RGCs. These results also suggest that TGF-β regulation of C1q may be unique to neurons, as suggested by our earlier in vitro data (Fig. 1c). Retinal TGF-β regulates complement levels in the thalamus C1q and C3 localize to synapses in the dLGN during the period of synaptic refinement, but whether RGC neurons are a key source of C1q in the dLGN is not known. One possibility is that neuronal C1q is locally secreted from RGC axons into the dLGN to initiate complement-dependent synaptic pruning. Alternatively, microglia, although sparse in the dLGN at this time, may be a primary source of secreted C1q in the postnatal dLGN during refinement. To address this question, we examined C1q protein levels by immunohistochemistry in cross sections of postnatal (P5) optic nerve. We found that C1q protein could be detected in optic nerve fiber bundles, identified by TUJ1 staining. Moreover, C1q immunoreactivity in the optic nerve was significantly reduced in retinal TGFβRII−/− mice versus WT littermates (Fig. 4e,f), suggesting that TGF-β signaling in RGCs regulates the levels of C1q in RGC axons in the optic nerve. If C1q in the dLGN is supplied by RGCs, we predicted that retinal TGFβRII−/− mice would have a reduction in C1q protein levels throughout the dLGN. Indeed, immunohistochemisy for C1q revealed a significant decrease in the intensity of C1q staining in the dLGN but not in primary visual cortex (V1) (Fig. 5a), which is not a direct target for RGCs. Quantification showed a significant reduction in fluorescence intensity in the retinal TGFβRII−/− as well as a significant decrease in the density of C1q puncta in the dLGN but not in V1 (Fig. 5b,c), indicating that RGC axons are a key source of C1q in the postnatal dLGN. Given previous findings that C1q and C3 localize to synapses in the dLGN, we examined synaptic localization of complement in the dLGN in retinal TGFβRII−/− mice and WT littermates to determine if synaptic localization of these proteins is dependent on retinal TGF-β signaling. Immunostaining in the dLGN using antibodies for C1q, C3, and vglut2 to label RGC terminals showed approximately 15% of vglut2-positive puncta co-labeled with C1q (Fig. 5d,e) and, ~25% of vglut2 puncta co-stained with the downstream complement protein, C3 (Fig. 5f,g). The increase in the number of RGC terminals labeled with C3 versus C1q is consistent with the placement of these molecules in the classical complement cascade, since multiple C3 molecules can be activated downstream of a single C1q molecule. Also, as expected given that C1q is upstream of C3 activation in the complement cascade, we found that C3 deposition in the dLGN was undetectable in the C1q−/− mouse (Supplemental Fig. 3f). In retinal TGFβRII−/− dLGNs, we also observed a significant reduction in synaptic localization of both C1q and C3 (Fig. 5d–g). Given that retinal TGFβRII−/− exhibit specific reduction in TGF-β signaling and C1q in the retina, a reduction in C1q and C3 synaptic localization in the dLGN suggests that a significant fraction of synaptically localized C1q is supplied by RGC terminals and that C1q and C3 localization to synapses is dependent on retinal TGF-β signaling. Thus, local microglia-derived C1q in the dLGN does not appear to localize to synapses to compensate for the loss of RGC-derived C1q. TGF-β signaling is required for eye specific segregation In C1q−/− mice, RGC axons do not segregate into eye specific territories normally as assessed by anterograde tracing techniques3. If C1q supplied by RGC axons is required for eye specific segregation and retinal TGF-β signaling regulates C1q expression, then retinal TGFβRII−/− mice should exhibit a phenotype similar to C1q−/− mice. To test this hypothesis, we used established anterograde tracing techniques to visualize the formation of eye specific territories in the dLGN and to assay eye specific segregation in retinal TGFβRII−/− mice and in mice injected with anti-TGF-β. Mice received intraocular injections of cholera toxin beta conjugated to Alexa 488 or 594 (CTB-488 or CTB-594) at P9 in the left and right eyes respectively and mice were sacrificed at P10. We assayed eye specific segregation using an unbiased assay in which the degree of segregation is represented by the variance of the distribution of the logarithm of the ratio of fluorescence intensity from each fluorescence channel (R value)20. Using this assay, high variance signifies a high degree of segregation in the dLGN, while low variance corresponds to increased overlap between contralateral and ipsilateral territories. Our results showed a significantly lower variance in C1q−/− mice compared to WT controls, as expected (Fig. 6a,b). Importantly, we observed a similar phenotype in retinal TGFβRII−/− (Fig. 6a,b) and mice injected with anti-TGF-β (Fig. 6a,c), suggesting a common pathway. Defects in eye-specific segregation were confirmed by quantifying the percent overlap using the original method used to identify the C1q−/− phenotype (Fig. 7a,b). No differences were observed in dLGN area (Fig. 7c) or in dLGN relay neuron number as determined by SMI32 staining (data not shown) in retinal TGFβRII−/− mice compared to WT. Our results showed that retinal TGF-β is required for eye specific segregation, but it was not clear whether this effect was through regulation of C1q. To determine if TGF-β could regulate synaptic refinement independently of C1q, we performed intraocular injection of anti-TGF-β neutralizing antibodies in WT and C1q−/− animals to determine if blocking TGF-β disrupted synaptic refinement in the absence of C1q. If TGF-β and C1q regulate pruning by different mechanisms, blocking TGF-β in C1q−/− mice should produce a more severe segregation defect, or increased overlap compared to vehicle-injected C1q−/− mice. If TGF-β and C1q are in the same pathway, blocking TGF-β signaling in the C1q−/− mouse should have no effect. Blocking TGF-β in the C1q−/− mouse did not result in a more severe phenotype, consistent with TGF-β exerting its effect on synaptic refinement via regulation of C1q (Fig. 6a–c; 7a,b). Taken together, these results show that retinal TGF-β signaling is required for eye specific segregation and that TGF-β and C1q likely work in the same pathway to regulate this process. Retinal TGF-β and C1q regulate microglia-mediated pruning Microglia engulfment of retinogeniculate synapses is thought to be the final step in complement-dependent synapse elimination. Recent work has demonstrated that microglia engulf RGC terminals in the dLGN during the pruning period in a complement (C3/CR3)-dependent manner7. Therefore, if RGC-derived C1q regulates this process, we predict retinal TGFβRII−/− mice to show a reduction in microglial engulfment of RGC terminals. To test this hypothesis, we used an established microglia engulfment assay7 to quantify microglial phagocytosis of RGC inputs in C1q−/− mice and retinal TGFβRII−/− mice. We found a significant reduction in microglial engulfment of RGC inputs at P5 in C1q−/− and TGFβRII−/− mice as compared to littermate controls (Fig. 6d). We found no differences in the number, distribution, or activation state of microglia in the dLGN in WT vs. C1q−/− or TGFβRII−/− conditions (supplemental Fig. 4a–d). These results show that reduction of retinal TGF-β signaling, and consequently of C1q expression in RGCs, significantly reduced microglial phagocytosis of RGC terminals in the dLGN. Together these data support the hypothesis that RGC-derived C1q is required to initiate the process of complement-dependent synapse elimination by microglia. Discussion This study establishes that TGF-β signaling in RGC neurons plays a key role in refinement of RGC synapses on their target relay neurons in the dLGN by regulating expression of C1q, the initiating protein of the classical complement cascade. We demonstrated that TGF-β signaling is necessary and sufficient for transcriptional upregulation of C1q in purified RGCs (Fig. 2). Consistent with in vitro studies, conditional knockout of TGFβRII in retinal neurons reduced C1q expression in RGCs during the period of active refinement of retinogeniculate synapses in the thalamus (Fig. 4). Furthermore, our data show that C1q and C3 localization to synapses in the dLGN during retinogeniculate refinement is dependent on retinal TGF-β signaling (Fig. 5). Importantly, inhibition of TGF-β signaling in the postnatal retina resulted in significant defects in eye specific segregation in the dLGN, mimicking the phenotype observed in global C1q−/− mice (Fig. 6a–c). We also observed defects in microglia engulfment of RGC terminals (Fig. 6d), which phenocopied the C3−/− and CR3−/− mouse microglial engulfment defects7. Taken together, our findings support a model in which retinal TGF-β signaling controls C1q expression and local release in the dLGN to regulate microglia-mediated, complement-dependent synaptic refinement (Supplemental Fig. 5). C1q regulates microglia-mediated pruning in the thalamus Emerging evidence implicates microglia in developmental synaptic refinement7,21,22; however, the mechanisms controlling the timing and location of microglia-mediated synaptic refinement in the brain are poorly understood. Process-bearing, phagocytic microglia in the postnatal dLGN engulf RGC inputs during retinogeniculate refinement in a manner dependent on complement C3-CR3 signaling and neuronal activity7. Upon complement cascade activation, C3 is cleaved to form the potent opsonin, C3b, a ligand for the phagocytic complement receptor CR3 expressed in the brain by microglia exclusively. Thus, as the initiator of the classical complement cascade, C1q is a critical regulator of C3 activation and C3-dependent phagocytosis. Our results suggest that C3 localization to synapses (Fig. 5) and microglia-mediated pruning (Fig. 6) are dependent on retina-derived C1q. Specifically, we observed a significant decrease in C3 localization to synapses in C1q−/− and in the retinal TGFβRII−/− mice. We also found that microglial engulfment of RGC terminals is decreased in C1q−/− (Fig. 6d) and in the retinal TGFβRII−/− mice (Fig. 6d), placing retina-derived C1q and retinal TGF-β signaling upstream of this process. Interestingly, recent work revealed that microglia-mediated pruning is regulated by neuronal activity7. During the retinogeniculate pruning period, microglia preferentially engulf less active RGC inputs in the dLGN7, suggesting that microglia can sense or read out local changes in synaptic activity. The engulfment defects observed in complement-deficient mice and the role for complement in the immune system suggest the intriguing possibility that complement proteins could be one cue guiding microglia to engulf less active synapses. Other immune molecules implicated in synaptic refinement, such as MHC class I23 and neural pentraxins24, show activity-dependent transcriptional regulation, but whether these molecules have any role in activity-dependent microglial engulfment is unknown. Interestingly, C1q expression corresponds to the onset of retinal wave activity, and the C1q−/− phenotype closely mimics the phenotype observed when spontaneous activity is disrupted in the retina. Whether and how neuronal activity regulates complement expression and function are open areas of investigation; however, based on our data, we envision a model in which C1q is secreted locally from RGC terminals in an activity-dependent manner to regulate C3-CR3 dependent microglial synaptic phagocytosis (Supplemental Fig. 5). Retinal C1q is required for retinogeniculate refinement In the postnatal retinogeniculate system, both RGCs and microglia express C1q. Although microglia express comparatively higher levels of C1q than neurons (Fig. 1c, 2g), several lines of evidence suggest that RGCs contribute significantly to C1q levels and complement-dependent synaptic refinement in the postnatal dLGN. In retinal TGFβRII−/− mice, which have reduced retinal C1q expression but normal microglial C1q expression, we observed defects in retinogeniculate refinement and microglial engulfment (Figs, 6 and 7), suggesting that microglial C1q expression cannot compensate functionally for the loss of RGC-derived C1q in complement-dependent synaptic refinement. If RGC inputs are a critical source of C1q in the dLGN during synaptic refinement, how does C1q reach RGC terminals in the dLGN far from the cell bodies in the retina? C1q is a large glycoprotein packaged and secreted by macrophages via traditional secretory pathways in the golgi25. C1q then is secreted directly into the bloodstream by circulating macrophages. In RGCs, however, proteins that are produced and packaged in the soma in the retina have a long distance to travel to axon terminals in the dLGN. Given that in the retinal TGFβRII−/− mice we observed reduced C1q levels compared to WT in the retina (Fig. 4a–d), the optic nerve (Fig, 4e–f), and the dLGN (Fig. 5), we hypothesize that C1q is transported along RGC axons to the dLGN within secretory vesicles, based on its packaging and secretion in macrophages. Alternatively, local translation and synthesis of C1q within RGC axons in the optic nerve or the dLGN is possible, although there have been no previous reports of local translation of C1q. Given the importance of spontaneous retinal activity in driving synaptic refinement and that C1q regulation by TGF-β seems to be unique to neurons (Fig. 1c,4b), it is intriguing to speculate that C1q secretion from RGC inputs may be regulated by neuronal activity and act as a molecular cue linking information about the retina to the dLGN to drive proper circuit development. TGF-β: regulating neuronal C1q and CNS development In the immune system, TGF-β is characterized as an anti-inflammatory cytokine that dampens the immune response. In the mammalian nervous system, TGF-β signaling pathways regulate diverse developmental processes, including neuronal survival and programmed cell death, axon specification, and synaptogenesis. TGF-β signaling modulates embryonic and postnatal periods of programmed cell death in the mouse retina26 and also amplifies the efficacy of neurotrophic factors critical for cell survival, such as GDNF27. In the developing rodent neocortex, TGF-β signaling is necessary and sufficient for axon specification28. Recent work also has shown that TGF-β regulates synaptogenesis in cortical neurons in vitro29 and at the Drosophila and Xenopus NMJs30. At the Drosophila NMJ, glia produce the TGF-β ligand31 and they direct synaptogenesis via regulating a neuron-derived TGF-β family member and the downstream RacGEF Trio32. Also, in Drosophila, TGF-β directs large scale neuronal and axonal remodeling during metamorphosis33,34. TGF-β also affects synaptic function at excitatory and inhibitory synapses in the pre-Bötzinger complex in the mouse brainstem35 and modulates sensory neuron excitability and synaptic efficacy at sensorimotor synapses in the sea slug Aplysia36. In the present study, we found that TGF-β signaling in postnatal RGCs is required for developmental synaptic refinement. Given the many important roles for TGF-β in development, we characterized other aspects of retinal development and found no significant deficits in the retinal TGFβRII−/− mouse. We found that disrupting TGFβRII in the retina did not dramatically affect the number of retinal neurons or retinal morphology. Moreover, despite the role for TGFβRII in axon specification in cortex, disruption of TGFβRII signaling in the retina does not alter axon specification in the retina or axon caliber in the optic nerve (Supplemental Fig. 2f–h), suggesting that other mechanisms underlie axon development and specification in RGCs. Although we have not quantified synapses in the dLGN of retinal TGFβRII−/− mice, it is unlikely that defective synaptogenesis could explain our phenotype, as we observed an increase in overlap between eye specific territories. Furthermore, previous studies have reported that TGF-β is not synaptogenic in RGC cultures10, further supporting that synaptogenesis is likely normal in these mice. Together our data implicate TGFβ as a key regulator of synaptic refinement in the mammalian CNS. Interestingly, in Drosophila, a class of TGF-β family members together with an immunoglobulin superfamily member protein, Plum, has been implicated in the dramatic remodeling that occurs during metamorphosis in the CNS and at the NMJ33,34. This finding combined with our findings raises the question of whether TGF-β signaling represents an important signal for synaptic remodeling throughout the nervous system. Could TGF-β initiate a synaptic refinement gene program in RGCs or other neurons that undergo refinement? Interestingly, C1q and TGF-β expression are expressed in embryonic and postnatal spiral ganglion neurons37 around the time when spiral ganglion cell inputs onto outer hair cells are refined38. Little is known about the synaptic refinement mechanisms in the auditory system; however, this developmental expression of C1q and TGF-β suggests that the same mechanism for synapse elimination may drive refinement in both the auditory and visual systems. Importantly, in both of these systems, C1q expression in sensory neurons (RGCs and spiral ganglion neurons) is developmentally regulated and restricted to periods of developmental synaptic remodeling. These findings are consistent with our hypothesis that neuron-derived C1q plays a critical role in synaptic refinement, although it remains unclear if TGF-β regulates C1q or other refinement genes in other brain regions. Implications of TGF-β regulation of C1q in disease Our findings also have important implications for understanding mechanisms underlying synapse elimination in the diseased brain. Dysregulation of immune system components including complement proteins and cytokines has been demonstrated in many CNS disorders including epilepsy, schizophrenia, and neurodegenerative diseases such as glaucoma and Alzheimer’s disease. In particular, TGF-β localizes to beta-amyloid plaques and has been linked to the formation of these plaques in Alzheimer’s disease39 and blocking TGF-β and Smad2/3 signaling mitigates plaque formation in Alzheimer’s mouse models40. C1q associates with plaques in Alzheimer’s brains as well41, and in mouse models of Alzheimer’s, C1q-deficiency has been shown to be neuroprotective42. Synapse loss and/or dysfunction have emerged as early hallmarks of neurodegenerative diseases, suggesting that aberrant complement upregulation may reactivate the developmental synapse elimination pathway in disease to promote synapse loss. Our work demonstrating a new link between TGF-β signaling, complement, and synapse elimination opens up new avenues of investigation into the role of this regulatory mechanism for C1q in these disorders and in other regions of the healthy CNS during development. ONLINE METHODS Mice Floxed TGFβRII mice (B6.129S6-TGF-βr2tm1Hlm) were obtained from the NCI Mouse Repository and crossed to the CHX10-Cre line, Tg(Chx10-EGFP/cre,-ALPP)2Clc/J (Jackson Lab), to generate retina-specific TGFβRII−/− mice. C1q−/− mice (C57BL6 background) were generously provided by M. Botto43. Experiments were approved by the institutional animal use and care committee in accordance with NIH guidelines for the humane treatment of animals. Neuron and Astrocyte Cultures Retinal ganglion cells were cultured from P8 Sprague-Dawley rats after serial immunopanning steps to yield >99.5% purity as described19. Cells were maintained in serum-free media as described44. Cortical astrocytes were prepared from P1–P2 rat cortices as previously described9. Retinal astrocytes were prepared from P8 Sprague Dawley rat or mouse retinas by adapting described methods for purification of cortical astrocytes12. After first immunopanning away microglia and RGCs, an anti-ITGB5-coated petri plate was used to isolate astrocytes from remaining cells in suspension. Purified cortical astrocytes were prepared as described12. Purified retinal and cortical astrocytes were maintained in a defined serum-free medium supplemented with hbEGF as described12. Astrocyte conditioned medium (ACM) was prepared as previously described9. Astrocytes were switched to minimal media (Neurobasal + glutamine, Pen/Strep, Sodium Pyruvate) once confluent and media was collected after 5 days and concentrated to 10× using Vivaspin columns (Sartorius). qPCR RGCs, microglia, and astrocytes were acutely isolated using immunopanning as described above from either mice or rats at indicated ages. For acute isolation experiments, RNA was collected directly from the immunopanning plate without culturing the cells. For cultured RGC experiments, total RNA was prepared, cDNA was synthesized, and qPCR performed using the Applied Biosystems Cells to Ct Power SYBR green kit as described by the manufacturer. Briefly, cell lysates were collected from 80,000 RGCs in provided lysis buffer and cDNA was synthesized directly from this lysate. QPCR reactions were assembled for the genes of interest (c1qa, c1qb, tieg, gapdh) using 4µL of cDNA per reaction and samples were run on the Rotogene qPCR machine (QIAGEN). Expression levels were compared using the ddCT method normalized to GAPDH. Immunohistochemistry Brains and eyes were harvested from mice after transcardial perfusion with 4% paraformaldehyde (PFA). Tissue was then immersed in 4% PFA for 2 hours following perfusion, cryoprotected in 30% sucrose, and embedded in a 2:1 mixture of OCT:20% sucrose PBS. Tissue was cryosectioned (12–14 microns), sections were dried, washed three times in PBS, and blocked with 2% BSA+ 0.2% Triton X in PBS for 1 hr. Primary antibodies were diluted in antibody buffer (+ 0.05% triton + 0.5% BSA) as follows: C1q (undiluted culture supernatant, Epitomics, validated in Stephan et al., 201314), C3 (Cappel,1:300), vglut2 (Millipore,1:2000), TGFβRII (R&D Systems goat antihuman, 1:200), pSmad (Millipore,1:200), Calretinin (Millipore,1:1000), Iba1 (Wako, 1:400), CD68 (Serotec,1:300), TUJ1 (Covance, 1:400), and incubated overnight at 4°C. Secondary Alexa-conjugated antibodies (Invitrogen) were added at 1:200 in antibody buffer for 2 hr at room temperature. Slides were mounted in Vectashield (+DAPI) and imaged using the Zeiss Axiocam, Zeiss LSM700, or Perkin Elmer Ultraview Vox Spinning Disk Confocal. ELISA The 7-plex Mouse Inflammatory Cytokine kit (MSD) was used to profile cytokines in ACM. Freshly prepared ACM was profiled according to manufacturer’s provided protocol. Standards were diluted in minimal media used to make ACM for greater accuracy. Plates were read and data were acquired and analyzed using the MSD Sector Imager 2400. ELISA kits were obtained for TGF-β1, 2, and 3 separately (R&D systems, MSD) and were performed according to manufacturer’s instructions. Western Blot RGC and whole retina lysates were collected and homogenized in RIPA buffer with complete protease inhibitors (Roche). Samples were boiled for 5 min in SDS sample buffer, resolved by SDS PAGE, transferred to PVDF membranes, and immunoblotted. Antibodies were diluted in 5% milk in PBS + 0.1% Tween. Antibodies: Rabbit anti-C1qA polyclonal (Epitomics, 1:5000, specificity validated in Stephan et al., 201314); Goat anti-TGFβRII (R&D Systems, 1:1000). In situ hybridization In situ hybridization for C1qb was performed on 12 um retinal sections as previously described3. Probes targeting the entire C1qb coding sequence (Open Biosystems clone: 5715633) were generated by digesting the plasmid with EcoRI, and performing in vitro transcription with T7 polymerase using the DIG RNA labeling kit (Roche Applied Science) as per the manufacturer’s instructions. 1.8kb probes were then cleaved to form 300 bp probes by alkaline hydrolysis before use. LGN analysis Mice received intraocular injection of cholera toxin-b subunit (CTB) and were sacrificed the following day. Tissue was processed and analyzed as previously described3,45. Mouse pups were anesthetized with inhalant isofluorane. Mice received intravitreal injections of cholera toxin-b subunit (CTB) conjugated to Alexa 488 (green label) in the left eye and CTB conjugated intraocular injection to Alexa 594 (red label) into the right eye as described2. Images were digitally acquired using the Zeiss Axiocam. All images were collected and quantified “blind,” and compared to age-matched littermate controls. Gains and exposures were established for each label. Raw images of the dLGN were imported to Photoshop (Adobe), and the degree of left and right eye axon overlap in dLGN was quantified using the multi-threshold protocol as previously described2 and using threshold independent R value analysis as described20. For threshold independent analysis, we performed background subtraction using a 200 pixel rolling ball radius filter and normalized the images. We then calculated the R value (log(FI/FC)) for each pixel and determined the variance of the R value distribution for each image (4 images/animal). Pseudocolored images representing the R value distribution were generated in ImageJ. Retinal cell counts Retinal flat mounts were prepared by dissecting out retinas whole from the eyecup and placing four relieving cuts along the major axis, radial to the optic nerve. Each retina was stained with DAPI (Vector Laboratories, Burlingame, CA) to reveal cell nuclei. Measurements of total cell density in the ganglion cell layer (which includes both ganglion cells and displaced amacrine cells) were carried out blind to genotype from matched locations in the central and peripheral retina for all four retinal quadrants of each retina. Quantification was limited to P30 retinas, which is an age subsequent to ganglion cell genesis and apoptosis in the mouse. For each retina (1 retina per animal; n=3 mice per treatment condition or genotype), 12 images of peripheral retina and 8 images of central retina were collected. For each field of view collected (20 per retina), Macbiophotonics ImageJ software (NIH) was used to quantify the total number of DAPI using the nuclei counter plugin and TUJ1-positive cells were counted using the cell counter plugin. All analyses were performed blind to genotype or drug treatment. Z stack Image and Microglial Engulfment Analysis In vivo microglia phagocytosis assay and analyses were performed as previously described in detail by Schafer et al., 2012. In brief, mice received intraocular injections of anterograde tracers at P4. All mice were sacrificed at P5 and brains were 4% PFA fixed overnight (4°C). Only those brains with sufficient dye fills were analyzed. For each animal, two sections of medial dLGN were chosen for imaging for reconstruction of RGC inputs and C1q staining as well as for microglia engulfment analysis. Images were acquired on a spinning disc confocal at 60× using 0.2 µm z-steps. For each dLGN, 4–8 fields were imaged in the ipsilateral territory and 4–8 fields were imaged in the contralateral territory (minimum of 8 fields per dLGN, 16 fields per animal). Subsequent images were processed and quantified using ImageJ (NIH) and Imaris software (Bitplane). For subsequent acquired z-stacks, ImageJ (NIH) was used to subtract background from all fluorescent channels (rolling ball radius=10) and a mean filter was used for the EGFP channel (stained for Iba1) of 1.5. Subsequently, Imaris software (Bitplane) was used to create 3D volume surface renderings of each z-stack. Surface rendered images were used to determine the volume of the microglia, all RGC inputs, and the volume of C1q staining. To visualize and measure the volume of engulfed inputs, any fluorescence that was not within the microglia volume was subtracted from the image using the mask function. The remaining engulfed/internal fluorescence was surface rendered using parameters previously determined for all RGC inputs/C1q and total volume of engulfed/internal inputs was calculated. To determine % engulfment (or %C1q-positive terminal), the following calculation was used: Volume of internalized RGC input (or volume of C1q) (µm3)/Volume microglial cell (or RGC inputs) (µm3). For all KO engulfment experiments, all analyses were performed blind. Microglia Density Quantification For quantification of cell density, 2 dLGN were imaged per animal (n=3 per treatment condition or genotype). To capture the entire dLGN, a 10× field was acquired. Microglia were subsequently counted from each 10× field. To calculate the density of microglia, the area of the dLGN was measured using ImageJ software (NIH). All analyses were performed blind to genotype or drug treatment. Quantification of Microglial Activation State Activation state was quantified based on established methods (Schafer et al, 2012). Immunohistochemistry was performed on 40um cryosections with antibodies against Iba1 and CD68. For each genotype, two 20× fields of view were analyzed on a spinning disk confocal using 2um z-steps. The activation state was then determined based on a maximum intensity projection. Iba1 staining was used to assess morphology based on the number of branches and the expression pattern of CD68 was analyzed and scored as 0 (no/scarce expression), 1 (punctate expression), or 2 (aggregated expression or punctate expression all over the cell). Scores from 0–3 for number of branches or 0–2 were assigned based on Iba1 and CD68 staining and these score were combined to give the most activated microglia an overall score of 5 and least activated scoring 0. For each genotype, 4 mice were analyzed and all analysis was performed blind. Statistical Analysis For all statistical analyses, data distribution was assumed to be normal but this was not formally tested. GraphPad Prism 5 software (La Jolla, CA) was used for all statistical tests. Analyses used include one-way ANOVA, two-way ANOVA, and Student’s t-test. For ANOVA analysis, Bonferonni post hoc tests were used. Igor was used to calculate the R value variance in Figure 6. No statistical methods were used to pre-determine sample sizes but our sample sizes are similar to those reported in previous publications3,7. Data collection and analysis were performed blind to the conditions of the experiments. Also, data for each experiment were collected and processed randomly and animals were assigned to various experimental groups randomly as well. All n and p values and statistical tests are indicated in figure legends. All error bars represent SEM and mean +/− SEM is plotted for all graphs except where noted. Supplementary Material 1 6 7 8 9 10 11 12 13 2 3 4 5 Acknowledgments We thank C. Chen, L. Benowitz, D.P. Schafer, and M. Buckwalter for helpful comments on the manuscript and critical discussion. In addition we thank Alexander Stephan, Ben Barres, and Andrea Tenner for assistance with anti-C1q antibody production and characterization. We also thank M. Rasband for the βIV spectrin antibody. Thank you to D.P. Schafer and E.K. Lehrman for technical expertise on the microglial engulfment assay and Imaris image analysis, T. Nelson for technical assistance, and the imaging core at Boston Children’s Hospital including T. Hill for technical support. This work was supported by grants from the Smith Family Foundation (B.S.), Dana Foundation (B.S.), the Ellison Foundation (B.S.), John Merck Scholars Program (B.S.), NINDS (RO1-NS-07100801; B.S.), NIDA (RO1-DA-15043; B.A.B.), NIH (P30-HD-18655; MRDDRC Imaging Core). Author Contributions A.R.B. conducted all experiments, performed data analysis, and wrote the manuscript. B.S. advised on and supervised the project and co-wrote the manuscript. Figure 1 C1q is rapidly upregulated in neurons in response to astrocyte-secreted factors (A) QPCR results for c1qb showed increased c1qb expression relative to controls (RGCs treated with control media) after 6-day treatment with astrocyte insert or astrocyte conditioned medium (ACM). ACM prepared from purified cortical astrocytes (cortical), purified retinal astrocytes (retinal), or astrocytes prepared using the McCarthy and Devellis protocol (MD) all showed a similar upregulation (one-way ANOVA, n=3 experiments, p<0.0001, F(5,12)=75.41). (B) C1q upregulation timecourse for all three C1q genes (c1qa, c1qb, and c1qc) (two-way ANOVA, n=4 experiments,**p<0.001, p<0.05, F(4,75)=34.37). (C) QPCR results for c1qb showed increased c1qb expression relative to control in RGC cultures but not in microglia or astrocyte cultures after treatment with ACM for 15 min. (two way ANOVA, n=3 experiments, p<0.005, F(2,18)=5.71). (D) Western blot showed an increase in C1q protein in RGC media after 6 days of treatment with ACM. Results shown are representative of 3 experiments. Full length blots are displayed in Supplemental Figure 6. (E) There is a corresponding increase in C1q protein within 6 hours of adding ACM to cultures detected by immunohistochemistry (rabbit anti-mouse C1qA14). Scale bar = 100um. (F) Quantification showed increased C1q levels in ACM treated cultures measured as a change in fluorescence intensity relative to control untreated cultures (t test, n=3 experiments, p<0.01, t(4)=37.48). Figure 2 TGF-β is necessary and sufficient for neuronal C1q upregulation in vitro (A) ACM cytokine profiling by ELISA. N=3 independent ACM batches. (B) Immunodepletion of TGF-β, but not other cytokines, significantly reduced ACM-induced C1q upregulation (one-way ANOVA, n= 3 experiments, *p<0.001, p<0.01, F(4,12)=22.15). (C) Validation of immunodepletion. %Neutralization represents ([TGF-β]initial – [TGF-β]depletion)/(initial TGF-β concentration). N=3 experiments. (D) Immunodepletion of each TGF-β isoform showed that depletion of pan-TGF-β or TGF-β3 blocked C1q upregulation (two-way ANOVA, n=3 experiments, *p<0.001, F(4,20)=79.52). (E) Concentration-response curves for TGF-β1, 2, and 3 (Two-way ANOVA, n= 3 experiments, p<0.0001, p<0.01, F(10,36)=23.56). (F) Glycine elution showed that either anti-pan TGF-β or anti-TGF-β3 eluates upregulate C1q (one-way ANOVA, n= 3 experiments, *p<0.001, F(4,10)=11.02). (G) QPCR results for c1qb in RGC cultures and in microglia or retinal astrocyte cultures after TGF-β3 treatment (50 pg/ml) for 15 min. (two way ANOVA, n=3 experiments, p<0.05, F(2,12)=415.96). (H) RGCs showed increased nuclear accumulation of pSmad2 (15–30 min. ACM treatment). Quantification showed a significant increase in pSmad (red) within the nuclear area (blue) (one-way ANOVA, n= 15 cells/condition, p<0.001, F(2,9)=19.83). Scale bar = 20um. (I) RT-PCR for tgfbr1, tgfbr2, and tgfbr3 in P5 retina. Data shown are representative of 4 samples tested. Full length gels are displayed in Supplemental Fig. 6. (J) Blocking TGFβRII signaling with neutralizing antibodies or with inhibitors of TGFβRI significantly reduced the effects of ACM or TGF-β3 (0.05 ng/ml) on C1q (two-way ANOVA, n= 3 experiments, p<0.01, p<0.001, F(4,18)=35.06). Figure 3 TGF-β expression corresponds to synaptic refinement period in the retinogeniculate system (A) RT-PCR showed expression of all three TGF-β isoforms in the P5 mouse retina. Data are representative of 4 mice. (B) Western blot for TGFβRII (goat anti-human TGFβRII, R&D systems) showed developmental expression of TGFβRII in the mouse retina. See Supplemental Fig. 6 for full length blot. (C) Relative intensity quantification normalized to beta-actin control for each age showed developmental TGFβRII expression in the postnatal mouse retina (one-way ANOVA, n=3 experiments, p<0.01, F(2,6)=26.36). (D) Immunostaining with antibodies against TGFβRII (R&D systems, goat anti-TGFβRII) showed that the receptor localizes to the RGC layer and the IPL (arrows) and that staining intensity is dramatically reduced at P15 relative to P5. Antibody staining was confirmed for specificity by staining retinal TGFβRII−/− mice. All images were obtained with set exposure times. Scale bar = 50um. (E) RT-PCR confirmed the absence of tgfbr2 mRNA in RGCs acutely isolated from P5 mice using immunopanning. Data shown are representative of 4 animals tested. Full length gel in Supplemental Fig. 6. (F) Immunohistochemistry for total Smad2 showed no difference in relative fluorescence intensity (RGC layer) in WT littermates and retinal TGFβRII−/− mice (t test, n=3 mice/genotype, p=0.96 (ns), t(4)=0.053). Scale bar = 50µm. (G) Immunohistochemistry for phosphorylated Smad (pSmad). Co-staining for an RGC marker, Brn3a, and pSmad2/3 showed a significant reduction in pSmad levels quantified in RGCs specifically (t test, n= 3 mice/group, p<0.001, t(4)=13.18). Scale bar = 50µm. Figure 4 TGF-β signaling is required for neuronal C1q expression in vivo (A) In situ hybridization for c1qb showed significantly reduced C1q expression in the RGC layer (arrows) in retinal TGFβRII−/− mice. Scale bar = 100µm. (B) RGCs acutely isolated from P5 WT (white bar) and retinal TGFβRII−/− (grey bar) retinas using immunopanning showed significantly reduced C1q expression. Microglia acutely isolated using CD45 immunopanning showed no significant difference in C1q levels (two-way ANOVA, n= 5 mice/group, *p<0.01, F(1,16)=19.11). (C) Immunostaining for C1q in P5 retina. (Scale bar = 50µm). In retinal TGFβRII−/− mice, C1q localization to the RGC layer (inset a vs. d), the IPL (b vs. e) is reduced relative to WT animals. Retinal microglia showed no change in C1q levels (inset c and f). (D) Quantification of the relative fluorescence intensity in each retinal area in WT littermates and retinal TGFβRII−/− mice showed significantly reduced C1q localization to the RGC layer and IPL when TGF-β signaling was blocked (two-way ANOVA, n= 4 mice/group, p<0.05, F(2,12)=11.69). (E) Immunohistochemistry for C1q in optic nerve cross sections showed C1q-immunopositive puncta co-localized with RGC axon fascicles labeled by TUJ1 (Green). C1q levels were significantly reduced in the retinal TGFβRII−/− mouse within the axon, as indicated by co-localization with TUJ1. C1q labeling of microglia remained unchanged (arrows). Scale bar = 10µm. (F) Quantification of the fluorescence intensity for C1q staining with axon bundles shows a significant decrease in C1q levels (t test, n=3 mice (two nerves per mouse), p=0.0055, t(6)=4.225). Figure 5 Retinal TGF-β signaling is required for complement localization in the dLGN (A) C1q immunohistochemistry in the dLGN and primary visual cortex (V1) shows reduced C1q fluorescence intensity in the dLGN but not in V1. Scale bar = 20µm. Inset shows 3× magnification. (B) Quantifying relative fluorescence intensity showed significant reduced intensity in the dLGN of retinal TGFβRII−/− vs. WT mice (two-way ANOVA, n=4 mice/group, *p<0.01, F(1,12)=11.21). No differences were observed in V1. (C) C1q puncta density quantification showed significantly reduced C1q puncta density in the dLGN in retinal TGFβRII−/− mice vs. WT littermates (two-way ANOVA, n=4 d C1q expression. Microglia acutely isolated using CD45 immunopanning showed no significant difference in C1q levels (two-way ANOVA, n= 5 mice/group, p<0.05, F(1,8)=7.48). No difference in C1q puncta density was observed in V1. (D) C1q localization to vglut2-positive RGC terminals is reduced in the retinal TGFβRII−/− mouse. Immunostaining for C1q and vglut2 in WT and retinal TGFβRII−/− P5 dLGNs showed a reduction in co-localization of C1q and vglut2. Scale bar = 20µm. Star indicates the enlarged synaptic puncta shown in the inset. (E) Quantification of C1q co-localization with vglut2 showed significantly reduced synaptic localization of C1q in retinal TGFβRII−/− mice. Co-localized puncta were identified and counted using ImageJ Puncta Analyzer (t test, n=4 mice, p=0.0226, t(6)=3.045). (F) C3 localization to vglut2-positive RGC terminals is reduced in the retinal TGFβRII−/− mice, similar to what we observed for C1q. Scale bar = 20µm. (G) Co-localized C3 and vglut2 puncta were identified and counted using ImageJ Puncta Analyzer (t test, n=4 mice, p<0.0395, t(6)=2.622). Figure 6 TGF-β signaling and C1q are required for eye specific segregation and microglia-mediated pruning in the retinogeniculate system (A) Representative dLGN images for WT, retinal TGFβRII−/−, C1q−/−, IgG1 control, and anti-TGF-β injected WT and C1q−/− mice pseudocolored to show the R-value for each pixel. R=log10(FIpsi/FContra). Scale bar = 100µm. (B) Quantification of the mean variance of the R-value for each group. A significant reduction in the mean variance of the R value is seen in mice deficient in TGF-β signaling or C1q (one-way ANOVA, n= 6 animals/group, *p<0.01, F(2,15)=8.228). (C) There is no additional decrease in the R-value variance when TGF-β signaling is blocked in C1q−/− mice. Data shown as mean R-value variance +/− SEM (one-way ANOVA, n= 6 animals/group, p<0.05, F(2,15)=8.567). (D) Microglia show reduced engulfment of RGC terminals in mice deficient in C1q or retinal TGF-β signaling. Volume of each microglia and the engulfed CTB was quantified in Imaris and the % engulfment defined as the volume of internalized CTB/volume of microglia. Results were normalized to WT engulfment levels and C1q−/− or retinal TGFβRII−/− mice both showed a significant reduction in % engulfment (one-way ANOVA, n= 6 mice/group, p<0.01, F(3,20)=13.66). Scale bar = 10µm. Insets show enlargement of boxed area. Figure 7 Mice deficient in C1q or retinal TGF-β signaling show increased overlap of contralateral and ipsilateral areas in the dLGN (A) Representative images of anterograde tracing (Alexa conjugated b-cholera toxin) of contralateral (top row) and ipsilateral (second row) retinogeniculate projections, merged channels (third row) and their overlap (yellow, bottom row) in the dLGN for WT, retinal TGFβRII−/−, C1q−/−, and anti-TGF-β injected WT and C1q−/− mice. Scale bar = 100µm. (B) Quantification of percentage of dLGN area receiving input from both contralateral and ipsilateral eyes (yellow area). Data shown as mean yellow area +/− SEM (two-way ANOVA, n= 6 animals/group, p<0.001, p<0.01, p<0.05, F(4,175)=101.00). (C) Measurements of dLGN area in P10 mice showed no significant difference between WT and retinal TGFβRII−/− mice. 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==== Front 04104626011NatureNatureNature0028-08361476-46872457235310.1038/nature13005nihpa554241ArticleDetection and replication of epistasis influencing transcription in humans Hemani Gibran 12Shakhbazov Konstantin 12Westra Harm-Jan 3Esko Tonu 456Henders Anjali K. 7McRae Allan F. 12Yang Jian 1Gibson Greg 8Martin Nicholas G. 7Metspalu Andres 4Franke Lude 3Montgomery Grant W. 7+Visscher Peter M 12+Powell Joseph E 12+1 Queensland Brain Institute, University of Queensland, Brisbane, QLD, Australia2 University of Queensland Diamantina Institute, University of Queensland, Princess Alexandra Hospital, Brisbane, Queensland, Australia3 Department of Genetics, University Medical Center Groningen, University of Groningen, Hanzeplein 1, Groningen, the Netherlands4 Estonian Genome Center, University of Tartu, Tartu, 51010, Estonia5 Medical and Population Genetics, Broad Institute, Cambridge, MA, 02142, US6 Divisions of Endocrinology, Children’s Hospital, Boston, MA, 02115, US7 Queensland Institute of Medical Research, Brisbane, Queensland, Australia8 School of Biology and Centre for Integrative Genomics, Georgia Institute of Technology, Atlanta, Georgia United States of America Corresponding author: [email protected]+ These authors contributed equally. 11 3 2014 26 2 2014 10 4 2014 10 10 2014 508 7495 249 253 Users may view, print, copy, download and text and data- mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: http://www.nature.com/authors/editorial_policies/license.html#termsEpistasis is the phenomenon whereby one polymorphism’s effect on a trait depends on other polymorphisms present in the genome. The extent to which epistasis influences complex traits1 and contributes to their variation2,3 is a fundamental question in evolution and human genetics. Though often demonstrated in artificial gene manipulation studies in model organisms4,5, and some examples have been reported in other species6, few examples exist for epistasis amongst natural polymorphisms in human traits7,8. Its absence from empirical findings may simply be due to low incidence in the genetic control of complex traits2,3, but an alternative view is that it has previously been too technically challenging to detect due to statistical and computational issues9. Here we show that, using advanced computation10 and a gene expression study design, many instances of epistasis are found between common single nucleotide polymorphisms (SNPs). In a cohort of 846 individuals with 7339 gene expression levels measured in peripheral blood, we found 501 significant pairwise interactions between common SNPs influencing the expression of 238 genes (p < 2.91 × 10−16). Replication of these interactions in two independent data sets11,12 showed both concordance of direction of epistatic effects (p = 5.56 ×10−31) and enrichment of interaction p-values, with 30 being significant at a conservative threshold of p < 0.05/501. Forty-four of the genetic interactions are located within 2Mb of regions of known physical chromosome interactions13 (p = 1.8 × 10−10). Epistatic networks of three SNPs or more influence the expression levels of 129 genes, whereby one cis-acting SNP is modulated by several trans-acting SNPs. For example MBNL1 is influenced by an additive effect at rs13069559 which itself is masked by trans-SNPs on 14 different chromosomes, with nearly identical genotype-phenotype (GP) maps for each cis-trans interaction. This study presents the first evidence for multiple instances of segregating common polymorphisms interacting to influence human traits. ==== Body In the genetic analysis of complex traits it is usual for SNP effects to be estimated using an additive model where they are assumed to contribute independently and cumulatively to the mean of a trait. This framework has been successful in identifying thousands of associations14. But to date, though its contribution to phenotypic variance is frequently the subject of debate1–3, there is little empirical exploration of the role that epistasis plays in the architecture of complex traits in humans7,8. Beyond the prism of human association studies there is evidence for epistasis, not only at the molecular scale from artificially induced mutations4 but also at the evolutionary scale in fitness adaptation15 and speciation16. Methods are now available to overcome the computational problems involved in searching for epistasis, but its detection still remains problematic due to reduced statistical power. For example, increased dependence on linkage disequilibrium (LD) between causal SNPs and observed SNPs17,18, increased model complexity in fitting interaction terms19, and more extreme significance thresholds to account for increased multiple testing9 all make it more difficult to detect epistasis in comparison to additive effects. Thus, with small genetic effect sizes, as is expected in most complex traits of interest14, the power to detect epistasis diminishes rapidly. There are two simple ways to overcome this problem. One is by using extremely large sample sizes20; another is by analysing traits that are likely to have large effect sizes among common variants. Because our focus was to ascertain the extent to which instances of epistasis arises from natural genetic variation we designed a study around the latter approach and searched for epistatic genetic effects that influence gene expression levels. Transcription levels can be measured for thousands of genes and like most complex diseases, these expression traits are typically heritable21. But unlike complex diseases, genetic associations with gene expression commonly have very large effect sizes that explain large proportions of the genetic variance22, making them good candidates to search for epistasis, should it exist. In our discovery dataset (Brisbane Systems Genetics Study, BSGS23) of 846 individuals genotyped at 528,509 SNPs, we used a two stage approach to identify genetic interactions. First, we exhaustively test every pair of SNPs for pairwise effects against each of 7339 expression traits in peripheral blood (1.03 × 1015 statistical tests, family-wise error rate of 5% corresponding to a significance threshold of p < 2.91 × 10−16, Methods). Second, we filtered the SNP pairs from stage 1 on LD and genotype class counts, and tested the remaining pairwise effects for significant interaction terms and used a Bonferroni correction for multiple testing (estimated type 1 error rate 0.05 ≤ α ≤ 0.14, Methods, Supplementary Figure S1). Using this design we identified 501 putative genetic interactions influencing the expression levels of 238 genes (Supplementary Table S1). We used strict quality control measures to avoid statistical associations being driven by technical artifacts (Methods). However it remains possible that unexplained technical artifacts may have led to the significant discovery interactions. Of the 501 discovery interactions, 434 had available data and passed filtering (Methods) in two independent replication datasets, Fehrmann12 and the Estonian Genomics Centre University of Tartu (EGCUT)11, in which we saw convincing evidence for replication. We used the summary statistics from the replication datasets to perform a meta analysis to obtain an independent p-value for the putative interactions, and 30 were significant after applying a Bonferroni correction for multiple testing (5% significance threshold p < 0.05/501, Table 1). To quantify the similarity of GP maps between the independent datasets (Figure 1) we decomposed the genetic effects of each of the SNP pairs into orthogonal additive, dominance and epistatic effects (A1, A2, D1, D2, A1 × A2, A1 × D2, D1 × A2, D1 × D2) and tested for concordance of the sign of the most significant effect (Supplementary Table S3, Methods). Sign concordance between the discovery and both replication datasets was observed in 22 out of the 30 significantly replicated interactions (expected value = 7.5 under the null hypothesis of no interactions, p = 3.76 × 10−8). In addition, using the meta analysis from the replication samples only, we observed that 316 of the remaining 404 discovery SNP pairs had replication interaction p-values more extreme than the 2.5% confidence interval of the quantile-quantile plot against the null hypothesis of no interactions where p-values are assumed to be uniformly distributed (p ≪ 1.0×10−16, Figure 2 and Supplementary Figure S2). Concordance of the direction of the effect of the largest variance component was also highly significant (p = 5.71 × 10−31, Supplementary Table S3). The congruence of the epistatic networks in discovery and replication datasets is shown in Figure 3, demonstrating that these complex genetic patterns are common even across independent datasets. A further replication was attempted using the Centre for Health Discovery and Wellbeing (CHDWB) dataset24, but only 20 of the SNP pairs passed filtering because the sample size was small (n = 139), and likely due to insufficient power we found no evidence for replication (Supplementary Figure S6). It should be noted that although it is a necessary step to establish the veracity of the interactions from the discovery set, replication of epistatic effects in independent samples is difficult in practice due to LD (Methods). Though seldom the focus of association studies, SNPs with known main effects are often tested for A×A genetic interactions9, but our analysis suggests this is unlikely to be the best strategy for its detection. The majority of our discovery interactions comprised of one SNP that was significantly associated with the gene expression level in the discovery dataset, and one SNP that had no previous association22 (439 out of 501, Methods). Only nine interactions were between SNPs that both had known main effects while 64 were between SNPs that had no known main effects. Additionally, we observed that the largest epistatic variance component for the 501 interactions was equally divided amongst A × A, A × D, D × A and D × D at the discovery stage (p = 0.22 for departure from expectation). This is not surprising because these patterns of epistasis used for statistical decomposition are simply convenient orthogonal parameterisations of a two locus model, and are not intended to model biological function25. Of the discovery interactions, 26 were cis-cis acting (within 1Mb of the transcription start site, mean distance between SNPs was 0.53Mb), 462 were cis-trans-acting, and 13 were trans-trans-acting. We observed a wide range of significant GP maps (Figure 1) but the most common pattern of epistasis that we detected involved a trans-SNP masking the effect of an additive cis-SNP. For example, MBNL1 (involved in RNA modification and regulation of splicing26) has a cis effect at rs13069559 which in turn is controlled by 13 trans-SNPs and one cis-SNP that each exhibit a masking pattern, such that when the trans-SNP is homozygous for the masking allele the decreasing allele of the cis-SNP no longer has an effect (Supplementary Figure S10). Each of these interactions has evidence for replication in at least one dataset and six are significantly replicated at the Bonferroni level (Supplementary Figure S3). We see similar epistatic networks involving multiple (eight or more) trans-acting SNPs for other gene expression levels too, for example TMEM149 (Supplementary Figure S11), NAPRT1 (Supplementary Figure S12), TRAPPC5 (Supplementary Figure S13), and CAST (Supplementary Figure S14). We observed that from pedigree analysis these five gene expression phenotypes had non-additive variance component estimates within the 95th percentile of the 17,994 gene expression phenotypes that were analysed previously22 (Supplementary Table S2, Methods). In total the 501 interactions comprised 781 unique SNPs, which we analysed for functional enrichment (Methods). We tested the SNPs for cell-type specific overlap with transcriptionally active chromatin regions, tagged by histone-3-lysine-4,tri-methylation (H3K4me3) chromatin marks, in 34 cell types27 (Supplementary Figure S5). There was significant enrichment for cis-acting SNPs in haematopoietic cell types only (p < 1 × 10−4 for the three tissues with the strongest enrichment after adjusting for multiple testing). However trans-acting SNPs did not show any tissue specific enrichment (p > 0.1 for all tissues). This difference between cis and trans SNPs suggests different roles in epistatic interactions where tissue specificity is provided by the cis SNPs. There is also enrichment for cis-SNPs to be localised in regions with regulatory genomic features as measured by chromatin states28 (Supplementary Figure S4). We also demonstrate physical organisation of interacting loci within the cell, suggesting a mechanism by which biological function can lead to epistatic genetic variance. It has been shown that different chromosomal regions spatially colocalise in the cell through chromatin interactions13. We cross-referenced our epistatic SNPs with a map of chromosome interacting regions (n = 96, 139) in K562 blood cell lines29 (Methods) and found that 44 epistatic interactions mapped to within 5Mb (p < 1.8 × 10−10), (Supplementary Figure S15). Interaction of distant loci may occur through physical proximity in transcriptional factories that organise across different chromosome regions and can regulate transcription of related genes30. Quantifying the importance of epistasis in complex traits in humans remains an open question. Here we are able to identify 238 gene expression traits with at least one significant interaction given our experiment-wide threshold, where the minimum estimated variance explained by the epistatic effects of any interaction was 2.1% of phenotypic variance. Taking results from our previously published eQTL23 we calculated that 1848 of the 7339 gene expression levels analysed were influenced by additive effects where the estimated additive variance of a locus was 2.1% or greater. Thus, we can infer that the number of instances of large additive effects is significantly greater than the number of instances of large epistatic effects. In terms of their contribution to complex traits a more important metric might be the proportion of the variance that the epistatic loci explain2. Taking all additive effects detected in Powell et al (2012) that have additive variance explaining 2.1% or greater of phenotypic variance, we calculated that the proportion of total phenotypic variance of all 7339 gene expression levels explained by additive effects alone was 2.16%. By contrast, the estimated epistatic variance from the interacting SNPs detected in this study on average explain a total of 0.22% of phenotypic variance, approximately ten times lower than the estimated additive variance. There are several caveats to this comparison which we discuss in the Methods. Overall, we have demonstrated that it is possible to identify and replicate epistasis in complex traits amongst common human variants, despite the relative contribution of pairwise epistasis to phenotypic variation being small. The bioinformatic analysis of the significant epistatic loci suggests that there are a large number of possible mechanisms that can lead to non-additive genetic variation. Further research into such epistatic effects may provide a useful framework for understanding molecular mechanisms and complex trait variation in greater detail. With computational techniques and data now widely available the search for epistasis in larger datasets for traits of broader interest is warranted. Online methods 1 Discovery data 1.1 Data description The Brisbane Systems Genetics Study (BSGS) comprises 846 individuals of European descent from 274 independent families23. DNA samples from each individual were genotyped on the Illumina 610-Quad Beadchip by the Scientific Services Division at deCODE Genetics Iceland. Full details of genotyping procedures are given in Medland et al.31 Standard quality control (QC) filters were applied and the remaining 528,509 autosomal SNPs were carried forward for further analysis. Gene expression profiles were generated from peripheral blood collected with PAXgene TM tubes (QIAGEN, Valencia, CA) using Illumina HT12-v4.0 bead arrays. The Illumina HT-12 v4.0 chip contains 47,323 probes, although some probes are not assigned to RefSeq genes. We removed any probes that did not match the following criteria: contained a SNP within the probe sequence with MAF > 0.05 within 1000 genomes data; did not map to a listed RefSeq gene; were not significantly expressed (based on a detection p-value < 0.05) in at least 90% of samples. After this stringent QC 7339 probes remained for 2D-eQTL mapping. These data are accessible through GEO Series accession number GSE53195. 1.2 Normalisation Gene expression profiles were normalised and adjusted for batch and polygenic effects. Profiles were first adjusted for raw background expression in each sample. Expression levels were then adjusted using quantile and log2 transformation to standardise distributions between samples. Batch and polygenic effects were adjusted using the linear model (1) y=μ+β1c+β2p+β3s+β4a+g+e where μ is the population mean expression levels, c, p, s and a are vectors of chip, chip position, sex and generation respectively, fitted as fixed effects; and g is a random additive polygenic effect with a variance covariance matrix (2) Gjk={σa2j=k2ϕjkσa2j≠k The parameter σa2 is the variance component for additive background genetic. Here, we are using family based pedigree information rather than SNP based IBD to account for relationships between individuals and so ϕjk is the kinship coefficient between individuals j and k. The residual, e, from equation 1 is assumed to follow a multivariate normal distribution with a mean of zero. Residuals were normalised by rank transformation and used as the adjusted phenotype for the pairwise epistasis scan to remove any skewness and avoid results being driven by outliers. The GenABEL package for R was used to perform the normalisation32. 2 Exhaustive 2D-eQTL analysis 2.1 Two stage search We used epiGPU10 software to perform an exhaustive scan for pairwise interactions, such that each SNP is tested against all other SNPs for statistical association with the expression values for each of the 7339 probes. This uses the massively parallel computational architecture of graphical processing units (GPUs) to speed up the exhaustive search. For each SNP pair there are 9 possible genotype classes. We treat each genotype class as a fixed effect and fit an 8 d.f. F-test to test the following hypotheses: (3) H0:∑i=13∑j=13(x¯ij-μ)2=0; (4) H1:∑i=13∑j=13(x¯ij-μ)2>0; where μ is the mean expression level and xij is the pairwise genotype class mean for genotype i at SNP 1 and genotype j at SNP 2. This type of test does not parameterize for specific types of epistasis, rather it tests for the joint genetic effects at two loci. This has been demonstrated to be statistically more efficient when searching for a wide range of epistatic patterns, although will also include any marginal effects of SNPs which must be dealt with post-hoc18. 2.1.1 Stage 1 The complete exhaustive scan for 7339 probes comprises 1.03 × 1015 F-tests. We used permutation analysis to estimate an appropriate significance threshold for the study. To do this we performed a further 1600 exhaustive 2D scans on permuted phenotypes to generate a null distribution of the extreme p-values expected to be obtained from this number of multiple tests given the correlation structure between the SNPs. We took the most extreme p-value from each of the 1600 scans and set the 5% FWER to be the 95% most extreme of these p-values, T* = 2.13 × 10−12. The effective number of tests in one 2D scan being performed is therefore N* = 0.05/T* ≈ 2.33 × 1010. To correct for the testing of multiple traits we established an experiment wide threshold of Te = 0.05/(N* × 7339) = 2.91 × 10−16. This is likely to be conservative as it assumes independence between probes. Filtering We used two approaches to filter SNPs from stage 1 to be tested for significant interaction effects in stage 2. Filter 1 After keeping SNP pairs that surpassed the 2.91 × 10−16 threshold in stage 1 only SNP pairs with at least 5 data points in all 9 genotype classes were kept. We then calculated the LD between interacting SNPs (amongst unrelated individuals within the discovery sample and also from 1000 genomes data) and removed any pairs with r2 > 0.1 or D′2 > 0.1 to avoid the inclusion of haplotype effects and to increase the accuracy of genetic variance decomposition. If multiple SNP pairs were present on the same chromosomes for a particular expression trait then only the sentinel SNP pair was retained, i.e. if a probe had multiple SNP pairs that were on chromosomes one and two then only the SNP pair with the most significant p-value was retained. At this stage 6404 filtered SNP pairs remained. Filter 2 We also performed a second filtering screen applied to the list of SNP pairs from stage 1 that was identical to filter 1 but an additional step was included where any SNPs that had previously been shown to have a significant additive or dominant effect (p < 1.29×10−11) were removed22, creating a second set of 4751 unique filtered SNP pairs. 2.1.2 Stage 2 To ensure that interacting SNPs were driven by epistasis and not marginal effects we performed a nested ANOVA on each pair in the filtered set to test if the interaction terms were significant. We did this by contrasting the full genetic model (8 d.f.) against the reduced marginal effects model which included the additive and dominance terms at both SNPs (4 d.f.). Thus, a 4 d.f. F-test was performed on the residual genetic variation, representing the contribution of epistatic variance. Significance of epistasis was determined using a Bonferroni threshold of 0.05/(6404+4751) = 4.48×10−6. This resulted in 406 and 95 SNP pairs with significant interaction terms from filters 1 and 2, respectively. 2.2 Type 1 error rate Using a Bonferroni correction of 0.05 in the second stage of the two stage discovery scan implies a type 1 error rate of α = 0.05. However, this could be underestimated because the number tests performed in the second stage depends on the number of tests in the first stage, and this depends on statistical power and model choice. We performed simulations to estimate the type 1 error rate of this study design. We assumed a null model where there was one true additive effect and 7 other terms with no effect. To simulate a test statistic we simulated 8 z-scores, z1~N(NCP,1) and z2..8 ~ N(0, 1). Thus zfull=∑i=18zi~χ28 (representing the 8 d.f. test) and zint=∑i=58zi~χ42 (representing the 4 d.f. test where the null hypothesis of no epistasis is true). For a particular value of NCP we simulated 100,000 z values, and calculated the pfull-value for the zfull test statistic. The nint test statistics with pfull < 2.31 × 10−16 were kept for the second stage, where the type 1 error rate of stage 2 was calculated as the proportion of pint < 0.05/nint. The power at stage 1 was calculated as nint/100, 000. This procedure was performed for a range of NCP parameters that represented power ranging from ~ 0 to ~ 1. 2.3 Population stratification We ruled out population stratification as a possible cause of inflated test statistics. To test for cryptic relatedness driving the interaction terms we tested for increased LD among the SNPs33. We calculated the mean of the off-diagonal elements of the correlation matrix of all unique SNPs from the 501 interactions (731 SNPs) using only unrelated individuals, r2¯=0.0039. This is not significantly different from the null hypothesis of zero (sampling error = 1/nunrelated = 0.0039). 2.4 Probe mapping To avoid possibility that epistatic signals might arise due to expression probes hybridising in multiple locations we verified that probe sequences for genes with significant interactions mapped to only a single location. As an initial verification we performed a BLAST search of the full probe sequence against 1000 genomes phase 1 version 3 human genome reference and ensured that only one genomic location aligned significantly (p < 0.05). As a second step, to mitigate the possibility of weak hybridisation elsewhere in the genome we divided the probe sequence into three sections (1–25bp, 13–37bp, 26–50bp) and performed a BLAST search of these probe sequence fragments. No probe sequemces or probe sequence fragments mapped to positions other than the single expected genomic target (p < 0.05). 3 Replication 3.1 Data description We attempted replication of the 501 significant interactions from the discovery set using three independent cohorts; Fehrmann, EGCUT, and CHDWB. It was required that LD r2 < 0.1 and D′2 < 0.1 between interacting SNPs (as measured in the replication sample directly), and all nine genotype classes had at least 5 individuals present in order to proceed with statistical testing for replication in both datasets. We also excluded any putative SNPs that had discordant allele frequencies in any of the datasets. Details of the cohorts are as follows. Fehrmann n = 1240 The Fehrmann dataset12 consists of peripheral blood samples of 1240 unrelated individuals from the United Kingdom and the Netherlands. Some of these individuals are patients, while others are healthy controls. Individuals were genotyped using the Illumina HumanHap300, Illumina Human-Hap370CNV, and Illumina 610 Quad platforms. RNA levels were quantified using the Illumina HT-12 V3.0 platform. These data are accessible through GEO Series accession numbers GSE20332 and GSE20142. EGCUT n = 891 The Estonian Genome Center of the University of Tartu (EGCUT) study11 consists of peripheral blood samples of 891 unrelated individuals from Estonia. They were genotyped using the Illumina HumanHap370CNV platform. RNA levels were quantified using the Illumina HT-12 V3.0 platform. These data are accessible through GEO Series accession number GSE48348. CDHWB n = 139 The Center for Health Discovery and Well Being (CD-HWB) Study24 is a population based cohort consisting of 139 individuals of European descent collected in Atlanta USA. Gene expression profiles were generated with Illumina HT-12 V3.0 arrays from peripheral blood collected from Tempus tubes that preserve RNA. Whole genome genotypes were measured using Illumina OmniQuad arrays. Due to the small sample size, most SNP pairs did not pass filtering in this dataset (20 SNP pairs remained) and so we have excluded it from the rest of the analysis. 3.2 Meta Analysis The 4 d.f. interaction p-values for each independent replication dataset were calculated using the same statistical test as was performed in the discovery dataset. We then took the interaction p-values from EGCUT and Fehrmann and calculated a joint p-value using Fisher’s method of combining p-values for a meta analysis as -2lnp1-2lnp2~χ4d.f.2. As in the discovery analysis, all gene expression levels were normalised using rank transformation to avoid skew or outliers in the distribution34. 3.3 Concordance of direction of effects We used four methods to calculate the concordance of the direction of effects between the discovery and replication datasets. Test 1 Is the most significant epistatic effect in the discovery set in the same direction as the same epistatic effect in the replication sets? We decomposed the genetic variance into 8 orthogonal effects, four of which are epistatic (A×A, A × D, D × A, D × D). The sign of the epistatic effect that had the largest variance in the discovery was recorded, and then was compared to the same epistatic effect in the two replication datasets (regardless of whether or not the same epistatic effect was the largest in the replication datasets). The probability of the sign being the same in one dataset is 1/2. The probability of the sign being the same in two is 1/4. Test 2 Is the most significant epistatic effect in the discovery the same as the largest epistatic effect in the replication set with the sign being concordant. As in Test 1, but this time we required that the largest effect was the same in the discovery and the replication, and that they had the same sign (e.g. if the largest effect in the discovery is A×A, with a positive effect, then concordance is achieved if the same is true in the replication). The probability of one replication dataset being concordant by chance is 1/8, and concordance in both is 1/64. Test 3 Do the epistatic effects that are significant at nominal p < 0.05 in the discovery have the same direction of effect as in the replication? Here we count all the epistatic variance components in the discovery that have p < 0.05 (1133 amongst the 434 discovery SNP pairs, i.e. each SNP pair has at least 1 and at most 4 significant epistatic variance components). Then we compare the direction of the effect in the replication dataset. The probability of the sign being the same in one dataset for any one significant effect is 1/2. The probability of the sign being the same in two is 1/4. Test 4 If we count how many of the 4 epistatic effects are concordant between the discovery and replication data for each interaction then is this significant from what we expect by chance? There can be either 0, 1, 2, 3 or 4 concordant signs at each interaction, each with expectation of p = 1/16, 4/16, 6/16, 4/16, 1/16 under the null, respectively. Observed counts are multinomially distributed, and we tested if the observed proportions were statistically different from the expected proportions using an approximation of the multinomial test35. The probability of observing the number of concordant signs in tests 1–3 is calculated using a binomial test. All variance decompositions were calculated using the NOIA method36. 4 Effects of LD on detection and replication The power to detect genetic effects, when the observed markers are in LD with the causal variants, is proportional to rx. For additive effects x = 2, but for non-additive effects x is larger, i.e. x = 4 for dominance or A × A, x = 6 for A × D or D × A, and x = 8 for D × D. Many biologically realistic GP maps may be comprised of all 8 variance components18. This is important for both detection and for replication of epistasis. For detection, if the epistatic effect includes the D × D term then if the two causal variants are tagged by observed markers that are each in LD r = 0.9, then if the true variance is Vt then the observed variance Vo at the markers will be 0.98Vt = 0.43Vt. Therefore, it is important to consider the sampling variation of r̂x in a sample given some true population value of r. 4.1 Simulation 1 For some values of fixed population parameters, p1 (minor allele frequency at observed marker), q1 (minor allele frequency at causal variant), and r (LD between marker and causal variant), the expected haplotype frequencies are (5) h11=rp1q1p2q2+p1q1 (6) h12=p1q2-rp1q1p2q2 (7) h21=p2q1-rp1q1p2q2 (8) h22=rp1q1p2q2+p2q2 where p2 = 1 − p1 and q2 = 1 − q1. For a range of population parameters we randomly sampled 2n haplotypes where the expected haplotype frequencies were h11, h12, h21, h22. From the sample haplotype frequencies we then calculated sample estimates of r̂ where (9) r^=h^11-p^1q^1p^1q^1p^2q^2 For each value of combination of the parameters p1, q1, r, n 1000 simulations were performed and the sampling mean and sampling standard deviation of r̂, r̂2, r̂4, r̂6, r̂8 were recorded. It was observed that sampling variance increases for increasing x in r̂x. 4.2 Simulation 2 We assume that the discovery SNP pairs are ascertained (from a very large number of tests) have high r̂ between observed SNPs and causal variants because otherwise power of detection would be low. We can hypothesis that the distribution of r̂ in this ascertained sample will be a mixture of r that is high and r that is lower but with ascertained higher values from sampling. Therefore, we would expect those with truly high r to have a higher replication rate in independent datasets, and those with ascertained high r̂ to have lower replication because resampling is unlikely to result in the same extreme ascertainment. To obtain empirical estimates of r̂ in discovery and replication datasets we conducted the following simulation. Using 1000 genomes data (phase 1, version 3, 379 European samples) we selected the 528,509 “markers” used in the original discovery analysis, plus 100,000 randomly chosen “causal variants” (CVs) with minor allele frequence > 0.05. The 379 individuals were split into discovery (190) and replication (189) sets. For each CV the marker with the maximum r^D2 from the marker panel was recorded in the discovery set. This marker was known as the “discovery marker” (DM). The r^R2 for each CV/DM pair was then calculated in the replication set where the discovery LD was ascertained to be high, such that r^D2>0.9. We observed that there was an average decrease in r^Rx relative to r^Dx, and that this decrease was larger with increasing x. We observed that (r^R2-r^D2)/r^D2=0.029 whereas (r^R8-r^D8)/r^D8=0.092. The average drop in in replication r̂8 was3 times higher than the drop in r̂2. 4.3 Interpretation Simulation 1 shows that sampling variance of rx increases as x increases. Detection of epistatis is highly dependent upon high r̂. Amongst the discovery SNPs there will be a mixture of interactions where observed SNPs are either in true high LD with causal variants, or will have highly inflated sample r̂x compared to the population rx. Simulation 2 shows that as x gets larger, the average decrease in r̂x between discovery and replication becomes larger, likely to be a result of ascertained high r̂ in the discovery and increased sampling variance with increasing x in the replication. These results demonstrate that if all else is equal, the impact of sampling variance of r alone will reduce the replication rate of epistatic effects compared to additive effects. 5 Additive and non-additive variance estimation 5.1 Fixed effects To compare the relative contribution to the phenotypic variance of gene expression levels between additive and epistatic effects we are constrained by the problem that non-additive variance components for a phenotype cannot be calculated directly. Here, we only have SNP pairs that exceed a threshold of p < 2.91 × 10−16 = Te. A strong conclusion cannot be made about the genome-wide variance contribution, but we can compare the variance explained by SNP effects at this threshold for additive scans and epistatic scans. In Powell et al 201223 an expression quantitative trait locus (eQTL) study was performed searching for additive effects in the same BSGS dataset as was used for the discovery here. Using the threshold Te for the additive eQTL study, 453 of the 7339 probes analysed here had at least one significant additive effect. Assuming that the phenotypic variance for each of the probes is normalised to 1, the total phenotypic variance of all 7339 explained by the significant additive effects was 1.73%. Following the same procedure, at the threshold Te there were 238 gene expression probes with at least one significant pairwise epistatic interaction out of the 7339 tested. In total the proportion of the phenotypic variance explained by the epistatic effects at these SNP pairs was 0.25%. 5.2 Limitations of this type of comparison Though it is useful to compare the relative variances of epistatic and additive effects, it must be stressed that our results here are approximations that are very limited by the study design. We estimate that additive effects explain approximately 10 times more variance than epistatic effects, but this could be an overestimate or an underestimate due to a number of different caveats. Firstly, the ratio of additive to epistatic variance may differ at different minimum variance thresholds, and our estimate is determined by the threshold used. Secondly, the power of a 1 d.f. test exceeds that of an 8 d.f. test. Thirdly, the non-additive variance at causal variants is expected to be underestimated by observed SNPs in comparison to estimates for additive variance. And forthly, the extent of winner’s curse in estimation of effect sizes may differ between the two studies. 5.3 Pedigree estimates The gene expression levels for MBNL1, TMEM149, NAPRT1, TRAPPC5 and CAST are influenced by large cis-trans epistatic networks (eight interactions or more). Though it is not possible to orthogonally estimate the non-additive genetic variance for non-clonal populations, an approximation of a component of non-additive variance can be estimated using pedigree information. The BSGS data is comprised of some related individuals and standard quantitative genetic analysis was used to calculate the additive and dominance variance components for each gene expression phenotype in Powell et al 201322. The dominance effect is likely to capture additive × additive genetic variance plus some fraction of other epistatic variance components. We found that the aforementioned genes had dominance variance component estimates within the top 5% of all 17,994 gene expression probes that were analysed in Powell et al 2013. 6 Functional enrichment analysis 6.1 Tissue specific transcriptionally active regions We employed a recently published method (http://www.broadinstitute.org/mpg/epigwas/)27 that tests for cell-type-specific enrichment of active chromatin, measured through H3K4me3 chromatin marks37 in regions surrounding the 731 SNPs that comprise the 501 discovery interactions. The exact method used to perform this analysis has been described previously38. Briefly, we tested the hypothesis that the 731 SNPs were more likely to be in transcriptionally active regions (as measured by chromatin marks) than a random set of SNPs selected from the same SNP chip. This hypothesis was tested for 34 cell types across four broad tissue types (haematopoietic, gastrointenstinal, musculoskeletal and endocrine, and brain). 6.2 Chromosome interactions It has been shown13 that different regions on different chromosomes or within chromosomes spatially colocalise within the cell. We shall refer to the colocalisation of two chromosome regions as a chromosome interaction. A map of pairwise chromosome interactions for K562 blood cell lines was recently produced29, and we hypothesised that part of the underlying biological mechanism behind some of the 501 epistatic interactions may arise from chromosome interactions. We found that 44 of the putative epistatic interactions were amongst SNPs that were within 5Mb of known chromosome interactions. This means that SNP A was no more than 2.5Mb from the focal point of the chromosome interaction on chromosome A, and SNP B was no more than 2.5Mb from the focal point on chromosome B. We performed simulations to test how extreme the observation of 44 epistatic interactions overlapping with chromosome interactions is compared to chance. Chromosome interactions fall within functional genomic regions13,29, and the SNPs in our epistatic interactions are enriched for functional genomic regions. Therefore, we designed the simulations to ensure that the null distribution was of chromosome interactions between SNPs enriched for functional genomic regions but with no known epistatic interactions. To do this we used the 731 SNPs that form the 501 putative epistatic interactions and randomly shuffled them to create new sets of 501 pairs, disallowing any SNP combinations that were in the original set. Therefore, each new random set was enriched for functional regions but had no genetic interactions. We scanned the map of chromosome interactions for overlaps with the new sets and then repeated the random shuffling process. We performed 1,000 such permutations to generate a null distribution of chromosome interaction overlaps. We repeated this process, searching for overlaps within 1Mb, 250kb, and 10kb. 6.3 SNP colocalisation with genomic features We tested for enrichment of genomic features for the 687 IndexSNPs that comprise the 434 epistatic interactions with data present in discovery and replication datasets. For each of the 687 IndexSNPs we calculated LD with all regional SNPs within a radius of 0.5Mb and kept all regional SNPs with LD r2 > 0.8. We then cross-referenced the remaining regional SNPs with the annotated chromatin structure reference28) querying whether the regional SNPs fell in Predicted promoter region including TSS (TSS), Predicted promoter flanking region (PF), Predicted enhancer (E), Predicted weak enhancer or open chromatin cis regulatory element (WE), CTCF enriched element (CTCF), Predicted transcribed region (T), or Predicted Repressed or Low Activity region (R) positions. Therefore a particular IndexSNP might cover multiple genomic features through LD. We then performed the whole querying process for each of the 528,509 SNPs present in the SNP chip used in the scan, and used the results from this second analysis to establish a null distribution for the expected proportion of SNPs for each genomic feature. We calculated p-values for enrichment of each of the seven genomic features independently, and for cis- and trans-SNPs separately, using a binomial test. For each genomic feature we used the expected proportion of SNPs as the expected probability of “success” (p). Here, a success is defined as an IndexSNP residing in a region that includes the genomic feature. The observed number of successes for each IndexSNP (k) out of the total count of IndexSNPs (n) was then modelled as Pr(X=k)=(nk)pk(1-p)n-k. 6.4 Transcription factor enrichment To test for enrichment of transcription factor binding sites (TFBS) we followed a procedure similar to that described in Section 6.3. For each of the 687 IndexSNPs we extracted regional SNPs as previously described. We then used the PWMEnrich package in Bioconductor (http://www.bioconductor.org/packages/2.12/bioc/html/PWMEnrich.html) to identify which TFBSs each of the regional SNPs for one IndexSNP falls in (within a radius of 250bp). Thus, the number of occurrences of a particular TFBS was counted for each IndexSNP. We used the “Threshold-free affinity” method for identifying TFBSs39. We constructed a null distribution of expected TFBS occurrences based on the same null hypothesis as described in Section 6.3 - the probability of an IndexSNP covering a particular TFBS is identical to any of the 528,509 SNPs in the discovery SNP chip. To do this, we performed the same procedure for each SNP in the discovery SNP chip as was performed for each IndexSNP to obtain an expected probability of covering a particular TFBS. We then tested the IndexSNPs for enrichment of each TFBS independently, and for cis- and trans-SNPs separately. p-values were obtained using Z-scores, calculated by using a normal approximation to the sum of binomial random variables representing motif hits along the sequence40. 6.5 Defining previously identified SNP associations The discovery dataset (BSGS) had previously been analysed for additive and dominant marginal effects for all gene expression levels22,23. To define SNPs that had been previously detected to have effects for a particular gene expression level we used a significance threshold accounting for multiple testing across SNPs and expression probes, Tm = 0.05/(528509 × 7339) = 1.29 × 10−11. From this, we found that only nine of the 501 discovery interactions had known main effects, 64 were between SNPs that had no known marginal effects, and 439 were between a SNP with a known marginal effect and a SNP with no known marginal effect. Supplementary Material 1 We are grateful to the volunteers for their generous participation in these studies. We thank Bill Hill, Chris Haley and Lars Ronnegard for helpful discussions and comments. This work could not have been completed without access to high performance GPGPU compute clusters. We acknowledge iVEC for the use of advanced computing resources located at iVEC@UWA (www.ivec.org), and the Multimodal Australian ScienceS Imaging and Visualisation Environment (MASSIVE) (www.massive.org.au). We also thank Jake Carroll and Irek Porebski from the Queensland Brain Institute Information Technology Group for HPC support. The University of Queensland group is supported by the Australian National Health and Medical Research Council (NHMRC) grants 389892, 496667, 613601, 1010374 and 1046880, the Australian Research Council (ARC) grant (DE130100691), and by National Institutes of Health (NIH) grants GM057091 and GM099568. The QIMR researchers acknowledge funding from the Australian National Health and Medical Research Council (grants 241944, 389875, 389891, 389892, 389938, 442915, 442981, 496739, 496688 and 552485), the and the National Institutes of Health (grants AA07535, AA10248, AA014041, AA13320, AA13321, AA13326 and DA12854). We thank Anthony Caracella and Lisa Bowdler for technical assistance with the micro-array hybridisations. The CHDWB study funding support from the Georgia Institute of Technology Research Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript The Fehrmann study was supported by grants from the Celiac Disease Consortium (an innovative cluster approved by the Netherlands Genomics Initiative and partly funded by the Dutch Government (grant BSIK03009), the Netherlands Organization for Scientific Research (NWO-VICI grant 918.66.620, NWO-VENI grant 916.10.135 to L.F.), the Dutch Digestive Disease Foundation (MLDS WO11-30), and a Horizon Breakthrough grant from the Netherlands Genomics Initiative (grant 92519031 to L.F.). This project was supported by the Prinses Beatrix Fonds, VSB fonds, H. Kersten and M. Kersten (Kersten Foundation), The Netherlands ALS Foundation, and J.R. van Dijk and the Adessium Foundation. The research leading to these results has received funding from the European Communitys Health Seventh Framework Programme (FP7/2007-2013) under grant agreement 259867. The EGCUT study received targeted financing from Estonian Government SF0180142s08, Center of Excellence in Genomics (EXCEGEN) and University of Tartu (SP1GVARENG). We acknowledge EGCUT technical personnel, especially Mr V. Soo and S. Smit. Data analyzes were carried out in part in the High Performance Computing Center of University of Tartu. Author contributions G.H., J.E.P., P.M.V., and G.W.M. conceived and designed the study. G.H., J.E.P., K.S., H-J.W., and J.Y. performed the analysis. T.E. and A.M. provided the EGCUT data. A.K.H., A.F.M., G.W.M., N.G.M., and J.E.P. provided the BSGS data. G.G. provided the CHDWB data. H-J.W. and L.F. provided the Fehrmann data. G.H. and J.E.P. wrote the manuscript with the participation of all authors. Author information The authors declare no financial competing interests. Figure 1 Replication of GP maps in two independent populations The GP maps for each epistatic interaction that is significant at the Bonferroni level in both replication datasets are shown. Each GP map consists of nine tiles where each tile represents the expression level for that two-locus genotype class. Phenotypes are for gene transcript levels (dark coloured tiles = high expression, light coloured tiles = low expression). Columns of GP maps are for each independent dataset. Rows of GP maps are for each of 30 significantly replicated interactions at the Bonferroni level, corresponding to the rows in Table 1. There is a clear trend of the GP maps replicating across all three datasets. Figure 2 Q-Q plots of interaction p-values from replication datasets The top panel shows all 434 discovery SNPs that were tested for interactions. Observed p-values (y-axis, −log10 scale) are plotted against the expected p-values (x-axis, −log10 scale). The multiple testing correction threshold for significance following Bonferroni correction is denoted by a dotted line. The bottom panel shows the same data as the top panel but excluding the 30 interactions that were significant at the Bonferroni level in the replication datasets. The shaded grey area represents the 5% confidence interval for the expected distribution of p-values. Dark blue points represent p-values that exceed the confidence interval, light blue are within the confidence interval. Figure 3 Discovery and replication of epistatic networks All 434 putative genetic interactions (edges) with data common to discovery and replication sets is shown, where black nodes represent SNPs and red nodes represent traits (gene expression probes). Three hundred and forty-five interactions had p-values exceeding the 2.5% confidence interval following meta analysis of the replication data The remaining 89 interactions that did not replicate are depicted in grey. It is evident that a large proportion of the complex networks identified in the discovery set also exist in independent populations. An interactive version of this graph can be found here: http://kn3in.github.io/detecting_epi/ Table 1 Epistatic interactions significant at the Bonferroni level in two replication sets Gene (chr.) SNP 1 (chr.) SNP 2 (chr.) BSGS2 Fehrmann3 EGCUT3 Meta4 1 ADK (10) rs2395095 (10) rs10824092 (10) 6.691 18.331 21.211 39.821 2 ATP13A1 (19) rs4284750 (19) rs873870 (19) 5.30 12.18 3.25 14.23 3 C21ORF57 (21) rs9978658 (21) rs11701361 (21) 9.42 6.08 16.36 21.67 4 CSTB (21) rs9979356 (21) rs3761385 (21) 11.99 25.20 16.72 42.27 5 CTSC (11) rs7930237 (11) rs556895 (11) 7.16 18.76 15.06 33.53 6 FN3KRP (17) rs898095 (17) rs9892064 (17) 16.16 28.24 29.39 59.95 7 GAA (17) rs11150847 (17) rs12602462 (17) 13.91 19.98 12.99 32.60 8 HNRPH1 (5) rs6894268 (5) rs4700810 (5) 15.38 8.55 3.01 10.37 9 LAX1 (1) rs1891432 (1) rs10900520 (1) 19.16 18.60 11.22 29.24 10 MBNL1 (3) rs16864367 (3) rs13079208 (3) 13.49 16.25 24.74 41.56 11 MBNL1 (3) rs7710738 (5) rs13069559 (3) 7.92 2.55 7.89 9.28 12 MBNL1 (3) rs2030926 (6) rs13069559 (3) 7.10 0.91 5.80 5.53 13 MBNL1 (3) rs2614467 (14) rs13069559 (3) 5.74 4.13 2.22 5.30 14 MBNL1 (3) rs218671 (17) rs13069559 (3) 7.63 0.62 5.82 5.23 15 MBNL1 (3) rs11981513 (7) rs13069559 (3) 7.71 0.43 5.36 4.58 16 MBP (18) rs8092433 (18) rs4890876 (18) 5.40 7.06 21.91 28.73 17 NAPRT1 (8) rs2123758 (8) rs3889129 (8) 8.45 15.12 16.08 30.77 18 NCL (2) rs7563453 (2) rs4973397 (2) 7.31 7.51 6.33 12.70 19 PRMT2 (21) rs2839372 (21) rs11701058 (21) 4.81 0.69 4.47 4.06 20 RPL13 (16) rs352935 (16) rs2965817 (16) 4.98 3.79 14.41 17.24 21 SNORD14A (11) rs2634462 (11) rs6486334 (11) 7.31 13.11 10.96 23.22 22 TMEM149 (19) rs807491 (19) rs7254601 (19) 12.16 81.55 45.78 145.78 23 TMEM149 (19) rs8106959 (19) rs6926382 (6) 5.80 3.06 8.80 10.72 24 TMEM149 (19) rs8106959 (19) rs914940 (1) 6.22 3.36 6.96 9.20 25 TMEM149 (19) rs8106959 (19) rs2351458 (4) 7.30 0.04 9.61 8.00 26 TMEM149 (19) rs8106959 (19) rs6718480 (2) 8.55 3.31 5.15 7.36 27 TMEM149 (19) rs8106959 (19) rs1843357 (8) 6.21 3.72 3.33 6.00 28 TMEM149 (19) rs8106959 (19) rs9509428 (13) 9.44 0.10 5.75 4.47 29 TRA2A (7) rs7776572 (7) rs11770192 (7) 8.23 3.19 1.89 4.09 30 VASP (19) rs1264226 (19) rs2276470 (19) 5.09 0.94 5.14 4.95 1 −log10 p-values for 4 d.f. interaction tests 2 Discovery dataset 3 Independent replication dataset 4 Meta analysis of interaction terms between replication datasets only ==== Refs 1 Carlborg O Haley CS Epistasis: too often neglected in complex trait studies? 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==== Front 87115626325OncogeneOncogeneOncogene0950-92321476-55942416650110.1038/onc.2013.444nihpa550260ArticleAurora-A is a determinant of tamoxifen sensitivity through phosphorylation of ERα in breast cancer Zheng XQ 1236Guo JP 16Yang H 1Kanai M 1He LL 1Li YY 1Koomen JM. 1Minton S. 4Gao M 2Ren XB 3Coppola D 5Cheng JQ 1‡1 Departments of Molecular Oncology, H. Lee Moffitt Cancer Center & Research Institute, Tampa, FL 336122 Department of Thyroid and Neck Tumour, Tianjin Medical University Cancer Institute and Hospital, Oncology Key Laboratory of cancer prevention and therapy, National Clinical Research Center of Cancer, Tianjin, P. R. China, 3000603 Department of Immunology, Tianjin Medical University Cancer Institute and Hospital, Oncology Key Laboratory of cancer prevention and therapy, National Clinical Research Center of Cancer, Tianjin, P. R. China, 3000604 Department of Women’s Oncology, H. Lee Moffitt Cancer Center & Research Institute, Tampa, FL 336125 Department of Pathology, H. Lee Moffitt Cancer Center & Research Institute, Tampa, FL 33612‡ To whom correspondence should be addressed, at H. Lee Moffitt Cancer Center, 12902 Magnolia Drive, SRB3, Tampa, FL 33612. Phone (813) 745-6915; [email protected] Contributed equally to this work. 4 2 2014 28 10 2013 16 10 2014 16 4 2015 33 42 4985 4996 Users may view, print, copy, download and text and data- mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: http://www.nature.com/authors/editorial_policies/license.html#termsDespite the clinical success of tamoxifen, its resistance remains a major challenge in breast cancer. Here we show that Aurora-A determines tamoxifen sensitivity by regulation of estrogen receptor (ER)α. Ectopic expression of Aurora-A decreases and depletion of Aurora-A enhances tamoxifen sensitivity in ERα-positive breast cancer. Elevated Aurora-A was significantly associated with the recurrence of ERα-positive tumours. Notably, Aurora-A inhibitor MLN8237, which is currently in clinical trial, synergizes with tamoxifen and overcomes tamoxifen-resistance. Furthermore, Aurora-A interacts with and phosphorylates ERα on serine-167 and -305, leading to increase in ERα DNA-binding and transcriptional activity. Elevated levels of Aurora-A are significantly associated with disease-free survival in ERα-positive but not -negative breast cancers. These data suggest that Aurora-A plays a pivotal role in tamoxifen resistance and ERα is a bona fide substrate of Aurora-A. Thus, Aurora-A represents a prognostic marker in ERα-positive tumor and a critical therapeutic target in tamoxifen-resistant breast cancer, and Aurora-A inhibitor could be used as either an independent or concurrent agent in tamoxifen-resistant tumour. Aurora-Atamoxifen resistanceERαphosphorylationtranscriptional activation ==== Body INTRODUCTION Aurora-A is a mitotic serine/threonine kinase, which is evolutionally conserved and is localized at the centrosome.1 Activation of Aurora-A is required for mitotic entry, centrosome maturation and separation and G2 to M transition.2 It has been shown that Aurora-A is frequently amplified and/or overexpressed in breast carcinoma.3 In animal models, estrogen-induced rat breast cancers expressed high levels of Aurora-A and displayed centrosome amplification and aneuploidy.4 Moreover, mouse mammary tumour virus (MMTV)-driven Aurora-A transgenic mice developed breast tumours and genetic instability occurred prior to tumorigenesis in mammary epithelial cells.5 In addition, recent studies have shown that Aurora-A promotes distant metastases only in ERα-positive breast cancer cells.6, 7 These findings indicate the critical role of Aurora-A in mammary carcinogenesis. However, the role of Aurora-A in endocrine-therapy resistance and ERα signaling remains largely unknown. ERα is a hormone-dependent nuclear transcription factor expressed in approximately 70% of breast tumours.8 The binding of estrogen to ERα results in ERα dimerization and its recruitment to the estrogen-responsive elements (EREs) on the promoters of ERα target genes.9, 10 Because ERα plays a major role in the development and progression of breast cancer, current endocrine therapies for breast cancer are mainly based on targeting the ERα signaling pathway. Among the endocrine therapies targeting ERα, the selective estrogen receptor modulator tamoxifen, which inhibits breast cancer growth through competitive binding of ERα, has been the principal endocrine therapy for breast cancer for the past 30 years.11 However, tamoxifen resistance presents a major challenge in treating the disease.12–14 ERα is a key determinant of breast cancer susceptibility to endocrine therapy, and post-translational modifications of ERα often lead to endocrine resistance.15 The phosphorylation of ERα has long been implicated in modulating endocrine response 16, 17 and has been shown to influence ERα-binding potential and gene expression profiles,16 resulting in tamoxifen resistance. In this study, we demonstrated a critical role of Aurora-A in regulation of tamoxifen sensitivity. Aurora-A inhibitor MLN8237 synergized with tamoxifen to inhibit cell growth and cell survival and to overcome tamoxifen resistance. Furthermore, Aurora-A interacted with ERα and phosphorylated ERα-Ser167/Ser305. The phosphorylation of ERα by Aurora-A resulted in activation of ERα in the absence of estrogen. These data indicate that Aurora-A plays an important role in regulation of ERα activity and tamoxifen resistance, and thus could be a therapeutic target in breast cancer endocrine-therapy. In addition, these findings underscore the potential of MLN8237 as either an independent or concurrent agent in tamoxifen-resistant breast cancer and the pERα-Ser167/Ser305 as potential clinical biomarkers in Aurora-A inhibitor therapy. RESULTS Knockdown of Aurora-A reverses tamoxifen resistance and ectopic expression of Aurora-A renders cells resistance to tamoxifen Previously studies showed frequent overexpression/activation of Aurora-A in breast cancer and a critical role of Aurora-A in mammary epithelial cell immortalization and transformation.5, 18 When we examined Aurora-A expression in a panel of breast cancer cell lines, we found elevated levels of Aurora-A protein and p-Aurora-A-T288 in tamoxifen-resistant BT474 and MCF7-TamR cells as compared to tamoxifen-responsive MCF7 cells (Figure 1a). These findings prompted us to assess the role of Aurora-A in tamoxifen resistance. We initially assessed the tamoxifen sensitivity in 3 ERα-positive and 1 ERα-negative cell lines (Figure S1) and subsequently knocked down Aurora-A in BT474 and MCF7-TamR cells using 2 siRNAs against different coding regions of Aurora-A (Figures 1b and S2a). Following incubation for 2 days, the cells were treated with and without increasing doses of tamoxifen for 72 hours and then subjected to MTT, Annexin V/FACS and colony formation assays. In agreement with previous reports, tamoxifen alone had minimal effect on cell death and colony growth in tamoxifen-resistant BT474 and MCF7-TamR cells.19 However, knockdown of Aurora-A significantly reduced cell survival and colony formation, and enhanced tamoxifen anti-tumour effects in both BT474 and MCF7-TamR cell lines (Figures 1c–e, and Figures S2b and 2c). To further examine the role of Aurora-A in tamoxifen sensitivity, we ectopically expressed Aurora-A in tamoxifen-responsive MCF7 cells (Figure 2a). The cells transfected with empty vector were used as controls. Following treatment with tamoxifen, we found that overexpression of Aurora-A rendered MCF7 cells resistance to tamoxifen (Figures 2b–d). As controls, we evaluated the effects of Aurora-A on response to tamoxifen in ERα-negative cells. Aurora-A was knocked down and ectopically expressed in MDA-MB-468 and MDA-MB-231 cells, respectively. After administration of tamoxifen, cell viability and colony formation were found no difference between Aurora-A-manipulated and control-siRNA/vector-treated cells in both ERα-negative cell lines (Figure S3). These data indicate that Aurora-A plays a role in tamoxifen resistance and could be a critical therapeutic target for overcoming the resistance in ERα-positive breast cancer cells. Aurora-A inhibitor MLN8237 cooperates with tamoxifen and overcomes tamoxifen-resistance in vitro and in vivo Since tamoxifen-resistant MCF7-TamR and BT474 cells expressed high levels of Aurora-A and knockdown of Aurora-A reduced the resistance, we further investigated whether Aurora-A inhibitor MLN8237, which is currently in clinical trial,20 is able to overcome tamoxifen resistance and to cooperate with tamoxifen. We first treated MCF7-TamR and BT474 cells with various concentrations of MLN8237 and tamoxifen alone and in combination and monitored cell survival. Figures 3a and 3c show that MLN8237 alone inhibited p-Aurora-A-T288 and caused a dose-dependent inhibition of cell survival with IC50 values of ~4.0 μm/L, whereas tamoxifen alone had much less effect on cell survival with IC50 values of greater than 5 μm/L. However, combination treatment of cells with Aurora-A inhibitor and tamoxifen resulted in significant cell death with IC50 values of tamoxifen from greater than 5 μm/L (in the absence of MLN8237) to 0.1 μm/L (in the presence of 0.5 μmol/L MLN8237; Figure S4). In addition, we found that combination of MLN8237 and tamoxifen increased cleavage of PARP, caspase-7 (MCF7-TamR) or caspase 3 (BT474) more significantly than either one alone (Figure 3b). Therefore, MLN8237 sensitized MCF7-TamR and BT474 cells to tamoxifen. To further examine if MLN8237 synergizes with tamoxifen, we treated cells with a combination of MLN8237 and tamoxifen and used Calcusyn software to generate Fa-CI plots as described under Methods. Figure S4 shows that almost all the experimental points have CI values of less than 1.0. The resulting Fa-CI plot curve falls well below 1 for the effect range, showing that the combination of MLN8237 and tamoxifen is synergistic in tamoxifen-resistant MCF7-TamR and BT474 cells. We next determined whether Aurora-A inhibitor MLN8237 overcomes tamoxifen resistance in vivo. Orthotopic tumours were established by injection of BT474 cells into the mammary fat pads of athymic nude mice. After implanted tumours reached approximately 100 mm3 in size, the mice were randomized in 4 groups (7 mice/group): vehicle, tamoxifen alone, MLN8237 alone, and both tamoxifen and MLN8237. As control, orthopotic tumours with tamoxifen-sensitive MCF7 cells were also established and administered with tamoxifen and vehicle (Figure S5). The mice were treated for 12 consecutive days and the tumour growth was monitored for an additional 1 week (Figure 3d). While the control group tumour reached 1500 mm3 in volume at the endpoint of the assay, the MLN8237-treated tumours grew much slower than control and tamoxifen-treated groups. Treatment with tamoxifen alone completely inhibited MCF7 tumour growth but only caused an approximately 10% reduction in BT474 tumour size compared with the control group (Figures 3d and S5), whereas the combination of both MLN8237 and tamoxifen significantly repressed BT474 tumour growth (Figure 3d). Similarly, the BT474 tumour weight in MLN8237- and the combination-treated groups was dramatically reduced as compared with control and tamoxifen-treated groups (Figure 3e). Immunoblotting analysis of the representative tumours revealed that p-Aurora-A-T288 was inhibited by MLN8237 and that PARP cleavage was induced in the tumours treated with MLN8237 alone and combination of MLN8237 and tamoxifen (Figure 3f). Moreover, immunostaining with cleavage caspase-3 and Ki-67 antibodies showed that inhibition of Aurora-A alone and combination with tamoxifen significantly activated caspase-3 and inhibited proliferation (Figure 3g). Taken together, these data indicate that Aurora-A inhibitor MLN8237 not only overcomes tamoxifen resistance but also synergizes with tamoxifen in vitro and in vivo. Aurora-A induces ERα transactivation activity Post-translational modification of ERα has been shown to be one of major mechanisms in tamoxifen resistance.21, 22 Thus, we next investigated if Aurora-A regulates ERα transactivation activity. Reporter assay was performed by introducing ERα responsive element reporter (ERE-Luc) combined with HA-Aurora-A into MCF7 cells, and the results showed that Aurora-A strongly induced ERα transactivation activity in a dose-dependent manner (Figure 4a). In addition, the reporter activity induced by E2 was further increased by expression of Aurora-A (Figure S6a). However, E2- but not Aurora-A-induced ERα activation was inhibited by tamoxifen (Figure S6b). Furthermore, the effect of Aurora-A on ERE-Luc activity was evaluated in ERα-negative MDA-MB-231 cells. Following transfection of ERE-Luc and ERα together with and without Aurora-A, luciferase assay revealed that expression of ERα alone moderately induced ERE-Luc activity. However, the reporter activity was significantly stimulated by co-expression of ERα and Aurora-A (Figure 4b). These data suggest that Aurora-A not only activates ERα activity but also enhances E2 action and that Aurora-A-induced ERα activation could not be inhibited by tamoxifen. Aurora-A interacts with and phosphorylates ERα To determine the mechanism by which Aurora-A induces ERα transactivation activity, we initially performed co-immunoprecipitation in MDA-MB-231 cells following transfection of GFP-ERα and HA-Aurora-A. Figure 4c shows that HA-Aurora-A interacted with GFP-ERα. We next examined if endogenous ERα and Aurora-A forms a complex. Co-immunoprecipitation was carried out in MCF7-TamR cells, which express elevated Aurora-A and ERα (Figure 1a). As shown in Figure 4d, ERα was readily detected in Aurora-A immunoprecipitates and vice versa. Furthermore, GST-pull down assay revealed that GST-Aurora-A but not GST could pull-down the recombinant ERα (Figure 4e). These data indicate that Aurora-A directly binds to ERα. To determine if Aurora-A co-localizes with ERα, we performed immunofluorescence staining in MCF7-TamR cells with anti-ERα and -Aurora-A antibodies and we found that Aurora-A co-localized with ERα not only in the nucleus but also in centrosome (Figure 4f). We next investigated if Aurora-A phosphorylates ERα. In vitro Aurora-A kinase assay was performed by incubation of full-length human recombinant ERα with and without recombinant Aurora-A. Figure 5a shows that ERα was highly phosphorylated in the reaction containing Aurora-A. To determine if Aurora-A phosphorylates ERα in vivo, MDA-MB-231 cells were co-transfected with Myc-ERα and either HA-Aurora-A or vector. After labeling with [32P]-orthophosphate for 3 hours, ERα was immunoprecipitated with anti-Myc antibody and the immunoprecipitates were separated in SDS-PAGE. Following exposure of an x-ray film, we observed that phosphorylation of ERα was induced by Aurora-A (Figure 5b). We further defined the amino acid(s) of ERα that is phosphorylated by Aurora-A. In vitro Aurora-A kinase assay was carried out using GST fusion proteins containing different portions of ERα as substrates (Figure S7a). Since ERα/1–200 and ERα/1–318 but not ERα/1–150 were phosphorylated by Aurora-A, a potential phosphorylation site(s) was mapped to the amino acid 150–318 region of ERα (Figure S7b). Mass spectrometry analysis revealed serine-167 (Ser167), Ser212 and Ser305 as putative Aurora-A phosphorylation sites. To verify if these 3 serine residues are phosphorylated by Aurora-A, we further created 3 different GST-ERα fusion proteins that contain Ser167, Ser212 or Ser305 and their serine-alanine mutation S167A, S212A and S305A individually (Figure S7a). In vitro kinase assays revealed that Aurora-A phosphorylated wild-type GST-ERα-S167, even it is not perfect match with Aurora-A phosphorylation consensus motif,23 and -S305 but not GST-ERα-S212, -S167A, and -S305A (Figure S7c). Furthermore, in vivo [32P]orthophosphate labeling and Western blotting analysis revealed that Aurora-A phosphorylation of wild-type ERα but not ERα-S167A/S305A (ERα-2A) mutant (Figures 5c and 5d), suggesting that Ser167 and Ser305 of ERα are phosphorylated by Aurora-A. These findings were further confirmed by immunoblotting of Aurora-A overexpressing MCF7 and Aurora-A knocking down BT474 cells (Figure 5e) as well as of cold in vitro Aurora-A kinase reaction (Figure S7d) using specific phospho-ERα-Ser167 and -Ser305 antibodies. In addition, we observed that Aurora-A inhibitor MLN8237 significantly inhibited p-ERα-Ser167/Ser305 levels in MCF7-TamR and BT474 cells (Figures S8a and S8b) and the xenografts (Figure S8c). Based on these findings, we conclude that ERα-Ser167 and -Ser305 are phosphorylated by Aurora-A in vitro and in vivo. Aurora-A-induced ERα transactivation activity and CCND1 expression via phosphorylation of ERα-Ser167 and Ser305 Since Aurora-A induces ERα transactivation, we next investigated whether Aurora-A-induced ERα activity depends on phosphorylation of ERα-Ser167 or/and Ser305. ERα-negative MDA-MB-231 cells were transfected with wild-type ERα, ERα-S167A, ERα-S305A or ERα-S167A/S305A (ERα-2A) together with ERE-Luc. Reporter assay showed that expression of Aurora-A significantly induced wild-type ERα but not ERα-S167A/S305A transactivation activity (Figure 6a). Furthermore, Aurora-A also stimulated ERα-S167A- and ERα-S305A-induced ERE-Luc activity to a much lesser extent than wild-type ERα (Figure 6a) Since CCND1 (cyclin D1) is a major target of ERα and overexpression of CCND1 has been implicated in tamoxifen resistance,25, 26 we also examined whether Aurora-A induces CCND1 expression and, if present, whether the induction depends on phosphorylation of ERα-Ser167/SerS305. ERα-positive MCF7 and ERα-negative MDA-MB-231 cells were transfected with Aurora-A. Following 72 hours of incubation, semi-quantitative RT-PCR and immunoblot analyses revealed that mRNA and protein levels of CCND1 were induced by Aurora-A in MCF7 but not in MDA-MB-231 cells (Figure 6b and Figure S9). Furthermore, knockdown of Aurora-A decreased CCND1 expression in BT474 cells (Figure 6c), suggesting Aurora-A induction of CCND1 through ERα. To determine if Aurora-A phosphorylation of ERα mediates this action, we expressed wild-type ERα, ERα-S167A/S305A, ERα-S167A and ERα-S305A together with and without Aurora-A in MDA-MB-231 cells. As shown in Figures 6d–f, co-expression of Aurora-A and wild-type ERα significantly increased CCND1 protein and mRNA levels and ERα transactivation and DNA-binding activity towards the CCND1 promoter. However, Aurora-A failed to exert these effects when co-transfected with ERα-S167A/S305A. Expression of ERα-S167A or ERα-S305A partially inhibited Aurora-A-induced CCND1 (Figure 6d and 6e). In addition, we examined the relationship between Aurora A and CCND1 mRNA levels in patient samples from GEO gene expression datasets and found the significant positive correlation of Aurora-A with CCND1 expression in ERα-positive but not ERα-negative breast cancers (Figure S10). Taken together, these data indicate that Aurora-A induces ERα and ERα-targeted gene CCND1 through phosphorylation of Ser167 and Ser305. Upregulation of Aurora-A correlates with p-ERα-Ser167/Ser305, endocrine therapy resistance and disease-free survival in ERα-positive breast cancer Having demonstrated Aurora-A phosphorylation of ERα-Ser167/Ser305 in cells, we next investigated if this event occurs in human breast tumour samples. Of the 167 ERα-positive breast cancers examined by immunohistochemical staining and Western blot, 73 had Aurora-A overexpression and 34 had elevated both p-ERα-Ser167 and p-ERα-Ser305 (Figures 7a–c and Table S1). Further, p-ERα-Ser167 and p-ERα-Ser305 were detected in 88 and 71 tumours, respectively, which include 34 cases with positive p-ERα-Ser167/Ser305 (Figure 7c). Of the 34 tumours with elevated levels of p-ERα-Ser167/Ser305, 32 (94.1%) also had elevated Aurora-A (p < 0.00001; Figure 7d). The other 2 cases with elevated p-ERα-Ser167/Ser305 could be resulted from activation of other kinases (Figure 8). Further analyses showed that Aurora-A expression level, p-ERα-Ser167 and p-ERα-Ser305 status were not related to tumour size, lymph node metastasis, tumour stage and grade (Table S1). However, p-ERα-Ser167/Ser305 and pERα-Ser167 alone, but not p-ERα-Ser305, are significantly associated disease-free survival (DFS; Figure S11a–c and Table S2). All 32 patients with elevated Aurora-A and positive pERα-Ser167/Ser305 relapsed from tamoxifen/nolvadex treatment and had poor DFS (Figure S11d). Notably, elevated levels of Aurora-A were found to be significantly associated with DFS (Figure 7d and Table S2). To further confirm these findings, we took advantage of the available gene expression datasets summing up to 854 ERα-positive primary breast cancers with associated clinical data, including endocrine therapy, disease recurrence and survival (Table S3). We defined each dataset into two groups of tumours with respectively high and low level of expression of Aurora-A (Figure S12). Strikingly, the univariate Kaplan-Merier survival analysis revealed that the group expressing high levels of Aurora-A displayed a significant higher probability to develop recurrence when compared to the “low” group (p values ranged from 0.0064 to 3E-01, depending on the datasets; Figure 7e, S12, S13 and Table S4). In addition, the KM Plotter database (http://www.kmplot.com;24 analysis of breast cancers also showed that Aurora-A levels were significantly associated with recurrence-free survival in ERα-positive breast cancers treated with endocrine therapy (Figure 7f and Table S5) but not in ERα-negative and basal breast tumours (Figure S14 and Table S5). Since antiestrogen therapy was used in ERα-positive tumours collected in these datasets, the recurrence largely represents the resistance to endocrine therapy. Therefore, these data suggest that elevated Aurora-A has significant implication in recurrence of ERα-positive breast cancers which are mostly due to Aurora-A inducing endocrine therapy-resistance by phosphorylation of ERα. DISCUSSION Here we show that overexpression of Aurora-A sufficed to induce tamoxifen resistance, whereas inhibition of Aurora-A by small molecule inhibitor MLN8237 or siRNA knockdown overcame the resistance. Previous reports demonstrated that overexpression/activation of Aurora-A is a recurrent event in human breast cancer.3 A recent study showed that Aurora-A promotes distant metastases by inducing epithelial-to-mesenchymal transition in ERα-positive breast cancer cells,6 suggesting a link between Aurora-A and ERα, which plays a critical role in disease progression in ERα-positive breast cancer. Our data showed that Aurora-A directly interacted with ERα and phosphorylated ERα-Ser167/Ser305 in vitro and in vivo (Figures 4 and 5). Co-existence of overexpression of Aurora-A and phospho-ERα-Ser167/Ser305 was detected in tamoxifen resistance breast cancer cells and primary tumours with poor prognosis (Figures 1, 7c and S11d). Furthermore, data mining analysis of ~2,400 breast cancers revealed that elevated Aurora-A was significantly associated with short recurrence-free survival only in ERα-positive and antiestrogen-treated breast cancers (Figures 7e, 7f and S12–S14, and Table S4 and S5). Thus, these findings indicate that ERα is a bona fide substrate of Aurora-A and that elevated level of Aurora-A is a causal factor of endocrine therapy-resistance and a valuable prognostic marker in ERα-positive breast cancer. Previous studies showed that at least 80% of tamoxifen resistant breast tumours retain ERα expression11 and that phosphorylation of ERα by protein kinases is one of major mechanisms that cause the resistance. Serine-167, which is located in the N-terminal activation function 1 domain (AF-1) of ERα, has been shown to be phosphorylated by Akt, S6K1, pp90rsk as well as IKBKE and the phosphorylation of Ser167 leads to increase of ERα transactivation activity.27–30 In a small study based on tamoxifen-treated breast cancer patients,31 the p-ERα-Ser167 was correlated with increased tamoxifen sensitivity,32 In contrast, other results have indicated that the phosphorylation of ERα-Ser167 is linked to reduce tamoxifen sensitivity.27, 28 In this study, our data show that p-ERα-Ser167 is associated with poor prognosis (Figure S11b). Serine-305 is located in the hinge region of ERα and the phosphorylation of this residue by PKA and PAK1 has been shown to result in ERα transcriptional activation of its target genes.33, 34 In addition, phosphorylation of Ser305 is correlated with tamoxifen resistance while it is not associated with DFS.33–37 We showed that Aurora-A phosphorylates both Ser167 and Ser305 of ERα, which is significantly related with short DFS (Figures 5, 7e S11a and S11d) and significantly induces ERα transactivation activity and CCND1 expression (Figure 6). Mutation of Ser167 and Ser305 to alanine abrogated the effect of Aurora-A on ERα activity and CCND1 expression (Figure 6). Collectively, these data suggest that Aurora-A-induced ERα activation, tamoxifen resistance and disease recurrence could be largely through phosphorylation of Ser167 and Ser305 (Figure 8). Aurora-A is a centrosome kinase, however, it has been shown to also phosphorylate the molecules outside centrosome, including TRF1, RalA, p53, HDAC6 etc.38–41 Our data showed that Aurora-A co-localized with ERα in both nucleus and centrosome (Figures 4f and S15b). We also observed the co-localization of p-ERα-Ser167 and p-ERα-Ser305 with Aurora-A in the centrosome (Figure S15a), implying that Aurora-A also phosphorylates ERα within the centrosome. The function of ERα in the centrosome is currently unknown. Further studies are required to characterize the role of ERα in Aurora-A-induced centrosome amplification. Finally, several small molecule inhibitors of Aurora kinases have been developed and are currently undergoing preclinical and early clinical testing. In particular, MLN8237 is a novel, orally bioavailable, second-generation selective inhibitor of Aurora-A. MLN8237 has exhibited efficacy against solid tumours and hematologic malignancies in preclinical models and are currently undergoing clinical evaluation in hematological and solid cancers.42, 43 In this report, we showed that MLN8237 not only inhibited tamoxifen-resistance breast cancer cell survival and tumour growth but also co-operated with tamoxifen in cell culture and orthotopic breast cancer model (Figures 3 and S4). In addition, MLN8237 abrogated Aurora-A phosphorylation of ERα-Ser167 and -Ser305 (Figure S8). These data underscore the potential of MLN8237 as either an independent or concurrent agent in tamoxifen-resistant breast cancer and the pERα-Ser167/Ser305 as potential clinical biomarkers in Aurora-A inhibitor therapy. MATERIALS AND METHODS Reagents and Plasmids Antibodies for Aurora-A and p-Aurora-A-T288 were purchased from Abcam and Cell Signaling, respectively. ERα and p-ERα-Ser167 antibodies were from Upstates and Santa Cruz. Antibody for p-ERα-Ser305 was purchased from Millipore. Anti-HA and -Flag antibodies were from Sigma. Recombinant ERα and Aurora-A proteins were from Stressgen and Cell Signaling, respectively. HA-tagged (pHM6) Aurora-A, GFP-tagged and Myc-tagged full-length and truncated ERα mutants as well as GST-ERα constructs were previously described.18, 27 Mutations of ERα-Ser167/Ser305 to alanine (ERα-2A) were prepared with QuikChange Site-directed Mutagenesis kit (Stratagene) and were confirmed by sequencing analysis. Small interfering RNAs (siRNA) of Aurora-A were from Qiagen. Tamoxifen and Aurora-A inhibitor MLN8237 were obtained from Sigma and Selleckchem, respectively. Cell Culture and Transfection Human breast cancer cell lines (T47D, BT474, MDA-MB-468, MDA-MB-231 and MCF-7) were obtained from ATCC. MCF-7-TamR was generated by chronic low dose treatment with tamoxifen.44 The cells were grown in RPMI-1640 or DMEM medium supplemented with 10% fetal bovine serum or in phenol red-free DMEM with charcoal stripped serum. Transfections were performed using Lipofectamine™ 2000 (Invitrogen) following the manufacturer’s instruction. Frozen and formalin-fixed paraffin embedded human primary breast cancer and normal breast tissues were obtained from the Tissue Procurement Facility at Moffitt Cancer Center under an approved IRB protocol. Western Blot, Immunoprecipitation, Immunohistochemistry (IHC) and Immuno-fluorescence Staining Western blot, immunoprecipitation, IHC and immunofluorescence were performed as previously described.45 Briefly, cell lysates were prepared in a lysis buffer and then subjected to immunoprecipitation and/or immunoblots with antibodies indicated in the Figure Legend. For IHC, breast cancer sections were immunostained with anti-Aurora-A (1:200), -ERα (1:250) and -ERα-Ser167/Ser305 (1:200) antibodies. The expression of Aurora-A and ERα was evaluated and scored as previously described.7 Immunofluorescence staining was carried out by fixing cells with 10% formalin/10% methanol for 20 min, and permeabilized in 1% NP-40 in phosphate-buffered saline. Cells were then blocked by 10% normal goat serum in phosphate-buffered saline, and incubated with primary antibodies against Aurora-A and ERα followed by secondary antibodies. The DNA was counterstained with 4′,6-diamidino-2-phenylindole. Luciferase Reporter Assay Cells were transiently transfected with EREα-Luc or CCND1-Luc, Aurora-A, wild-type or mutant ERα and β-galactosidase. The amount of DNA in each transfection was kept constant by the addition of empty pHM6 vector. After 48 hours of transfection, luciferase activity was measured using a luciferase assay reagents (Promega). Luciferase activities were normalized with respect to parallel β-galactosidase activities, to correct for differences in transfection efficiency. β-galactosidase assays were performed using the β-Galactosidase Enzyme Assay System (Promega). Mass spectrometry, GST Pull-down, in vitro Kinase Assay and in vivo [32P]Pi Cell Labeling Mass spectrometry was performed as previously described.27 Glutathione-Sepharose beads coupled with recombinant GST-Aurora-A or GST, were incubated with recombinant ERα (Abcam) in a binding buffer (50 mM HEPES, pH 7.2, 150 mM NaCl, 1 mM MgCl2, 1% Triton-X-100) for 2 h at 4°C. After washing the beads three times with the binding buffer, proteins bound to the beads were analyzed by 10% SDS–PAGE followed by immunoblotting with ERα antibody. In vitro Aurora-A kinase assay was performed by incubation of recombinant Aurora-A and recombinant ERα, as a substrate, in an in vitro kinase buffer for 25 min and followed by SDS–PAGE.18 For in vivo labeling, MDA-MB-231 cells were transfected with ERα and together with and without HA-Aurora-A. After serum starvation overnight, cells were labeled with [32P]Pi (0.5 mCi/ml) in phenol red-free MEM without phosphate for 4 hours. ERα was immunoprecipitated and separated in SDS-PAGE. The phospho-ERα was detected and quantified. RT-PCR and Chromatin Immunoprecipitation (ChIP) RT-PCR and ChIP assays were performed as previously described.27 Primers are: ERα, 5′-GGTGCCACCTGTGGTCCACCTG-3′ (sense) and 5′-CTTCACTTGTGGCCCAGATAGG-3′ (antisense); GAPDH, 5′-CATGTTCGTCATGGGTGTGAACCA-3′ (sense) and 5′-AGTGATGGCATGGACTGTGGTCAT-3′ (antisense); CCND1, 5′-GAACAGAAGTGCGAGAAGGAG-3′ (sense), and 5′-AGGCGGTAGTAGGACAGGAAG-3′ (antisense). For ChIP assay, the primers for CCND1 were used as following: forward (−1039) AACAAAACCAATTAGGAACCTT, reverse (−770) ATTTCCTTCATCTTGTCCTTCT. The PCR products were analyzed by electrophoresis on a 1.5% agarose gel and visualized by ethidium bromide staining. Colony Formation, Cell Viability, Apoptosis Assays and Synergy Analysis Colony formation, MTT and apoptotic assays were performed as previously described.28 Each experiment was repeated three times in triplicate. The results are expressed as the enrichment factor relative to the untreated controls. The synergistic effects of drug combinations were evaluated with Calcusyn software (Biosoft). This software uses the Chou-Talalay combination index method, which is based on the median–effect equation, itself a derivation from the mass-action law.46 For this analysis, Drug1 was combined with Drug2 at a constant ratio determined by IC50 Drug1/IC50 Drug2. We entered the resulting proliferation data, along with the data obtained from single drug treatments, into Calcusyn to determine a combination index value (CI) for each combination point, which quantitatively defines additivity (CI = 1.0), synergy (CI < 1.0), and antagonism (CI > 1.0). The resulting values were used to construct a plot of CI values over a range of fractions affected (FaCI plot). Orthotopic Breast Cancer Mouse Model Experimental procedures involving animals were reviewed and approved by the Institutional Animal Care and Use committee. Animal care was in accord with institution guidelines. Cells (5 × 106) were injected into mammary fat pads of 6 weeks old female nude mice (Charles River, Wilmington, MA). Tumour growth was monitored twice weekly by caliper measurements (LxWxD). When the tumours reached the average size of 100 mm3, the mice were divided into four groups, each with seven mice and an even distribution of tumour sizes, and treated as follows. Tamoxifen (at a dose of 5 mg) and Aurora-A inhibitor MLN8237 (at a dose of 30 mg/kg in a final formulation in 10% 2-hydroxypropyl-β-cyclodextrin/1% sodium bicarbonate) were orally given alone and combination daily for 26 days. Control group was orally given 100 μL vehicle control daily. Breast Cancer Datasets To assess the relation of overexpression of Aurora-A with recurrence of ERα-positive breast cancer treated with endocrine therapy, 674 ERα-positive breast cancers that were treated with antiestrogen agents were analyzed in the KM Plotter database (http://www.kmplot.com; Table S5). We also collected 4 different datasets from (Table S1). For each data set, we performed survival analysis to test if the Aurora-A levels are associated with recurrence. Each dataset has been processed independently from the other to preserve the original differences among the various studies (e.g., patient cohort, microarray type, sample processing protocol, etc.). We downloaded ERα-positive breast cancer gene expression datasets with clinical information from Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/GEO/), or author’s individual web pages. Table S3 reports the complete list of datasets and their sources. The datasets included both Affymetrix and dual-channel cDNA microarray platforms. When CEL files were available, expression values were generated from intensity signals using the RMA algorithm; values have been background adjusted, normalized using quantile normalization, and expression measure calculated using median polish 7 summarization. Statistic Analysis Statistic significance of differences between groups was analyzed by unpaired Student’s t test. The correlation of Aurora-A with ERα phosphorylation was examined by Chi-square. Hazard ratios (HR) and 95% confidence intervals (95% CI) were estimated using the Cox proportional hazards model. Recurrence-free survival time was calculated as the time between diagnosis and any of the events: locoregional recurrence, distant metastasis, or breast cancer death. Recurrence-free survival time distributions were compared with the log-rank test and plots were drawn using the Kaplan-Meier technique. Multivariate analysis of recurrence rates and breast cancer mortality rates was done with Cox proportional hazard regression, a method also used for the interaction analysis of different factors. All analyses were performed using the SPSS 11.5 Statistical Software, and p ≤ 0.05 was considered to be statistically significant. Supplementary Material 1 We appreciate Dr. Kenji Fukasawa for his scientific input. We are grateful for Tissue Procurement, DNA Sequence, Proteomics and Image Core Facilities at H. Lee Moffitt Cancer Center for providing cancer specimens, sequencing and cell apoptosis analysis. Grant Support This work was partially supported by grants from NIH grant CA160455 (JQC) and Florida James & Esther King Biomedical Research Program 1KG02 (JQC). CONFLICT OF INTEREST The authors disclose no potential conflicts of interest. Figure 1 Knockdown of Aurora-A sensitizes tamoxifen-resistant cells to tamoxifen (A) Western blot was performed with indicated antibodies in a panel cell lines. Note: elevated Aurora-A was detected in tamoxifen-resistant MCF7-TamR and BT474 cells. (B) BT474 cells were transfected with two siRNAs of Aurora-A and control siRNA targeting eGFP. After 72 hours of incubation, immunoblot was carried out with indicated antibodies. (C–E) Aurora-A siRNA- and control siRNA-treated BT474 cells were treated with and without tamoxifen (0–2.0 μM). Total cell viability (C), apoptosis (D) and cell growth (E) were assessed by MTT assays, Annexin V/FACS and focus formation, respectively. Experiments were repeated three times, each experiment was triplicates. Error bars represent mean plus standard deviation. The asterisks denote significance (P < 0.05 and * P < 0.01). Figure 2 Expression of Aurora-A induces tamoxifen-resistance (A) Immunoblot analysis of MCF7 cells, which were stably transfected with HA-Aurora-A and pHM6 vector, with anti-HA and, -ERα and -actin antibodies. (B–D) Aurora-A- and pHM6 vector-transfected MCF7 cells were treated with and without tamoxifen for 72 hours and then were assayed for cell viability (B), apoptosis (C), and focus formation (D). Experiments were repeated three times, each experiment was triplicated. Error bars represent mean plus standard deviation. The asterisks denote significance (P < 0.05 and * P < 0.01). Figure 3 Aurora-A inhibitor MLN8237 overcomes tamoxifen resistance (A) Following treatment with and without MLN8237, MCF7-TamR and BT474 cells were immunoblotted with indicated antibodies. (B and C) Following treatment of MCF7-TamR and BT474 cells with and without various concentrations of MLN8237 and tamoxifen for 72 hours, cells were processed for MTT assay (C) and Western blot (B, note: cleaved caspase 7 for MCF7-TamR and cleaved caspase 3 for BT474). (D–G) MLN8237 alone and combination with tamoxifen inhibited tamoxifen-resistant tumour growth in orthotopic breast cancer model. BT474 cells were injected to mammary fat pads of nude mice. When tumour reached ~100mm3, mice were treated with MLN8237 or/and tamoxifen as described in “Materials and Methods”. The tumour growth was monitored (D). At the end of experiment, the tumour weight was calculated (E). Representative tumour tissues were proceeded to Western blot (F) and immunohistochemical staining (F) with indicated antibodies. Figure 4 Aurora-A induces ERα transactivation activity and interacts/co-localizes with ERα (A and B) ERα-positive MCF7 (A) and ERα-negative MDA-MB-231 (B) cells were transfected with ERE-Luc and other indicated plasmids. Following 48 hours of incubation, luciferase activity was measured and normalized to β-galactosidase. Results are the mean ± S.E. of three independent experiments performed in triplicate. (C–E) Aurora-A directly binds to ERα. MDA-MB-231 cells were transfected with HA-Aurora-A and GFP-ERα. After 48 hours of incubation, cells were lysed, immunoprecipitated with anti-GFP antibody and immunoblotted with anti-HA antibody (top panel) and vice versa (panel 2). Panels 3 and 4 show the expression of transfected plasmids (C). For endogenous Aurora-A and ERα interaction, MCF-7 cells were immunoprecipitated with anti-ERα and detected with anti-Aurora-A antibody and vice versa (D). GST pull-down assay was performed by incubation of recombinant ERα with GST-Aurora-A or GST protein (E). (F) MCF7 cells were immunostained with anti-Aurora-A (green; b) and -ERα (red; c) antibodies, and counterstained with DAPI (blue; a). The merged pictures (green, red and blue) were shown as d. The magnified images of the indicated areas in panels i-iii are shown at the right side; Aurora-A: top panel (green), ERα: middle panel (red), overlay: bottom panel. Figure 5 Aurora-A phosphorylates ERα-Ser167/Ser305 in vitro and in vivo (A) In vitro kinase was performed by incubation of recombinant ERα with and without recombinant Aurora-A (top panel). Bottom panels are immunoblots showing the proteins used for in vitro kinase assay. (B and C) In vivo labeling. MDA-MB-231 cells were transfected with Myc-ERα or -ERα-2A (Ser167A/Ser305A) together with and without HA-Aurora-A. After 36 hours of transfection, cells were labeled with [32P]Pi (0.5 mCi/ml) in phenol red-free MEM without phosphate and serum for 4 hours. Myc-ERα was immunoprecipitated, separated on SDS-PAGE and exposed (top panel). NS stands for non-specific band. Bottom panels show expression of the transfected plasmids. (D) MDA-MB-231 cells were transfected with indicated plasmids. After 48 hours of transfection, cells were immunoprecipitated with anti-ERα antibody and immunoblotted with indicated antibodies. (E) MCF7 cells were transfected with Aurora-A and BT474 cells were treated with siRNA of Aurora-A. Following 72 hours of incubation, cells were immunoprecipitated with anti-ERα antibody and then immunoblotted analysis with indicated antibodies (upper 2 panels). Bottom panels are Western blots probed with indicated antibodies. Figure 6 Aurora-A induces ERα transactivation and upregulates CCND1 through phosphorylation of ERα-Ser167/Ser305 (A) Luciferase assay. MDA-MB231 cells were transfected with indicated plasmids. Following 48 hours of incubation, luciferase assay was performed as described in Figure 4. (B and C) Semi-quantitative RT-PCR (upper panels) and Western blot (lower panels) analyses were carried out in Aurora-A transfected MCF7 and Aurora-A-knockdown BT474 cells. (D–F) ERα-negative MDA-MB-231 cells were transfected with indicated plasmids and subjected to RT-PCR and Western blot (D), luciferase (E) and ChIP (F) assays. Figure 7 Expression of Aurora-A correlates with p-ERa-Ser167/Ser305 and is associated with recurrence-free survival (A and B) Immunohistochemical staining (A) and immunoblot (B) analyses were performed with indicated antibodies in ERα-positive human breast tumours. (C) Chi-square test analysis of Aurora-A expression and p-ERα-Ser167/Ser305 in ERα-positive breast cancer specimens. (D – F) Kaplan-Meier curves show the recurrence free-survival in ERα-positive breast cancer patients from Moffitt Cancer Center patients (D), 4 breast cancer datasets (E) and the KM Plotter database (F; note: there are 8 datasets in KM Plotter including 3 datasets from panel E). The log-rank test p values reflect the significance of association between high Aurora-A and shorter survival. 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==== Front Biochem Res IntBiochem Res IntBRIBiochemistry Research International2090-22472090-2255Hindawi Publishing Corporation 10.1155/2014/657189Review ArticleExploring Drug Targets in Isoprenoid Biosynthetic Pathway for Plasmodium falciparum Qidwai Tabish 1 http://orcid.org/0000-0001-8184-2354Jamal Farrukh 2 Khan Mohd Y. 3 Sharma Bechan 4 1Department of Biotechnology, Faculty of Engineering & Technology, Raja Balwant Singh, Engineering Technical Campus, Agra 283105, India2Department of Biochemistry, Dr. Ram Manohar Lohia Avadh University, Faizabad 224001, India3Department of Biotechnology, Babasaheb Bhimrao Ambedkar University, Lucknow, India4Department of Biochemistry, University of Allahabad, Allahabad, IndiaFarrukh Jamal: [email protected] Editor: Andrei Surguchov 2014 23 4 2014 2014 65718924 12 2013 7 2 2014 7 2 2014 Copyright © 2014 Tabish Qidwai et al.2014This 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.Emergence of rapid drug resistance to existing antimalarial drugs in Plasmodium falciparum has created the need for prediction of novel targets as well as leads derived from original molecules with improved activity against a validated drug target. The malaria parasite has a plant plastid-like apicoplast. To overcome the problem of falciparum malaria, the metabolic pathways in parasite apicoplast have been used as antimalarial drug targets. Among several pathways in apicoplast, isoprenoid biosynthesis is one of the important pathways for parasite as its multiplication in human erythrocytes requires isoprenoids. Therefore targeting this pathway and exploring leads with improved activity is a highly attractive approach. This report has explored progress towards the study of proteins and inhibitors of isoprenoid biosynthesis pathway. For more comprehensive analysis, antimalarial drug-protein interaction has been covered. ==== Body 1. Introduction Falciparum malaria is a well-known major killer, causing approximately one million deaths per year and 300–500 million clinical cases [(WHO 2010) World malaria report. World Health Organization, Geneva]. The malaria parasite belongs to apicomplexan phylum and has a plastid-like structure “apicoplast.” The metabolic pathways in apicoplast differ from the host and therefore apicoplast opens up new possibilities of targeting P. falciparum. The isoprenoid metabolic pathway is crucial for the P. falciparum. In plants more than 30000 isoprenoids are known. However the number of isoprenoids in malaria parasites is low. Isoprenoid includes cholesterol, bile acids, steroid hormones, dolichol, ubiquinone, prenylated proteins, and a wide variety of plant terpenoids [1]. The isoprenoid compounds are widespread in the three domains of archaebacteria, eubacteria, and eukaryotes. The “cyclisation reactions” are unknown in malaria parasites; however, rearrangements and oxidation of the carbon skeleton are responsible for the enormous structural diversity. Although they are produced from the condensation of the same precursors in all organisms (isopentenyl pyrophosphate and dimethylallyl diphosphate), the evolutionary origin of their biosynthesis remains controversial. Currently, two different routes have been identified to biosynthesize isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP). The well-known mevalonate pathway in most eukaryotes include mammals, higher plants, and archaea and the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway, also known as 1-deoxy-D-xylulose-5-phosphate (DOXP) or nonmevalonate pathway, in bacteria and plant plastids, especially in several pathogenic microorganisms [2]. Antimicrobial drug resistance is the most common problem in the control and treatment of many serious infections, including P. falciparum malaria, tuberculosis, and other infectious diseases. The nonmevalonate pathway of isoprenoid biosynthesis is essential in eubacteria (not all) and P. falciparum. As this pathway is absent in humans, consequently there is a great interest in targeting the enzymes of nonmevalonate metabolism for antiparasitic drug development. Fosmidomycin is a broad-spectrum antimicrobial agent and has been used in clinical trials of combination therapies for the treatment of malaria [3]. In vitro, fosmidomycin is known to inhibit the deoxyxylulose phosphate reductoisomerase (DXR) enzyme of isoprenoid biosynthesis from multiple pathogenic organisms. Isoprenoid metabolism proceeds through DXR even in the presence of fosmidomycin but is inhibited at the level of the downstream enzyme, methylerythritol phosphate cytidylyltransferase (IspD). Overexpression of IspD in E. coli conferred fosmidomycin resistance, and fosmidomycin was found to inhibit IspD in vitro [3]. Under in vivo conditions fosmidomycin may inhibit IspD directly or may cause other changes within the cell that reduce IspD activity [3]. Mycobacterium tuberculosis synthesizes isoprenoids via the nonmevalonate or DOXP pathway. Previous work had demonstrated that three enzymes, namely, Dxr, IspD, and IspF, are all required for growth in vitro. The DOXP biosynthetic pathway of M. tuberculosis is specific and essential and represents an attractive potential target for the design of new antimycobacterial agents. The work by Brown et al. (2010) demonstrated that three enzymes in the pathway (Dxr, IspD, and IspF) are all required for in vitro growth of M. tuberculosis [4]. Most eubacteria (not all) synthesize their isoprenoids using the methylerythritol-4-phosphate pathway, whereas a minority uses the unrelated mevalonate pathway and only a few have both [5]. Isoprenoids are a large and highly diverse group of natural products with many functions and their synthesis is essential for the parasite's survival [6]. This paper attempts to cover the different enzymes involved in isoprenoid biosynthesis and their importance in P. falciparum and antimalarial drug against these enzymes. In addition, an insight into host-parasite genetic polymorphisms is also presented. 2. Scope of Apicoplast as Antimalarial Drug Target The parasites such as Plasmodium spp., Babesia spp., Toxoplasma gondii, Cryptosporidium spp., Isospora belli, and Cyclospora cayetanensis infect humans [9]. With the exception of Cryptosporidium spp., these parasites possess apicoplast, a nonphotosynthetic plastid-like organelle. The apicoplast genome houses only 68 open reading frames (ORFs) for rRNAs, tRNAs, ribosomes, RNA polymerase, translational elongation factor (EF-Tu), ClpC chaperone, and Fe-S cluster protein (SufB) [1, 9]. Most of the proteins involved in the apicoplast metabolic pathways are encoded in the nucleus, synthesized in the cytoplasm, and subsequently imported into the apicoplast [2]. The apicoplast contains metabolic pathways critical for liver-stage and blood-stage development. During the blood stages, parasites lacking an apicoplast can grow in the presence of isopentenyl pyrophosphate, demonstrating that isoprenoids are the only metabolites produced in the apicoplast which are needed outside of the organelle. Two of the isoprenoid biosynthesis enzymes are predicted to rely on iron-sulfur (FeS) cluster cofactors; however, little is known about FeS cluster synthesis in the parasite or the roles that FeS cluster proteins play in parasite biology [10]. In P. falciparum, the protein SufS and its partner SufE were found exclusively in the apicoplast and SufS was shown to have cysteine desulfurase activity in a complementation assay. IscS and its effector Isd11 were solely mitochondrial, suggesting that the Isc pathway cannot contribute to apicoplast FeS cluster synthesis. The Suf pathway was disrupted with a dominant negative mutant resulting in parasites that were only viable when supplemented with IPP. These parasites lacked the apicoplast organelle and its organellar genome—a phenotype not observed when isoprenoid biosynthesis was specifically inhibited with fosmidomycin. These results demonstrate that the Suf pathway is essential for parasite survival [10]. Fatty acid, isoprenoid, haem biosynthesis, Fe-S clusters, and DNA transactions are prominent pathways in apicoplast. The most important metabolic functions and the mevalonate independent 1-deoxy-D-xylulose-5-phosphate (DOXP) pathway of isoprenoid synthesis and the type II fatty acid synthesis system operate inside the apicoplast. Classical antibacterial drugs such as ciprofloxacin, tetracycline, doxycycline, clindamycin, and spiramycin inhibit the apicoplast-located gyrase and translation machinery, respectively, and are currently used for the treatment of infections with apicomplexan parasites. Fosmidomycin, an inhibitor of isoprenoid synthesis, was proven to be effective against acute falciparum malaria in clinical phase II studies. Fosmidomycin alone or in combination with clindamycin was evaluated for the treatment of acute uncomplicated falciparum malaria. Monotherapy using fosmidomycin led to a fast parasite clearance but was inefficient in radical elimination of the parasites [11]. Triclosan, an inhibitor of fatty acid synthesis, was active in a malaria mouse model. In vitro antimalarial activity was shown for inhibitors of peptide deformylase and the import of apicoplast-targeted proteins. In the drug discovery against P. falciparum, the presence of apicoplast has been a milestone [12]. 3. Isoprenoid Biosynthesis It is required for the production of isopentenyl pyrophosphate and dimethylallyl pyrophosphate that plays a role in the biosynthesis of molecules used in protein prenylation, cell membrane maintenance, hormones, protein anchoring, and N-glycosylation. Plants and apicomplexan protozoa such as malaria parasites have the ability to produce their isoprenoids by means of an alternative pathway (nonmevalonate pathway) which takes place in their plastids. Interestingly, most bacteria including important pathogens such as M. tuberculosis synthesize IPP and DMAPP via the nonmevalonate pathway. 3.1. Nonmevalonate (MEP/DOXP Pathway) Plants and apicomplexan protozoa such as malaria parasites produce their isoprenoids by utilizing an additional alternative pathway called the methylerythritol phosphate (MEP) or nonmevalonate pathway, which takes place in their plastids (Figure 1). Higher plants possess the MEV pathway in the cytosol, in addition to the MEP pathway in the plastids. In contrast, the MEV pathway has not yet been detected in the cytosol of apicomplexan parasites. The nonmevalonate pathway or 2-C-methyl-D-erythritol-4-phosphate/1-deoxy-D-xylulose-5-phosphate pathway (MEP/DOXP pathway) of isoprenoid biosynthesis is an alternative metabolic pathway leading to the formation of IPP and DMAPP [7]. The MEP pathway consists of 8 steps and 7 enzymes (Figures 2(a) and 2(b)). 3.2. Enzymes of MEP Pathway Cassera et al. (2004) suggested that two genes encoding the enzymes of MEP pathway, namely, 1-deoxy-D-xylulose-5-phosphate synthase and 1-deoxy-D-xylulose-5-phosphate reductoisomerase, play a role in isoprenoid biosynthesis in P. falciparum. Fosmidomycin could inhibit the activity of 1-deoxy-D-xylulose-5-phosphate reductoisomerase [13]. The metabolite 1-deoxy-D-xylulose-5-phosphate is not only an intermediate of the MEP pathway for the biosynthesis of isopentenyl diphosphate but is also involved in the biosynthesis of thiamin (vitamin B1) and pyridoxal (vitamin B6) in plants and many microorganisms. An added advantage of targeting 1-deoxy-D-xylulose-5-phosphate synthase is its influence on vitamins B1 and B6 biosynthesis in malaria parasite [14]. Most of the downstream intermediates (1-deoxy-D-xylulose-5-phosphate, 2-C-methyl-D-erythritol-4-phosphate, 4-(cytidine-5-diphospho)-2-C-methyl-D-erythritol,4-(cytidine-5-diphospho)-2-C-methyl-D-erythritol-2phosphate, and 2-C-methyl-D-erythritol-2,4-cyclodiphosphate) of the MEP pathway in the three intraerythrocytic stages of P. falciparum have been unfolded [15]. The effect of fosmidomycin on the biosynthesis of each intermediate of this pathway and isoprenoid biosynthesis (dolichols and ubiquinones) has been explored. MEP pathway is functionally active in all intraerythrocytic forms of P. falciparum, and de novo biosynthesis of pyridoxal in a protozoan has been reported [16]. Its absence in the human host makes both pathways potentially very attractive targets for antimalarial drug development. Another enzyme of the nonmevalonate pathway, 1-deoxy-D-xylulose-5-phosphate reductoisomerase which is involved in the transformation of 1-deoxy-D-xylulose 5-phosphate to 2-C-methyl-D-erythritol-4-phosphate. DXR protein from the human malaria parasite P. falciparum (PfDXR) was overproduced in Escherichia coli and crystallized using the hanging-drop vapour-diffusion method in the presence of nicotinamide adenine dinucleotide (NADPH) [16]. Cassera et al. (2007) reported that, in P. falciparum, the formation of isopentenyl diphosphate and dimethylallyl diphosphate intermediates in the biosynthesis of isoprenoids and occurs via the methylerythritol phosphate pathway [17]. Fosmidomycin is a specific inhibitor of the 1-deoxy-D-xylulose-5-phosphate reductoisomerase. The effect of fosmidomycin on the levels of each intermediate and its metabolic requirement for the isoprenoid biosynthesis, such as dolichols and ubiquinones, throughout the intraerythrocytic cycle of P. falciparum have been analyzed. Fosmidomycin treatment resulted in a decrease of the intermediate levels in the MEP pathway as well as in ubiquinone and dolichol biosynthesis. The MEP pathway associated transcripts were modestly altered by the drug, indicating that the parasite is not strongly responsive at the transcriptional level. This was the first study that compares the effect of fosmidomycin on the metabolic and transcript profiles in P. falciparum, which has only the MEP pathway for isoprenoid biosynthesis. 3.3. Dolichol Phosphate Mannose Synthase The intraerythrocytic stages of P. falciparum use dolichol and its phosphorylated derivatives as carrier lipids in biosynthesis of several glycoconjugates. Anchors and N-linked glycoproteins require dolichyl phosphate and dolichyl pyrophosphate as carriers of different monosaccharide constituents. Kimura et al. (1996) demonstrated the effect of N-linked glycoproteins on differentiation of intraerythrocytic stages of P. falciparum [18]. The reaction between dolichol phosphate (Dol-P) and guanosine diphosphate mannose (GDP-Man) to form dolichol-phosphate-mannose (Dol-P-Man) is catalyzed by dolichol phosphate mannose synthase (DPM) enzyme [19]. This molecule acts as mannose donor for N-glycosylation and glycosylphosphatidylinositol (GPI) biosynthesis. P. falciparum DPM1 (Pfdpm1) possesses a single predicted transmembrane region near the N-terminus but not the C-terminus. Pfdpm1 was unable to complement a mouse mutant deficient in DPM but efficiently complemented the Schizosaccharomyces pombe fission yeast mutant, indicating a difference between fission yeast and mammalian DPM genes. Many eukaryotic cells, such as yeast and a number of mammalian cells, are unable to incorporate more complex isoprenoid precursors such as FPP and GGPP. In contrast, intraerythrocytic forms of P. falciparum easily metabolise these compounds when they are added to the culture medium, permitting the subsequent identification of higher isoprenoids. Walter (1986) reported that dolichol kinase, a rate limiting enzyme for the supply with dolichyl monophosphate as glycosyl carrier lipid in P. falciparum, and inhibition of this enzyme (dolichol kinase) occurred by mefloquine [20]. The enzyme was found to be associated with the pellet fraction and to depend on cytidine triphosphate as phosphoryl donor. Couto et al. (1999) identified dolichol, dolichyl phosphate, and dolichyl pyrophosphate of 55 and 60 carbons (11/12 isoprenic units) [21]. Biochemical studies in P. falciparum indicated that, in addition to the pathway for synthesis of phosphatidylcholine from choline (CDP-choline pathway), the parasite synthesizes this major membrane phospholipid via an alternative pathway named the serine-decarboxylase- phosphoethanolamine-methyltransferase (SDPM) pathway using host serine and ethanolamine as precursors [22]. The pathogenic stages of P. falciparum are those that invade mature erythrocytes, which are devoid of internal organelles and incapable of de novo lipid biosynthesis. Despite this, P. falciparum undergoes dramatic morphological and metabolic developmental changes and asexually divides to form new daughter cells within a human erythrocyte [23]. This rapid multiplication of P. falciparum within host erythrocytes entails the active production of new plasma membranes in which phospholipids are the major architectural and functional components. Phosphatidylcholine (PtdCho)2 and phosphatidylethanolamine (PtdEtn) comprise 40–50% and 35–45%, respectively, of the parasite's total plasma membrane phospholipid content [24]. Genome data predicts that P. falciparum possesses enzymatic pathways for the synthesis of all the necessary phospholipids from precursors transported from host milieu, such as serine, choline, inositol, glycerol, and fatty acids [24, 25]. The PfPMT gene in P. falciparum encodes the phosphoethanolamine methyltransferase that specifically methylates phosphoethanolamine to phosphocholine (p-Cho) via the SDPM pathway [26]. The studies show that PfPMT is important for membrane biogenesis, development, survival, and propagation of the parasite. Compounds that target PfPMT could thus be combined with those that specifically target choline transport, choline phosphorylation, or other steps of the CDP-choline pathway in order to completely block PtdCho biosynthesis and kill the parasite. In P. falciparum, the biosynthesis of ubiquinone or coenzyme Q involves two major steps: synthesis of the benzoquinone by the shikimate pathway and synthesis of the isoprene side chain by the MEP pathway. The presence of dolichol and isoprenylated proteins has been detected in P. falciparum, but no studies are available about the biosynthesis of the isoprenic side chain attached to the benzoquinone ring of coenzyme Q. De Macedo et al. (2002) showed that P. falciparum synthesizes different homologs (coenzyme Q(8) and coenzyme Q(9)), depending on the given intermediate [27]. The authors also demonstrated that nerolidol treatment of P. falciparum parasites resulted in a reduced ability to synthesise CoQ and inhibited P. falciparum growth in vitro. Treatment with nerolidol arrested development of the intraerythrocytic stages of the parasites, indicating that the drug may have an antimalarial potential. 4. Antimalarial Compounds against Isoprenoid Biosynthetic Pathway Biosynthesis of several isoprenoids in P. falciparum was studied and terpenes (molecules with a similar chemical structure to the intermediates of the isoprenoids pathway) as potential antimalarial drugs were evaluated [28–30]. Different terpenes and S-farnesylthiosalicylic acid were tested on cultures of the intraerythrocytic stages of P. falciparum, and the 50% inhibitory concentration for each one was found. Farnesol, nerolidol, limonene, and linalool terpenes have been used against P. falciparum [30]. All the terpenes tested inhibited dolichol biosynthesis in the trophozoite and schizont stages. Farnesol, nerolidol, and linalool showed stronger inhibitory activity in the biosynthesis of the isoprenic side chain of the benzoquinone ring of ubiquinones in the schizont stage. The inhibitory effect of terpenes and S-farnesylthiosalicylic acid on the biosynthesis of both dolichol, the isoprenic side chain of ubiquinones, and the isoprenylation of proteins in the intraerythrocytic stages of P. falciparum appears to be specific, because overall protein biosynthesis was not affected. A variety of proteins undergo posttranslational modification such as prenylation near the carboxyl terminus with farnesyl (C15) or geranylgeranyl (C20) groups. Protein farnesyltransferase (PFT) transfers the farnesyl group from farnesyl diphosphate to the SH of the cysteine near the C-terminus of proteins such as Ras. PFT inhibitors (PFTIs) have been extensively developed as anticancer agents because of their ability to block tumor growth in experimental animals. PFTIs have been explored as antimalarial and antitrypanosome agents because these compounds are much more toxic to these parasites than to mammalian cells, and there are a large number of lead compounds which have been explored as part of the antiparasite drug discovery program [31]. MEP pathway inhibition with fosmidomycin reduces protein prenylation, confirming that de novo isoprenoid biosynthesis produces the isoprenyl substrates for protein prenylation. One important group of prenylated proteins is small GTPases, such as Rab family members, which mediate cellular vesicular trafficking [32]. Substituted tetrahydroquinolines (THQs) have been previously identified as inhibitors of mammalian protein farnesyltransferase. Fletcher et al. (2008) designed and synthesized a series of inhibitors that are selective for P. falciparum farnesyltransferase (PfPFT). Several PfPFT inhibitors have been found to inhibit the malarial enzyme with IC50 values down to 1 nM, and that blocks the growth of P. falciparum in infected whole cells (erythrocytes) with ED50 values down to 55 nM. Potent, Plasmodium-selective farnesyltransferase inhibitors that arrest the growth of malaria parasites have been explored [33]. A new synthetic pathway was devised to reach tetrasubstituted 3-arylthiophene 2-carboxylic acids in a three-step solid-phase synthesis. This very efficient methodology provided more than 20 new compounds that were evaluated for their ability to inhibit protein farnesyltransferase from different species as well as Trypanosoma brucei and P. falciparum proliferation [34]. Strong inhibition of P. falciparum PFT (PfPFT) by peptidomimetics illustrated the potential of targeting these enzymes in developing drug therapy for malaria. Ohkanda et al. (2001) have recently demonstrated the potency of a variety of other peptidomimetics as inhibitors of P. falciparum growth and PfPFT activity [35]. Moura et al. (2001) have also shown that the monoterpene, limonene, inhibits parasite development and prenylation of P. falciparum proteins [36]. The enzymes of the nonmevalonate pathway for isoprenoid biosynthesis are attractive targets for the development of novel drugs against malaria and tuberculosis. Fosmidomycin and FR900098 (an N-acetyl derivative of fosmidomycin) are inhibitor of DOXP reductoisomerase which showed antimalarial activity in vitro and in vivo [15, 29](Figure 3). Reverse hydroxamate-based inhibitor for IspC enzyme was evaluated by Behrendt et al. (2011) [37]. Fosmidomycin has been proven to be efficient in the treatment of P. falciparum malaria by inhibiting 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR), an enzyme of the nonmevalonate pathway, which is absent in humans (Figure 4). 1-Deoxy-D-xylulose-5-phosphate reductoisomerase (Dxr) represents an essential enzyme of the mevalonate-independent pathway of the isoprenoid biosynthesis. Using fosmidomycin as a specific inhibitor of Dxr, this enzyme was previously validated as target for the treatment of malaria and bacterial infections. The replacement of the formyl residue of fosmidomycin by spacious acyl residues yielded inhibitors active in the micromolar range. As predicted by flexible docking, evidence was obtained for the formation of a hydrogen bond between an appropriately placed carbonyl group in the acyl residue and the main-chain NH of Met214 located in the flexible catalytic loop of the enzyme [38, 39]. Specific inhibition of enzymes of the nonmevalonate pathway is a promising strategy for the development of novel antiplasmodial drugs. α-Aryl-substituted β-oxa isosteres of fosmidomycin with a reverse orientation of the hydroxamic acid group were synthesized and evaluated for their inhibitory activity against recombinant 1-deoxy-d-xylulose 5-phosphate reductoisomerase (IspC) of Plasmodium falciparum and for their in vitro antiplasmodial activity against chloroquine-sensitive and resistant strains of P. falciparum. The most active derivative inhibits IspC protein of P. falciparum (PfIspC) with an IC50 value of 12 nM and shows potent in vitro antiplasmodial activity [40]. The structure-activity relationships for 43 inhibitors of 1-deoxyxylulose-5-phosphate (DOXP) reductoisomerase, derived from protein-based docking, ligand-based 3D QSAR, and a combination of both approaches as realized by AFMoC (adaptation of fields for molecular comparison) have been presented by Silber et al. (2005) [41]. A series of novel 3′-amido-3′-deoxy-N(6)-(1-naphthylmethyl) adenosines was synthesized applying a polymer-assisted solution phase (PASP) protocol and was tested for antimalarial activity versus the Dd2 strain of Plasmodium falciparum. Further, this series and 62 adenosine derivatives were analyzed regarding 1-deoxy-D-xylulose-5-phosphate reductoisomerase inhibition. Biological evaluations revealed that the investigated 3′,N(6)-disubstituted adenosine derivatives displayed moderate but significant activity against the P. falciparum parasite in the low-micromolar range [42]. 5. Genetic Polymorphism in P. falciparum P. falciparum malaria is an example of evolutionary selection. Both host and parasite show the phenomenon of natural selection. Several polymorphisms in human host and parasite have been found. Table 1 shows details of genes involved in isoprenoid metabolism and their polymorphisms in P. falciparum. In the drug discovery process the polymorphisms in parasite genes encoding the target protein should be considered. In P. falciparum the genetic polymorphisms at 10 loci are considered potential targets for specific antimalarial vaccines [43]. The polymorphism is unevenly distributed among the loci; loci encoding proteins expressed on the surface of the sporozoite or the merozoite (AMA-1, CSP, LSA-1, MSP-1, MSP-2, and MSP-3) are more polymorphic than those expressed during the sexual stages or inside the parasite (EBA-175, Pfs25, PF48/45, and RAP-1). Comparison of synonymous and nonsynonymous substitutions indicates that natural selection may account for the polymorphism observed at seven of the 10 loci studied. This inference depends on the assumption that synonymous substitutions are neutral. The authors obtained evidence for an overall trend towards increasing A+T richness but no evidence of mutation. Although the neutrality of synonymous substitutions is not definitely established, this trend towards an A+T rich genome cannot explain the accumulation of substitutions at least in the case of four genes (AMA-1, CSP, LSA-1, and PF48/45) because the G→ C transversions are more frequent than expected. Predicted polymorphisms in genes encoding isoprenoid biosynthesis may play an important role in drug response (Table 1). Nonsynonymous single nucleotide polymorphisms in 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (LytB), 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase, putative (IspD), 1-deoxy-D-xylulose 5-phosphate synthase, 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DOXR), and 4-diphosphocytidyl-2c-methyl-D-erythritol kinase (CMK) proteins may change the amino acids. Such change may affect the proteins and interaction with antimalarial drugs. 5.1. DNA Sequence Variation(s) and Evolution Mutations or genetic variations that have ability to alter the activity or availability of transcription factors, as well as mutations that alter the cis-regulatory sequences (transcription factor binding sites) to which they bind, can change expression of gene and both types of changes contribute to evolution. Several studies have suggested that mutations affecting cis-regulatory activity (transcription factor binding site) are the predominant source of expression divergence between species [44, 45]. Changes in gene expression often alter phenotypes; mutations that affect gene expression can affect fitness and contribute to adaptive evolution. DNA sequence variations reported in genes involved in dolichol pathway of P. falciparum were shown. 5.2. DNA Sequence Variation and Drug Response In the genome different types of variations are reported such as copy number variations (CNV), microsatellite repeats (SSR), and single nucleotide polymorphisms (SNP) which are known to play role in regulation of gene expression. Presence of such variations in the transcription factor binding site (TFBS) may influence the binding of transcription factor to their respective binding sites and is responsible for the alteration of gene product in the cell. The presence of several polymorphisms in the upstream region gives rise to different haplotypes. These haplotypes in the transcription factor binding sites alter the interaction of transcription factor (TF) to TFBS and may cause transcriptional modification of gene, altering the level of protein. Genetic variation associated with increased susceptibility to complex diseases can elucidate genes and underlying biochemical mechanisms linked to disease onset and progression. It has been suggested that when gene shows association with disease, the most common condition is that genetic variation or causal mutations alter an encoded protein sequence. But a significant number of undiscovered causal mutations may alter the regulation of gene transcription. However, it remains a challenge to separate causal genetic variations from linked neutral variations. One of the most important applications of genetic polymorphisms is the drug response. Earlier, we have detected that peoples from different ethnic backgrounds have shown different susceptibility/resistance to falciparum malaria [46, 47]. It has been clear from several studies that polymorphisms are important factor for determining the drug response. The most studied example of polymorphisms and drug response in the P. falciparum is the multidrug resistance gene (pfmdr-1) that contributes to variability and is known to play role in drug response. The peoples varying in the genotype at a particular locus may have different responses to a particular drug which is illustrated in Figure 5. 6. Discussion It is very difficult to treat malaria because of the development of resistance of the parasite to antimalarial(s). Currently the main drug available is artemisinin (ART) and its derivatives and serious efforts are underway to develop ART-based combination therapies. The new pharmacophores attacking different targets in the parasite can bring in new strategies to combat malaria. Apart from efficacy, toxic side effects, pharmacokinetic compatibility, and potential to develop resistance would all be the major parameters in the eventual development of a successful drug. Therefore, all the targets and molecules discussed in this review are important. Apicoplast itself contains a small circular genome, most of the proteome of this organelle is encoded in the nuclear genome, and the proteins are subsequently transported to the apicoplast. It is assumed to contain a number of unique metabolic pathways not found in the vertebrate host, making it an ideal “playground” for those interested in drug targets. The apicoplast contains metabolic pathways critical for liver-stage and blood-stage development. During the blood stages, parasites lacking an apicoplast can grow in the presence of isopentenyl pyrophosphate (IPP), demonstrating that isoprenoids are the only metabolites produced in the apicoplast which are needed outside of the organelle. Two of the enzymes involved in isoprenoid biosynthesis are predicted to rely on iron-sulfur (FeS) cluster cofactors [48]. Since the nonmevalonate pathway of isoprenoid biosynthesis is essential in eubacteria and P. falciparum and this pathway is not present in humans, there is a great interest in targeting the enzymes of nonmevalonate metabolism for antiparasitic drug development. Fosmidomycin is currently in clinical trials of combination therapies for the treatment of malaria [49]. Cell-penetrating peptides, such as oligoarginines, improved the intracellular delivery of the drug fosmidomycin [50]. Analogues of fosmidomycin that is substituted in both the Cα and the hydroxamate positions have been found more potent than fosmidomycin in terms of killing P. falciparum in an in vitro assay [51]. A screen of host cell proteins that might facilitate lipid scavenging by the parasite during the liver stage has pointed toward the surface-expressed cholesterol ester receptor, scavenger receptor class B type 1 (SR-BI) [42]. A decrease or complete loss of SR-BI surface availability via treatment with siRNA (short interfering RNA), antibodies, drugs, or gene deletion results in a reduction in parasite invasion and growth in hepatocytes [52, 53]. P. falciparum mitochondrion presents structural and physiological characteristics different from mitochondria in other eukaryotes [54]. One of the reasons is the adaptation of the parasite to different environments, in particular, the great differences in oxygen tension between the host and the mosquito. Mitochondrial role in the intraerythrocytic environment particularly focuses on mitochondrial metabolic pathways that relate to oxidative phosphorylation, including the tricarboxylic acid cycle, de novo pyrimidine biosynthesis via dihydroorotate dehydrogenase, and the particularities of the electron transport chain. Such unique particularities of parasite mitochondria could be promising targets for development of a new therapy. The elucidation of the role of this organelle in aerophilic respiratory metabolism and the association of antimalarial drugs with hyperbaric oxygen therapy might provide new treatments for infection by P. falciparum. Howe et al. (2013) demonstrate that protein prenylation depends on de novo isoprenoid biosynthesis in P. falciparum [32]. When isoprenoid biosynthesis is inhibited, the digestive food vacuole of the parasite is structurally and functionally disrupted. These results suggest that a key function of isoprenoids in malaria is protein prenylation and support a role for prenylated proteins, such as Rab GTPases, in FV maintenance. Dolichol and its derivatives are the lipids discovered most recently in eukaryotic cells. They serve as inducers of membrane fusion. The DOXP pathway provides precursors for protein farnesylation. Plasmodial farnesyl transferase activity has unique biochemical features and inhibitors of this process have in vitro antimalarial activity [35, 55]. The resistance of malaria parasites to available drugs continues to grow, increasingly limiting our ability to control this serious disease. Recent increases in the pace of progress in this area will be helpful in the development of novel antimalarial therapies. As outlined in this review, a number of pathways and inhibitory substances have already been identified which hold great promise for the future. With the completion of the Plasmodium genome project new biosynthetic routes in the apicoplast are discovered and efforts are on to explore them as candidate drug targets. The development of a number of genetic tools to validate these possible targets in transgenic parasites and also for drug screening purposes will have an enormous impetus on the development of drugs against this pathogen. The analysis of the utility of isoprenoid biosynthesis pathway in P. falciparum reveals that the proteins of this pathway might be important drug targets. The study of host genetic factors and polymorphisms in parasite isoprenoid biosynthetic genes will be helpful in the analysis of disease susceptibility and drug response. Acknowledgments The authors acknowledge the Council of Scientific & Industrial Research, New Delhi, India, for providing research facility and supportive environment to carry out doctoral research work at Central Institute of Medicinal & Aromatic Plants, Lucknow, India. Supportive facilities and conducive environment at Dr. Ram Manohar Lohia Avadh University, Faizabad, India, are thankfully acknowledged. Conflict of Interests The authors declare that there is no conflict of interests regarding the publication of this paper. Figure 1 The role of the apicoplast in production of isoprenoid precursors, IPP and DMAPP, which are exported into the cytoplasm and used to synthesize small molecule isoprenoids and prenylated proteins. P. falciparum is unable to synthesize isoprenoid precursors either due to inhibition of the biosynthetic pathway by fosmidomycin or loss of the apicoplast following doxycycline inhibition which can be chemically rescued by addition of exogenous IPP. The exogenous IPP enters the host cell through unknown membrane transporters and fulfills the missing biosynthetic function [7]. Figure 2 (a) Synthesis of isoprenoid compound by nonmevalonate pathway: a flow chart; (b) chemical reactions involved in biosynthesis of isoprenoid via nonmevalonate pathway. Figure 3 Structures of diphosphate and bisphosphonates (inhibitors of pathway) [8]. Figure 4 (a) Interaction of fosmidomycin with PfDXR. FR900098 complex. (b) The carbon atoms of the FR900098 molecule are shown in magenta, (c) fosmidomycin, and (d) FR900098. Figure 5 Diagrammatic representation of role of polymorphisms in drug response. Table 1 Details of genes involved in isoprenoid metabolism and their polymorphisms in P. falciparum 3D7. Gene ID Previous ID Chromosome Product description Protein length Nonsynonymous SNPs in all strains Synonymous SNPs in all strains PF3D7_0104400 PFA0225w 1 4-Hydroxy-3-methylbut-2-enyl diphosphate reductase (LytB) 535 3 3 PF3D7_0106900 PFA0340w 1 2-C-Methyl-D-erythritol 4-phosphate cytidylyltransferase, putative (IspD) 734 6 2 PF3D7_0209300 PFB0420w 2 2-C-Methyl-D-erythritol 2,4-cyclodiphosphate synthase (IspF) 240 0 0 PF3D7_0318800 PFC0831w 3 Triosephosphate isomerase, putative 357 0 0 PF3D7_0503100 PFE0150c 5 4-Diphosphocytidyl-2c-methyl-D-erythritol kinase (CMK), putative 537 4 0 PF3D7_0508300 PFE0410w 5 Triose phosphate transporter (oTPT) 342 0 0 PF3D7_0530200 PFE1510c 5 Triose phosphate transporter (iTPT) 524 1 0 PF3D7_0623200 PFF1115w 6 Ferredoxin NADP reductase (FNR) 371 1 1 PF3D7_1022800 PF10_0221 10 4-Hydroxy-3-methylbut-2-en-1-yl diphosphate synthase (GcpE) 824 1 0 PF3D7_1037100 PF10_0363 10 Pyruvate kinase 2 (PyKII) 745 2 1 PF3D7_1318100 MAL13P1.95 13 Ferredoxin, putative 194 2 0 PF3D7_1337200 MAL13P1.186 13 1-Deoxy-D-xylulose 5-phosphate synthase 1205 13 2 PF3D7_1439900 PF14_0378 14 Triosephosphate isomerase (TIM) 248 0 0 PF3D7_1467300 PF14_0641 14 1-Deoxy-D-xylulose 5-phosphate reductoisomerase (DOXR) 488 2 2 ==== Refs 1 Poulter CD Bioorganic chemistry: a natural reunion of the physical and life sciences Journal of Organic Chemistry 2009 74 7 2631 2645 2-s2.0-64549118232 19323569 2 Lombard J Moreira D Origins and early evolution of the mevalonate pathway of isoprenoid biosynthesis in the three domains of life Molecular Biology and Evolution 2011 28 1 87 99 2-s2.0-78650467548 20651049 3 Zhang B Watts KM Hodge D A second target of the antimalarial and antibacterial agent fosmidomycin revealed by cellular metabolic profiling Biochemistry 2011 50 17 3570 3577 2-s2.0-79955399200 21438569 4 Brown AC Eberl M Crick DC Jomaa H Parish T The nonmevalonate pathway of isoprenoid biosynthesis in Mycobacterium tuberculosis is essential and transcriptionally 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==== Front 04104626011NatureNatureNature0028-08361476-46872404847610.1038/nature12535nihpa514597ArticleGenomic organization of human transcription initiation complexes Venters Bryan J. 12Pugh B. Franklin 11 Center for Eukaryotic Gene Regulation, Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA 168022 Current Address: Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, TN 37232 Correspondence and requests for materials should be addressed to B.F.P. ([email protected]).6 12 2013 18 9 2013 3 10 2013 13 5 2014 502 7469 53 58 Users may view, print, copy, download and text and data- mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: http://www.nature.com/authors/editorial_policies/license.html#termsThe human genome is pervasively transcribed, yet only a small fraction is coding. Here we address whether this noncoding transcription arises at promoters, and detail the interactions of initiation factors TBP, TFIIB, and RNA polymerase (Pol) II. Using ChIP-exo, we identify ~160,000 transcription initiation complexes across the human K562 genome, and more in other cancer genomes. Only ~5% associate with mRNA genes. The remaining associate with non-polyadenylated noncoding transcription. Regardless, Pol II moves into a transcriptionally paused state, and TBP/TFIIB remain at the promoter. Remarkably, the vast majority of locations contain the four core promoter elements: BREu, TATA, BREd, and INR, in constrained positions. All but the INR also reside at Pol III promoters, where TBP makes similar contacts. This comprehensive and high resolution genome-wide detection of the initiation machinery produces a consolidated view of transcription initiation events from yeast to humans at Pol II/III TATA-containing/TATA-less coding and noncoding genes. ChIP-seqChIP-exoGTFshumanpromoter elementsnoncoding transcription ==== Body The classic paradigm for assembling the minimal core transcription machinery at mRNA promoters starts with the recruitment of the TATA binding protein (TBP) to the TATA box core promoter element1. Next is the docking of TFIIB, which straddles TBP and locks onto flanking TFIIB-responsive elements (BREu and BREd)2,3. Together with TFIIF, TFIIB then engages Pol II in its active site to help set the start site of transcription (TSS) at an Initiator element (INR)4-6. The recruitment of the transcription machinery has long been thought to be an important rate-limiting step in gene expression7. Concepts in transcription initiation by all three RNA polymer-ases (I, II, and III) have been guided by this basic theme8. Clashing with this seemingly simplified view is that the TATA box has been identified at only ~10% of human promoters9,10, with most genes ostensibly being classified as “TATA-less” in all three RNA polymerase systems. The other core promoter elements are apparently equally rare. A second complication of the classic view, particular to multi-cellular eukaryotes, is that the general transcription factors may be largely pre-assembled at promoters. There Pol II is in a transcriptionally engaged but paused state, approximately 30-50 bp downstream from the TSS11-13. A third complication is that transcription of genomes is not restricted to coding genes, but appears to be quite pervasive, without clear evidence of being coupled to definable promoters14. These complications, together, paint a seemingly complex picture of eukaryotic transcription initiation. Towards reconciling simplistic models against complex data, we recently developed the ChIP-exo assay to map sites of protein-DNA interactions at near single-base resolution15. We discovered in yeast that so-called TATA-less promoters actually possess degenerate versions of the TATA-box, and that most yeast promoters assemble the transcription machinery fundamentally in accord with the classic paradigm16, although a deep dichotomy between the TATA/SAGA/stress-induced genes and TATA-less/TFIID/housekeeping genes remains. This led us to consider whether similar simplicity was true in humans, albeit with additional complications of paused polymerase and pervasive noncoding transcription. TBP/TFIIB separation from paused Pol II Using ChIP-exo, we detected 159,117 TFIIB locations (peak pairs) in K562 cells, of which 36% were associated with ENCODE-defined transcriptional domains (Extended Data Fig. 1a)17. Remarkably, half were associated with heterochromatic regions, which are generally thought to be devoid of stable RNA production. However, heterochromatic transcription may be more pervasive. We assigned a TBP/TFIIB location to >50% of all annotated protein-coding K562-expressed genes (Extended Data Fig. 1b), thereby providing independent validation. Seemingly expressed genes that lacked a TBP/TFIIB location may have arisen from multiple sources including rare but stable mRNAs, detection noise, and antisense transcription arising from a more distal promoter. TBP/TFIIB/Pol II occupancy and mRNA levels were correlated (Extended Data Fig. 1c), as expected of recruitment being at least partially rate-limiting in gene expression. We initially focused on all 8,364 K562 TFIIB locations near the TSS of 6,511 coding RNAs as defined by RefSeq18. Fig. 1a provides one example of the raw tag distribution and the identified core promoter elements concentrated ~25 bp upstream of the RPS12 ribosomal protein gene TSS. When individual genes were examined (Fig. 1b), or averaged across all 6,511 genes (Fig. 1c), two regions of high TFIIB/TBP/Pol II occupancy were observed. The major right-ward peaks corresponded to primary promoter transcription initiated complexes (Fig. 1c, upper panel). Those in the left-ward direction matched divergent TSSs19-22, although the resulting RNA was less abundant than expected from TFIIB/TBP/Pol II occupancy levels (Fig. 1c, lower vs upper panel; Note that 2° TSS represents only 24% of the total TSS signal). This may result from RNA instability, as seen in yeast. The clear spatial separation of complexes indicates that divergent transcripts arise from distinct initiation complexes, most (78%) of which were in CpG islands. On average two complexes were detected per CpG island23, regardless of island length, with the center of the island being enriched ~100 bp downstream of the primary TSS (Extended Data Fig. 2a,b). Complexes tended to be separated by 70-180 bp (Extended Data Fig. 2c, red), and had un-correlated occupancies (Extended Data Fig. 2c, black), which suggests that they are regulated independently. For the vast majority of transcription units, Pol II crosslinked 50 bp downstream of the primary TSS (Fig. 1b, c), where it is expected to pause after initiating transcription13. Pol II was most depleted over the core promoter, indicating that it does not stably reside there in proliferating K562 cells. Therefore when Pol II enters the core promoter, it rapidly initiates transcription and then moves into a paused state ~50 bp downstream, thereby preventing any new polymerase from detectably engaging the core promoter. The crosslinking pattern of human TFIIB was of particular interest since TFIIB in budding yeast crosslinks broadly across the relatively stable single-stranded DNA region within the Pol II active site at core promoters16, in accord with crystallographic models of “open” complexes24. Remarkably, human TFIIB maintained its contact within this region, despite the absence of polymerase (Fig. 1c, upper panel). Mechanistically, this might occur via TFIIB contacts with BREd3 (see below), which are absent in budding yeast. The coincidence of TBP and TFIIB crosslinking at the BREd suggests that TBP may be predominantly crosslinking to TFIIB there, rather than directly to DNA. BREu TATA, BREd and INR are common We looked for core promoter elements (illustrated in Fig. 2a) within the narrow intervals defined by 8,364 mRNA TSS-proximal TFIIB locations. Remarkably, and consistent with yeast16, nearly 85% of them had a sequence with 0-3 mismatches to the TATA-box consensus (TATAWAWR)25 (Fig. 2b-c). Less than 3% had a perfect match to the consensus. Deviations from the TATA box consensus inversely correlated with TFIIB and TBP occupancy levels (Extended Data Fig. 3a), indicating that TATA element sequence quality contributes to their occupancy level, consistent with previous observations26 on their in vivo functionality. Several controls put the false positive rate for TATA elements at ~20% (Fig. 2c). First, 10,000 randomly generated sequences having the same human genome sequence bias found that only 16% were called by chance. Second, a scrambled version of the motif (having 0-3 mismatches) was identified only 20% of the time, and had no positional relationship with TFIIB/TBP (not shown). Third, coordinates having a single isolated tag were used to generate an essentially random set of false-positive locations, and the analysis repeated. TATA elements (0-3 mismatches) were identified only 20% of the time. Fourth, whereas control sequences were distributed randomly across the query space, the distribution of TATA elements was not random. Instead it displayed a tight peak 20 bp upstream of TFIIB and TBP locations (Fig. 2d, and data not shown). TFIIB in complex with TBP makes sequence-specific contacts with BREu and BREd, which flank the TATA box2,3 and are upstream of the INR (Fig. 2a). However, these elements are essentially nonexistent in yeast, and ill-defined across mammalian genomes. Using the identified TATA elements as a reference point, we searched upstream for the BREu and downstream for the BREd and INR. Strikingly, in nearly every instance a sequence with three or less mismatches to the literature-derived consensus for BRE (SSRCGCC)2, BREd(RTDKKKK)3, and INR (YYANWYY)27 was found (Fig. 3a-c). Remarkably, sequences within each element appeared to co-vary. For example, the BREd consensus tended towards either GTKGGGG or ATKTTTT, rather than an equal mixture of all possible combinations (Fig. 3b), making them less degenerate than the consensus would suggest. Similarly, the INR consensus tended towards either CCANWCC or TTANWTT (Fig. 3c). Sequence bifurcation was not observed with TATA or BREu elements. Given the strong bias towards either strong (G/C) or weak (A/T) base-pairing, this sequence dimorphism may reflect selection for distinct thermodynamic stabilities towards helix melting, which is an essential first step in initiation at these elements. Consistent with this, A/T-rich BREd and INR elements had substantially higher crosslinking levels of TFIIB than their G/C-rich counterparts (not shown). However, this may not explain the strand bias of the sequences. Similar to our TATA analysis, the TFIIB peak density was tightly focused at a fixed distance from each core promoter element (Fig. 3d), and randomized controls were rarely found (Fig. 3e), thereby validating them. TFIIB peak-pairs were centered over BREd, suggesting that the primary crosslinking point is through the BREd. Unlike the TATA element, the BRE and INR elements deviated relatively little from their consensus (compare Figs. 2c and 3e) and such deviations did not correlate with TBP and TFIIB occupancy levels (not shown). Thus, BRE and INR sequence variability may regulate occupancy of the basal initiation complex to a lesser extent than TATA. Within their search space, the locations of each core promoter element peaked at previously-defined canonical positions (Fig. 3f and Extended Data Fig. 3b), thereby providing cross-validation and a core promoter consensus: SSRCGCCNNNTATAWAWRNNRTDKKKKNNNNYYANWYY. The tolerance for mismatches in these elements appears to be 2-3-2-1, respectively. 150,000 noncoding initiation complexes We next examined the remaining 150,753 putative TFIIB locations that were far (>500 bp) from a protein-coding gene (Extended Data Table 1). At a 20% false discovery rate per element, we identified at least 3 of the 4 core promoter elements at 97% of all non-mRNA TFIIB locations (Extended Data Fig. 4a). Deviations from the consensus were no more than at mRNA genes (average of 5 deviations across 28 positions within the four core promoter elements). TBP, TFIIB, and Pol II peaked at the same canonical distances from each motif as found at mRNA promoters (Extended Data Fig. 4b,c). They were also embedded in the same chromatin environment as mRNA promoters (Fig. 4a,b), but displayed comparatively lower TFIIB occupancy (Extended Data Fig. 4d). Remarkably, TBP/TFIIB/Pol II complexes were linked to the production of nonpolyadenylated RNA (87% had them) rather than polyadenylated transcripts (Fig. 4c and Extended Data Fig. 5), which is in agreement with the finding of enhancer RNAs28. Their locations mapped precisely to the location of TFIIB. Nonpolyadenylated transcript levels also correlated more strongly with “noncoding” TFIIB occupancy than did polyadenylated levels (Fig. 4d), further validating the link. Taken together, we conclude that the vast majority of all 159,117 TFIIB locations (noncoding plus coding) detected in K562 cells represent bona fide and fundamentally identical core promoter initiation complexes of which ~5% produce mRNA and ~95% produce RNA that is non-polyadenylated and noncoding. Restricted motif spacing in promoters We searched for an overall core promoter element (CPE) consensus (SSRCGCCNNNTATAWAWRNNRTDKKKKNNNNYYANWYY) and ~40 spacing variants within 100 bp of all TFIIB locations, and plotted their distribution relative to TFIIB (Extended Data Fig. 6). Remarkably, the consensus spacing defined in Fig. 3f displayed the strongest positional relationship with TFIIB (Fig. 5a). For example, a consensus having the spacing between BREd and INR reduced by 1 bp displayed almost no positional relationship with TFIIB (red vs black thick traces in Fig. 5a), as would be expected of a random/nonfunctional sequence. There was very little or no tolerance for variable spacing between core promoter elements (Fig. 5b), which reflects structural constraints of the initiation complex5. Surprisingly, proper spacing was accompanied by greater sequence deviations within individual core promoter elements (thick vs thin black line in Fig. 5a), whereas small (~1 bp) spacing deviations were accompanied by stronger elements (thick vs thin red line in Fig. 5a, and summarized in Fig. 5b,c). In short, core promoters may be weak by design, through a compensatory balance of sequence and spacing deviations from the consensus. This allows for greater dependence on transcriptional activators, but also provides for a specified basal output. We conducted ChIP-exo mapping of TFIIB locations across four ENCODE cancer cell lines: HeLa S3, HepG2, and MCF7 in addition to K562 (cervical, liver, breast, and blood, respectively). We detected TFIIB at 9,074 mRNA genes in at least one cell line, and at 1,691 genes in all lines (group 1 in Extended Data Fig. 9). Cluster analysis suggested that while TFIIB occupancy levels varied from gene to gene, most were relatively constant at individual genes across cell lines. About a third displayed noticeable cell-type specificity (e.g., group 3 in Extended Data Fig. 9). For noncoding initiation complexes, we focused on those present in two or more cell types, and found 100,349 such locations (376,074 locations were found in at least one cell type). Noncoding complexes appeared to have more cell-type specificity and were bimodally distributed at high and low occupancy levels. This heterogeneity may reflect more numerous and diverse roles for the resulting noncoding transcription and/or RNA in cell-type specific physiology compared to proteins. tRNA genes have TATA and BRE With some exception29, tRNA genes have been classically defined as TATA-less, where TFIIIC recognizes specific sequences downstream of the TSS, then recruits TFIIIB to a region immediately upstream of the TSS that lacks apparent sequence specificity30,31. Pol III then binds to form an initiation complex. TFIIIB contains TBP (and BRF, a factor related to TFIIB) and thus it has been enigmatic as to how TBP in TFIIIB engages the upstream region without a TATA box. Remarkably, TBP crosslinked ~21 bp upstream of 386 tRNA genes (Fig. 6a, left panel), as seen at Pol II promoters. In nearly every instance we found a TATA element (Fig. 6a, middle) that was ~18 bp further upstream (Fig. 6b). Similar to TBP crosslinking through TFIIB, we suspect that TBP crosslinks through BRF. Indeed, the peaks of BRF and TBP crosslinking are coincident at Pol III genes in mice32. As with Pol II promoters, we found a BREd centered between each TBP peak pair (Fig. 6a, right panel) and a BREu immediately upstream of TATA (not shown). Enrichment of these elements, but not the Pol II-specific INR33, were statistically significant (Fig. 6c). Thus, TBP in complex with a TFIIB family member engages a set of BREu-TATA-BREd core promoter elements similarly in Pol II and III systems. Consolidated genomic view of initiation Genome-wide mapping of the general transcription machinery at near single-base resolution offers a consolidated model of certain transcription initiation events from yeast to humans, Pol II to Pol III, TATA-containing to TATA-less, and mRNA to ncRNA. In general, a TFIIB/BRF family member is recruited to all coding or noncoding core promoters via a TBP family member and spatially-constrained core promoter elements. Sequence-specific (BREd) contact with the DNA a few bp downstream of TATA, might “bookmark” the site of DNA melting for a rapidly departing Pol II or III. Yeast Pol II is relatively slow to depart, and so it produces equivalent TFIIB-“open” promoter contacts in the absence of a BREd. Pol II then scans downstream several bp, where it encounters an INR that allows for productive transcription, which subsequently pauses 30-50 bp further downstream. In yeast, where an INR and pausing appear absent, a nucleosome border may help set the start site of productive transcription. Although core promoters are seemingly long (~38 bp in human) for sequence-specific binding, they are designed to be inherently low in specificity, presumably to keep basal transcription low and to maintain high dependence on transcriptional activators. Appropriate specificity is achieved via a blend of degeneracy in motif sequence and spacing. Broad clusters of TSSs at mammalian genes4 can therefore be explained in terms of clusters of core promoters, many of which may fall below bioinformatic detection. The discovery that transcription of the human genome is vastly more pervasive than what produces coding mRNA raises the question as to whether Pol II initiates transcription promiscuously through random collisions with chromatin as biological noise or whether it arises specifically from canonical Pol II initiation complexes in a regulated manner. Our discovery of ~150,000 noncoding promoter initiation complexes in human K562 cells and more in other cell lines suggests that pervasive noncoding transcription is promoter-specific, regulated, and not much different from coding transcription, except that it remains nuclear and nonpolyadenylated. An important next question is the extent to which transcription factors regulated this ncRNA. We detected promoter transcription initiation complexes at 25% of all ~24,000 human coding genes, and found that there were 18-fold more noncoding complexes than coding. We therefore estimate that the human genome potentially harbors as many as 500,000 potential promoter initiation complexes, corresponding to an average of about one every 3 kb in the non-repetitive portion of the human genome. This number may vary more or less depending on what constitutes a meaningful transcription initiation event. The finding that these initiation complexes are largely limited to locations having well-defined core promoters and measured TSSs indicates that they are functional and specific, but it remains to be determined to what end. Their massive numbers would appear to provide an origin for the so-called dark matter RNA of the genome34, and could house a substantial portion of the missing heritability35. METHODS Cell Culture Human chronic myelogenous leukemia cells (K562, ATCC) were maintained between 1×105 – 1×106 cells/milliliter in DMEM media supplemented with 10% bovine calf serum at 37°C with 5% CO2. Human adenocarcinoma cells from the cervix (HeLa S3, ATCC), liver (HepG2, ATCC), and breast (MCF7, ATCC) were grown in a similar manner as K562 cells except that they were maintained between 25-90% confluence. Cells were washed and phosphate buffered saline (1× PBS, 8 mM Na2HPO4, 2 mM KH2PO4, 150 mM NaCl, and 2.7 mM KCl) before incu bation with formaldehyde in a final concentration of 1% for 10 minutes. Cells were lysed (10 mM Tris pH 8, 10 mM NaCl, 0.5% NP40, and complete protease inhibitor cocktail (CPI, Roche), and then the nuclei lysed (50 mM Tris pH 8, 10 mM EDTA, 0.32% SDS, CPI). Purified chromatin was resuspended in IP dilution buffer (40 mM Tris pH 8.0, 7 mM EDTA, 56 mM NaCl, 0.4% Triton x-100, 0.2% SDS, and CPI) and sonicated with a Bioruptor (Diagenode) to obtain fragments with a size range between 100 and 500 bp. ChIP-exo and Antibodies With the following modifications, ChIP-exo was carried out as previously described36 with chromatin extracted from 10 million cells, ProteinG MagSepharose resin (GE Healthcare), and 3 ug of either TFIIB (Santa Cruz Biotech, sc-225), TBP (Santa Cruz Biotech, sc-204), or Pol II (Santa Cruz Biotech, sc-899, directed against the N-terminus of the Pol II large subunit encoded by POL2RA). Alignment to Genome, Peak Calling, and Data Access Libraries were sequenced on an Illumina HiSeq sequencer. The entire length of the sequenced tags were aligned to the human hg18 reference genome using BWA41 using default parameters. Raw sequencing data are available at NCBI Sequence Read Archive (SRA067908). The resulting sequence read distribution was used to identify peaks on the forward (W) and reverse (C) strand separately using the peak calling algorithm in GeneTrack (sigma = 20, exclusion zone = 40 bp)42. For strand-specific and strand-merged plots, sequencing tags were normalization to input. All 11,458 locations that were present in the ENCODE designated blacklist were removed from the analysis. Peaks were paired if they were 0-80 bp in the 3’ direction from each other and on opposite strands. Since patterns described here were evident among individual biological replicates, and replicates were well correlated, we merged all tags from biological replicate data sets to make final peak-pair calls. Peak pairs were considered to be TFIIB if they had a tag count of >4 in the merged datasets. 159,117 locations met these criteria. Peak pair matches across cell lines required that their midpoints be within 80 bp of each other. NCBI-curated RefSeq TSSs (n=26,987)18 comprising 23,181 nonredundant mRNA genes were considered. Assignment of TFIIB (8,364 peak-pairs) and TBP (7,642 peak-pairs) to the nearest RefSeq TSS in Extended Data Table 1 required that they be within 500 bp of the TSS, yielding 6,511 nonredundant mRNA genes. Importantly, using a more stringent interval only marginally changed these numbers and did not alter our conclusions. If a gene had >1 TSS, then the TSS nearest to the bound location (peak-pair midpoint) was used as the primary TSS, and other nearby TSSs were considered secondary (Fig. 1f, lower panel). Motif analysis At each of these 6,511 promoters, using the MEME suite of tools37, we searched for TATA elements within 80 bp of the midpoint of TFIIB-bound locations on the sense strand, first by searching for the consensus TATAWAWR (Extended Data Table 1), then sequentially for one to three mismatches to the consensus, if an element was not found. In rare cases where multiple elements were found, we chose the one closest to the TFIIB peak. This rule had no qualitative impact on the data since such events were rare and choosing the furthest element gave the same result (not shown). Moreover, peak motif detection for BREu, TATA, and INR were not centered over TFIIB, indicating that this distance criteria was not driving the observed motif enrichment at TFIIB locations. Using a similar strategy, we searched for candidate BREu element (Supplementary Table 4) within 40 bp upstream of the 5,546 identified TATA elements, and searched for candidate BREd and INR elements (Extended Data Table 1) within 40 bp and 60 bp downstream of the 5,546 TATA elements, respectively. At Pol III promoters, candidate BREd elements were required to be within 20 bp of a TBP peak pair midpoint, and in the same orientation as the TATA element. Our searches infrequently picked up multiple motif instances within the search window. Where this did occur, we chose the motif with the best match to the published consensus (not the closest to TFIIB). In the situation where we obtained more than one motif with the same number of mismatches, we chose the one closest to TFIIB. Third, when we discard these multiple occur-rences, the results qualitatively did not change. Fourth, the peak locations that we obtained for BREu, TATA, and INR were not centered over TFIIB. Instead they peaked at the canonical location that had been established in the literature. This provided independent validation. Using a PSPM matrix derived from individual core promoter element (CPE) logos from Figs. 2 and 3 (the matrices and data processing details are presented in Extended Data Table 1), FIMO37 was used to find 37-40 bp sequences within 100 bp of a TFIIB peak pair, and had either a p-value of <10-4 (thick trace in Fig. 5 and Extended Data Fig. 6) or between 10-4 and 10-3 (thin trace). Any CPE <50 bp from a stronger CPE (defined by motif and spacing similarity to the consensus) was eliminated. Distances between the two (TFIIB peak-pair midpoint to consensus BREd midpoint, i.e. 13 bp upstream of the CPE 3’ end) were then calculated for those CPE spacing variants listed at the top of Extended Data Fig. 6. Their frequency distribution was then plotted as a 5 bp moving average. Distributions were transformed into enrichment scores by calculating the ratio of occurrences near TFIIB (0-15 bp) to those far from TFIIB (55-70 bp), then log2-transforming the data. Supplementary Material Table S1 Table S2 Table S3 Table S4 Table S5 Acknowledgements We thank Pindi Albert and Yunfei Li for bioinformatic assistance, and Michael Cousar and Ka-Yim Chan-Salis for experimental support. This work was supported by National Institutes of Health grant GM059055. Author Contributions B.J.V. performed the experiments and conducted data analyses. B.J.V. and B.F.P. conceived the experiments, analyses, and co-wrote the manuscript. Author Information Sequencing data have been deposited at the NCBI Sequence Read Archive under accession number SRA067908. The authors declare no competing financial interests. Readers are welcome to comment on the online version of this article at www.nature.com/nature. Supplementary Information is linked to the online version of the paper at www.nature.com/nature. One sentence summary Widespread coding and noncoding transcription across the human genome arises from discrete transcription initiation complexes assembled at four core promoter elements. Figure 1 Transcription machinery organization at human mRNA promoters a, Smoothed distribution of strand-separated ChIP-exo tag 5’ ends at the RPS12 gene. Core promoter elements are shown with lower case denoting mismatches to the consensus. b, Peak-pair distribution or RNA at RefSeq genes (rows). Rows are linked, and sorted by TFIIB occupancy. c, Upper panel: Averaged ChIP-exo patterns around the closest (1°) RefSeq TSS. The “spikes” of TBP and TFIIB are indiscernible (vertically offset in inset). Lower panel: Distribution of 2° polyadenylated RNA38, with traces separated by sense (blue) and antisense (red, inverted trace) orientations relative to the corresponding mRNA TSS. Figure 2 TATA elements at most mRNA genes a, Core promoter schematic. b, Nucleotide distribution for TATA elements with 0-3 mismatches (panels) to the consensus, and sorted by ascending p-value. Colors are reflected in the logo color. c, Cumulative percent of TFIIB locations having a TATAWAWR sequence with 0-3 mismatches (solid line). Controls include a randomized sequence (60% GC, dashed black line), a scrambled consensus (dashed red line), and 8,364 locations represented by a single background tag (dashed gray line). d, Distance of strand-specific TFIIB peaks (exonuclease stop sites) from TATA element midpoints. Opposite-strand peaks are in red and inverted. Figure 3 BRE and INR at most mRNA genes a-c, Nucleotide distribution for BREu, BREd, and INR, vertically separated by 0-3 mismatches to the consensus, and sorted by ascending p-value within panels. d, Distance of strand-specific TFIIB peaks from BREu, BREd, and INR. Opposite-strand peaks are in red and inverted. e, Cumulative percent of genes with 0-3 mismatches to each motif in panels a-c. Controls were randomized sequences (60% GC, dashed lines). f, Distribution of core promoter elements relative to TATA box borders. Figure 4 Noncoding TFIIB locations have chromatin marks and non-polyadenylated RNA a, Distribution of chromatin marks around TFIIB at RefSeq genes (left) and ncRNA (right). b, TFIIB locations that overlap with chromatin marks and epigenetic regulators39. c, Distribution of polyadenylated38 and non-polyadenylated40 RNA-seq tags around TFIIB >500 bp from a RefSeq TSS. Percentages reflect TFIIB having an RNA tag <2 kb away. Left panels include sense (blue) and antisense (red and inverted) strands for RefSeq genes, which was not applied to ncRNA (right panels). d, 100-gene moving average of polyadenylated and nonpolyadenylated RNA levels versus TFIIB occupancy at mRNA and ncRNA genes (left and right panels, respectively) on a median-centered log2 scale. Figure 5 Restricted spacing of core promoter elements a, Candidate core promoter enrichment at varying distances from all 159,117 TFIIB locations, for spacing variants “323” and “324”, for motifs with weak (thick lines) and strong (thin lines) p-values. b, Traces from panel a and Extended Data Fig. 6, were transformed into enrichment scores and shown as a table, sectored by element spacing, and at two motif p-value intervals. Values are heat-map colored from green to light gray. Configurations in white were not examined. c, Schematic of core promoters having the strongest positional correlation with TFIIB, rank ordered by opacity. “324” (***) stood out as the strongest. Figure 6 TATA and BRE elements at most tRNA genes a, Left panel: TBP peak density separated by forward and reverse strand orientation (blue and red colors, respectively) relative to each tRNA TSS. Corresponding sequences are shown in the right two panels. b, Average distribution of TBP peaks around all identified tRNA TATA elements. c, Cumulative percent of tRNA genes with the indicated promoter element having 0, 1, 2, or 3 mismatches to the consensus. Dashed lines represent calculations for an equivalent number of randomized sequences for the color-linked solid traces. Extended Data Figure 1 Validation of ChIP-exo data and association with ENCODE annotated regions a, Pie chart of all 159,117 TFIIB-bound locations in K562 cells parsed into ENCODE-annotated regions. b, Venn overlap among mRNA genes having TBP or TFIIB locations (<500 bp from its TSS) and genes with measured polyadenylated mRNA levels detected by RNA-seq38. Data thresholding may contribute to nonoverlapping sets. c, Moving average (100-gene) of mRNA levels versus TFIIB/TBP/Pol II occupancy levels on a median-centered log2 scale. Extended Data Figure 2 Distribution of the TFIIB/TBP/Pol II in CpG islands that overlap mRNA TSSs a, Peak-pair distribution for TFIIB, TBP and Pol II at the 5,095 CpG islands that overlap with the mRNA TSSs from Figure 1b (78% overlap), and with the direction of transcription to the right. Rows are linked, and sorted by CpG island length. CpG island borders are indicated by blue and red bars, respectively. b, Shown is the averaged data from panel a. c, All 159,117 TFIIB locations were sorted by location, and inter-TFIIB distances calculated (red trace). Data were then sorted by distance, and the standard deviation of TFIIB occupancy was calculated on a sliding window of 30 values. Peak calling parameters preclude detection of two separate TFIIB locations <~40 bp apart. Those that were 40-70 bp apart were correlated, whereas those >~70 bp apart were uncorrelated. Extended Data Figure 3 Properties of core promoter elements associated with RefSeq genes a, Average TFIIB and TBP occupancy parsed by the number of mismatches to the TATA consensus. b, Distribution of each candidate core promoter element relative to each other. Extended Data Figure 4 Core promoter elements at noncoding loci bound by TFIIB a, Bar graph showing the percentage of all 150,754 putative “noncoding” TFIIB binding locations (>500 bp from an annotated RefSeq TSS) that have the indicated number of core promoter elements. b, Distribution of ChIP-exo peaks on each strand relative to the indicated core promoter element, for 150,754 putative “noncoding” TFIIB locations. Opposite strand traces (red) are inverted. c, Distribution of TBP (purple) and Pol II (black) peak-pair midpoints relative to the TATA motif midpoint derived from the 150,754 TFIIB putative “noncoding” locations. d, TFIIB occupancy versus percentage of locations that code for proteins. All 159,117 TFIIB locations were sorted by occupancy level, then the percentage of locations linked to an annotated RefSeq feature was plotted as a moving average. Extended Data Figure 5 Enrichment of different RNA fractions at 159,117 TFIIB locations throughout the human genome Frequency distribution RNA 5’ ends for Poly(A)+38 (top plots) and ENCODE project RNA fractions40 as indicated to the far left. Traces in the left panels are separated by sense (blue) and anti-sense (red, inverted) orientations relative to the corresponding mRNA TSS, which is directed to the right. Since the TSS orientation is not known for the Poly(A)- ncRNA loci, positive and negative strand tags were plotted relative to the TFIIB midpoint. The percent of putative TFIIB locations that exist within 2 kb of an RNA tag are indicated in the upper right corner of each plot. Extended Data Figure 6 TFIIB-core promoter distances Candidate CPE at varying distances from all 159,117 TFIIB locations, for the indicated spacing variants (not all possible combination were tested). Digits within spacing variant names reflect the bp spacing (N) between elements (e.g., “324” denotes BREu-NNN-TATA-NN-BREd-NNNNINR). The collection of core promoters illustrated at the top had the strongest positional enrichment, and thus were used to associate candidate core promoters with TFIIB (Extended Data Table 1). Extended Data Figure 7 Promoter complexes across cancer cell lines Occupancy levels for TFIIB linked to coding genes (a) and noncoding regions (b) in the indicated cell type were normalized by column. The color scales represent the range of average-centered, log2 transformed values within each respective column. Detection in all four cell types define Group 1. Groups 2-4 were parsed by k-means clustering. Rows were sorted within groups based on TFIIB occupancy averaged across the four cell types (yellow-black-cyan-gray, denote high, medium, low, and zero occupancy, respectively). For clarity in panel b, TFIIB locations that were detected in only one cell line were excluded from clustering. Columns were hierarchically clustered. The MCF7 dataset had 20-30% of the coverage of other cell lines (reported in Extended Data Table 1), which likely accounts for excessive number of zero-occupancy loci (gray). Extended Data Table 1 Illumina sequencing statistics Summary of uniquely mapped sequencing reads for each biological replicate. Factor Antibody Cell Line Total Reads Uniquely Mapped Reads Unique Mapping Rate Input none K562 126,007,656 104,591,819 83% Input none K562 109,745,112 91,160,835 83% Totals: 235,752,768 195,752,654 TBP sc-204 K562 97,896,951 60,581,579 62% TBP sc-204 K562 181,420,753 132,655,896 73% TBP sc-204 K562 200,167,837 115,213,419 58% Totals: 479,485,541 308,450,894 TFIIB sc-225 K562 64,473,390 43,727,825 68% TFIIB sc-225 K562 129,513,614 80,930,721 62% Totals: 193,987,004 124,658,546 Pol II sc-899 K562 40,833,504 31,260,456 77% Pol II sc-899 K562 119,799,682 88,431,598 74% Totals: 160,633,186 119,692,054 TFIIB sc-225 HeLa-S3 62,249,055 41,815,431 67% TFIIB sc-225 HeLa-S3 185,240,056 123,002,393 66% Totals: 247,489,111 164,817,824 TFIIB sc-225 HepG2 78,313,847 50,505,201 64% TFIIB sc-225 HepG2 264,530,278 172,112,282 65% Totals: 342,844,125 222,617,483 TFIIB sc-225 MCF7 25,615,261 14,780,271 58% TFIIB sc-225 MCF7 120,958,757 28,600,410 24% Totals: 146,574,018 43,380,681 ==== Refs REFERENCES 1 Buratowski S Hahn S Guarente L Sharp PA Five intermediate complexes in transcription initiation by RNA polymerase II. 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==== Front J Am Chem SocJ. Am. Chem. SocjajacsatJournal of the American Chemical Society0002-78631520-5126American Chemical Society 10.1021/ja501979gCommunicationChemoselective Reactions of (E)-1,3-Dienes: Cobalt-Mediated Isomerization to (Z)-1,3-Dienes and Reactions with Ethylene Timsina Yam N. Biswas Souvagya RajanBabu T. V. Department of Chemistry and Biochemistry, The Ohio State University, Columbus, Ohio 43210, United [email protected] 04 2015 08 04 2014 30 04 2014 136 17 6215 6218 25 02 2014 Copyright © 2014 American Chemical Society2014American Chemical Society In the asymmetric hydrovinylation (HV) of an E/Z-mixture of a prototypical 1,3-diene with (S,S)-(DIOP)CoCl2 or (S,S)-(BDPP)CoCl2 catalyst in the presence of Me3Al, the (E)-isomer reacts significantly faster, leaving behind the Z-isomer intact at the end of the reaction. The presumed [LCo–H]+-intermediate, especially when L is a large-bite angle ligand, similar to DIOP and BDPP, promote an unusual isomerization of (E/Z)-mixtures of 1,3-dienes to isomerically pure Z-isomers. A mechanism that involves an intramolecular hydride addition via an [η4-(diene)(LCo–H)]+ complex, followed by π–σ–π isomerization of the intermediate Co(allyl) species, is proposed for this reaction. National Institutes of Health, United Statesdocument-id-old-9ja501979gdocument-id-new-14ja-2014-01979gccc-price ==== Body We recently reported a new protocol for a highly enantioselective Co(II)-catalyzed asymmetric hydrovinylation (HV) of unactivated 1,3-dienes that involves the use of a [1,n-bis-diphenylphosphinoalkane]CoCl2 and Me3Al (Scheme 1).1,2 Scheme 1 Ni(II)- and Co(II)-Catalyzed Hydrovinylation of 1,3-Dienes In order to explain the improved selectivity in the Co-catalyzed reactions as compared to the corresponding Ni-catalyzed reactions3 (Scheme 1), we invoked1a an η4-cobalt-hydride complex 4 that restricts the conformations of the reactive intermediates in the former (Scheme 2). Complex 4 subsequently forms an allyl-cobalt intermediate 5, which undergoes ethylene insertion and β-hydride elimination to regenerate the presumed [LCo–H]+ catalyst 3(4) to complete the catalytic cycle. When an E/Z-mixture of a prototypical 1,3-diene was subjected to our standard HV conditions (except for the presence of ethylene), the terminal (Z)-1,3-diene was found to be mostly unreactive at low temperature. The attendant implication is that the (Z)-isomer is unreactive toward [LCoH]+ or the equivalent catalyst. Since such hydride species are also known to be capable of isomerization of alkenes, we wondered if conditions can be found to maximize the Z-isomer from an E/Z-mixture under kinetic conditions.5,6 The results of these studies are described in this paper. The enantioselectivity in the asymmetric HV of a mixture of (E)-8 and (Z)-8 (Z:E = 53:47) using [(S,S)-DIOP]CoCl2/Me3Al (TMA) was found to depend on the conversion, with the (E)-isomer reacting at a significantly faster rate (eq 1, Table 1).6,7 At low conversions (entries 1–3, Table 1), only E-8 undergoes hydrovinylation giving a maximum of ∼83% ee. As conversion increases, the proportion of Z-8 increases, leaving behind, at 23 h, essentially pure (Z)-8 (Z:E = 49:1, entry 5). A minor product (<5%), tentatively identified as a linear hydrovinylation product (10), is also formed at higher conversions. A similar behavior is observed with (S,S)-2,4-BDPP ligand, except a notable decrease in enantioselectivity (from 85% ee at 21% conversion to 73% ee at 61% conversion, entries 6–10) results. These results are most readily rationalized if one assumes that the (Z)-isomer is a reluctant partner in the HV reaction, while the (E)-isomer undergoes a fast reaction giving the (S)-9 [with (S,S)-DIOP] as the major product. With the more reactive BDPP complex, at higher temperatures (−15 °C), upon complete conversion of the (E)-isomer, the (Z)-isomer undergoes the reaction, giving enantiomeric product, along with 10. The (DIOP)CoCl2 complex shows much more discrimination in the reactions of the (Z)- and (E)-isomers of the starting diene, leaving behind almost all of the (Z)-isomer unreacted (46%, Z:E = 49:1, entry 5) at the end of the reaction (entry 5, compared to 25% in entry 10 using 2,4-BDPP). The lower enantioselectivity at higher conversions using the BDPP ligand (entries 9 and 10) also suggests that the 1,4-HV of the (Z)-8 gives predominantly the opposite enantiomer.6 Implicit in these observations is also the striking absence of the isomerization of the (Z)-(8) into the thermodynamically favored (E)-isomer in the presence of the presumed Co(II)-hydride intermediates. Since (E)-8 appears to readily engage the [LCo–H]+ in the facile hydrovinylation, in the absence of ethylene, under proper conditions, kinetic control might prevail in the (E)–(Z)-isomerization of 8, and if so, using ligand effects could one make the (Z)-isomer?5 These expectations have indeed been borne out, and here we report the details of these investigations. While there are excellent methods for the formation of the (E)-1,3-diene isomer,8 there is a dearth of methods for the synthesis of isomerically pure (Z)-1,3-dienes,9 and in many biologically important compounds this configuration is important.10 1 Scheme 2 Working Hypothesis on the Mechanism of Co(II)-Catalyzed Hydrovinylation Table 1 Chemoselectivity in the Asymmetric HV of (Z/E)-8a entry time (h) % (9) ee %b (9) % (8) Z:Eb (8) 10 (S,S)-DIOPc 1 0.5 16 80 84 2.1:1.0 – 2 1.2 23 81 77 2.7:1.0 – 3 3 26 83 71 3.4:1.0 3 4 6 31 82 65 4.4:1.0 4 5 23 49 84 46 49:1 5 (S,S)-BDPPd 6 1 21 85 79 1.9:1.0 <1 7 2 30 85 70 2.6:1.0 <1 8 5.2 42 82 49 7.1:1.0 9 9 8 47 77 43 13.3:1.0 10 10 23 61 73 25 49:1 13 a See eq 1 for procedure. b Determined by GC. See Supporting Information for chromatograms. c At −45 °C. (S)-9 major. d At −15 °C. (R)-9 major. Before we describe our results, attention should be drawn to a related paper by Hilt et al., who documented a distinctly different protocol for the E/Z isomerization of 1,3-dienes.5d In this report (Scheme 3), a Co(I) complex prepared under reducing conditions from a tridentate pyridine-imine ligand (11), CoBr2, Zn, and ZnI2 gave moderate yields of (Z)-1,3-dienes (14) from a 1:1 mixture of (Z)- and (E)-dienes (13). Behavior of the 1,3-diene was found to be highly dependent on the ligand, with a bis-phosphine-CoBr2 under identical conditions (i.e., with Zn/ZnI2) giving modest yields of a product (15), arising via a 1,5-hydrogen migration. The difference between Hilt’s [bis-phosphine]Co-chemistry and the [bis-phosphine]CoCl2/Me3Al-mediated reactions described in this paper is highlighted by the total absence of the 1,5-hydrogen migration in the latter and the higher yields of Z-isomerization products obtained. Scheme 3 Co(I)-Catalyzed Isomerization Reactions The initial scouting experiments were carried out on a mixture of (Z)- and (E)-8 using CoCl2 complexes of chelating bis-diphenylphosphinoalkane ligands under conditions similar to the HV except for the presence of ethylene (eq 2). The composition of isomers and identification of the product(s) were determined by 1H and 13C NMR spectroscopy and gas chromatography.6 The results are presented in Table 2. 2 Table 2 Isomerization of 1,3-Diene (Z/E)-8: Ligand Effectsa entry start mat (Z):(E)-8 ligand bite angle temp (°C)/time (h) product (Z):(E)- 8 1 33:67 [DPPM] 72 –15/14 37:63 2 33:67 [DPPE] 85 –10/14 74:26 3 33:67 [DPPP] 91 –16/22 82:18 4 33:67 [DPPP]b 91 –4/16 33:67 5 33:67 [DPPB] 98 –15/14 >99:<1 6 33:67 [DPPpent]c – –15/14 >99:<1 7 33:67 (S,S)-DIOP 98 –10/12 100:0d a See eq 2 for procedure. b Using CoBr2. c Bis-1,5-diphenyphos-phinopentane. d Isomeric product not seen in GC or NMR. As shown in Table 2, the Z/E composition of the products is highly dependent on the ligand. A Co-complex containing ligand with a small bite angle,11 1,1-bis-diphenylphosphinomethane (DPPM, bite angle β = 72), showed little tendency to effect the isomerization (entry 1), whereas complexes of large bite angle ligands, 1,4-bis-diphenylphosphinobutane (entry 5, β = 98), 1,5-bis-diphenylphosphinopentane (entry 6), and DIOP (entry 7, β = 98), gave quantitative conversion to the (Z)-isomer at low temperature. Bis-diphenylphosphinopropane (DPPP, bite angle 91) gave up to 82% of the (Z)-isomer at −15 °C (entry 3). The reaction is specific for the chloride complex; as shown in the entry 4, the corresponding bromide complex is ineffective for the isomerization reaction. We have examined the isomerizations of the mixtures of a number of 1,3-dienes under the optimized conditions (eq 2), and the most significant results are listed in Table 3. An expanded list of complexes and their effect on the isomerization of each of the dienes is included in the Supporting Information. As can be seen in entries 1–9, the isomerization reaction is broadly applicable giving excellent yields of the (Z)-isomers of most dienes. As inferred from the scouting studies (Table 2), ligands with relatively large bite angles, DPPB and DPPPent, were found to be the most generally applicable for this reaction. For substrates where the diene is conjugated to an aromatic moiety (entries 5, 6, and 7), DPPPent is the ligand of choice, giving excellent conversion to the expected (Z)-isomer. DPPB leads to slightly lower selectivities. The isomerization reaction gives satisfactory results even in substrates that contain Lewis basic centers (entries 6 and 7). A preparative scale experiment (3 mmol) using 16 as the starting material gave 91% isolated yield of the expected product.6 Table 3 Co(II)-Catalyzed Isomerization of (Z)- and (E)-1,3-Dienesa a See eq 2 for procedure. For an expanded list of ligand effects for each substrate, see Supporting Information. b Yield of volatile products were estimated from NMR and GC. c Product contains an unidentified impurity from the starting material. The isomerization reaction appears to be limited to terminal 1,3-dienes as illustrated by the examples shown in eqs 3 and 4. Substrates 24 and 25 failed to undergo the reaction under a variety of conditions using the ligands discussed previously. The starting material was recovered virtually unchanged. 3 The experiments listed in previous sections suggest that one reason for the poor reactivity/selectivity of the Z-substrates might be their reluctance to form an η4- complex.12 A plausible explanation for the observed results, based on the assumption that the initial [LCo–H]+ addition to the 1,3-diene is reversible, is shown in Scheme 4. An intramolecular hydride delivery via an η4-complex 4E gives the syn-anti-Co(allyl)-complex 5as.13 This species could undergo the familiar π–σ–π isomerization14 to give, among others, an anti–anti complex (5aa). Hydride elimination from this species would generate a diene complex 4Z, which for steric reasons, might dissociate to give the (Z)-diene. The (Z)-diene, once formed, will most likely exist in the (s)-trans conformation, precluding any further η4-complexation with the Co(II)-catalyst.15 Scheme 4 Plausible Mechanism for the E- to Z-Isomerization of a 1,3-Diene In summary, attempts to effect asymmetric hydrovinylation of a mixture of (Z)- and (E)-1,3-dienes using (P ∼ P)CoCl2/Me3Al reveal that there is a significant difference in the relative rates of ethylene incorporation, with the (E)-isomer reacting significantly faster. In the absence of ethylene, under otherwise identical conditions, this Co-catalyst promotes an unusual isomerization of an (E)/(Z)-mixture of 1,3-dienes almost exclusively to the (Z)-isomer. This result is strikingly different from the related reaction mediated by the reagent combination [(P ∼ P)CoBr2/Zn/ZnI2), where a product of 1,5-hydrogen shift is the major.5d,16 A mechanism that involves an intramolecular hydride addition via an η4-complex and subsequent π–σ–π isomerization of the intermediate Co(allyl) species is proposed for this reaction. Supporting Information Available Experimental details and characterization data. This material is available free of charge via the Internet at http://pubs.acs.org Supplementary Material ja501979g_si_001.pdf The authors declare no competing financial interest. Acknowledgments Financial assistance for this research provided by US National Science Foundation (CHE-1057818) and National Institutes of Health (General Medical Sciences, R01 GM075107) is gratefully acknowledged. We are grateful to Mr. William Coldren and Professor Chris Hadad for help with the DFT calculations. ==== Refs References a Sharma R. K. ; RajanBabu T. V. J. Am. Chem. Soc. 2010 , 132 , 3295 .20163120 b Page J. P. ; RajanBabu T. V. J. Am. Chem. Soc. 2012 , 134 , 6556 .22452442 For other reports of Co-catalyzed hydroalkenylations, see: a Hilt G. Eur. J. Org. Chem. 2012 , 4441 . b Grutters M. M. P. ; Müller C. ; Vogt D. J. Am. Chem. Soc. 2006 , 128 , 7414 .16756275 c Hilt G. ; Lüers S. Synthesis 2002 , 609 . d Hilt G. ; du Mesnil F.-X. ; Lüers S. Angew. Chem., Int. Ed. 2001 , 40 , 387 . e Iwamoto M. ; Yuguchi S. Bull. Chem. Soc. Jpn. 1968 , 41 , 150 . f Wittenberg D. Angew. Chem., Int. Ed. Engl. 1964 , 3 , 153 . For recent reviews of hydrovinylation of alkenes, see: a RajanBabu T. V. Synlett 2009 , 853 .19606231 b RajanBabu T. V. Chem. Rev. 2003 , 103 , 2845 .12914483 Reports of asymmetric hydrovinylation of 1,3-dienes: c Saha B. ; Smith C. R. ; RajanBabu T. V. J. Am. Chem. Soc. 2008 , 130 , 9000 .18570419 d Zhang A. ; RajanBabu T. V. J. Am. Chem. Soc. 2006 , 128 , 54 .16390118 e Bogdanović B. ; Henc B. ; Löser A. ; Meister B. ; Pauling H. ; Wilke G. Angew. Chem., Int. Ed. Engl. 1973 , 12 , 954 . For other reports of a [LCoH]+ intermediate in related reactions, see: a Britovsek G. J. P. ; Bruce M. ; Gibson V. C. ; Kimberley B. S. ; Maddox P. J. ; Mastroianni S. ; McTavish S. J. ; Redshaw C. ; Solan G. A. ; Strömberg S. ; White A. J. P. ; Williams D. J. J. Am. Chem. Soc. 1999 , 121 , 8728 . b Bianchini C. ; Giambastiani G. ; Meli A. ; Toti A. Organometallics 2007 , 26 , 1303 . c Tellmann K. P. ; Gibson V. C. ; White A. J. P. ; Williams D. J. Organometallics 2005 , 24 , 280 . d Tellmann K. P. ; Humphries M. J. ; Rzepa H. S. ; Gibson V. C. Organometallics 2004 , 23 , 5503 . e Zhang G. ; Scott B. L. ; Hanson S. K. Angew. Chem., Int. Ed. 2012 , 51 , 12102 .A complex [(DPPE)2CoH]+, isoelectronic with 4 has been described in the literature: f Ciancanelli R. ; Noll B. C. ; DuBois D. L. ; DuBois M. R. J. Am. Chem. Soc. 2002 , 124 , 2984 .11902890 Several examples of metal and ligand-dependent isomerization of alkenes to seemingly less stable isomers have been described in the literature. a Ni-catalyzed isomerization of allyl ethers to (Z)-vinyl ethers: Wille A. ; Tomm S. ; Frauenrath H. Synthesis 1998 , 305 and references cited therein . b Corresponding Ir(I)-catalyzed reaction give the (E)-isomers: Nelson S. G. ; Bungard C. J. ; Wang K. J. Am. Chem. Soc. 2003 , 125 , 13000 .14570453 c Larsen C. R. ; Grotjahn D. B. J. Am. Chem. Soc. 2012 , 134 , 10357 .22702432 Correction: J. Am. Chem. Soc. 2012 , 134 , 15604 . d Co-catalyzed isomerization of an E/Z-mixture of a 1,3-diene to a (Z)-I,3-diene: Pünner F. ; Schmidt A. ; Hilt G. Angew. Chem., Int. Ed. 2012 , 51 , 1270 . e Co-catalyzed isomerization of internal to terminal alkene: Obligacion J. V. ; Chirik P. J. J. Am. Chem. Soc. 2013 , 135 , 19107 .24328236 f Co-catalyzed terminal to internal Z-selective isomerization: Chen C. ; Dugan T. R. ; Brennessel W. W. ; Weix D. J. ; Holland P. L. J. Am. Chem. Soc. 2014 , 136 , 945 .24386941 For a review, see: g Donohoe T. J. ; O’Riordan T. J. C. ; Rosa C. P. Angew. Chem., Int. Ed. 2009 , 48 , 1014 . Precise proportion of isomeric compounds were determined by gas chromatography and NMR. See Supporting Information for details including chromatograms of products from various reactions. At higher temperatures (−10 °C, 1 atm ethylene) (DPPB)CoCl2/MAO converts both (Z)- and (E)-8 to racemic 9 in quantitative yield. See Supporting Information for details. a Ikeda Y. ; Ukai J. ; Ikeda N. ; Yamamoto H. Tetrahedron 1987 , 43 , 731 . b Wang S. ; West F. G. Synthesis 2002 , 99 . c de Vicente J. ; Huckins J. R. ; Rychnovsky S. D. Angew. Chem., Int. Ed. 2006 , 45 , 7258 . d Billard F. ; Robiette R. ; Pospíŝil J. Org. Chem. 2012 , 77 , 6358 .22764737 a Ikeda Y. ; Ukai J. ; Ikeda N. ; Yamamoto H. Tetrahedron 1987 , 43 , 723 . b Paterson I. ; Schlapbach A. Synlett 1995 , 498 .Syntheses of (Z)-dienyl alcohols and amines via Rh-catalyzed reductive coupling of acetylene with aldehydes and imines have been reported. c Skucas E. ; Kong J. R. ; Krische M. J. J. Am. Chem. Soc. 2007 , 129 , 7242 .17511459 d Kong J. R. ; Krische M. J. J. Am. Chem. Soc. 2006 , 128 , 16040 .17165749 a Discodermolide: Gunasekera S. P. ; Gunasekera M. ; Longley R. E. ; Schulte G. K. J. Org. Chem. 1990 , 55 , 4912 . b Dictyostatin: Pettit G. R. ; Cichacz Z. A. ; Gao F. ; Boyd M. R. ; Schmidt J. M. J. Chem. Soc., Chem. Commun. 1994 , 1994 , 1111 . van Leeuwen P. W. N. M. ; Kamer P. C. J. ; Reek J. N. H. ; Dierkes P. Chem. Rev. 2000 , 100 , 2741 .11749304 We have carried out Co-catalayzed asymmetric HV of E/Z-mixtures of 16 and 17 and observed results similar to what is documented in Table 1 for E/Z-8. See Supporting Information for details. Kinetic preference for the formation of an anti-crotyl-η3- complex, and its subsequent isomerization to the more stable syn-isomer is known in {[P(OEt)3]4Ni-H}+ additions. See: Tolman C. A. J. Am. Chem. Soc. 1970 , 92 , 6777 . a Consiglio G. ; Waymouth R. M. Chem. Rev. 1989 , 89 , 257 .b Trost B. M. ; Lee C. Asymmetric Allylic Alkylation Reactions . In Catalytic Asymmteric Synthesis ; Ojima I. , Ed.; Wiley-VCH : New York , 2000 ; pp 593 –649 . We have carried out high-level DFT calculations (Gaussian 09, geometries optimized with the 6-31G basis set in conjunction with the B3LYP) on two of the dienes (8) and (16). Not surprisingly, the E-isomer is the more stable one (KE/Z = 3924 and 24.8, respectively, 298 K), and both isomers exist almost exclusively in the s-trans form. The (Z)-isomer, once generated, will also exist exclusively in the s-trans conformation (Ks-trans/s-cis = 1998 and 612, respectively), preventing a stable η4-coordination to Co(II). See Supporting Information for details of these calculations and references to experimental data on E/Z-isomerization of 1,3-pentadiene. We have also observed up to 69% conversion of a (Z/E)-mixture (46:54) of 16 to a product of 1,5-H-shift (15, R = C7H14) by using (DPPE)CoBr2 (20 mol%)/Zn/ZnI2 (40 mol%) for 72 h (see Supporting Information for details).	24712838	PMC4046774	NO-CC CODE	2021-01-05 01:08:57	yes	J Am Chem Soc. 2014 Apr 30; 136(17):6215-6218
==== Front Med Sci MonitMed. Sci. MonitMedical Science MonitorMedical Science Monitor : International Medical Journal of Experimental and Clinical Research1234-10101643-3750International Scientific Literature, Inc. 2506033010.12659/MSM.890641890641Animal StudyBrain Susceptibility Weighted Imaging Signal Changes in Acute Hemorrhagic Anemia: An Experimental Study Using a Rabbit Model Xia Jun 1AEXie Ni 2AEFeng Yuning 1CDYin Anyu 1CDLiu Pinni 1BDZhou Ruming 3BCLin Fan 1BCTeng Guozhao 4BDLei Yi 1BF1 Department of Radiology, Second People’s Hospital of Shenzhen City, First Affiliated Hospital of Shenzhen University, Shenzhen, Guangdong province, China2 Biobank, Second People’s Hospital of Shenzhen City, First Affiliated Hospital of Shenzhen University, Shenzhen, Guangdong province, China3 Department of Interventional Radiology, Second People’s Hospital of Shenzhen City, First Affiliated Hospital of Shenzhen University, Shenzhen, Guangdong province, China4 Medical Record and Statistics Room, Second People’s Hospital of Shenzhen City. First Affiliated Hospital of Shenzhen University, Shenzhen, Guangdong province, ChinaCorresponding Author: Yi Lei, e-mail: [email protected], [email protected] Study Design B Data Collection C Statistical Analysis D Data Interpretation E Manuscript Preparation F Literature Search G Funds Collection * The two authors are listed as co-first authors with equal contribution to the paper 2014 25 7 2014 20 1291 1297 06 3 2014 09 4 2014 © Med Sci Monit, 20142014This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported LicenseBackground The aim of this study was to investigate susceptibility-weighted imaging (SWI) signal changes in different brain regions in a rabbit model of acute hemorrhagic anemia. Material/Methods Ten New Zealand white rabbits were used for construction of the model of acute hemorrhagic anemia. Signal intensities of SWI images of the bilateral frontal cortex, frontal white matter, temporal lobe, and thalamic nuclei were measured. In addition, the cerebral gray-white contrast and venous structures of the SWI images were evaluated by an experienced physician. Results Repeated bloodletting was associated with significant reductions in red blood cell count, hemoglobin concentration, hematocrit, pH, and PaCO2, and elevations of blood lactate and PaO2. In normal status, the SWI signal intensity was significantly higher in the frontal cortex than in the frontal white matter (63.10±22.82 vs. 52.50±20.29; P<0.05). Repeated bloodletting (5 occasions) caused significant (P<0.05) decreases in the SWI signals of the frontal cortex (from 63.10±22.82 to 37.70±4.32), temporal lobe (from 52.50±20.29 to 42.60±5.54), and thalamus (from 60.40±20.29 to 39.40±3.47), but was without effect in the frontal white matter. The cerebral white-gray contrast and venous structures were clearer after bloodletting than before bloodletting. Conclusions The effect of hemorrhage on the brain is reflected by SWI signal changes in the cerebral cortex and gray matter nuclei. MeSH Keywords AnemiaCholangiopancreatographyMagnetic ResonanceHemoglobin, Sickle ==== Body Background A number of clinical conditions cause acute hemorrhagic anemia, including massive gastrointestinal tract hemorrhage (caused by traumatic surgery, trauma, rupture of gastroesophageal varices, and duodenal and gastric ulcer), pulmonary or bronchial hemoptysis, the sudden bleeding resulting from tumor erosion into the blood vessel wall, and hemostatic defect-related diseases (such as hemophilia, Von Willebrand’s disease, and platelet dysfunction) [1–3]. Each of these diseases can decrease blood flow and oxygen supply to the brain, and affect the physiological function of the brain, which often manifests as a wide variety of neurological symptoms, including dizziness, tinnitus, and even stroke and paralysis, and high disability and fatality rates, especially for children. In addition, anemia concurrent with brain trauma and a ruptured aneurysm also easily induces delayed cerebral infarction, thereby affecting treatment effect. Therefore, the detection of changes in brain function in patients with acute hemorrhagic anemia is helpful in preventing neurological complications and evaluating therapeutic effect. However, the clinical changes of the nervous system in patients with anemia have not received much research attention. One important reason is that the current means can only indirectly detect changes in the brain function following anemia and lag behind the real condition of the brain function. Some methods, such as transcranial Doppler ultrasound, can detect the changes in cerebral blood flow velocity resulting from compensatory response of deficient oxygen delivery in anemia, including the increase in the state of anemia and returning to normal level when anemia is improved, but cannot reflect the actual situation of the oxygen supply and metabolism in the brain or organic brain changes such as stroke [4]. The other means, such as conventional computed tomography (CT) and magnetic resonance imaging (MRI), can detect early cerebral infarction and thus facilitate early treatment, but cannot monitor the physiological changes in the brain from the occurrence of anemia [5]. A non-invasive method, near-infrared spectroscopy (NIRS), is able to measure total levels of blood oxygen in the brain [6–8], but is unable to provide fine details concerning the alterations in different cerebral tissues. Susceptibility-weighted imaging (SWI) is a newly developed, contrast-enhanced MRI technique that has been used widely in the clinical diagnosis and research of many nervous system diseases, including cerebral hemorrhage and brain tumors [9–12]. SWI is known as high-resolution blood oxygenation level-dependent (BOLD) venographic imaging [13], and can reflect oxygen metabolism-related blood protein concentrations. This technology can produce clear images of cerebral venules, and may be used to detect strokes and microbleeds [11,14–16]. SWI is potentially a novel, non-invasive method for sensing changes in cerebral oxygen levels and may provide more detailed information on cerebral blood flow in patients with hemorrhage [17]. However, there is a lack of studies in animal models assessing the utility of SWI for monitoring pathophysiologic changes in the brain following acute hemorrhagic anemia. In the present study, we have used SWI to detect cerebral changes in an animal model of acute hemorrhagic anemia. We found that the SWI images and signals showed changes following hemorrhage, and the extent of the change in the SWI signal following acute hemorrhagic anemia differed between brain parenchyma and white matter regions. This indicates that the partial pressure of oxygen and carbon dioxide, and the concentration of deoxygenated hemoglobin in cerebral blood, are altered following acute hemorrhagic anemia. Our results suggest that SWI may be used to assess various regions of the brain after acute post-hemorrhagic anemia, and that it provides valuable information concerning the pathophysiologic changes in the brain. Material and Methods New Zealand white rabbits Ten male New Zealand white rabbits (weight, 2.1–2.4 kg; age, 3–4 months) were obtained from the Experimental Animal Center of Guangdong Medical College, Guangzhou, China. The study was approved by the Animal Care and Ethics Committee of the First Affiliated Hospital, Shenzhen University, Guangzhou, China. All rabbits were given free access to food and water. Construction of a rabbit model of acute hemorrhagic anemia Construction of the rabbit model of acute hemorrhagic anemia was based on the method used by Morimoto et al. [18]. After a 12-h fast, the rabbits were anesthetized by intramuscular injection of 0.2 ml/kg xylazine hydrochloride (Su-Mian-Xin, Veterinary Institute, Academy of Military Medicine Science, Changchun, China). Once the respiratory rate had stabilized at 12–18 breaths/min, the skin of the groin was incised along the groin fold. A catheter was then inserted into the femoral artery, for bloodletting and blood sampling. The blood samples obtained were used for whole blood tests and blood gas analysis (for determination of PaCO2, PaCO2, lactic acid, and pH); in addition, the mean arterial pressure (MAP) was measured. An additional catheter was inserted through the femoral vein to the atrium, for infusion of fluid and assessment of red blood cell count (RBC), hemoglobin concentration (HGB), hematocrit (HCT%), and central venous pressure (CVP). Blood sampling Before the first MRI scan was performed, blood samples were drawn for blood gas analysis and whole blood tests. After the first MRI scan had been carried out, a 40-mL blood sample was drawn through the arterial catheter. To compensate for the effects of simple blood volume loss, the same volume of a 6% hydroxyethyl starch in 0.9% sodium chloride solution was injected through the femoral vein catheter. Following this, a blood sample was drawn again for blood gas analysis and whole blood tests, and the head of the rabbit was re-scanned by MRI. The bloodletting, fluid infusion, and scanning processes were repeated continuously 5 times before the animal recovered from anesthesia; on the fifth occasion, the bloodletting volume and fluid infusion volume were both 50 mL. MRI scanning The rabbits were anesthetized, fixed to a special board in the supine position, and scanned by a Siemens Magnetom Avanto 1.5T MRI Scanner (Siemens), using a body coil (excitation) and wrap-around surface coil (reception). T2 dual-echo fast spin-echo with fat-suppression (FSE-T2WI/PD) and SWI 3D sequences were used. The scan extended downward from a plane passing through the superior orbital margin to the medulla oblongata of the rabbit. FSE-T2WI/PD acquisition was conducted using the following parameters: repetition time (TR)=2800 ms; echo time (TE)=33/78 ms; field of view (FOV)=12×12 cm; matrix size =256×256; and acquisition time =3.09 min. SWI acquisition was performed with a 3D gradient echo sequence, as follows: TR=49 ms; TE=40 ms; flip angle (FA)=15°; FOV=15×15 cm; bandwidth =80 KHz; and IPAT factor =2. SWI image processing Additional processing was carried out on the phase-corrected SWI sequences. The third ventricle and the olfactory bulb parallel to the corpus callosum were measured. The bilateral frontal cortex, frontal white matter, temporal lobe, and thalamus were selected manually as regions of interest (ROI). The signal intensity in each of these regions (area fixed at 0.08±0.01 cm2) was measured, and the average was calculated. The ROI was positioned so as to avoid blood vessels and the skull. Histology The rabbits were sacrificed immediately after the fifth MRI scan. The skin was incised and the skull opened. Brain and cerebellum were harvested, immediately fixed in 4% formalin, and embedded in paraffin. Sliced sections (3–5 μm) were stained with hematoxylin and eosin (HE) and examined under an Olympus BX41 microscope. Statistical analysis All measurement data are expressed as the mean ± standard deviation. Data analysis was performed using SPSS 21.0 software (IBM). The difference between the 2 groups was analyzed using Students t-test. For all analyses, a value of P<0.05 was considered to indicate statistical significance. In addition, a physician with five years of experience of MRI interpretation was invited to evaluate the cerebral white-gray contrast and vein structure of the SWI minimum intensity projection (MIP) images, without knowledge of the sequence in which the images were acquired. Results Blood test results Comparisons of the blood test results before and after bloodletting are shown in Figure 1. There was an approximate halving of the RBC, HGB and HCT% values after the first bloodletting, with further progressive decreases in the values of these parameters following each of the 4 subsequent bloodletting procedures (Figure 1A). The RBC, HGB, and HCT% values after the fifth bloodletting (4.72±0.43×1012/L, 98.20±10.22 g/L and 32.54±3.88%, respectively) were significantly lower than the corresponding control (pre-bleed) values (0.27±0.11×1012/L, 6.01±2.31 g/L and 1.97±1.02%, respectively). The bloodletting procedures were associated with substantial increases in lactic acid concentration as well as a small, but statistically significant, change in blood pH (Figure 1B). After the fifth bloodletting, the lactic acid concentration rose to 14.47±6.30 compared to 3.60±2.48 at pre-bleed. The pH value was decreased from 7.4±0.06 of pre-bleed to 7.25±0.01. PaO2 increased and PaCO2 decreased progressively with each bloodletting procedure (Figure 1C), such that following the fifth bloodletting, PaO2 was significantly elevated (150.40±9.78 vs. 80.53±19.24 mmHg) and PaCO2 was significantly reduced (22.00±7.35 vs. 42.65±4.13 mmHg) compared with the corresponding pre-bleed values. Bloodletting was not associated with any changes in CVP or MAP (Figure 1D). These results suggest the successful construction of acute hemorrhagic anemia in experimental rabbits. SWI signals and images The SWI signals of rabbit brains were acquired before and after bloodletting. Figure 2 shows representative SWI and corresponding T2-weighted images of the brain of a rabbit, obtained at the superior aspect of the olfactory bulb, the border of the olfactory bulb, the thalamus, and the cerebellum; also evident are examples of the ROIs chosen for analyses of the SWI images (see Materials and Methods). The mean SWI signal intensities (arbitrary units) of the frontal cortex, frontal white matter, temporal lobe, and thalamus, before and after bloodletting, are presented in Figure 3. The control (pre-bleed) SWI signal intensity of the frontal white matter was significantly lower than that of the frontal cortex (52.50±20.29 vs. 63.10±22.82; P<0.05). Bloodletting was not associated with any significant changes in the SWI signal of the frontal white matter. In contrast, there were significant (P<0.05) decreases in the SWI signals of the frontal cortex, temporal lobe, and thalamus after the second, third, fourth, and fifth bloodletting procedures, compared with the corresponding control (pre-bleed) values. Following the fifth bloodletting, the SWI signal intensities of the frontal cortex, temporal lobe, and thalamus were 37.70±4.32, 42.60±5.54, and 39.40±3.47, compared with corresponding control (pre-bleed) values of 63.10±22.82, 52.50±20.29, and 60.40±20.29, respectively (P<0.05). The value of the ROI only reflects the signal intensity of a localized region of the brain, and may be influenced by the volume effect. Therefore, we also evaluated the overall cerebral white-gray contrast and vein structure by inviting a physician with 5 years of experience to examine the SWI MIP images recorded before and after bloodletting (Figure 4). The physician was blinded to the sequence in which the images were taken. The interpretation of the physician was that, compared with the control (pre-bleed) images, the contrast between the cerebral gray and white matter was higher after bloodletting, particularly after the fourth and fifth bloodletting procedures, and that venous structure was more abundant and clearer after bloodletting. Histology Histological sections of brain tissues after bloodletting revealed that degeneration and necrosis of neurons and glial cells were not evident (Figure 5). However, spaces had formed around the blood vessels and cells, consistent with the occurrence of cerebral edema. Discussion Recent studies have demonstrated that the cerebral venous contrast of SWI images and the SWI signal intensity of brain tissues change when certain drugs (such as narcotics, caffeine, and contrast agents) are applied or certain pathophysiologic conditions are present [19,20]. In the present study, SWI was used to investigate the association between the SWI signal intensities of various regions of the rabbit brain and the partial pressures of blood oxygen and carbon dioxide in these regions, after acute hemorrhagic anemia. The rabbits in our study were anesthetized during MRI scanning. To remove the possible influence of the anesthetic on the partial pressures of oxygen and carbon dioxide in the blood, and thereby on the BOLD signal intensity, we compared the SWI signal intensities of different brain regions of the anesthetized rabbits before and after bloodletting. Our results showed that following the hemorrhage, the RBC count, hemoglobin, and hematocrit of the rabbits decreased, indicative of a state of hemorrhagic shock. After bloodletting, the rabbits were injected with hydroxyethyl starch in sodium chloride solution to maintain blood volume and venous return in the short term and hence improve organ perfusion, resulting in little or no change in the CVP and MAP values of the rabbits. This supplement of fluid is often used as emergent treatment of patients in hemorrhagic or extensive burn situations. However, although the hydroxyethyl starch/sodium chloride solution was able to increase blood volume, it did not replenish the red blood cells that carry oxygen, and hence it could not correct the problem of inadequate tissue oxygenation. As a result, the PaO2 increased significantly and the PaCO2 decreased significantly. The blood lactate levels initially increased, then decreased to some extent, and finally increased again. A possible explanation for this is that at the early stage of blood loss, a rapid decrease in blood volume led to tissue hypoperfusion and an increase in anaerobic metabolism; subsequently, the injection of hydroxyethyl starch/sodium chloride solution caused an increase in blood volume that partially replenished the blood oxygen, thereby reducing lactic acid levels; and finally, as more hydroxyethyl starch/sodium chloride solution was injected into the rabbits, the acidic substances deposited in the tissues were able to enter the bloodstream and cause an elevation of the lactic acid level. The BOLD signal is closely related to intravenous oxygen concentration and can indirectly reflect changes in cerebral blood flow (CBF), thereby allowing the monitoring of oxygen saturation. SWI calculates the oxygen saturation based on the differences in the magnetic sensitivities of oxyhemoglobin and deoxyhemoglobin [21]. The SWI images of venous structures depend on the T2* time, which may be shortened by deoxyhemoglobin-induced non-uniformity of the magnetic field, and phase differences between the surrounding tissue and blood vessels [22]; therefore, changes in CBF can also cause changes in the SWI signals [21]. The R2* parameter of BOLD may be affected by blood volume, the red blood cell volume ratio, oxygen consumption, and small arteries [23], as well as by other factors such as subject age [24]. Since SWI is an imaging technique with full flow compensation, it has a higher sensitivity than BOLD MRI when the magnetic field is uneven [25], and therefore can minimize the interference of small arteries in the measurement and can accurately detect deoxygenated hemoglobin. The results presented here demonstrate that after repeated bloodletting in rabbits, the SWI signal intensities of gray matter structures in the bilateral frontal cortex, temporal lobe, and thalamus were significantly decreased. However, the SWI signal intensity of the frontal white matter was not significantly affected by bloodletting. The reductions in the SWI signal intensities of the bilateral frontal cortex, temporal lobe, and thalamus after bloodletting may have been the result of decompensation induced by the repeated loss of blood. Bloodletting may have resulted in a fall in blood pressure, an elevation of heart rate, and hyperventilation, further increasing the emission of carbon dioxide and thus decreasing its partial pressure, as was evident from our observations. The resulting hypocapnia may have led to cerebral artery contractions and reductions in CBF. To meet the oxygen needs of the brain, the proportion of oxygen extracted from the blood must increase, thereby decreasing cerebral venous oxygen, elevating levels of intravenous deoxyhemoglobin, and reducing the SWI signal intensity. Previous studies have revealed that the overall metabolic rate, the number of capillaries in gray matter, and the flow rate of the cerebral cortex are 4 times that of the white matter [26]. Our study also demonstrated that before bloodletting the SWI signal intensity of the frontal cortex of rabbits was significantly higher than that of the frontal white matter, suggesting higher perfusion and more perfusion-induced oxyhemoglobin in the gray matter than in the white matter. Furthermore, the results presented here imply that the gray matter was more sensitive to changes in PaO2 and PaCO2 caused by acute hemorrhage, consistent with a previous report using arterial spin-labeling MR imaging [27]. In our study, blood loss led to a decrease in the SWI signal intensities of the gray matter structures of the cortex and thalamus, but little or no change in the white matter structures, suggesting that in the hypocapnic state associated with acute hemorrhage, the reductions in cerebral perfusion in the cortex and thalamic nuclei were greater than that in the white matter. In contrast to our findings, Rostrup et al. [27] reported that in conscious volunteers scanned using functional MRI, elevations in blood carbonic acid content were associated with increases in the BOLD signal intensity of only the cortical gray matter, with no rise detected in the gray matter nuclei and white matter. A possible explanation for this discrepancy is that the use of anesthetics in our study may have inhibited gray matter structures; in addition, SWI is more sensitive and accurate at measuring alterations in BOLD signal intensities, allowing detection of changes in the gray matter nuclei that may not have been detectable in the Rostrup et al. study. Moreover, we evaluated the cerebral white-gray contrast of the SWI images. The gray matter of the brain contains more veins than the white matter, which may explain our observation that the SWI signal of the white matter was not significantly decreased. During the hemorrhage, cerebral venous structures became clearer, and the cerebral white-gray contrast was markedly improved. Conclusions This study has revealed that SWI is an effective tool for detecting PaO2- and PaCO2-induced changes in the cerebral oxygenation of different brain regions after acute hemorrhagic anemia. Therefore, SWI may be a useful technique for monitoring the pathophysiological changes and related complications associated with acute anemia. Conflict of interests All authors declare that they have no conflict of interest. Source of support: This study was supported by the Science and Technology Project of Shenzhen (JCY20120613170958482), and the First Affiliated Hospital of Shenzhen University breeding program (2012015) Figure 1 The effects of repeated bloodletting on the results of whole blood tests, blood gas analyses, mean arterial pressure, and central venous pressure. (A) Repeated bloodletting was associated with progressive reductions in red blood cell count (RBC), hemoglobin concentration (HGB), and hematocrit (HCT%). (B) Repeated blood loss resulted in an increase in blood lactic acid concentration and a decrease in pH. (C) Bloodletting resulted in a progressive increase in the arterial partial pressure of oxygen (PaO2), and a progressive decrease in the arterial partial pressure of carbon dioxide (PaCO2). (D) Blood loss was not associated with changes in mean arterial pressure (MAP) or central venous pressure (CVP). Data are presented as mean ± standard deviation, * P<0.05. Figure 2 Representative SWI (panels 1a–4a) and corresponding T2-weighted (1b–4b) images of the brain of a rabbit, obtained at the superior aspect of the olfactory bulb (1a, 1b), the border of the olfactory bulb (2a, 2b), the thalamus (3a, 3b), and the cerebellum (4a, 4b). In the SWI images, ROIs are indicated by numbers as follows: 1a (squares 1 and 2): frontal lobe cortex (left and right sides); 2a (squares 1 and 2): frontal lobe brain parenchyma (left and right sides); 3a (squares 1 and 2): thalamus (left and right sides); and 4a (squares 1 and 2): temporal lobe cortex (left and right sides). For all images, the anterior aspect of the brain is toward the top of the image, and the left side of the brain is toward the right, as indicated in panel 1a. Figure 3 The effects of repeated bloodletting on the SWI signal intensities of selected regions of the brain. Repeated bloodletting was associated with reductions in the mean SWI signal intensities of the frontal cortex, temporal lobe, and thalamus, but not of the frontal white matter. Data are shown as means ± standard deviations. Compared with the control (pre-bleed) value, significant (P<0.05) decreases in SWI signal intensity were observed for the frontal cortex, temporal lobe, and thalamus following the second, third, fourth, and fifth bloodlettings; the values after the first bloodletting were not significantly different from the control (pre-bleed) values. For the frontal white matter, none of the values after bloodletting were significantly different from the control (pre-bleed) value. Figure 4 Images of the thalamus and cerebral veins obtained by magnetically sensitive scanning. Images of the thalamus are shown before bloodletting (A), after the first bloodletting (B), and after the fifth bloodletting (C). The cerebral white-gray contrast of the gray matter becomes clearer with repeated blood loss. Images of the central veins are shown before bloodletting (D), after the first bloodletting (E), and after the fifth bloodletting (F). The vein structure (arrows) also becomes clearer with repeated blood loss. Figure 5 Histological section of the brain after bloodletting (HE, ×100). Spaces (arrows) had formed around the blood vessels and cells, indicative of the development of cerebral edema. ==== Refs References 1 Raphaeli T Menon R Current treatment of lower gastrointestinal hemorrhage Clin Colon Rectal Surg 2012 25 219 27 24294124 2 Jin S Fu Q Wuyun G Wuyun T Management of post-hepatectomy complications World J Gastroenterol 2013 19 7983 91 24307791 3 Hurt K Bilton D Haemoptysis: diagnosis and treatment Acute Med 2012 11 39 45 22423349 4 Purkayastha S Sorond F Transcranial Doppler ultrasound: technique and application Semin Neurol 2012 32 411 20 23361485 5 Allmendinger AM Tang ER Lui YW Spektor V Imaging of stroke: Part 1, Perfusion CT – overview of imaging technique, interpretation pearls, and common pitfalls Am J Roentgenol 2012 198 52 62 22194479 6 Yamazaki K Suzuki K Itoh H Cerebral oxygen saturation evaluated by near-infrared time-resolved spectroscopy (TRS) in pregnant women during caesarean section – a promising new method of maternal monitoring Clin Physiol Funct Imaging 2013 33 109 16 23383688 7 Navarro LH Lima RM Khan M Continuous measurement of cerebral oxygen saturation (rSO(2)) for assessment of cardiovascular status during hemorrhagic shock in a swine model J Trauma Acute Care Surg 2012 73 S140 46 22847085 8 Murkin JM Arango M Near-infrared spectroscopy as an index of brain and tissue oxygenation Br J Anaesth 2009 103 Suppl 1 i3 13 20007987 9 Huang P Chen CH Lin WC Clinical applications of susceptibility weighted imaging in patients with major stroke J Neurol 2012 259 1426 32 22186853 10 Niwa T de Vries LS Benders MJ Punctate white matter lesions in infants: new insights using susceptibility-weighted imaging Neuroradiology 2011 53 669 79 21553013 11 Lee YJ Shon YM Yoo WJ Diminished Visibility of Cerebral Venous Vasculature in Subclinical Status Epilepticus by Susceptibility-Weighted Imaging: A Case Report Clin Neuroradiol 2014 24 1 69 72 23392581 12 Roelcke U Boxheimer L Fathi AR Cortical hemosiderin is associated with seizures in patients with newly diagnosed malignant brain tumors J Neurooncol 2013 115 463 68 24045969 13 Boeckh-Behrens T Lutz J Lummel N Susceptibility-weighted angiography (SWAN) of cerebral veins and arteries compared to TOF-MRA Eur J Radiol 2012 81 1238 45 21466929 14 Santhosh K Kesavadas C Thomas B Susceptibility weighted imaging: a new tool in magnetic resonance imaging of stroke Clin Radiol 2009 64 74 83 19070701 15 Goos JD van der Flier WM Knol DL Clinical relevance of improved microbleed detection by susceptibility-weighted magnetic resonance imaging Stroke 2011 42 1894 900 21566235 16 Cheng AL Batool S McCreary CR Susceptibility-weighted imaging is more reliable than T2-weighted gradient-recalled echo MRI for detecting microbleeds Stroke 2013 44 2782 86 23920014 17 Li M Hu J Miao Y In vivo measurement of oxygenation changes after stroke using susceptibility weighted imaging filtered phase data PLoS One 2013 8 e63013 23675450 18 Morimoto Y Mathru M Martinez-Tica JF Zornow MH Effects of profound anemia on brain tissue oxygen tension, carbon dioxide tension, and pH in rabbits J Neurosurg Anesthesiol 2001 13 33 39 11145476 19 Rauscher A Sedlacik J Barth M Nonnvasive assessment of vascular architecture and function during modulated blood oxygenation using susceptibility weighted magnetic resonance imaging Magn Reson Med 2005 54 87 95 15968657 20 Sedlacik J Helm K Rauscher A Investigations on the effect of caffeine on cerebral venous vessel contrast by using susceptibility-weighted imaging (SWI) at 1.5, 3 and 7 T Neuroimage 2008 40 11 18 18226553 21 Shen Y Kou Z Kreipke CW In vivo measurement of tissue damage, oxygen saturation changes and blood flow changes after experimental traumatic brain injury in rats using susceptibility weighted imaging Magn Reson Imaging 2007 25 219 27 17275617 22 Haacke EM Xu Y Cheng YC Reichenbach JR Susceptibility weighted imaging (SWI) Magn Reson Med 2004 52 612 18 15334582 23 Fan Z Elzibak A Boylan C Noseworthy MD Blood oxygen level-dependent magnetic resonance imaging of the human liver: preliminary results J Comput Assist Tomogr 2010 34 523 31 20657219 24 Haacke EM Miao Y Liu M Correlation of putative iron content as represented by changes in R2 and phase with age in deep gray matter of healthy adults J Magn Reson Imaging 2010 32 561 76 20815053 25 Guyton AC Hall JE Cerebral blood flown cerebrospinal fluid and brain metabolism Philadelphia Elsevier Saunders 2006 26 Floyd TF Clark JM Gelfand R Independent cerebral vasoconstrictive effects of hyperoxia and accompanying arterial hypocapnia at 1 ATA J Appl Physiol (1985) 2003 95 2453 61 12937024 27 Rostrup E Larsson HB Toft PB Functional MRI of CO2 induced increase in cerebral perfusion NMR Biomed 1994 7 29 34 8068522	25060330	PMC4116343	NO-CC CODE	2021-01-05 00:49:52	yes	Med Sci Monit. 2014 Jul 25; 20:1291-1297
==== Front Ann NeurosciAnn NeurosciANSAnnals of Neurosciences0972-75310976-3260Indian Academy of Neurosciences 252058881017030610.5214/ans.0972-7531.1017306Research ArticleTubercular MeningitisDiagnostic efficacy of adenosine deaminase levels in cerebrospinal fluid in patients of tubercular meningitis: A comparison with PCR for Mycobacterium Tuberculosis Chacko Fiju 1Modi Manish 1Lal Vivek 1Prabhakar S DM1Rana S.V. 2Arora S.K. 31 Departments of Neurology2 Gastroenterology3 lmmunopathology Post Graduate Institute of Medical Education and Research, Chandigarh, INDIA. Corresponding Author Department of NeurologyPostgraduate Institute of Medical Education and Research, Chandigarh-160012, [email protected] 2010 17 3 126 130 26 11 2009 13 1 2010 25 2 2010 Copyright © 2010, Annals of Neurosciences2010Background The rapid diagnosis of Tubercular meningitis (TBM) is fundamental to clinical outcome. The key to diagnosis lies in Cerebrospinal fluid (CSF) analysis and radiological investigations. There are numerous lacunae in the confirmation of diagnosis of TBM from CSF. Purpose The aim of present study was to compare the efficacy of CSF adenosine deaminase (ADA) level assays and Polymerase chain reaction (PCR) for Mycobacterium tuberculosis (M. tuberculosis) in the diagnosis of TBM. Methods Fifty four adult patients with suspected TBM and 37 controls were included in the study and CSF analyzed for ADA and PCR for M. tuberculosis. The cases were subdivided into definite (5), highly probable (22), probable (22) and possible TBM (5) as per previously validated criteria. The first two were grouped as "most likely" TBM (27) and last two as "unconfirmed" TBM (27). Results The mean ADA of the "most likely" TBM was 29±24, "unconfirmed" TBM was 21 ± 15 and controls were 4.8±2.2 U/L. The ADA levels correlated with CSF proteins, absolute lymphocyte count and the staging of the disease. Using a cut off level of >L10 U/L, CSF ADA had a sensitivity of 92.5% and specificity of 97%. PCR for M. tuberculosis was positive in 12 out of 27 "most likely" TBM cases, 5 out of 27 "unconfirmed" TBM cases and 3 out of 37 controls. PCR for M. tuberculosis had a sensitivity of 44.5% and specificity of 92% in the "most likely" TBM cases. Conclusions: ADA is a rapid, inexpensive and sensitive test in the diagnosis of TBM. It is more sensitive than AFB smear and culture. PCR is another rapid test in the diagnosis of TBM with a good specificity, even in those patients already on presumptive anti-tuberculous treatment. However, despite the sensitivity and specificity of CSF ADA, it should be corroborated with AFB smear and CSF PCR. Adenosine deaminasePCRTubercular meningitis ==== Body Introduction TBM is the most dangerous form of extra pulmonary TB and occurs in about 7-12% of all TB patients in developing countries. Despite availability of effective chemotherapy, mortality and morbidity remain unacceptably high. Delay in the diagnosis and institution of proper treatment is directly related to poor outcome and sequelae, which are severe in 20-25% of cases.1 Early diagnosis by demonstration of AFB in CSF smear is not consistently positive, since there are only a few organisms in the CSF sample. The delay of 6-8 weeks to obtain culture results and 10-14 days with rapid culture is critical in planning treatment.2 In smear negative cases with high clinical suspicion of TBM, other rapid diagnostic methods need to be relied upon, or treatment has to be started on the basis of presumptive diagnosis. ADA, a polymorphic enzyme involved in purine metabolism is found to be elevated in the CSF of TBM patients and gradually returns to normal values after 2-6 weeks of specific treatment. The estimation is easy, fast, inexpensive and can be done in ordinary laboratories. 3 PCR is a powerful tool for rapid diagnosis of infectious diseases. Its advantage lies in the fact that the starting DNA is amplified several times, leading to a high sensitivity. The decrease in specificity due to contamination is a cause for concern. Several authors concluded that PCR cannot be used to start or stop ATT in suspected cases of TBM. 4,5 With improvement in methods to reduce contamination and by increasing the sample volume, both specificity and sensitivity were found to be increased.6 The present study compares CSF ADA levels and PCR as rapid methods in diagnosis of suspected TBM. Methods Informed consent was obtained as per Institute Ethical Committee before study. Fifty four adult patients with suspected TBM and thirty seven controls were included in the study and the CSFwasanalysedforADAand PCRforM. tuberculosis. The cases were evaluated in detail and subdivided into definite, highly probable, probable and possible TBM cases as per in the clinical criteria (Table 1).7 ATT and steroids were started in all cases and response evaluated. Controls were taken from patients in Neurology ward in whom TB was not suspected and CSF was analysed for diagnostic purposes like Acute Inflammatory Demyelinating Polyradiculoneuropathy, Multiple Sclerosis, Polyneuropathy, Idiopathic Intracranial Hypertension, etc. ADA estimation in CSF was done using the method of Guisti.8 PCR was done on CSF placed in microcentrifuge tube (in aliquots) and centrifuged for 10 minutes at 10,000 rpm. To the pellet formed after centrifugation (which contains cells) 20jul of 0.1 %Triton-x-100 is added and incubated for 15 minutes. Primers as used by Scarpellini et al9 were used for amplication of a conserved repetitive insertional sequence IS 6110. 35 cycles of PCR were carried out (denaturation at 95°C, annealing at 56°C, and extension at 72°C for 1 minutes each). PCR product was analysed by gel electrophoresis. Genomic DNA of M. tuberculosis was used as a positive control. Statistical analysis was done using Pearsons co-efficient of correlation, chi-square test and student 't' tests for comparison, with p value <0.05 considered as significant. Table 1: Criteria for Diagnosis of TBM 7 I. Clinical symptoms and signs Mandatory: Fever and headache >2 weeks Optional: Vomiting, neck stiffness, altered sensorium, seizures or focal Neurologic deficit II. Supporting critera 1. CSF: Cells : >20/cmm, lymphocytes : >60% proteins : 100 mg%, sugar: <60% of corresponding blood sugar, negative gram stain, India stain and VDRL where relevant 2. CECT/MRI Showing one or more of : exudates in basal cisterns / Sylvian fissures, gyral enhancement, hydrocephalus, Infarcts, tuberculoma 3. Active extraneural TB : as evidenced by appropriate mycobacterial tests, radiology, histopathological examination 4. Clinical response to ATT and relief of symptoms The cases were classified as being: •Highly probable TBM (met clinical criteria and 3 of the 4 supporting criteria) •Probable TBM (met clinical criteria and 2 of the 4 supporting criteria) •Possible TBM (met clinical criteria and 1 out of 4 supporting criteria) •Confirmed TBM (AFB stain or/and culture of CSF was positive) Results All the patients had fever for a mean duration of 52.3±73.1 days (median 30). Headache was present in all patients and 49 patients (91%) had vomiting. Forty seven patients (87%) had alteration in sensorium of a mean duration of 6±4 days. Seizures were present in 18 patients (33%) with focal seizures in five. Signs of meningeal irritation were present in 50 patients (93%). Papilledema was seen in 17 patients (31 %) and abducens nerve palsy was observed in 17 patients (31%) of which it was bilateral in eight. Thirty one patients had ventriculomegaly and 11 of them required ventriculoperitoneal shunt insertion. Eighteen patients had other cranial nerves palsies (oculomotor 17%, facial 13%, vagus 4%, optic 2%, vestibulocochlear 2%, glossopharyngeal 2%). Eight patients had multiple cranial nerve palsies. Five patients had hemiparesis. Chest X-ray abnormalities were seen in 15 patients (28%) (military mottling 8%, cavities 8%, hilar nodes 6%, pleural effusion 6%). CECT head showed basal exudates in 41%, hydrocephalus in 39%, infarcts in 13% and tuberculoma in 13% of all cases studied. No abnormality was detected in cranial imaging of 7 patients (13%). Twelve patients had received ATT prior to admission to hospital and five of them had received the treatment for more than one week. All patients were started on ATT. Five patients developed drug induced hepatitis and were managed accordingly. Four patients expired while in the hospital. The CSF cell count ranged from 20-1320 cells per cu.mm with a mean of 275.5±357 cells (median 145). The absolute lymphocyte count was 153±178 (median 102). The mean protein level was 297±350 mg% (median 150) and the mean CSF sugar level was 37.3±17.1 mg% (median 38.5). The ratio of CSF glucose to blood sugar was 0.33±0.14 (median 0.32). AFB smear was positive in only one patient and AFB culture was positive in 5 patients (9%). The mean CSF ADA level was 26.1 ± 18.8 μ/L (median 19). The major CSF findings of the TBM groups are shown in Table 2. CSF ADA showed a positive correlation with absolute lymphocyte count and V value was 0.15 using Pearson's coefficient of correlation. However, this was not significant on the students 't' test. CSF ADA showed a stronger positive correlation with CSF protein ('r' value 0.55 using Pearson's formula). This was statistically significant (p <0.01). With increasing severity of disease (as per the Medical Research Council Staging), the CSF ADA showed a increasing trend, but it was not statistically significant. There was no significant correlation with the duration of disease. The ADA level tended to decrease within one week of ATT. The ADA levels decreased further with ATT of more than one week and the difference was statistically significant p<0.02). Overall, PCR positivity was seen in 17 cases of TBM, it being positive in 4 out of 5 cases of definite TBM (80%), 8 out of 22 cases of highly probable TBM (39%), 5 out of 22 cases of probable TBM (17%) and none of the 5 cases of possible TBM. Out of the 37 patients in the control group, 10 patients had demyelinating illness, 5 each had polyneuropathy and idiopathic intracranial hypertension, 3 had acute inflammatory demyelinating polyradiculoneuropathy, 2 had subacute sclerosing Panencephalitis and 12 had other neurologic disorders. The major CSF findings of the control groups are shown in Table 3. The comparison between CSF ADA levels of TBM group and controls was statistically significant (p <0.001). The comparison of PCR in the TBM cases with control group was statistically significant (p <0.01). The difference between highly probable TBM and unconfirmed TBM cases was also statistically significant (p <0.05). Table 2: CSF findings in the various subgroups of TBM All cases of TBM (n=54) Definite (Culture +ve) (n=5) Highly probable (n=22) Probable (n=22) Possible (n=5) Total cells (per cumm) Mean±SD (Median) 276±357 (145) 338±401 (300) 290±384 (165) 260±355 (145) 135±177 (60) Absolute lymphocyte count (per cumm) Mean ± SD (Median) 153±178 (102) 135±147 (100) 198±226 (140) 116±123 (82) 98±123 (45) Protein (mg%) Mean±SD (Median) 297±350 (150) 163±81 (125) 465±452 (300) 214±241 (92) 89 ±34 (800) Ratio of CSF sugar to blood sugar Mean±SD (Median) 0.33±0.14 (0.32) 0.32±0.12 (0.28) 0.29±0.12 (0.28) 0.35±0.14 (0.33) 0.48±0.18 (0.48) CSF ADA levels (U/L) Mean±SD (Median) 26±19 (19) 23.2±8.6 (19) 30.3±24.8 (21) 24.0±15.9 (19) 14.0±4.5 (14) PCR positivity 17 (32%) 4 (80%) 8 (36%) 5 (23%) 0 (0%) Table 3: Control CSF samples (n=37) showing the CSF findings All control (n=37) Demyelination (n=10) Idiopathic Intracranial Hypertension (n=5) Polyneuropathy (n=5) Acute Inflammatory Demyelinating Polyneuropathy (n= 3) SSPE (n=2) Others (n= 12) Total cells (per cumm) Mean±SD (Median) 4.6±26.3 (br/>(0) - - - - - 14±46 (0) Absolute lymphocyte count(per cumm) Mean ± SD (Median) 3.7±3.5 (0) - - - - - 11.5±36.8 (0) Protein (mg%) Mean±SD (Median) 46.5±29.5 (40) 37±5 (38) 24±8 (25) 74±50 (25) 52±11 (56) 40±14 (40) 52±34 (40) Ratio of CSF sugar to blood sugar Mean±SD (Median) 0.62±0.14 (0.62) 0.66±0.14 (0.66) 0.60±0.09 (0.63) 0.6±0.14 (0.66) 0.6±0.14 (0.60) 0.67±0.08 (0.67) 0.58±0.18 (0.60) CSF ADA levels (U/L) Mean±SD (Median) 4.8±2.2 (5) 4.0±1.6 (3) 4.4±1.3 (5.0) 5.0±1.9 (6) 5.3±0.6 (5) 5.0±0 (5.0) 5.4±3.3 (5.0) PCR (false positivity) 3/37 (8%) - - 1/5 (20%) - - - - 2-12 (17%) The sensitivity and specificity of PCR was 37% and 92% respectively in the diagnosis of TBM. The sensitivity was 44.5% for the most likely TBM cases. The positive and negative predictive values were 85% and 48% respectively. Discussion Early treatment of TBM is essential to prevent both morbidity and mortality. Rapid diagnostic tests with good sensitivity and specificity are required to aid the presumptive diagnosis, as AFB staining is not sensitive enough to help the clinician in ruling out the possibility of TBM. The CSF smear was positive for AFB in only one patient in the study group. Since the sensitivity is very low, it cannot be relied upon to pick up cases of TBM. Meticulous examination of smear from large volume CSF samples and serial taps is reported to yield better results.6 The CSF culture grew AFB in 5 patients (9%) in this study. This is in comparison with the previous studies by Shankar et al(12%)10, Miorner et al(17%)11 and Nguyen etal(19%)12. Even though this is the gold standard, the delay of 6-8 weeks to obtain a positive result coupled with the low sensitivity makes this test less useful to the clinician in the diagnosis of TBM. The mean ADA in TBM group was 26.1 ±18.8 U/L which is comparable to that in adults with TBM observed by Malen et al(26.2 U/L).3 The CSF ADA levels correlated with absolute lymphocyte counts and CSF proteins (more with the latter), as reported in previous studies.3,13 ADA levels tend to decrease with therapy significantly if treatment duration was beyond one week (p<0.02). A cut off value of >.10 U/L gives a good sensitivity and specificity. The overall results are in comparison with the previous studies as shown in Table 4. However, elevated levels of CSF ADA are not specific for TBM. Diseases like pyogenic meningitis, CNS lymphoma, and fungal meningitis were shown to have elevated CSF ADA.3 In the most probable TBM group, the sensitivity of PCR was 44.5% and specificity was 92%. The sensitivity of PCR is low when compared with most of the previous studies (Table 5). In the present study, both TBM and control samples were marked as suspected TBM and thus were blinded for processing of PCR. Previous studies tested a smaller number of patients and used a selected patient group7 or had a higher number of false positives in PCR.10 Out of the 5 culture positive cases, one was PCR negative. This discrepancy can be explained by the differences in the volumes of sample tested. It would thus be advisable to send a large volume of CSF for PCR analysis. False negative reactions can also be caused by inhibitors of Taq polymerase,4 when the CSF is contaminated with blood. Contamination of CSF sample with blood may not always be detected by the naked eye. A better DNA extraction procedure that could take care of the inhibitory proteins in the CSF may be helpful in increasing the sensitivity of PCR. Table 4: Comparison of Sensitivity and Specificity of CSF ADA in the diagnosis of TBM in the present study with previous studies Sensitivity (%) Specificity (%) Year (Ref) 90 NA Malan et al, 1984 73 71 Coovadia et al, 1986 99 100 Ribera et al, 1987 50 90 Kaur et al, 1992 44 75 Gambhir et al, 1999 87 86 Pushpa et al, 2000 92.5 97 Present Study Table 5: Comparison of Sensitivity and Specificity of PCR in diagnosis of TBM In the present study with the previous studies Sensitivity (%) Specificity (%) Study Year (Ref) 83 100 Kaneko et al, 1990 65 89 Shankar et al, 1991 76 100 Lin et al, 1995 48 100 Kox et al, 1995 54 94 Miorner et al, 1995 82 100 Nguyen et al, 1996 60 100 Bonington et al, 1998 44.5 92 Present Study Prior treatment with ATT decreased the sensitivity of PCR in this study. Still, there were 29% PCR positive cases, in those treated for a week and one case was positive even after 3 months of ATT. Lin etal concluded that it is still valuable to apply PCR in clinically suspected TBM who have already received a therapeutic trial of ATT even upto 3 weeks.19 In a study by Kaneko etal, a CSF sample was positive for TB even after one month of ATT.18 The persistent presence of M. tuberculosis DNA in CSF might indicate that ATT induces release of mycobacterial DNA in CSF. This is especially important in tertiary care centers, where many cases at presentation are already on presumptive ATT which further decreases the chances of obtaining an AFB stain or culture. It can be suggested that PCR is still useful in those who have already received a trial ATT, even upto 3 months. Some studies have already shown that a nested PCR protocol could also improve the sensitivity of detecting mycobacterial DNA in clinical samples.22 Conventional method using a one step amplification of DNA is associated with a low sensitivity. Two step nested amplification of DNA enhances the sensitivity by several folds.22 Even though nested amplification protocol could not be used in our study due to technical reasons, this can be recommended for further studies as it has been shown to increase the sensitivity. In a recent study from Vietnam, it was shown that the number of copies of insertional sequence IS6110 in 168 strains of M. tuberculosis varied from 0 to 23. Three strains from North Vietnam lacked IS6110 strains and other had 3 to 14 copies of IS6110 while Southern strains had 15-23 copies of IS6110. If the strain does not have IS6110, then PCR cannot give a positive result even when there is bacterial DNA in the CSF. Whether similar variations in insertional sequence exist in our patients require further studies and confirmation in each areas.23 In the present study, CSF samples from three controls were positive by PCR. Since these patients had no cells in the CSF, silent tubercular lesions in the meninges are unlikely in the absence of symptoms. These positive results may be attributed to possible cross contamination of samples during initial handling, as has been reported in the literature.12,18,21 Conclusion ADA is a rapid, inexpensive and sensitive test in the diagnosis of TBM. It is sensitive than AFB smear and culture and can be suggestive of the diagnosis of TBM. PCR is a rapid test in the diagnosis of TBM with a good specificity. Even in those patients already on presumptive ATT upto 3 months, PCR can be helpful. By doing PCR with a larger volume of uncontaminated CSF, better DNA extraction techniques and a nested PCR protocol, the sensitivity can be increased. CSF ADA level • 10 μ/L is sensitive and can suggest the diagnosis of TBM, especially if the clinical suspicion is high. Other techniques like AFB smear, PCR for M.tuberculosis and rapid culture would be required for confirmation of the diagnosis. Competing interests - None, Source of Funding - None ==== Refs References 1 Prabhakar S Thussu A CNS Tuberculosis. Neurology India 1997 45 132 40 2 Venkataraman P Herbert D Paramasivan CN Evaluation of BACTEC radiometric method in early diagnosis of TB. Indian J Med Res 1998 108 120 27 9805840 3 Malan C Donald PR Golden M ADA levels in CSF in the diagnosis of TBM. Journal of Tropical Medicine and Hygiene 1984 87 33 40 6716540 4 Garcia JE Losada JP Gonzalez Villaron L Reliability of polymerase chain reaction in the diagnosis of mycobacterial infection. Chest 1996 110 300 301 8681652 5 Doucet-Populaire F Lalande V Carpentier E A blind study of PCR for detection of M. tuberculosis DNA. Tuber Lung Dis 1996 77 4 358 62 8796253 6 Ma TS Reliability of PCR in the diagnosis of M. tuberculosis. Chest 1996 110 301 302 7 Ahuja GK Mohan KK Behari M Diagnostic criteria for TBM and their validation. Tuber Lung Dis 1994 75 2 149 152 8032049 8 Guisti G Adenosine de aminase. In: Methods of Enzymatic Analysis Bergmeyer HU (ed) New York Acad Press 1974 1092 96 9 Scarpellini P Racca S Clinque P Nested PCR for diagnosis and monitoring treatment response in AIDS patients with TBM. AIDS 1995 9 895 900 7576324 10 Shankar P Manjunath N Mohan KK Rapid diagnosis of TBM by PCR. Lancet 1991 5 337 1670668 11 Miorner H Sjobring U Nayak P Diagnosis of TBM. A comparative analysis of 3 immunoassays, an immune complex assay and the PCR. Tubercle and Lung Dis 1995 76 381 12 Nguyen LN Kox LF Pham LD The potential contribution of polymerase chain reaction to the diagnosis of tubercular meningitis. Arch Neurol 1996 53 771 76 8759984 13 Gambhir IS Mehta M Singh DS Evaluation of CSF adenosine deaminase activity in tubercular meningitis. J Assoc Physicians India 1999 47 192 94 10999088 14 Coovadia YM Dawood A Ellis ME Evaluation of ADA activity and antibodies to M. tuberculosis antigen 5 in CSF and the radioactive bromide partition test for the early diagnosis of TBM. Arch Dis Child 1986 61 428 35 3087296 15 Ribera E Jose M Martinez V Activity of ADA in CSF for the diagnosis and follow up of TBM in adults. J Infec Dis 1987 155 4 603 07 3102627 16 Kaur A Basha A Ranjan M Poor diagnostic value of ADAin pleural, peritoneal and cerebrospinal fluids in TB. Indian J Med Res 1992 95 270 77 1291460 17 Pushpa C Jaishree V Harinath BC ADA levels in CSF and serum in the diagnosis of TBM. J Trap Ped 2000 46 378 79 18 Kaneko K Onodera O Miyatake T Rapid diagnosis of TBM by PCR. Neurology 1990 40 1617 18 2120615 19 Lin JJ Hain HJ Application of PCR to monitor M. tuberculosis DNA in the CSF of patients with TBM after antibiotic treatment. J Neurol Neurosurg Psychiat 1995 59 175 77 7629533 20 Kox LFF Der SK Kollis AHJ Early diagnosis of TBM by PCR. Neurology 1995 45 2228 32 8848198 21 Bonington A Strang Jl Klapper PE TB PCR in the early diagnosis of TBM. Tubercle and Lung Dis 2000 80 191 96 22 Miyazaki Y Koga H Kohno S Nested PCR for detection of M. tuberculosis in clinical samples. J Clin Microbiol 1993 31 2228 32 8370757 23 Tuyet LT Hoa BK Ly HM Molecular finger printing of M. tuberculosis strains isolated in Vietnam using IS 6110 as probe. Tuber Lung Dis 2000 80 75 83 10912282	25205888	PMC4116978	NO-CC CODE	2021-01-05 02:02:22	yes	Ann Neurosci. 2010 Jul; 17(3):126-130
==== Front 04104626011NatureNatureNature0028-08361476-46872504305510.1038/nature13375nihpa584334ArticlemiR-34a Blocks Osteoporosis and Bone Metastasis by Inhibiting Osteoclastogenesis and Tgif2 Krzeszinskia Jing Y. 1Wei Wei 1Huynh HoangDinh 1Jin Zixue 1Wang Xunde 1Chang Tsung-Cheng 2Xie Xian-Jin 34He Lin 5Mangala Lingegowda S. 67Lopez-Berestein Gabriel 78Sood Anil K. 679Mendell Joshua T. 23Wan Yihong 131 Department of Pharmacology, The University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA2 Department of Molecular Biology, The University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA3 Simmons Cancer Center, The University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA4 Department of Clinical Sciences, The University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA5 Division of Cellular and Developmental Biology, Molecular and Cell Biology Department, University of California at Berkeley, Berkeley, California 94705, USA6 Department of Gynecologic Oncology and Reproductive Medicine, The University of Texas MD Anderson Cancer Center, Houston, Texas 77030, USA7 Center for RNA Interference and Non-coding RNA, The University of Texas MD Anderson Cancer Center, Houston, Texas 77030, USA8 Department of Experimental Therapeutics, The University of Texas MD Anderson Cancer Center, Houston, Texas 77030, USA9 Department of Cancer Biology, The University of Texas MD Anderson Cancer Center, Houston, Texas 77030, USACorresponding author: Yihong Wan ([email protected])5 6 2014 25 6 2014 28 8 2014 28 2 2015 512 7515 431 435 Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:http://www.nature.com/authors/editorial_policies/license.html#termsThe bone resorbing osteoclasts significantly contribute to osteoporosis and cancer bone metastases1-3. MicroRNAs (miRNAs) play important roles in physiology and disease4,5, and present tremendous therapeutic potential6. Nonetheless, how miRNAs regulate skeletal biology is underexplored. Here we identify miR-34a as a novel and critical suppressor of osteoclastogenesis, bone resorption and the bone metastatic niche. miR-34a is down-regulated during osteoclast differentiation. Osteoclastic miR-34a over-expressing transgenic mice exhibit lower bone resorption and higher bone mass. Conversely, miR-34a knockout and heterozygous mice exhibit elevated bone resorption and reduced bone mass. Consequently, ovariectomy-induced osteoporosis, as well as bone metastasis of breast and skin cancers, are diminished in osteoclastic miR-34a transgenic mice, and can be effectively attenuated by miR-34a nanoparticle treatment. Mechanistically, we identify Tgif2 (transforming growth factor-beta-induced factor 2) as an essential direct miR-34a target that is pro-osteoclastogenic. Tgif2 deletion reduces bone resorption and abolishes miR-34a regulation. Together, using mouse genetic, pharmacological and disease models, we reveal miR-34a as a key osteoclast suppressor and a potential therapeutic strategy to confer skeletal protection and ameliorate bone metastasis of cancers. ==== Body We examined the levels of several cancer-related miRNAs during a time course of bone marrow osteoclastogenesis assay (Fig. 1a). While the expression of an osteoclast marker tartrate-resistant acid phosphatase (TRAP) was rapidly increased by RANKL and further elevated by rosiglitazone7,8 (Fig. 1b), miR-34a was rapidly down-regulated by RANKL and further diminished by rosiglitazone (Fig. 1c). The levels of miR-34b/c, two other members in the miR-34 family, were unaffected and expressed at much lower levels than miR-34a (Fig. 1d). The sequence of miR-34a is evolutionally conserved and identical in mice and humans. Osteoclast differentiation from both mouse bone marrow precursors (Fig. 1e-f) and human peripheral blood mononuclear cells (hPBMN) (Fig. 1g-j) was inhibited by a miR-34a precursor (pre-miR-34a) but enhanced by an antisense miR-34a inhibitor (anti-miR-34a), indicating that miR-34a regulation of bone resorption in mice will likely translate to human pathophysiology. We generated osteoclastic miR-34a transgenic mice using CAG34a mice (Fig. 2a) and Tie2-cre mice7,8. FACS and imaging showed that osteoclast progenitors from the 34a-Tie2-Tg (CAG34a+Cre+) mice were converted to GFP+LacZ- whereas the controls (CAG34a+Cre-) remained GFP-LacZ+ (Extended Data Fig. 1a-b). Northern blot confirmed the over-expression of mature-miR-34a in the bone marrow of 34a-Tie2-Tg mice (Extended Data Fig. 1c). Osteoclast differentiation assay reveal that the higher levels of mature miR-34a in the 34a-Tie2-Tg cultures resulted in a lower induction of osteoclast markers, diminished number/size of mature osteoclasts, and reduced resorptive activity, whereas precursor proliferation or survival was unaltered (Fig. 2b, Extended Data Fig. 1d-g). Consequently, serum bone resorption marker CTX-1 (C-terminal telopeptides of Type I collagen) and osteoclast number were decreased, whereas osteoblast number, bone formation rate (BFR) and mineral apposition rate (MAR) were unaltered (Fig. 2c, Extended Data Fig. 1h-i). μCT analysis of the proximal tibiae showed that 34a-Tie2-Tg mice had increased bone mass and decreased structure model index (SMI), which quantifies the relative amount of plates (SMI=0, strong) and rods (SMI=3, fragile) (Fig. 2d-e). Cortical BV/TV was also higher (Fig. 2f). Moreover, miR-34a transgenic mice generated by three other osteoclast-targeting cre drivers also exhibited a similar phenotype (Extended Data Fig. 2, 3). Thus, miR-34a in the osteoclast lineage augment bone mass by suppressing osteoclastogenesis and bone resorption. To determine whether miR-34a is a physiologically relevant regulator of bone resorption, we next examined miR-34a knockout (34a-KO) and heterozygous (34a-Het) mice (Fig. 2g). Northern blot confirmed the diminished levels of miR-34a in 34a-KO (Extended Data Fig. 4a). Consistent with prior reports9,10, miR-34a deletion had no overt effect on mouse development. Osteoclast differentiation was augmented in 34a-Het and 34a-KO cultures, while precursor proliferation or survival was unaffected (Fig. 2h, Extended Data Fig. 4b-e). As a result, serum CTX-1 and osteoclast number were elevated (Fig. 2i, Extended Data Fig. 4g-h). μCT revealed that 34a-KO and 34a-Het mice exhibited a low-bone-mass with decreased connectivity density (Conn.D) and increased SMI (Fig. 2j-l). Global miR-34a deletion also decreased bone formation as the serum marker P1NP (N-terminal propeptide of type I procollagen), osteoblast number, BFR and MAR were reduced (Fig. 2m, Extended Data Fig. 4g-h). The increased resorption in 34a-Het indicates that miR-34a function is haploinsufficient and sensitive to dosage reduction. The recently published miR-34abc triple KO (34abc-TKO)10 and full miR-34a KO9 also showed a similar phenotype (Extended Data Fig. 5a-h), which validate our miR-34a gene trap mice and strengthen the finding that miR-34a loss-of-function elevates bone resorption. Bone marrow transplantation showed that WT mice receiving 34a-KO marrow also exhibited higher CTX-1 (Extended Data Fig. 4f) compared to WT mice receiving WT marrow. Furthermore, osteoclastic miR-34a conditional KO mice (34a-Tie2-KO) also exhibited elevated osteoclast differentiation and bone resorption, but unaltered bone formation, leading to a decreased bone mass (Extended Data Fig. 5i-n). Thus, miR-34a deletion in the osteoclast lineage elevates bone resorption. Our genetic findings prompt us to investigate whether pharmacological administration of a miR-34a mimic can attenuate postmenopausal osteoporosis using an ovariectomy (OVX) mouse model and a chitosan (CH) nanoparticle vehicle. Reduction of uterine weight in all ovariectomized mice indicated effective estrogen depletion (Fig. 3a). Unaltered body weight indicates the absence of obvious toxicity from CH nanoparticles (Fig. 3b). Compared to sham controls, OVX mice treated with miR-Ctrl-CH showed increased CTX-1 and decreased P1NP, whereas both effects were largely prevented in OVX mice treated with miR-34a-CH (Fig. 3c-d). Consequently, OVX-induced bone loss was attenuated by miR-34a-CH (Fig. 4e-f; Extended Data Fig. 6a). miR-34a-CH also decreased bone resorption and increased bone formation in sham controls, leading to a higher bone mass (Extended Data Fig. 6b-d). Biodistribution analysis showed that miR-34a level in the bone marrow was the highest, and further increased by 5-fold by miR-34a-CH, indicating an efficient miR-34a delivery (Fig. 3g). In addition to acute systemic miR-34a treatment, we also examined the effects of chronic osteoclastic miR-34a over-expression. OVX-induced bone resorption and bone loss were also attenuated in 34a-Tie2-Tg mice without altering OVX effects on bone formation (Fig. 3h-k, Extended Data Fig. 6e). These results indicate that osteoclastic miR-34a over-expression is sufficient to impede osteoporosis, and osteoclast is a key site for miR-34a therapeutic benefit. To determine if osteoclastic miR-34a confers protection from bone metastases, we employed two cancer-cell-cardiac-injection models. First, a human breast cancer cell line (MDA231-BoM-1833) was xenografted into female nude mice. This model allows us to assess cancer cells from human. Second, a mouse melanoma cell line (B16-F10) was allografted into immunocompetent male mice. This model took consideration of adaptive immunity. In both models, bone metastases were attenuated in 34a-Tie2-Tg and 34a-PT-Tg mice but exacerbated in 34a-KO and 34a-Het mice (Fig. 3l-o, Extended Data Fig. 7a-f). Since miR-34a remained intact in the exogenous cancer cells, the altered bone metastases resulted from the altered miR-34a in the bone microenvironment of the host. Pharmacologically, we tested both a treatment protocol using the human breast cancer model and a prevention protocol using the mouse melanoma model. In both cases, bone metastases were diminished by miR-34a-CH (Fig. 3p-s). Systemic miR-34a-CH delivery affected neither tumor growth nor metastasis to other organs such as lung (Extended Data Fig. 8a-b). Moreover, treating only the cancer cells with miR-34a-CH before injection had no effect (Extended Data Fig. 8c-d). Consistent with the published finding that miR-34abc deletion does not increase tumorigenesis10, our 34a-KO mice also showed unaltered cancer susceptibility (Extended Data Fig. 8e). Since systemic miR-34a-CH treatment not only decreases bone resorption but also increases bone formation, we examined the effects of miR-34a over-expression in osteoblasts. We bred the CAG34a mice with Osterix-CreER mice to generate 34a-Osx-Tg mice. Osteoblast differentiation was reduced for 34a-KO and 34a-Het mice, but increased for 34a-Osx-Tg mice (Extended Data Fig. 9a-d). Consequently, 34a-Osx-Tg mice exhibited a higher bone formation but unaltered bone resorption, leading to an increased bone mass (Extended Data Fig. 9e-g). Importantly however, the elevated bone formation alone in the 34a-Osx-Tg mice was insufficient to attenuate either OVX-induced bone loss or cancer bone metastases (Extended Data Fig. 9h-i). Together, our conditional miR-34a transgenic mouse models pinpointed the mechanisms underlying the therapeutic benefits of miR-34a by revealing that osteoclast, rather than cancer cell or osteoblast, is the critical and essential player. To elucidate the mechanisms, we identified Tgif2 as a novel direct miR-34a target in the osteoclast lineage (Extended Data Fig. 10a-c). Tgif2 expression was suppressed by miR-34a gain-of-function, but increased by miR-34a loss-of-function, in both mouse and human osteoclast cultures (Fig. 4a-b, Extended Data Fig. 10d-e). The miR-34a seed region in Tgif2 3′UTR is evolutionally conserved in mammals (Fig. 4c). Luciferase reporter assay showed that Tgif2 3′UTR is sufficient to confer miR-34a regulation (Fig. 4d-e). Importantly, when the miR-34a seed region in the Tgif2 3′UTR was mutated, miR-34a regulation was abolished (Fig. 4d-e). Tgif2 expression was increased during WT osteoclast differentiation (Fig. 4b). Tgif2-KO and Tgif2-Het mice had lower bone resorption and higher bone mass (Fig. 4f-h, Extended Data Fig. 10f). Tgif2 deletion reduced osteoclast differentiation, and abolished the anti-osteoclastogenic effects of miR-34a (Fig. 4i-j). Moreover, Tgif2/miR-34a double knockout mice (DKO) could no longer increase osteoclast differentiation or bone resorption (Fig. 4k-l) compared to Tgif2-KO mice. These results indicate that Tgif2 is pro-osteoclastogenic and essential for miR-34a regulation. We next investigated how Tgif2 potentiates RANKL signaling. Transfection assays revealed that NFATc1, c-fos and c-jun, also to a lesser extent NFκB (p65), could induce Tgif2 expression (Fig. 4m). Response elements for NFATc1 and AP-1, but not NFκB, were identified in the Tgif2 promoter region (-5Kb to +5Kb). ChIP analysis in osteoclast cultures showed that NFATc1, c-jun and c-fos bound to these sites upon RANKL stimulation, leading to activated Tgif2 transcription shown by the elevated H3K4me3 level at the transcription start site (Fig. 4n). This indicates that NFATc1 and AP-1 induce Tgif2 expression during osteoclastogenesis. Luciferase reporter assay showed that Tgif2 augmented the activity of NFATc1, NFκB and c-Jun, but not c-fos (Fig. 6o). Consistently, the activity of endogenous NFATc1, NFκB and c-Jun, but not c-fos, was reduced in Tgif2-KO cultures and enhanced in 34a-KO cultures (Fig. 4p). Furthermore, NFATc1 mRNA, c-Jun phosphorylation and IκBα degradation were decreased in Tgif2-KO cultures and increased in 34a-KO cultures (Fig. 4q-r). Therefore, Tgif2 potentiates osteoclastogenesis via a positive feedback loop in which RANKL-induced transcription factors activate Tgif2 expression, and Tgif2 in turn promotes their activity. Collectively, these findings reveal Tgif2 as a novel yet critical regulator of osteoclastogenesis and bone resorption, as well as a key miR-34a direct target that is essential for miR-34a regulation (Fig. 4s). The roles of miRNAs in bone physiology have just begun to emerge. Recent studies show that osteoblast-specific gain-of-function of miR-34b/c decreases bone mass by suppressing osteoblastogenesis and bone formation11,12. Here we show that osteoclast-specific miR-34a gain-of-function protects bone by suppressing osteoclastogenesis and bone resorption. These findings uncover an interesting functional divergence among the miR-34 family members. Our study paves the road for future discovery of other miRNAs that may be regulated by RANKL and/or control Tgif2 expression, as well as future epidemiological and clinical studies to explore the pathological and therapeutic roles of this miR-34a-Tgif2 pathway in human. Most systemically delivered drugs can target multiple tissues and cell types. We found that miR-34a also enhances bone formation; although miR-34a over-expression in osteoblast is neither sufficient nor essential for the therapeutic benefits of miR-34a in osteoporosis or bone metastasis, miR-34a may be a superior new therapy that exerts both anti-catabolic and anabolic effects compared to the current drugs that are solely anti-catabolic. Our identification of miR-34a, as well as the recent report of miR-141 and miR-21913, opens an exciting avenue for the development of a whole new generation of RNA-based osteo-protective medicine. Other miR-34a target genes have been reported in different biological context, such as SIRT114, SIRT615 and PNUTS16. Although miR-34a may also target genes other than Tgif2 in osteoclasts, our genetic rescue ex vivo and in vivo show that Tgif2 is the key miR-34a target, suggesting that other genes are likely secondary or functionally irrelevant to osteoclastogenesis. miR-34abc are commonly deleted in human cancers17. In vitro studies suggest that miR-34abc may be critical mediators of p53 function and potential tumor suppressors18. Surprisingly, in vivo studies reveal that miR-34abc triple KO mice exhibit intact p53 function without increased tumorigenesis10. Nonetheless, systemic miR-34a administration can indeed attenuate cancer malignancy19. This raises the intriguing possibility that its anti-cancer effects may reside in other cells that constitute the tumor microenvironment such as the osteoclasts in the bone metastatic niche. Indeed, our findings illustrate that bone metastases are effectively blocked by miR-34a in osteoclasts, thus providing the first in vivo genetic evidence that miR-34a opposes malignant progression of cancer by disarming the metastatic niche. METHODS SUMMARY Conditional miR-34a transgenic mice were generated using the CAG-Z-EGFP vector. miR-34a knockout mice were generated using a gene-trap ES cell line. METHODS Mice To generate cre-flox controlled conditional miR-34a transgenic mice (CAG34a), a 431 base-pair genomic sequence containing 168 base pairs 5′ and 161 base pairs 3′ of the pre-miR-34a sequence was inserted into the CAG-Z-EGFP vector20. Transgenic founders on pure C57BL/6J background were established by pronuclear injection at the UT Southwestern transgenic core. From 14 founders that carry the LacZ and GFP transgenes, we selected 6 founders that had the highest tail lacZ expression, and bred them to cre transgenic mice. Representative results from at least two independent founders are reported here. To establish osteoclastic miR-34a transgenic mice, CAG34a mice were bred with the previously described Tie2cre mice7,8, PPARγ-tTA;TRE-cre (PT-cre) mice21, lysozyme-cre (Lys-cre) mice22 or Ctsk-cre mice23. To establish osteoblastic miR-34a transgenic mice, CAG34a mice were bred with the previously described Osx-CreER mice24. All conditional miR-34a transgenic mice were on pure C57BL/6J background, and compared to littermate controls that carry only the transgene allele or only the Cre allele; representative results for “transgene only” group is shown as “Ctrl” group; consistent with previous studies, these Cre lines alone do not exhibit bone phenotype. miR-34a knockout mice in a C57BL/6-129P2 mixed genetic background were generated using a mouse embryonic stem cell line (International Gene Trap Consortium clone YHA350) harboring a gene-trap integration in the miR-34a transcription unit, and backcrossed to C57BL/6J mice for at least five generations. In this gene-trap allele, a splice-acceptor followed by a β-geo cassette (fusion of β-galactosidase and neomycin transferase) was inserted between exon 2 and 3 of the mouse miR-34a gene, leading to a truncated and non-functional pri-miR-34a transcript. miR-34abc triple knockout mice and WT controls in a C57BL/6-129SvJae mixed background were provided by Dr. Andrea Ventura (Memorial Sloan-Kettering Cancer Center)10. miR-34a full knockout mice and WT controls on a pure C57BL/6 background were provided by Dr. Lin He (University of California at Berkeley)9. Tgif2-KO mice on a C57BL/6-129 mixed background were provided by Dr. David Wotton (University Of Virginia)25. miR-34a flox mice on a pure C57BL/6 background were from Jackson Laboratory9. Osteoclastic miR-34a conditional KO mice (34a-Tie2-KO) were generated by breeding miR-34a flox mice with Tie2-cre mice. Bone marrow transplantation was performed as described7. Briefly, bone marrow cells from 2-month-old male donor (WT or 34a-KO) were intravenously transplanted into five 2-month-old male C57BL/6J recipients that were irradiated at lethal dose (1000 roentgen); the mice were analyzed 3 month post transplantation. Ovariectomy or sham operation was performed on 10-20 week old female mice. miRNA-carrying chitosan (CH) nanoparticles26 were delivered by intravenous injections at 5μg/mouse or 10μg/mouse twice per week for 4-5 weeks. Sample size estimate was based on power analyses performed using SAS 9.3 TS X64_7PRO platform. With the observed group differences which is of great biological value, and the relatively small variation of the in vivo measurements, a sample size of four per group (n=4) will provide higher than 90% power at type I error rate of 0.05 (two-sided test), and a sample size of three per group (n=3) will provide higher than 80% power at type I error rate of 0.05 (two-sided test). For example, on BV/TV measures with a mean difference of 0.12 between the WT and mutant groups (standard deviation of 0.035 and 0.03 for each of the two groups), 4 mice per group will yield 98% power and 3 mice per group will yield 83% power using two sample t-test. Samples were randomly allocated to each group. Analyses were conducted in a blind fashion to the operator. All experiments were conducted using littermates. All protocols for mouse experiments were approved by the Institutional Animal Care and Use Committee of University of Texas Southwestern Medical Center. Reagents Mouse Tgif2 siRNA or control siRNA were from Santa Cruz Biotechnology. miR-34a precursor (pre-miR-34a) and negative control (pre-control), miR-34a inhibitor (anti-miR-34a) and negative control (anti-control) were from Life Technologies. All miRNA and siRNA were transfected with Lipofectamine™ RNAiMAX (Life Technologies) into bone marrow osteoclast progenitors. For in vivo miRNA delivery, HPLC-purified mirVana™ miR-34a mimic or negative control (Life Technologies) was packaged into chitosan nanoparticles as described26. RAW264.7 mouse macrophage cell line was from ATCC (TIB-71). Anti-Tgif2 antibody (catalog # 09-718) was from Millipore; anti-NFATc1 (7A6), anti c-Jun (H-79) anti IκBα (C-21) antibodies were from Santa Cruz Biotechnologies; anti-H3K4me3 (ab8580) and anti-c-fos (ab7963) antibodies were from Abcam; anti-p-c-Jun (Ser73) (catalog # 9164) was from Cell Signaling. Bone Analyses Micro-Computed Tomography (μCT) was performed to evaluate bone volume and architecture using a Scanco μCT-35 instrument (SCANCO Medical) as described8. Mouse tibiae were fixed in 70% ethanol and scanned at several resolutions for both overall tibial assessment (14 micron resolution) and the structural analysis of trabecular and cortical bone (7 micron resolution). Trabecular bone parameters were calculated using the Scanco software to analyze the bone scans from the trabecular region directly distal to the proximal tibial growth plate. As a bone resorption marker, serum CTX-1 was measured with the RatLapsTM EIA kit (Immunodiagnostic Systems)27. As a bone formation marker, serum amino-terminal propeptide of type I collagen (P1NP) was measured with the Rat/Mouse P1NP enzyme immunoassay kit (Immunodiagnostic Systems)27. Static and dynamic histomorphometry were performed with femurs and vertebrae as described27. Calcein (20mg/kg) were injected into 2 month old mice 2 and 10 days before bone collection. Ex Vivo Osteoclast and Osteoblast Differentiation Osteoclasts were differentiated from bone marrow cells as described7,8. Briefly, hematopoietic bone marrow cells were purified with 40μm cell strainer, and differentiated with 40ng/ml of mouse M-CSF (R&D Systems) in α-MEM containing 10% FBS for 3 days, then with 40ng/ml of mouse MCSF and 100ng/ml of mouse RANKL (R&D Systems) for 3-9 days, in the presence or absence of rosiglitazone (1μM). Mature osteoclasts were identified as multinucleated (>3 nuclei) TRAP+ cells. Osteoclast differentiation was quantified by the RNA expression of osteoclast marker genes using RT-QPCR analysis, as well as number and size of mature osteoclasts. For osteoclast resorptive function analyses, bone marrow osteoclast differentiation was conducted in OsteoAssay bone plates (Lonza), and osteoclast activity was quantified as calcium release from bone into culture medium using CalciFluo ELISA assay (Lonza). Osteoclast precursor proliferation was quantified using a bromodeoxyuridine (BrdU) cell proliferation assay kit (GE Healthcare) as described27. Osteoclast apoptosis was quantified using Annexin V: PE Apoptosis Detection Kit I (BD Biosciences). Human PBMN cells (ReachBio) were differentiated into osteoclasts in α-MEM containing 10% FBS, 25ng/ml MCSF, 50ng/ml hRANKL, 1μM Dexamethasome and 1μM rosiglitazone for 14 days; pre-miR or anti-miR were transfected on day 0 and day 6; human RANKL was added on day 7. Osteoblasts were differentiated from bone marrow cells as described27. Gene Expression Analyses For mRNA expression, RNA was reverse transcribed into cDNA using an ABI High Capacity cDNA RT Kit (Life Technologies) and then analyzed using real-time quantitative PCR (SYBR Green) in triplicate. All mRNA expression was normalized by L19. For mature miRNA expression, RNA was reverse transcribed into cDNA using NCode VILO miRNA cDNA Synthesis Kit (Life Technologies) and then analyzed in triplicate using real-time quantitative PCR (SYBR Green) and a primer specific for the mature miRNA. All miRNA expression was normalized by sno251. Identification of miR-34a Targets in the Osteoclast Lineage To elucidate the molecular mechanisms for miR-34a inhibition of osteoclastogenessi and bone resorption, we identified key direct miR-34a target genes that are pro-osteoclastogenic. First, we used the TargetScan bioinformatic tool to predict all the miR-34a targets by searching for conserved 8mer or 7mer sites that match the miR-34a seed region. Second, we searched databases such as BioGPS to select secondary targets that are expressed in the macrophage-osteoclast lineage. Third, we performed RT-QPCR to select tertiary targets that can be inhibited by miR-34a during osteoclast differentiation. Fourth, we performed luciferase reporter assay to test if the 3′UTR of each tertiary target could directly suppress gene expression in response to miR-34a. To generate a CMV-Luc-3′UTR reporter, a ~300bp Tgif2 3′UTR region centering the miR-34a target sequence was cloned into the pMIR-REPORT™ vector (Life Technologies) downstream of the luciferase open reading frame. To generate a mutant reporter with miss-matched miR-34a binding site, the miR-34a target sequence was altered using QuikChange II XL site-directed mutagenesis kit (Stratagene). The reporters were co-transfected with CMV-β-gal (as an internal transfection control), together with pre-miR-34a or pre-miR-control, anti-miR-34a or anti-miR-control using FuGENE HD reagent (Roche). The transfection assay was conducted in human embryonic kidney 293 cells and CV-1 monkey kidney cells to assess the intrinsic properties of the 3′UTR in different cellular context, and representative results for 293 cells are shown. Luciferase activity was normalized by β-gal activity. Bone Metastasis Analyses Using a VisualSonics Vevo770 small animal ultrasound device, luciferase-labeled cancer cells were injected into the left cardiac ventricle so that they can bypass the lung and efficiently migrate to the bone28. Bone metastases were detected and quantified weekly post injection by bioluminescence imaging (BLI) using a Caliper Xenogen Spectrum instrument at UTSW small animal imaging core facility. The osteolytic metastatic lesions were imaged by radiography using Faxitron Cabinet X-ray System with the X-ray tube voltage fixed at 26 kVp and the exposure time at 15 s. The luciferase-labeled bone-metastasis-prone MDA-MB-231 human breast cancer cell sub-line (MDA231-BoM-1833)29 was generously provided by Joan Massagué (a Howard Hughes Medical Institute Investigator at Memorial Sloan-Kettering Cancer Center) and injected into 6-week-old female nude mice (NCI) at 1×105 cells/mouse in 100μl PBS. The luciferase-labeled B16-F10 mouse melanoma cell line30 was generously provided by Katherine Weilbaecher (Washington University) and injected into 8-week-old male C57BL/6J mice at 5×104/mouse in 100μl PBS. Statistical Analyses All statistical analyses were performed with Student's t-Test and represented as mean ± standard deviation (SD) unless noted otherwise. No animal or sample was excluded from the analysis. The p values were designated as: , p<0.05; , p<0.01; , p<0.005; **, p<0.001; n.s. non-significant (p>0.05). ACKNOWLEDGEMENTS We thank UT Southwestern transgenic core and small animal imaging core for their assistance in our studies; Drs. Paul Dechow, Jerry Feng and Chunlin Qin (Baylor College of Dentistry) for assistance with μCT, histomorphometry and X-ray analysis; Dr. Andrea Ventura (Memorial Sloan-Kettering Cancer Center) for miR-34abc triple KO mice; Dr. David Wotton (University Of Virginia) for Tgif2-KO mice; Dr. Hank Kronenberg (Harvard Medical School) for Osx-CreER mice. Dr. Yuji Mishina (University of Michigan) for CAG-Z-EGFP vector. Y. Wan is a Virginia Murchison Linthicum Scholar in Medical Research. This work was in part supported by CPRIT (RP130145, YW; R1008, JM), DOD (BC122877, YW), NIH (R01 DK089113, YW; R01 CA120185 and P01 CA134292, JM; U54 CA151668 and UH2 TR000943, AS; R01 CA139067, LH), The Welch Foundation (I-1751, YW) and UTSW Endowed Scholar Startup Fund (YW). The UTSW Small Animal Imaging Resource is supported in part by the Harold C. Simmons Cancer Center through an NCI Cancer Center Support Grant (1P30 CA142543) and The Department of Radiology. The VisualSonics Vevo 770 was purchased with NIH ARRA stimulus funds 1S10RR02564801. The authors declare that they have no financial conflict of interest. AUTHOR CONTRIBUTIONS J.Y.K. and Y.W. conceived the project and designed the experiments. All experiments, except the ones listed below, were performed by J.Y.K. W.W. assisted with μCT, ELISA and histomorphometry analyses. H.D.H. assisted with bone marrow transplantation and injection. Z.J. assisted with FACS analyses. X.W assisted with western blot analyses. T.C.C. assisted with northern blot analyses and life-span experiments. X.J.X. assisted with statistical analyses. L.H. provided the full miR-34a KO mice. L.S.M., G.L.B. and A.K.S. assisted with nanoparticle packaging. J.T.M. provided the miR-34a gene trap KO mice. Y.W. wrote the manuscript. Figure 1 miR-34a Suppresses Osteoclastogenesis Ex Vivo a, A diagram of bone marrow osteoclast differentiation assay. Rosi, rosiglitazone. b-d, TRAP expression (b) and mature miRNA levels (c-d) (n=3). e-f, Osteoclast differentiation was decreased by pre-miR-34a (e) but increased by anti-miR-34a (f) (n=3). Left, mature miR-34a levels; Right, TRAP expression; Bottom, images of TRAP-stained cultures; mature osteoclast numbers (black) and resorptive activity (blue). Scale bar, 25μm. R, RANKL. g-j, Human RANKL-mediated osteoclast differentiation from human peripheral blood mononuclear (hPBMN) cells (n=4). g, Mature miR-34a levels. h, TRAP expression. i. Mature osteoclast numbers. j. TRAP-staining and resorptive activity. Scale bar, 25μm. Error bars, SD. Figure 2 miR-34a Inhibits Bone Resorption and Increases Bone Mass In Vivo a, A diagram of the conditional miR-34a transgene (CAG-34a). b, 34a-Tie2-Tg cultures showed decreased osteoclast differentiation (n=3). Left, miR-34a levels; Middle, TRAP expression; Right, TRAP staining, osteoclast numbers (black) and resorptive activity (blue). Scale bar, 25μm. c, Serum CTX-1 (2-month-old, male, n=5). d-f, μCT of the tibiae (2-month-old, male, n=4). d, Images of the trabecular bone of the tibial metaphysis (top) (scale bar, 10μm) and the entire proximal tibia (bottom) (scale bar, 1mm). e, Trabecular bone parameters. BV/TV, bone volume/tissue volume ratio; BS/BV, bone surface/bone volume ratio; Tb.Th, trabecular thickness; Tb.Sp, trabecular separation; Conn.D., connectivity density; SMI, structure model index. f, Cortical BV/TV. g, A diagram of miR-34a gene-trap knockout. h, 34a-KO and 34a-Het cultures showed enhanced osteoclast differentiation (n=3). i, Serum CTX-1 (2-month-old, male, n=6). j-l, μCT of the tibiae (2-month-old, male, n=4). j, Images. k, Trabecular bone parameters. l, Cortical BV/TV. m, Serum P1NP (2-month-old, male, n=6). Error bars, SD. Figure 3 miR-34a Attenuates Osteoporosis and Cancer Bone Metastases a-f, OVX or sham operation was performed on 10-week-old female mice. Three days post-surgery, the OVX mice were treated with miR-34a-CH (34a) or miR-Ctrl-CH (Ctrl) at 5μg/mouse twice/week for 5 weeks (n=5). a, Uterine weight. b, Body weight. c, Serum CTX-1. d, Serum P1NP. e, μCT images. f, Trabecular bone parameters. g, miR-34a levels in each tissue from miR-34a-CH- vs. miR-Ctrl-CH-treated mice 72 hrs after a single injection (n=3). Top, mature miR-34a levels; Bottom, fold induction. h-k, 34a-Tie2-Tg mice or controls (3-month-old, female, n=7) were subjected to OVX and analyzed 5 weeks post-surgery. h, Uterine weight. i, Serum CTX-1. j, Serum P1NP. k, BV/TV by μCT. l, Xenograft of MDA231-BoM-1833 cells into 34a-Tie2-Tg (n=8) or control (n=9). m, Allograft of B16-F10 cells into 34a-Tie2-Tg (n=4) or control (n=6). n, MDA231-BoM-1833 cells in 34a-KO (n=6), 34a-Het (n=6) or control (n=6). o, B16-F10 in 34a-KO (n=6), 34a-Het (n=4) or control (n=6). p-r, Bone metastasis of MDA231-BoM-1833 cells was attenuated by miR-34a-CH delivered 3 days post-xenograft at 10μg/mouse twice/week for 5 weeks (n=5). p, BLI signal. q, Left, BLI images; Right, bone metastases number and size. r, X-ray images and histology images for TRAP and ALP (alkaline phosphatase) staining. Arrows indicate osteolytic lesions. s, Bone metastasis of B16-F10 cells was attenuated by miR-34a-CH delivered at 5μg/mouse twice/week for 4 weeks starting 1 week before cancer cell injection (n=8). l-s, Statistical analyses were performed with Mann Whitney Test and are shown as mean ± SD with p value illustrated. a,c,d,f,h-k,l,n,o,p p<0.05 by ANOVA. Figure 4 Tgif2 is an Essential miR-34a Direct Target and a Pro-Osteoclastogenic Factor a, Tgif2 expression was inhibited by pre-miR-34a in osteoclast cultures (n=3). b, Tgif2 expression in WT and 34a-PT-Tg osteoclast cultures (n=3). c, Sequence alignment of the Tgif2 3′UTR. d, A diagram of Tgif2 3′UTR reporters. e, Luciferase readout from WT or mutant Tgif2 3′UTR reporter co-transfected in HEK293 cells with pre-miR-34a or anti-miR-34a (n=3). f-h, Comparison of Tgif2-KO, Tgif2-Het and WT control mice (1.5-month-old, male, n=7). f. Serum CTX-1. g-h, μCT of tibiae. g, Trabecular BV/TV. h, Images of the trabecular bone of the tibial metaphysis (scale bar, 10μm). i, Decreased osteoclast differentiation in Tgif2-KO and Tgif2-Het cultures (n=3). j, Tgif2-KO cultures were resistant to the anti-osteoclastogenic effects of premiR-34a (n=3). i-j, Top, TRAP expression; Bottom, TRAP staining, osteoclast number (black) and resorptive activity (blue). k-l, Tgif2/34a double knockout (DKO) mice were compared with WT, Tgif2-KO or 34a-KO (2-month-old, male, n=4). k, Osteoclast differentiation. l, Serum CTX-1. m, Tgif2 mRNA in RAW264.7 cells following transfection of transcription factors (n=3). n, ChIP of transcription factor binding and H3K4me3 levels at the endogenous Tgif2 promoter in RAW264.7 cells 3d after RANKL treatment (n=6); txn, transcription. o, Transcription factor was co-transfected into 293 cells with its luciferase reporter, together with Tgif2 or a GFP control (n=6). p, Luciferase reporter was transfected into WT, Tgif2-KO or 34a-KO osteoclast cultures (n=6). q-r, NFATc1 mRNA (q, n=3), c-Jun phosphorylation and IκBα degradation (r) in WT, Tgif2-KO or 34a-KO osteoclast cultures. Ratios of p-c-Jun/total-c-Jun and IκBα/β-actin are shown. s, A model for how miR-34a suppresses osteoclastogenesis. Error bars, SD. Extended Data Figure 1 Additional analyses of 34a-Tie2-Tg mice a-c, Further characterization of the transgene expression in 34a-Tie2-Tg mice. a, FACS analysis of the percentage of GFP+ bone marrow osteoclast progenitors (c-Fms+RANK+) in 34a-Tie2-Tg mice and “transgene only, no cre” control (n=3). b, Images showing GFP and LacZ expression in osteoclast progenitors from 34a-Tie2-Tg mice (GFP+LacZ-) and “transgene only, no cre” control mice (GFP-LacZ+). Scale bar, 100μm. c, Northern blot analysis confirmed miR-34a over-expression in the hematopoietic bone marrow cells of 34a-Tie2-Tg mice. Ct, control; Tg, 34a-Tie2-Tg; EtBr, ethidium bromide. d, QPCR analysis of mRNA expression of additional osteoclast marker genes (n=3). e, Osteoclast function analysis. Bone marrow osteoclast differentiation was conducted in OsteoAssay bone plates (Lonza), and osteoclast activity was quantified as calcium release using CalciFluo ELISA assay (Lonza) (n=8, mean ± s.e.). f, Osteoclast proliferation was not affected, quantified by BrdU incorporation (n=6). g, Osteoclast apoptosis was not affected, quantified by FACS analysis of AnnexinV+7-AAD- cells (n=6). h-i, Static and dynamic histomorphometry. h, Representative images of distal femur sections (2 month old, male). Scale bars, 1mm for Von Kossa images; 10μm for TRAP, ALP and Calcein images. i, Quantification of parameters at distal femur and vertebrae in 2-month-old male and female mice. Extended Data Figure 2 Effects of miR-34a over-expression using additional cre driver targeting osteoclast progenitors 34a-PT-Tg mice were generated using PPARγ-tTA-TRE-cre driver. a, Bone marrow osteoclast differentiation assays. Left, mature miR-34a level (n=3); middle, TRAP mRNA expression (n=3); right, TRAP staining of differentiation cultures, quantification of mature osteoclast numbers per well in 24-well plates (black, n=3), and quantification of bone resorptive activity by calcium release from bone plate into culture medium (μM) (blue, n=6). b, Serum CTX-1 bone resorption marker (2-month-old males, n=10). c, μCT analysis of the trabecular bone in proximal tibiae (2-month-old males, n=4). d, Histomorphometry of the distal femur and vertebrae in 2-month-old mice. Extended Data Figure 3 Effects of miR-34a over-expression using additional osteoclastic cre drivers a-d, 34a-Lys-Tg mice were generated using Lysozyme-cre driver. e-h, 34a-Ctsk-Tg mice were generated using Ctsk-cre driver. a,e, Bone marrow osteoclast differentiation assays. Left, mature miR-34a level (n=3); middle, TRAP mRNA expression (n=3); right, TRAP staining of differentiation cultures, quantification of mature osteoclast numbers per well in 24-well plates (black, n=3), and quantification of bone resorptive activity by calcium release from bone plate into culture medium (μM) (blue, n=6). b,f, Serum CTX-1 (2-month-old males; b, n=5, f, n=8). c,g, Trabecular BV/TV of proximal tibiae by μCT (2-month-old males; c, n=4; g, n=4). d,h, Histomorphometry of the distal femur and vertebrae in 2-month-old mice. Extended Data Figure 4 Additional analyses of gene-trap miR-34a knockout mice a, Northern blot analysis confirmed decreased miR-34a expression in the miR-34a gene trap KO mice. Six-week-old female mice with corresponding genotypes were irradiated with a dose of 6 Gy, and 4 h later the spleen was collected for RNA extraction. Northern blotting for miR-34a was performed as described (Chang TC et al. 2008, Nature Genetics 40:43-50). b, QPCR analysis of mRNA expression of additional osteoclast marker genes (n=3). c, Osteoclast function analysis. Bone marrow osteoclast differentiation was conducted in OsteoAssay bone plates (Lonza), and osteoclast activity was quantified as calcium release using CalciFluo ELISA assay (Lonza) (n=8, mean ± s.e.). d, Osteoclast proliferation was not affected, quantified by BrdU incorporation (n=6). e, Osteoclast apoptosis was not affected, quantified by FACS analysis of AnnexinV+7-AAD- cells (n=6). f. WT mice transplanted with 34a-KO bone marrow cells exhibited higher serum CTX-1 levels compared to WT mice transplanted with WT bone marrow cells (n=5 recipients per group). g-h, Static and dynamic histomorphometry. g, Representative images of distal femur sections (2 month old, male). Scale bars, 1mm for Von Kossa images; 10μm for TRAP, ALP and Calcein images. h, Quantification of parameters at distal femur and vertebrae in 2-month-old male and female mice. Extended Data Figure 5 Effects of targeted miR-34a deletion a-d, Targeted miR-34a/b/c triple KO (34abc-TKO) mice were compared with WT control mice (5 month males, n=4). a-c, Bone marrow osteoclast differentiation assay. a, Expression of miR-34a was diminished while expression of miR-34b and miR-34c remained absent/low in osteoclast precursors on d3. b, Expression of osteoclast markers were increased. c, Number, size and resorptive activity of mature osteoclasts were increased. d, Serum CTX-1 was increased. e-h, Targeted full miR-34a KO (34a-full-KO) mice were compared with WT control mice (2 month females, n=3). e-g, Bone marrow osteoclast differentiation assay. e, Expression of miR-34a was diminished in osteoclast precursors on d3. f, Expression of osteoclast markers were increased. g, Number, size and resorptive activity of mature osteoclasts were increased. h, Serum CTX-1 was increased. i-n, Conditional miR-34a KO mice by Tie2-cre (34a-Tie2-KO) were compared with littermate miR-34af/f control mice (2 month males, n=6). i-k, Bone marrow osteoclast differentiation assay. i, miR-34a expression was reduced in osteoclast precursors on d3. j, Expression of osteoclast markers were increased. k, Number, size and resorptive activity of mature osteoclasts were increased. l, Serum CTX-1 was increased. m, Trabecular BV/TV of proximal tibiae by μCT. n, Histomorphometry of the distal femur and vertebrae. For c, g, k, Mature osteoclasts were identified as multinucleated (>3 nuclei) TRAP+ (purple) cells. Scale bar, 25μm. Quantification of osteoclast number/well is shown in black. Quantification of osteoclast resorptive activity by calcium release from bone to culture medium (μM) is shown in blue. Extended Data Figure 6 Anti-osteoporosis effects of miR-34a a, Histomorphometry of the distal femur and vertebrae in OVX mice treated with miR-34a-CH nanoparticles. OVX or sham operation was performed on 10-week-old WT female C57BL/6J mice. Three days post surgery, the OVX mice were intravenously injected with miR-34a-CH (34a) or miR-Ctrl-CH (Ctrl) at 5μg/mouse twice a week for 5 weeks (n=5). b-d, Osteoprotective effects of miR-34a-CH in sham control mice. WT female C57B/6J mice (n=5, 10 week old) were subjected to sham operation and then treated with miR-34a-CH or miR-34a-Ctrl at 5μg/mouse twice a week for 5 weeks. b, Serum CTX-1. c, Serum P1NP. d, BV/TV of proximal tibiae by μCT. e, Histomorphometry of the distal femur and vertebrae in WT and 34a-Tie2-Tg mice after OVX. 34a-Tie2-Tg mice or controls (3-month-old, female, n=7) were subjected to OVX or sham operation and analyzed 5 weeks post-surgery. Extended Data Figure 7 Additional characterization of bone metastases a, Representative BLI images. b, Quantification of the number of metastasis. c, Quantification of the size of metastasis. For a-c, n=9 for Ctrl, n=8 for 34a-Tie2-Tg, n=6 for WT and 34a-KO; results are shown as average ± s.e.. d, Xenograft of MDA231-BoM-1833 human breast cancer cells into 34a-Tie2-Tg nude mice (n=8) or littermate control nude mice (n=9). Results from each week are shown separately to better visualize the difference. e, Xenograft of MDA231-BoM-1833 human breast cancer cells into 34a-PT-Tg nude mice (n=8) or littermate control nude mice (n=8). Results from each week are shown separately to better visualize the difference. f, Allograft of B16-F10 mouse melanoma cells into 34a-PT-Tg (n=7) or littermate control mice (n=7). Extended Data Figure 8 Effects of miR-34a on cancer cells a, Systemic miR-34a-CH delivery did not affect the growth of B16-F10 melanoma cells injected subcutaneously (n=5, male, 8 week old). Tumors were collected 18 days after cell injection, result is shown as average ± s.e. b, Systemic miR-34a-CH delivery did not affect cancer metastasis to other organs such as lung (n=5, male, 8 week old). B16-F10 cells were i.v. injected retro-orbitally, BLI signals were quantified 2 weeks later and the result is shown as average ± s.e. c-d, MiR-34a-CH treatment of cancer cell alone was not sufficient to inhibit bone metastasis. BoM-1833 cells were treated with miR-34a-CH or miR-Ctrl-CH in cultures for 24hrs before cardiac injection (n=5, male, 6 week old), and the mice were not treated with nanoparticles. c, Quantification of bone metastasis BLI signal 5 weeks after injection, shown as average ± s.e. d, MiR-34a over-expression in BoM-1833 cells persisted for 5 weeks in cultures. e, Loss-of-function in 34a-KO and 34a-Het mice did not result in significantly increased susceptibility of cancer and mortality. Left, Kaplan-Meier survival curve for WT (n=29), 34a-Het (n=35) and 34a-KO (n=29); p=0.223 by log-Rank (Mantel-Cox) test. Right, the 34a-KO allele was transmitted at normal Mendelian frequency. Extended Data Figure 9 Osteoblastic miR-34a over-expression is not sufficient to inhibit osteoporosis or bone metastases a, A schematic diagram of the ex vivo bone marrow osteoblast differentiation assay. MSC GF, mesenchymal stem cell growth factors; GP, β-glycerophosphate; AA, ascorbic acid. b, Osteoblast differentiation was decreased for bone marrow from 34a-KO and 34a-Het mice compared to WT controls, quantified by osteoblast marker genes osteocalcin and Col1a1 on day 13 (n=6). c-h, Characterization of osteoblastic miR-34a transgenic mice. CAG34a mice were bred with Osterix-CreER mice to generate miR34a-Osx-transgenic (34a-Osx-Tg) mice or littermate control mice that carry only CAG34a transgene; all mice (1-month-old, male) received tamoxifen injection on two consecutive days and analyzed 2 months later. c, Elevated levels of mature miR-34a in 34a-Osx-Tg osteoblast differentiation cultures on day 13 (n=6). d, Osteoblast differentiation was increased for bone marrow from 34a-Osx-Tg mice compared to control mice, quantified by osteoblast marker genes osteocalcin and Col1a1 on day 13 (n=6). e, Serum P1NP was increased in 34a-Osx-Tg mice (n=6). f, Serum CTX-1 was unaltered in 34a-Osx-Tg mice (n=6). g, Histomorphometry of distal femur and vertebrae in 34a-Osx-Tg and control mice. h, OVX-induced bone resorption and bone loss was unaltered in 34a-Osx-Tg mice. 34a-Osx-Tg mice or controls (3-month-old and 2 months after tamoxifen injection, female, n=5) were subjected to OVX or sham operation and analyzed 5 weeks post-surgery. i, Cancer bone metastasis was unaltered in 34a-Osx-Tg mice (n=8). Statistical analyses in i were performed with Mann Whitney Test and are shown as mean ± standard error. Extended Data Figure 10 Additional characterization of Tgif2 as a key miR-34a direct target gene a, A list of potential miR-34a target genes in the osteoclast lineage and characterization of miR-34a regulation. N.D., not determined. b, Fold changes in the expression of each candidate target gene after transfection with pre-miR-34a vs. pre-miR-ctrl in WT bone marrow osteoclast differentiation culture (n=3) . c, Fold changes in the luciferase readout from 3′UTR reporter for each candidate target gene co-transfected in HEK293 cells with pre-miR-34a or pre-control. The results were normalized by internal control β-galactosidase (β-gal) readout (n=3). d, Western blot analysis showing that Tgif2 protein expression is decreased in the bone marrow osteoclast progenitors from 34a-Tie2-Tg transgenic mice compared with control mice (left), but increased in the bone marrow osteoclast progenitors from 34a-KO and 34a-Het mice compared with WT control mice (right). e, Human Tgif2 expression in hPBMN osteoclast differentiation cultures was suppressed by pre-miR-34a but enhanced by anti-miR-34a via transfection (n=4). f, Histomorphometry of the distal femur and vertebrae in 1.5 month old Tgif2-KO, Tgif2-Het and WT control mice. ==== Refs REFERENCES 1 Coleman RE Bone cancer in 2011: Prevention and treatment of bone metastases. Nature reviews. Clinical oncology 2012 9 76 78 22182971 2 Ell B Kang Y SnapShot: Bone Metastasis. Cell 2012 151 690 690 e691 23101634 3 Novack DV Teitelbaum SL The osteoclast: friend or foe? 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Journal of cellular biochemistry 2008 104 1311 1323 18260128	25043055	PMC4149606	NO-CC CODE	2021-01-05 05:23:27	yes	Nature. 2014 Aug 28; 512(7515):431-435
==== Front DiabetesDiabetesdiabetesdiabetesDiabetesDiabetes0012-17971939-327XAmerican Diabetes Association 25024373002610.2337/db14-0026Obesity StudiesMyeloperoxidase Deletion Prevents High-Fat Diet–Induced Obesity and Insulin Resistance Wang Qilong Xie Zhonglin Zhang Wencheng Zhou Jun Wu Yue Zhang Miao Zhu Huaiping Zou Ming-Hui Section of Molecular Medicine, Department of Internal Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, OKCorresponding author: Ming-Hui Zou, [email protected] 2014 13 11 2014 63 12 4172 4185 07 1 2014 08 7 2014 © 2014 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.2014Activation of myeloperoxidase (MPO), a heme protein primarily expressed in granules of neutrophils, is associated with the development of obesity. However, whether MPO mediates high-fat diet (HFD)-induced obesity and obesity-associated insulin resistance remains to be determined. Here, we found that consumption of an HFD resulted in neutrophil infiltration and enhanced MPO expression and activity in epididymal white adipose tissue, with an increase in body weight gain and impaired insulin signaling. MPO knockout (MPO−/−) mice were protected from HFD-enhanced body weight gain and insulin resistance. The MPO inhibitor 4-aminobenzoic acid hydrazide reduced peroxidase activity of neutrophils and prevented HFD-enhanced insulin resistance. MPO deficiency caused high body temperature via upregulation of uncoupling protein-1 and mitochondrial oxygen consumption in brown adipose tissue. Lack of MPO also attenuated HFD-induced macrophage infiltration and expression of proinflammatory cytokines. We conclude that activation of MPO in adipose tissue contributes to the development of obesity and obesity-associated insulin resistance. Inhibition of MPO may be a potential strategy for prevention and treatment of obesity and insulin resistance. ==== Body Introduction Obesity, the most common cause of insulin resistance, is characterized by chronic, low-grade inflammation in insulin target tissues, including the liver, skeletal muscle, and adipose tissue (1,2). This is consistent with observations that the adipose tissue of obese mice and humans is infiltrated with many immune cells, including macrophages, T cells, B cells, and eosinophils (3–5). Adipose tissue macrophages secrete a variety of cytokines, such as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), that directly impair insulin signaling and lead to low insulin sensitivity. Recruitment of macrophages into adipose tissue is the initial event in obesity-induced inflammation and insulin resistance. In the early stage of obesity, neutrophils are recruited to adipose tissue, where they produce chemokines and cytokines, thereby promoting macrophage infiltration (6,7). Neutrophils can also promote an inflammatory response by releasing neutrophil elastase, a proteolytic enzyme produced during inflammation. Recently, several studies demonstrated that deletion of neutrophil elastase prevents high-fat diet (HFD)-induced insulin resistance and inhibits adipose tissue inflammation, with a reduction in adipose tissue neutrophils and macrophages (8,9), suggesting that neutrophils may participate in inflammation-induced metabolic disorders. In addition to neutrophil elastase, myeloperoxidase (MPO) is abundantly expressed in neutrophils and has been implicated in the initiation of the inflammatory response in adipose tissue. We therefore hypothesized that MPO might participate in obesity-induced insulin resistance. MPO is a chlorinating oxidant-generating enzyme that initiates an acute inflammatory response and propagates chronic inflammation through the generation of pro-oxidants, including hypochlorous acid (HOCl) and tyrosyl radicals (10,11). Bacterial invasion or pathological stress induces MPO-catalyzed synthesis of HOCl from H2O2 and Cl ions in neutrophils. MPO-derived HOCl directly modifies lipoproteins and enhances their affinity for macrophages and the vessel wall, facilitating the development of vascular inflammation (12). A recent animal study demonstrated that neutrophils infiltrate adipose tissue in the early stage of HFD administration and release various substances, including reactive oxygen species, TNF-α, and MPO, all of which have the capacity to induce inflammation (13). Observations in humans also show that neutrophil counts in peripheral blood are increased in obese individuals and in type 2 diabetic patients, and importantly, these patients have higher plasma levels of MPO (14), suggesting a positive correlation between activation of MPO and metabolic disorders (15,16). Thus, MPO was considered an early biomarker of inflammation and cardiovascular risk factor in obese individuals; however, the functional roles of elevated MPO expression in adipose tissue in the pathogenesis of insulin resistance remain to be determined. In the current study, we sought to determine whether MPO activation is involved in the development of obesity-associated insulin resistance. We found that consumption of an HFD induced neutrophil infiltration and MPO activation in adipose tissue and that knockout of MPO prevented HFD-induced obesity and insulin resistance. Research Design and Methods Reagents HOCl and 4-aminobenzoic acid hydrazide (ABAH) were purchased from Sigma-Aldrich (St. Louis, MO). Protein A/G-agarose, radioimmunoprecipitation assay (RIPA) lysis buffer, and antibodies against β-actin were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antibodies against phospho-IR-β (Tyr1150/1151), IR-β, phospho-Akt (Ser473), phospho-Akt (Thr308), Akt, and horseradish peroxidase–linked secondary antibodies were purchased from Cell Signaling Technology, Inc. (Beverly, MA). Antibody against MPO was from R&D Systems (Minneapolis, MN). Antibodies against nitrotyrosine (NT), uncoupling proteins 1 (UCP1), and UCP3 were from Millipore Corp. (Billerica, MA). The enhanced chemiluminescence detection kit was obtained from Pierce (Rockford, IL). Experimental Animals MPO knockout (MPO−/−) mice from The Jackson Laboratory (Bar Harbor, ME) were originally generated on the 129/SvJ background and were backcrossed onto C57BL/6J background for more than 10 generations (17). C57BL/6J mice were used as the wild-type (WT) control. Mice were housed in temperature-controlled cages under a 12-h light/12-h dark cycle. Starting at 6 weeks of age, male mice were fed an HFD (D12492; Research Diets, New Brunswick, NJ) consisting of 60% fat, 20% protein, and 20% carbohydrate, or a normal chow diet (ND) consisting of 13% fat, 29% protein, and 58% carbohydrates (LabDiet, St. Louis, MO) for up to 16 weeks. Body weight and blood glucose were monitored every 2 weeks. At the end of the experiments, mice were fasted for 6 h. Plasma, epididymal white adipose tissue (WAT), brown adipose tissue (BAT), and the liver were collected and stored at −80°C. The animal protocol was reviewed and approved by the University of Oklahoma Institutional Animal Care and Use Committee. Metabolic Studies Mice were fasted overnight, and a glucose tolerance test (GTT) was performed by intraperitoneal injection of 1 g/kg body weight of 10% d-glucose, followed by measurement of blood glucose levels at 0, 30, 60, 90, and 120 min with a glucometer. To assess insulin tolerance, mice were fasted for 6 h and intraperitoneally injected with 0.75 units/kg insulin (Humulin R; Eli Lilly and Company, Indianapolis, IN). Blood glucose was measured at 0, 15, 30, 60, 90, and 120 min after injection. Plasma insulin concentrations were measured using an ELISA kit (ALPCO, Salem, NH). Plasma concentrations of triglyceride and cholesterol were measured using an Infinity kit (Thermo Fisher Scientific, Waltham, MA). Isolation of Neutrophils, Monocytes, and Macrophages WT and MPO−/− mice were intraperitoneally injected with 4% thioglycollate (1.5 mL) to induce chemical peritonitis. The lavage was collected 6 hours after the injection, and neutrophils were isolated by Ficoll gradient centrifugation (13). Monocytes were collected 12 h after the injection and purified by adherence to culture dish in DMEM medium containing 10% FBS. Peritoneal macrophages were collected 3 days after the injection and purified by adherence to culture dish in DMEM medium containing 10% FBS. Nonadherent cells were removed by washing with PBS. Peroxidase Activity Assay Peroxidase activity in neutrophils and adipose tissue was measured by the chemiluminescence assay using luminol plus near-infrared quantum dots, as previously described (18). Briefly, epididymal WAT and neutrophils were homogenized with RIPA buffer containing 1 mmol/L Na3VO4, 1 µg/mL leupeptin, and 1 mmol/L phenylmethylsulfonyl fluoride. Protein concentration was determined using the bicinchoninic acid method. The protein concentrations of homogenates were adjusted to 2 mg/mL and 1 mg/mL, respectively. Eighty microliters protein lysate were placed in a Costar 96-well black plate with 80 µL 2.3 mmol/L luminol (Thermo Fisher, Rockford, IL) and 1 µL 8 μmol/L QD800 (Invitrogen, Grand Island, NY). Then, 80 μL 2 mmol/L H2O2 were added to the mixtures to trigger production of HOCl. Luminescence was recorded for 20 seconds after the H2O2 addition to estimate peroxidase activity by using an M1000 microplate reader (Tecan Group Ltd., Männedorf, Switzerland). Measurement of BAT Mitochondrial Oxygen Consumption Mitochondria were isolated from BAT by differential centrifugation as described (19). The mitochondrial fraction was suspended in buffer consisting of 5 mmol/L MgCl2, 215 mmol/L d-mannitol, 6.25 mmol/L KH2PO4, 20 μmol/L EGTA, 75 mmol/L sucrose, 20 mmol/L HEPES, and 0.1% BSA (pH 7.4). Forty microliters of 3 mg/mL mitochondrial protein were placed in a sealed chamber for measurement of oxygen consumption using a Clark-type oxygen electrode at 37°C (782 oxygen meter; Strathkelvin Instruments, North Lanarkshire, Scotland). Mitochondrial activation was initiated by addition of 0.25 mmol/L succinate and 1 mmol/L ADP. Oxygen consumption was monitored for 5 min before addition of 1 mmol/L KCN to stop the reaction. To determine the UCP1-dependent oxygen consumption rate, mitochondrial respiration was initiated by addition of the substrates, including 30 μmol/L palmitoyl CoA or 5 mmol/L pyruvate, for 2 min, and then inhibited by adding 2 mmol/L guanosine 5′ diphosphate (GDP) for 2 min. UCP1-dependent respiration was calculated as the differences between substrate-stimulated and GDP-inhibited oxygen consumption rates. Maximal oxygen consumption rates were calculated by adding 1.5 μmol/L FCCP (carbonyl cyanide 4-[trifluoromethoxy]phenylhydrazone); finally, the reaction was stopped by adding 5 µg/mL antimycin A plus 2 μmol/L rotenone. Measurement of ATP BAT mitochondria were incubated with 0.25 mmol/L succinate and 1 mmol/L ADP, as described previously. After the incubation, reaction mixture was centrifuged at 10,000g for 5 min. ATP in the supernatant was assayed by Jasco high-performance liquid chromatography, as described previously (20). Histological Analysis Hematoxylin and eosin staining of WAT, BAT, and liver sections, and immunohistochemical staining using antibodies against CD68 and neutrophil elastase (Abcam) were performed as described previously (21). Cell Treatment Mouse 3T3-L1 preadipocytes from the American Type Culture Collection (Manassas, VA) were cultured and differentiated into adipocytes using 3T3-L1 adipocyte differentiation medium DMEM/F12 50:50 (Zen-Bio, Inc., Durham, NC) supplemented with isobutylmethylxanthine (0.5 mmol/L), dexamethasone (1 μmol/L), and insulin (1.5 µg/mL; pH 7.4) (22). Adipocytes were used 8–12 days after differentiation, when 90–95% of the cells exhibited adipocyte phenotype. After overnight incubation in DMEM (pH 7.4) supplemented with 0.1% BSA, penicillin (100 units/mL), and streptomycin (100 µg/mL), the 3T3-L1 adipocytes were treated with HOCl in culture medium (DMEM, pH 7.4) for 1 h at 37°C. The concentrations of HOCl were determined at 292 nm in 0.1 mol/L NaOH (ε = 350 [mol/L]-1 ⋅ cm−1) before use. Immunoprecipitation and Western Blot Analysis Proteins were extracted from 3T3-L1 cells or mouse tissues with RIPA buffer containing 1 mmol/L Na3VO4, 1 µg/mL leupeptin, and 1 mmol/L phenylmethylsulfonyl fluoride, as described previously (21). Protein concentration was determined using the bicinchoninic acid method. A total of 500 μg protein was incubated with NT antibody overnight at 4°C. Immunoprecipitates were washed four times with lysis buffer, boiled in Laemmli buffer (Boston BioProducts, Inc., Ashland, MA) for 5 min, and analyzed by Western blotting. For the Western blot analysis, tissue or cell lysates were resolved by SDS-PAGE and transferred to polyvinylidene difluoride membrane (Millipore Corp.). Membranes were probed with specific antibodies and subsequently incubated with horseradish peroxidase–linked secondary antibodies. Proteins were visualized by using an enhanced chemiluminescence detection system. Quantitative Real-Time PCR Total RNA was isolated from BAT and WAT using the Qiagen RNeasy Mini Kit (Germantown, MD). PCR primers were designed to specifically span an intron of target genes to ensure only cDNA but not genomic DNA was amplified. The primer sequences are detailed in Supplementary Table 1. To validate the primers, we ran the PCR products on agarose gel, which revealed single bands of the correct size. To determine the efficiency of the primers, we ran standard curves. The slopes of the standard curves (Ct vs. concentration) were close to −3.32. For reverse transcription, 1 µg total mRNA was converted to first-strand cDNA in 20-µL reactions using a cDNA synthesis Kit (Promega). Quantitative real-time PCR was performed using SYBR Green (Invitrogen). The relative RNA amount was calculated with the 2−ΔΔCt method and normalized with 18S rRNA as the internal control. Peroxynitrite Measurement To determine HOCl-induced generation of peroxynitrite (ONOO−) in 3T3-L1 adipocytes, the cells were cultured in DMEM (pH 7.4), treated with HOCl (200 μmol/L) for 1 h, and then incubated in the culture medium containing dihydrorhodamine (5 μmol/L). After incubation for 1 h, 200 μL medium was collected to a 96-well plate. The formation of rhodamine 123 was monitored by fluorescence spectroscopy, and the excitation and emission wavelengths were 500 and 570 nm, respectively. Statistical Analysis Values are expressed as mean ± SEM. One-way or two-way ANOVA was used to compare the differences among three or more groups. P < 0.05 was considered statistically significant. Results HFD Consumption Results in Neutrophil Infiltration and MPO Activation in Adipose Tissues Obesity-associated tissue inflammation is a major cause of insulin resistance. Consistent with previous findings that adipose tissue of obese mice and humans is infiltrated with large numbers of immune cells, many neutrophils with pale pink cytoplasm and lobed nuclei were observed in epididymal WAT from HFD-fed mice, whereas infiltration of neutrophils was rarely found in adipose tissue from ND-fed mice (Fig. 1A). Because MPO is a major enzyme in neutrophils, we examined whether HFD feeding regulates peroxidase activity. MPO expression was upregulated in epididymal WAT from mice after 16 weeks of HFD feeding (Fig. 1B). The increase in MPO protein levels was associated with an increase in peroxidase activity, as determined by luminescent excitation of near-infrared nanoparticles (Fig. 1C) (18). Figure 1 High-fat feeding is associated with neutrophil accumulation and MPO activation in epididymal fat. C57BL/6J (WT) mice were fed the ND or HFD for 16 weeks. A: Epididymal fat was formalin-fixed and embedded in paraffin. Sections were stained with hematoxylin and eosin. Representative neutrophils are identified by arrows. Scale bars, 100 µm. Images are representative of triplicate samples. B: MPO protein expression in epididymal fat was determined by Western blotting. Data are representative of three independent experiments. C: Peroxidase activity was measured in epididymal fat (n = 4). P < 0.05 vs. ND. D: Neutrophils (NEU), monocytes (MONO), and macrophages (MAC) were isolated from the peritoneal lavage of WT mice treated with thioglycollate, and peroxidase activity was measured and normalized to cell numbers. Peritoneal cells isolated from the lavage of mice treated with normal saline were used as a control (Con; n = 4). P < 0.05 vs. Con. RLU, relative luminescence units. E: Neutrophils and macrophages in stromal vascular fraction were isolated from ND-fed and HFD-fed WT mice. Neutrophils were labeled by anti-Ly6G and CD11b antibodies, macrophages were labeled by anti-F4/80 and CD11b antibodies, and MPO was detected by anti-MPO antibody. The expression of MPO in neutrophils (Ly6G+) and macrophages (F4/80+) was measured by flow cytometry and expressed as median fluorescence intensity (MFI). F: Neutrophils were isolated from WT mice fed the ND or HFD for 16 weeks, and peroxidase activity was assayed and normalized to cell numbers (n = 4). P < 0.05 vs. ND-fed WT. Representative immunohistochemical staining for neutrophil elastase in liver (G) and BAT (I) from ND- or HFD-fed WT mice. Quantification of immunohistochemical staining of neutrophil elastase in liver (H) and BAT (J) sections (n = 5). P < 0.05 ND vs. HFD. Representative immunohistochemical staining for CD68 in liver (K) and BAT (M) from ND- or HFD-fed WT mice. Quantification of immunohistochemical staining of CD68 in liver (L) and BAT (N) sections (n = 5). P < 0.05 ND vs. HFD. MPO is not expressed in adipocytes; therefore, the increases in MPO protein levels and high peroxidase activity most likely results from infiltrated immune cells. To identify which immune cells contributed to the HFD-enhanced peroxidase activity, we first isolated neutrophils, monocytes, and macrophages from peritoneal lavage after thioglycollate administration and measured peroxidase activity in these cells. Peroxidase activity in neutrophils was 15-fold higher than that in peritoneal cells isolated from the mice treated with normal saline. Meanwhile, peroxidase activity was increased by 3.5-fold in monocytes and by 1.7-fold in macrophages (Fig. 1D). Next, we analyzed MPO expression in neutrophils and macrophages isolated from the stromal vascular fraction of epididymal WAT in HFD-fed WT mice by using flow cytometry. FACS revealed that the percentages of neutrophils (Ly6G+CD11b+ cells) and macrophages (F4/80+CD11b+ cells) in HFD-fed mice were higher than those in ND-fed mice (neutrophils: 0.72 ± 0.27% vs. 0.07 ± 0.005%; macrophages: 10.1 ± 0.6% vs. 6.7 ± 1.5%, n = 6; P < 0.05). The MPO level was significantly higher in neutrophils than in macrophages (Fig. 1E). We further examined whether HFD feeding enhanced peroxidase activity in neutrophils. As expected, the peroxidase activity of neutrophils in HFD-fed mice was twofold higher than that in ND-fed mice (Fig. 1F). These data suggest that infiltration of neutrophils in WAT is the major source of HFD-enhanced peroxidase activity and that activation of MPO may be a mechanism underlying HFD-induced obesity and insulin resistance. In addition to increasing infiltration of neutrophils in WAT, consumption of the HFD also increased neutrophil infiltration in the liver (Fig. 1G and H) and BAT (Fig. 1I and J). Similarly, the HFD also increased infiltration of macrophages in the liver (Fig. 1K and L) and BAT (Fig. 1M and N). MPO Deletion Attenuates HFD-Induced Obesity Next, we examined whether deletion of MPO would affect basic metabolic function using MPO−/− mice. Deletion of the MPO gene abolished MPO protein expression (Fig. 2A) and peroxidase activity (Fig. 2B) in neutrophils. Although HFD feeding did not enhance MPO protein levels in WT mouse neutrophils (Fig. 2A), it significantly increased peroxidase activity. This increase was absent in HFD-fed MPO−/− mouse neutrophils (Fig. 2B). Metabolic cages were used to monitor the metabolic changes after HFD feeding. WT and MPO−/− mice had similar energy and water intake (Supplementary Fig. 1A and B). Although there was no difference in body weight gain between ND-fed WT and MPO−/− mice, deletion of MPO significantly attenuated HFD-enhanced body weight (Fig. 2C). The body weight gains of WT and MPO−/− mice were similar in the early stage of HFD feeding; however, the WT mice gained more body weight than MPO−/− mice after 10 weeks of HFD feeding. Accordingly, HFD-fed WT mice gained more epididymal WAT, but not perirenal fat, than HFD-fed MPO−/− mice (Fig. 2D). Thus, MPO deficiency was resistant to HFD-induced obesity even though it did not affect energy intake. Figure 2 Deletion of MPO diminishes HFD-induced obesity via upregulation of UCP1 and mitochondrial oxygen consumption in BAT. A: MPO−/− and WT mice were fed the ND or HFD for 16 weeks. After thioglycollate treatment, neutrophils were isolated, and expression of MPO was determined by Western blotting. B: Neutrophils were isolated from WT and MPO−/− mice fed the ND or HFD, and peroxidase activity was assayed (n = 4). P < 0.05 vs. ND-fed WT. RLU, relative luminescence units. C: Weight gain in WT and MPO−/− mice fed the ND or HFD (n = 8–10). P < 0.05 vs. ND-fed WT; †P < 0.05 vs. HFD-fed WT. D: Fat pad weight in WT and MPO−/− mice fed the HFD for 16 weeks. P < 0.05 vs. WT. E: Rectal temperature of WT and MPO−/− mice fed the HFD for 16 weeks (n = 8). P < 0.05 vs. WT. Levels of UCP1 (F) and UCP3 (G) mRNA in BAT from WT and MPO−/− mice fed the HFD for 16 weeks were measured by quantitative real-time PCR. P < 0.05 vs. WT. H: Protein levels of UCP1 and UCP3 in BAT isolated from HFD-fed WT and MPO−/− mice were measured by Western blotting. I: UCP1 level was quantified by densitometry. P < 0.05 vs. WT. J: Mitochondrial oxygen consumption in HFD-fed WT and MPO−/− mouse BAT was measured after stimulation with succinate and ADP (n = 3). P < 0.05 vs. WT. K: BAT mitochondrial fractions were treated with succinate and ADP, and ATP production was assayed by high-performance liquid chromatography. L–N: BAT mitochondria were isolated from HFD-fed WT and MPO−/− mice, and oxygen consumption rates were measured as described in research design and methods (n = 4). P < 0.05 vs. WT. AM+Ro, antimycin A + rotenone; Pal. CoA, palmitoyl CoA. Upregulation of UCP1 and Body Temperature in MPO−/− Mice Because deletion of MPO attenuated HFD-enhanced body weight without affecting energy intake, we examined whether deletion of MPO resulted in less body weight gain in HFD-fed mice through increasing energy expenditure in BAT, a major tissue in which energy is dissipated to maintain body temperature. In agreement with the findings that neutrophil-specific elastase knockout mice have a higher core temperature and oxygen consumption (23), the rectal temperature of HFD-fed MPO−/− mice was higher than that of WT mice (Fig. 2E). To determine the mechanism underlying the higher temperature in HFD-fed MPO−/− mice, we examined the mRNA and protein levels of UCP1, UCP2, and UCP3, three major uncoupling proteins in BAT, which dissipate oxidative energy as heat in BAT mitochondria (24). UCP1 and UCP3, but not UCP2 (data not shown), mRNA levels were significantly higher in HFD-fed MPO−/− mice than in HFD-fed WT mice (Fig. 2F and G). At the protein level, UCP1 expression was higher in MPO−/− mice than in WT mice; however, UCP3 protein levels were not significantly different in WT and MPO−/− mice (Fig. 2H and I). In BAT, UCP1 acts as a proton carrier that dissipates the proton gradient generated in oxidative phosphorylation. Activation of UCP1 enhances mitochondrial respiration and uncouples the respiratory chain from ATP synthase, leading to the dissipation of oxidation energy as heat (25). We, therefore, measured oxygen consumption in mitochondria isolated from HFD-fed WT and MPO−/− mouse BAT using succinate as a substrate. The mitochondrial oxygen consumption of MPO−/− mice was higher than that of WT mice (Fig. 2J); however, mitochondria produced similar amounts of ATP in WT and MPO−/− BAT (Fig. 2K). UCP1 in BAT was activated by free fatty acids (e.g., palmitoyl-CoA) and carbohydrate (e.g., pyruvate) but inhibited by GDP (26,27). We further analyzed the oxygen consumption rate after addition of palmitoyl-CoA, pyruvate, and GDP. Addition of palmitoyl-CoA or pyruvate significantly increased oxygen consumption rate in MPO−/− mice compared with WT mice (Fig. 2L and M). The increase in the oxygen consumption rate was inhibited by addition of GDP, which inhibits UCP1 activity (Fig. 2L and M). In addition, the UCP1-dependent oxygen consumption rate, estimated by the GDP-inhibitable oxygen consumption rate, was higher in MPO−/− mice than in WT mice after addition of palmitoyl-CoA or pyruvate (Fig. 2N). These results suggest that upregulation of UCP1 promotes mitochondrial oxygen consumption and increases body temperature in MPO−/− mice. MPO Deficiency Alleviates the HFD-Induced Inflammatory Response in WAT MPO has been reported to initiate acute inflammation and propagate chronic inflammation in multiple organs. We therefore investigated whether deletion of MPO alleviates HFD-induced inflammation in WAT. As demonstrated by immunohistochemical staining of the macrophage marker CD68, the extent of macrophage infiltration in epididymal WAT was greater in HFD-fed WT than in HFD-fed MPO−/− mice. A characteristic crown-like structure that contains neutrophils and macrophages could often be observed in HFD-fed mice but was rarely observed in HFD-fed MPO−/− mice (Fig. 3A and B). Consistently, high-fat feeding dramatically increased the expression of F4/80 mRNA, another macrophage marker, in WT epididymal WAT, and the increase was significantly diminished in HFD-fed MPO−/− WAT (Fig. 3C). Figure 3 Deletion of MPO reduces inflammatory response in WAT. WT and MPO−/− mice were fed the HFD for 6 weeks. A: Representative images of WAT immunostained for the macrophage marker CD68. B: Quantification of immunohistochemical staining of CD68 in WAT sections (n = 4). P < 0.05 vs. WT. C: Expression of F4/80 mRNA was measured by quantitative real-time PCR (n = 4). P < 0.05 HFD vs. ND; †P < 0.05 MPO−/− vs. WT. D: Cytokine mRNAs in WAT were measured by quantitative real-time PCR (n = 4). P < 0.05 MPO−/− vs. WT. E: WT and MPO−/− mice were fed the HFD for 16 weeks. Expression of iNOS in WAT was analyzed by using immunohistochemistry. We further analyzed the mRNA levels of proinflammatory cytokines, including TNF-α, IL-6 and IL-1β, MCP-1, chemokine (C-X-C motif) ligand 14 (CXCL14), and chemokine receptor type 2 (CCR2), in WAT of HFD-fed WT and MPO−/− mice, and found that the deletion of MPO reduced HFD-enhanced proinflammatory cytokine expression (Fig. 3D). After 6 weeks of high-fat feeding, the expression of inducible nitric oxide synthase (iNOS) mRNA in MPO−/− mice tended to be lower than that in WT mice, although there was no statistical significance between these mice. We further analyzed protein expression of iNOS in WAT after 16 weeks of high-fat feeding. Immnohistochemical staining demonstrated that the expression of iNOS protein was significantly lower in MPO−/− mice than that in WT mice (Fig. 3E). MPO−/− Mice Are Resistant to HFD-Induced Insulin Resistance Obesity is a major risk factor for insulin resistance. Because MPO−/− mice were resistant to HFD-enhanced obesity, we investigated whether deletion of MPO affects insulin sensitivity. In WT mice, 16 weeks of HFD feeding resulted in an increase in blood glucose and a reduction in plasma insulin levels. Conversely, MPO−/− mice showed significantly lower levels of blood glucose compared with WT mice (Fig. 4A). In addition, after 6 weeks of HFD feeding, MPO−/− mice had lower levels of plasma insulin and fasting blood glucose than did WT mice (Fig. 4B and C); however, plasma concentrations of triglycerides and cholesterol were not significantly different in the two groups (Fig. 4D and E). We further analyzed insulin sensitivity by performing GTTs and insulin tolerance tests in WT and MPO−/− mice after 6 or 16 weeks of HFD feeding. The HFD-fed MPO−/− mice showed greater tolerance to the glucose challenge (Fig. 4F, G, I, and J) and better sensitivity to insulin stimulation (Fig. 4H and K). These results suggest that MPO deficiency attenuates HFD-impaired insulin sensitivity. Figure 4 Lack of MPO improves insulin sensitivity in HFD-fed mice. A: WT and MPO−/− mice were fed the ND or HFD for 16 weeks. A glucometer was used to continuously monitor the blood glucose level in tail vein blood of randomly selected mice (n = 8–10). P < 0.05 WT vs. MPO−/−; †P < 0.05 HFD vs. ND. Fasting blood glucose (B), plasma insulin (C), triglyceride (D), and cholesterol (E) levels were measured at baseline (0) and after 6 and 16 weeks of HFD feeding (n = 8–10). P < 0.05 vs. ND-fed WT; †P < 0.05 WT vs. MPO−/−. GTTs were performed in WT and MPO−/− mice fed the HFD for 6 weeks (F) or in WT and MPO−/− mice (n = 8–10) fed the ND or HFD for 16 weeks (I). P < 0.05 WT vs. MPO−/−; †P < 0.05 HFD vs. ND. Areas under the curve (AUC) were calculated for WT and MPO−/− mice fed the HFD for 6 (G) and 16 (J) weeks. P < 0.05 vs. WT. Insulin tolerance tests (ITT) were performed in WT and MPO−/− mice fed the HFD (n = 8–10) for 6 (H) or 16 weeks (K). P < 0.01 WT vs. MPO−/−. MPO Deletion Protects Against HFD-Impaired Insulin Signaling in Epididymal WAT and Liver To determine whether MPO affects insulin signaling in epididymal WAT of HFD-fed mice, we analyzed insulin-stimulated phosphorylation of insulin receptor-β (IR-β) and Akt in the animals. As shown in Fig. 5A–D, insulin stimulation increased phosphorylation of Akt at Thr308 and Ser473 in WAT isolated from ND-fed WT and MPO−/− mice. HFD feeding inhibited insulin-stimulated phosphorylation of Akt in WT mice. However, the inhibitory effect of the HFD on Akt phosphorylation was absent in MPO−/− mice, confirming that deletion of MPO improves insulin sensitivity in HFD-fed mice. Similarly, insulin-stimulated phosphorylation of IR-β was inhibited in HFD-fed WT mice, and inhibition of IR-β phosphorylation was absent in HFD-fed MPO−/− mice (Fig. 5C and D). Thus, MPO deletion prevented HFD-impaired insulin signaling. Figure 5 MPO deletion attenuates insulin resistance in epididymal WAT and liver. WT and MPO−/− mice fed the ND or HFD for 16 weeks were intraperitoneally injected with 2 units/kg insulin or saline alone. At 15 minutes after injection, homogenates of epididymal WAT were prepared, and levels of phosphorylated (P)-Akt-S473, P-Akt-T308, and P-IR-β were analyzed by Western blotting (A and C) and quantitated by densitometry (B and D). P < 0.05 vs. WT control; †P < 0.05 WT vs. insulin-injected MPO−/−. The expression of P-Akt-S473, P-Akt-T308 (E), P-IR-β, and IR-β (F) in liver homogenates were measured by Western blotting. We also analyzed the effect of the HFD on insulin signaling in the liver. Similar to the observations in epididymal WAT, insulin stimulation increased phosphorylation of Akt at Thr308 and Ser473 in the livers of ND-fed WT and MPO−/− mice. The increase in Akt phosphorylation was attenuated in HFD-fed WT mice but not in MPO−/− mice (Fig. 5E). Insulin-stimulated phosphorylation of IR-β was also inhibited in HFD-fed WT mice, and the inhibition was absent in HFD-fed MPO−/− mice (Fig. 5F). MPO Inhibitor Improves Insulin Sensitivity We further investigated whether pharmacological inhibition of MPO prevents HFD-induced insulin resistance. Chronic administration of the MPO inhibitor ABAH dose-dependently inhibited peroxidase activity of neutrophils in WT mice (Fig. 6A), with 50% inhibition at 40 mg/kg. This dose was then intraperitoneally injected into HFD-fed WT mice. After 1 or 2 weeks of treatment, GTT and insulin tolerance test analysis was done. The results showed that ABAH treatment improved insulin sensitivity and enhanced glucose tolerance in insulin-resistant, obese WT mice compared with DMSO-treated mice (Fig. 6B–E). Moreover, ABAH did not improve glucose tolerance and insulin sensitivity in MPO−/− mice (Fig. 6F and G). Figure 6 MPO inhibitor prevents HOCl production and insulin resistance. A: Male WT mice (8 weeks old) were intraperitoneally injected with the indicated amounts of ABAH (mg/kg) or vehicle (DMSO) for 1 week. At 16 h after the last treatment, the mice were treated with thioglycollate for 6 h. Neutrophils were isolated from the peritoneal lavage, and peroxidase activity was measured as described in research design and methods (n = 4). P < 0.05 vs. DMSO. WT mice fed the HFD for 16 weeks were treated with ABAH (40 mg/kg) or vehicle for the last week (B and C) or for 2 weeks (D and E). At 16 h after the last injection, an insulin tolerance test (B and D) or GTT (C and E) was performed (n = 8 per group). P < 0.05 vs. DMSO. F and G: MPO−/− mice were treated with ABAH (40 mg/kg) or DMSO for 1 week. At 16 hours after the last injection, an insulin tolerance test (F) and GTT (G) were performed (n = 4). MPO Deletion Prevents HFD-Induced IR-β Protein Nitration and Reduction Next, we determined whether deletion of MPO prevents HFD-reduced phosphorylation of IR-β through regulation of IR-β expression. As shown in Fig. 7A, IR-β levels were decreased in epididymal WAT from HFD-fed WT mice compared with ND-fed controls. Meanwhile, deletion of MPO attenuated the reduction in IR-β protein levels associated with HFD. However, IR-β mRNA expression was not affected by HFD feeding in WT and MPO−/− mice (Fig. 7B). Thus, HFD may regulate IR-β expression through posttranscriptional modification of the protein (28). MPO was reported to oxidize and nitrate proteins at tyrosine. Tyrosine nitration of a protein may alter the protein structure and accelerate its degradation (29). Thus, we hypothesized that activation of MPO induces tyrosine nitration of IR-β and subsequently accelerates its degradation. To test this hypothesis, we first detected 3-NT (3-NT) formation in epididymal WAT by using immunohistochemistry. High-fat feeding significantly increased 3-NT formation in WT mice, but this increase was attenuated in MPO−/− mice (Fig. 7C and D). Next, 3-NT was immunoprecipitated from homogenates of epididymal WAT, and IR-β was detected by Western blotting. The level of tyrosine nitration of IR-β was higher in HFD-fed WT mice than in HFD-fed MPO−/− mice (Fig. 7E). To determine whether MPO-produced HOCl mediates tyrosine nitration of IR-β in HFD-fed mice, tyrosine nitration of IR-β was detected in ABAH-treated and HFD-fed mice. Inhibition of HOCl production by administration of ABAH reduced tyrosine nitration of IR-β in HFD-fed mice (Fig. 7F). Figure 7 MPO deletion prevents HFD-induced IR-β nitration and reduction. A: Epididymal WAT was collected from WT and MPO−/− mice fed the ND or HFD for 16 weeks, and IR-β levels were measured by Western blotting and quantitated by densitometry. P < 0.05 vs. ND-fed WT; †P < 0.05 WT vs. MPO−/−. B: Expression of IR-β mRNA in epididymal fat was determined by quantitative real-time PCR. C: Representative immunohistochemical images of 3-NT formation in epididymal WAT from ND- or HFD-fed MPO−/− and WT mice. D: Quantification of immunohistochemical staining of 3-NT in WAT sections (n = 5). P < 0.05 ND vs. HFD; †MPO−/− vs. WT. E: Tyrosine nitration of IR-β in epididymal WAT was determined by immunoprecipitation of 3-NT and Western blotting. IgG was used as an internal control. F: WT mice fed the HFD for 16 weeks were intraperitoneally injected with ABAH (40 mg/kg) and vehicle for 2 weeks. Tyrosine nitration of IR-β in epididymal WAT was determined by immunoprecipitation and Western blotting. G: 3T3-L1 adipocytes were serum-starved overnight, treated with or without 200 μmol/L HOCl for 1 h, and then stimulated with or without 100 nmol/L insulin for 15 min. Western blotting was used to analyze the indicated proteins in the cell extracts. 3T3-L1 adipocytes were serum-starved overnight, treated with or without 200 μmol/L HOCl for 1 h in the absence or presence of SOD1 (150 U/mL), l-NAME (1 mmol/L), or uric acid (50 μmol/L), and then stimulated with insulin (100 nmol/L) for 15 min. H: IRS-1 was immunoprecipitated (IP) and immunoblotted (IB) with anti-IRS-1 and phosphatidylinositide 3-kinase p85 antibodies, respectively. I: Tyrosine nitration of IR-β was determined by immunoprecipitation and Western blotting. Data are representative of four independent experiments. J: 3T3-L1 cells were pretreated with HOCl for 1 h and then incubated in the medium containing dihydrorhodamine (5 μmol/L) for 1 h. After the treatment, ONOO− production was determined by dihydrorhodamine oxidation (n = 5). *P < 0.01 vs. control; †P < 0.01 vs. HOCl. K: Schematic description for MPO promoting obesity and insulin resistance in HFD-fed mice. HOCl Induces Insulin Resistance and Tyrosine Nitration of IR-β HOCl is a major chlorinating oxidant product of MPO; thus, we investigated whether MPO-derived HOCl contributes to insulin resistance in vitro. In 3T3-L1 adipocytes, HOCl pretreatment inhibited insulin-stimulated phosphorylation of IR-β, insulin receptor substrate-1 (IRS-1), and Akt, indicating impairment of insulin signaling (Fig. 7G). HOCl also inhibited insulin-induced binding of IRS-1 to P85, the regulatory subunit of phosphatidylinositide 3-kinase, which is the key enzyme to activate phosphoinositide-dependent kinase-1 and Akt during insulin signaling (Fig. 7H). HOCl has been reported to induce the production of ONOO−, a potent oxidant that nitrates proteins at tyrosine residues in endothelial cells (30). We therefore investigate if endogenous ONOO− production was involved in HOCl-induced tyrosine nitration of IR-β in 3T3-L1 adipocytes. We found that HOCl treatment dramatically increased IR-β tyrosine nitration in the presence or absence of insulin. In contrast, HOCl treatment had little effect on tyrosine nitration of IRS-1 and Akt (data not shown). Administration of superoxide dismutase 1 (SOD1) to remove superoxide, L-NAME to inhibit nitric oxide production, and uric acid to scavenge ONOO−, all prevented HOCl-induced IR-β tyrosine nitration (Fig. 7I) although they had no effects on the binding between IRS-1 and P85 (Fig. 7H). We further examined the effect of HOCl on ONOO− production in 3T3-L1 adipocytes. Exposure of cells to HOCl significantly enhanced ONOO− production in the presence and absence of insulin. The overproduction of ONOO− was abolished by overexpression of SOD1 or administration of L-NAME or uric acid. However, overexpression of catalase failed to prevent HOCl-induced overproduction of ONOO− (Fig. 7J). Taken together, our data suggest that ONOO− mediates HOCI-induced tyrosine nitration of IR-β. Discussion Obesity is characterized by immune cell infiltration in adipose tissue and high levels of proinflammatory molecules in the circulation. These proinflammatory mediators may impair insulin signaling, resulting in insulin resistance. MPO is an enzyme released from neutrophils during inflammation and has been implicated in the development of obesity and insulin resistance (15). However, the molecular mechanisms by which MPO promotes the development of obesity and insulin resistance have not been established. The current study demonstrates that obese mice have high MPO protein levels and activity in adipose tissues and neutrophils. Genetic deletion of MPO led to less body weight gain, inhibition of inflammation, and improvement of insulin sensitivity and glucose tolerance in HFD-fed mice. The improvement of insulin sensitivity is associated with the restoration of IR-β protein levels in HFD-fed mice (Fig. 7K). Emerging evidence suggests that activation of neutrophils is involved in the development of obesity and obesity-associated insulin resistance. In obese and type 2 diabetic patients, circulating neutrophil counts are significantly increased (31) in association with increased oxidative stress and inflammation (7). In HFD-fed mice, adipose tissue was infiltrated with neutrophils after 3 days of HFD feeding (13,32), and neutrophil infiltration was maintained throughout the 90-day study period (9). Suppression of HFD-induced neutrophil infiltration by deletion of neutrophil elastase led to improved glucose tolerance and increased insulin sensitivity. Consistent with these findings, we observed neutrophil accumulation in adipose tissue of HFD-fed mice, which was accompanied by enhanced body weight gain and impaired insulin sensitivity. Many inflammatory stimuli can stimulate neutrophils to release the contents of their granules, including MPO, neutrophil elastase, and proteinases, into the surrounding tissues to induce acute inflammation. Our data indicate that the infiltrating polymorphonuclear neutrophils are the major source of MPO activity in adipose tissue of HFD-fed mice and that deletion of MPO attenuated the HFD-enhanced inflammatory response and inhibited HFD-induced insulin resistance. Thus, all evidence supports the view that neutrophil infiltration of adipose tissue contributes to insulin resistance in obesity (33). MPO may promote tyrosine nitration of proteins, leading to alteration of protein structure and function. Nitration of a tyrosine residue either prevents subsequent phosphorylation of the residue or stimulates its phosphorylation, resulting in constitutively active protein. In addition, tyrosine nitration may alter the rate of proteolytic degradation of nitrated proteins, leading to faster degradation or accumulation of the nitrated proteins in cells. Tyrosine nitration of proteins by MPO has been reported in lung (34), blood, and vasculature (35). MPO chlorinates and nitrates Tyr192 of apolipoprotein A-I, leading to impaired cholesterol efflux (35,36). In vascular endothelial cells, MPO and HOCl increase ONOO− production (37), resulting in endothelial NOS uncoupling and exacerbating oxidative stress (30). Moreover, tyrosine nitration of protein has been shown to be associated with a strong inflammatory response in human atherosclerotic plaques (38) and retina (39). In agreement with the previous findings that tyrosine nitration of the insulin-signaling molecules, including IRS-1 and Akt, contributes to HFD-induced insulin resistance, we found that HFD feeding resulted in tyrosine nitration of IR-β, which was associated with a reduction in IR-β protein and phosphorylation levels and also with impaired insulin signaling. Deletion of MPO prevented HFD-induced tyrosine nitration of IR-β, restored IR-β protein expression, and improved insulin sensitivity. Whether tyrosine nitration of IR-β accelerates its degradation warrants further investigation. Consistent with the findings that high-fat feeding results in less body weight gain and higher body temperature and oxygen consumption in neutrophil elastase knockout mice than in control mice (8), we observed less body weight gain in MPO−/− mice after HFD feeding, with no effect on energy intake. Deletion of MPO also led to higher levels of UCP1 expression in BAT. UCP1 is a proton transporter of the mitochondrial inner membrane that uncouples oxidative metabolism from ATP synthesis and dissipates energy as heat. UCP1 has been reported to play important roles in energy homeostasis, and ablation of UCP1 prevented Western diet–induced obesity (40). In agreement with the upregulation of UCP1, rectal temperature and oxygen consumption measured in isolated BAT mitochondria were increased in the current study, but ATP production did not increase in HFD-fed MPO−/− mice, suggesting the uncoupling of oxidative metabolism and ATP synthesis and increase in thermogenesis, which may account for the smaller body weight gain in HFD-fed MPO−/− mice. Further investigations are warranted to elucidate the mechanism by which MPO regulates UCP1 expression and consequent thermogenesis and energy metabolism. Because loss of body weight and fat mass almost always improves insulin sensitivity (41), which inhibits the Jun NH2-terminal kinase and inhibitor of κB kinase–nuclear factor-κB pathways and reduces inflammation (42), the smaller body weight gain in HFD-fed MPO−/− mice may also contribute to the improvement of insulin resistance in these mice. In summary, activation of MPO was a critical event in HFD-induced obesity and insulin resistance. Deletion of MPO reduced body weight gain through the upregulation of UCP1 in BAT. In addition, MPO deficiency is associated with a restoration of IR-β protein levels and improved insulin sensitivity in HFD-fed mice. Thus, inhibition of MPO may be a potential strategy for prevention and treatment of obesity and its complications. Supplementary Material Supplementary Data This article contains Supplementary Data online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db14-0026/-/DC1. See accompanying article, p. 4001. Article Information Funding. This work was supported by National Institutes of Health grants (HL-079584, HL-080499, HL-074399, HL-089920, HL-096032, HL-10488, AG047776, and HL-105157), a research award from the American Diabetes Association, and funds from Warren Chair in Diabetes Research from the University of Oklahoma Health Sciences Center. Q.W. is a recipient of an American Heart Association Postdoctoral fellowship. M.-H.Z. is a recipient of the National Established Investigator Award of the American Heart Association. Duality of Interest. No potential conflicts of interest relevant to this article were reported. Author Contributions. Q.W. contributed to the study design, performed experiments, and wrote the manuscript. Z.X. designed experiments and wrote the manuscript. W.Z., J.Z., Y.W., M.Z., and H.Z. performed some experiments. M.-H.Z. contributed to the study design and interpretation and wrote the manuscript. 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==== Front Open J Cardiovasc SurgOpen J Cardiovasc SurgOpen Journal of Cardiovascular SurgeryOpen Journal of Cardiovascular Surgery1179-0652Libertas Academica 10.4137/OJCS.S16156ojcs-7-2014-001Original ResearchBedside Surgery to Treat Patent Ductus Arteriosus in Low-Birth-Weight Premature Infants Albayrak Gökhan 1Aykut Koray 1Karacelik Mustafa 2Soylar Ramazan 3Karaarslan Kemal 4Abud Burçin 4Guzeloglu Mehmet 1Hazan Eyup 11 Department of Cardiovascular Surgery, Izmir University, Medical Park Hospital, Izmir, Turkey.2 Department of Cardiovascular Surgery, Dr. Behcet Uz Children Hospital, Izmir, Turkey.3 Department of Pediatry, Izmir University, Medical Park Hospital, Izmir, Turkey.4 Department of Cardiovascular Surgery, Tepecik Research Hospital, Izmir, Turkey.CORRESPONDENCE: [email protected] 17 8 2014 7 1 4 21 4 2014 03 6 2014 05 6 2014 © 2014 the author(s), publisher and licensee Libertas Academica Ltd.2014This is an open-access article distributed under the terms of the Creative Commons CC-BY-NC 3.0 License.BACKGROUND Patent ductus arteriosus (PDA) is commonly seen in premature infants with low birth weights (LBW). It is a condition that has high mortality and morbidity rates. Early closure of the ductus arteriosus may require surgery or medical treatment. However, the decision of first medical approach for symptomatic PDA closure is still debated. In this study, we compared the surgical and medical treatments for the closure of PDA in premature LBW infants. METHODS This study included 27 premature infants whose birth weights were lower than 1500 g, who were born in the period between 2011 and 2013 and had symptomatic PDA. Patients were separated into two groups: groups A and B. Group A included patients whose PDAs were closed with medical treatment (n = 16), and group B included patients who had undergone surgical operations for PDA closure (n = 11). RESULTS There were no statistically significant differences between groups A and B when the groups were compared in terms of birth weight, gestational age, respiratory distress syndrome (RDS), necrotizing enterocolitis (NEC), sepsis, intraventricular hemorrhage (IVH), retinopathy of prematurity (ROP), and pneumothorax. Although the mortality rate was determined to be lower in group B (2 out of 11, 18.1%) than in group A (7 out of 16, 43.7%), no statistically significant difference was found between the two groups. A statistically significant increase was determined in the incidence of kidney function loss in patient group that received Ibuprofen, a medical treatment, in comparison to the patients who had surgery. CONCLUSION In conclusion, surgery is a safe method to repair PDA in premature LBW infants. Although there is no remarkable difference between surgery and medical treatment, we suggest that a surgical approach may be used as a first choice to repair PDA considering the lower rate of mortality and morbidity and higher rate of closure compared to medical treatment. patent ductus arteriosuslow birth weightsurgery ==== Body Introduction Patent ductus arteriosus (PDA) is a common disease that is seen in premature low-birth-weight (LBW) infants. It affects approximately 40–55% of preterm infants born at less than 29 weeks gestation and/or weighing less than 1500 g at birth. PDA may lead to a number of complications including chronic pulmonary disease (CPD), intracranial hemorrhage, necrotizing enterocolitis (NEC), renal failure, and metabolic acidosis. Surgical approaches and medical treatment may be applied for the early closure of PDA. However, there is no consensus on the first choice of treatment.1–3 Medical agents such as intravenous (i.v.) or oral ibuprofen and i.v. indomethacin may be used to treat PDA.4 In this study, we compared the reliability and results of surgical and medical treatments for the closure of PDA in premature LBW infants. Materials and Methods In this study, premature infants (n = 27) diagnosed with symptomatic PDA and LBW (<1500 g) and followed in an intensive between 2011 and 2013 were retrospectively analyzed. Ethics committee approval was not required because of the retrospective nature of the study. Patients were separated into two subgroups according to the type of treatment given for PDA repair: group A included patients who only had medical treatment while patients who had only undergone surgery were included in group B. Patients were not included in this study if they had surgery after unsuccessful PDA closure with medical treatments. The parameters evaluated included gestational age, birth weight, the presence of NEC, respiratory distress syndrome (RDS), intraventricular hemorrhage, and length of hospital stay. Patients in group A were treated with i.v. ibuprofen for three days, receiving a dosage of 10 mg/kg on the first day, and 5 mg/kg on days 2 and 3. Transthoracic echocardiography (TTE) was performed in patients at the end of the third day of treatment. Patients in group B had directly undergone surgery without taking any medicine for the PDA repair. Bedside surgery was performed for all patients in the premature intensive care unit. A “bedside surgery” team, including cardiovascular surgeons, specialists of neonatology and anesthesiology, nurses, and other assisting personnel, has performed the bedside interventions. Main reason of bedside surgery is poor thermal regulation of prematures. All surgical interventions have been performed in the incubator. Simplification of devices and lines with complete monitorization is essential. A heater is also an important part of surgical field. Safe lines for medications were created for anesthesiologist and neonatologists. The iodine solution for skin cleaning was preheated to 37°C before surgery. Placement of the electrocautery plaque is a very important issue. Avoiding of getting wet and adequate sizing is essential. A limited standard left posterolateral thoracotomy incision under general anesthesia was chosen in all cases. PDAs were doubly ligated with ligasure clips. Results The total number of patients included in this study was 27, of which 16 were in group A and 11 were in group B. The gender distribution in the study groups was 14 males and 13 females. The average birth weight was 916.6 ± 225 g in group A and 915.7 ± 278 g in group B. The mean date of birth (DOB) was 189.4 ± 19 days in group A and 191.5 ± 24 days in group B. The general characteristics and preoperative conditions of the patients are listed in Table 1. There was no statistically significant difference between the two groups when patients were compared in terms of birth weight, gestational age, presence and severity of RDS, NEC, sepsis, intraventricular hemorrhage (IVH), retinopathy of prematurity (ROP), and pneumothorax (P > 0.05) (Table 1). Although the death rate was lower (2/11, 18.1%) in group B than in group A (7/16, 43.7%), there was no significant difference between the two groups. In group A, three patients died from sepsis, one from NEC, one from heart failure, and two from pulmonary disorders, while in group B, one patient died from sepsis and one patient died from CPD. Hospital stay was determined to be longer in group B than in group A, but this difference was not found to be statistically significant (Table 2). Furthermore, in group A, the levels of creatinine were increased from 0.78 ± 0.30 to 1.23 ± 0.50 in response to medical treatment. The difference was statistically significant (P < 0.01) (Table 3). Increases in the values of creatinine were also observed in the postoperative period of the group B (Cr: 0.84 ± 0.36) as compared to the preoperative period (Cr: 0.76 ± 0.2). However, the difference was not found to be statistically significant (Table 3). Discussion The ductus arteriosus is a blood vessel that connects the pulmonary artery to the aorta. PDA affects approximately 31% of infants whose birth weight is between 501 and 1500 g. Functional closure occurs in the majority of term neonates by 9–12 hours after birth.5 The incidence of PDA is inversely proportional to gestational age and infants with the lowest gestational ages are the most exposed to the complications of prematurity. The major factor closing the ductus arteriosus is the tension of oxygen, which increases significantly after birth. The patency of the ductus arteriosus has ever been considered as a pathological situation in preterm infants and one likely cause of mortality and morbidity, including bronchopulmonary dysplasia, necrotizing enterocolitis, intraventricular hemorrhage, and ROP.6 Prostaglandin E2 has the opposite effect to that of oxygen; it relaxes smooth muscle and tends to inhibit the closure of the ductus arteriosus. Non-steroidal anti-inflammatory agents, such as indomethacin or ibuprofen, have been shown to be effective in closing or preventing PDA, with differences in side effects. Indomethacin has long been the drug of choice to treat PDA. However, indomethacin inhibits the synthesis of all prostaglandins, a wide variety of adverse effects can be expected, including NEC, intestinal perforation, renal failure, thrombocytopenia, and renal disfunction. More recently, ibuprofen has been proposed for the treatment of PDA as it was shown to induce less adverse effects on cerebral blood flow, intestinal, and renal hemodynamics, while retaining similar efficacy to indomethacin.7 Ibuprofen significantly impairs renal function in preterm infants with a GA ≤ 26 weeks and/or in ELBW neonates, while it may be considered safe for infants with a BW > 1000 g and/or GA > 26 weeks.8 Factors affecting the outcomes of surgical ligations, indications, and optimal time of surgery in very low-birth-weights infant (VLBWI) are unclear. It was concluded that PDA ligation can be considered at any time in VLBWI when medical therapy either failed to close or was contraindicate.9 There is a risk of complication with the surgical repair of PDA. These complications include pneumothorax, intraoperative hemorrhage, phrenic nerve paralysis, vocal cord paralysis, and wound infection. However, of the patients in the surgical group B, only one case was observed to develop the complication of pneumothorax. A recent study revealed that there was no significant difference between patients (n = 154) who had surgical treatment and those who had medical treatment for PDA closure with respect to the presence of hospital mortality, CPD, NEC, sepsis, IVH, and levels of creatinine. Additionally, while a low rate of failure was found in surgical closures of PDA, an increase was observed in the incidence of pneumothorax and ROP. Moreover, it was emphasized that more studies should be conducted to clarify the issue of choosing the first approach of either surgical or medical treatment in symptomatic PDA in LBW infants.1 Mosalli et al suggested that the prophylactic surgical ligation of the duct does not result in any significant differences of mortality, IVH, CPD, and ROP among the two study groups. Interestingly, the study reported a significant reduction in severe NEC, in the prophylactic surgical group, as compared to infants who received selective treatment for hemodynamically significant PDA.10 We found that there were no significant differences, among the two groups, when it came to hospital mortality and RDS, NEC, sepsis, and IVH. However, a statistically significant increase was determined in the incidence of kidney function loss in patient group that received Ibuprofen, a medical treatment, in comparison to the patients who had surgery (P < 0.05). The preoperative risk scores were high for all the patients, therefore an increased mortality in hospital and a prolonged hospital stay was observed in both patient groups. Conclusion Bedside surgery is a safe therapeutic approach for the closure of PDA in premature LBW infants. Although it was not statistically significant, our results demonstrated that the rate of mortality and morbidity was lower in surgically treated patients than in medically treated patients. Therefore, we suggest that a surgical approach may be used as a first choice to repair PDA considering the lower rate of mortality and morbidity and higher rate of closure compared to medical treatment. Author Contributions Conceived and designed the experiments: GA, KA. Analyzed the data: GA, MK, RS, KK, BA, MG, EH. The first draft of the manuscript: GA. Contributed to the writing of the manuscript: KA, RS, MG. Agree with manuscript results and conclusions: GA, KA, EH. Made critical revisions and approved final version: GA, KA. All authors reviewed and approved of the final manuscript. ACADEMIC EDITOR: Hendrick Barner, Editor in Chief FUNDING: Authors disclose no funding sources. COMPETING INTERESTS: Authors disclose no conflict of interest. This paper was subject to independent, expert peer review by a minimum of two blind peer reviewers. All editorial decisions were made by the independent academic editor. All authors have provided signed confirmation of their compliance with ethical and legal obligations including (but not limited to) use of any copyrighted material, compliance with ICMJE authorship and competing interests disclosure guidelines and, where applicable, compliance with legal and ethical guidelines on human and animal research participants. Table 1 General characteristics and preoperative conditions of the patients. GROUP A (n = 11) GROUP B (n = 16) P VALUE Sex Male: 7 (%63) Male: 7 (%44) Female: 4 (%37) Female: 9 (%56) Gestasyonel age (day) 191.5 ± 24 189.4 ± 19 0,132 Birth weight (gr) 915.7 ± 278 gr 916.6 ± 225 gr 0,693 RDS 10 (90%) 11 (68.7%) 0,342 NEC 1 (9%) 2 (12.5%) 0,904 SEPSIS 10 (90%) 13 (81.2%) 0,680 IVH 3 (27.2%) 5 (31%) 0,577 ROP 10 (90.9%) 12 (75%) 0,512 Pneumothorax 1 (9%) – 0,716 Abbreviations: RDS, respiratory distress syndrome; NEC, necrotising enterocolitis; IVH, intraventricular hemorrhage; ROP, retinopathy of prematurity. Table 2 Mortality rates and length of in-hospital stay. GROUP A (n = 11) GROUPB (n = 16) P VALUE Death 2 (18.18%) 7 (43.75%) 0,422 Length of in-hospital stay (days) 80 ± 2.3 46,5 ± 1.2 0,349 Table 3 Creatinine levels of the study groups. PREOPERATIVE CREATININE LEVELS POSTOPERATIVE CREATININE LEVELS P VALUE Group A 0,76 + 0,2 0,84 + 0,36 0,37 CREATININE LEVELS BEFORE MEDICAL THERAPY CREATININE LEVELS AFTER MEDICAL THERAPY P VALUE Group B 0,78 + 0,3 1,23 + 0,5 <0,01 ==== Refs REFERENCES 1 Malviya MN Ohlsson A Shah SS Surgical versus medical treatment with cyclooxygenase inhibitors for symptomatic patent ductus arteriosus in preterm infants Cochrane Database Syst Rev 2013 28 3 CD0039 2 Ko SM Yoon YC Cho KH Primary surgical closure should be considered in premature neonates with large patent ductus arteriosus Korean J Thorac Cardiovasc Surg 2013 46 3 178 184 23772404 3 Reller MD Rice MJ McDonald RW Review of studies evaluating ductal patency in the premature infant J Pediatr 1993 122 6 59 62 4 Yang EM Song ES Choi YY Comparison of oral ibuprofen and intravenous indomethacin for the treatment of patent ductus arteriosus in extremely low birth weight infants J Pediatr (Rio J) 2013 89 1 33 39 23544808 5 Poon G Ibuprofen lysine (NeoProfen) for the treatment of patent ductus arteriosus Proc (Bayl Univ Med Cent) 2007 20 1 83 85 17256050 6 Mercanti I Ligi I Boubred F Ibubrofen in the treatment of patent ductus arteriosus in preterm infants: what we know, what we still do not know Curr Pharm Des 2012 18 21 3007 3018 22564295 7 Mercanti I Boubred F Simeoni U Therapeutic closure of the ductus arteriosus: benefits and limitations J Matern Fetal Neonatal Med 2009 22 14 20 19925358 8 Bagnoli F Rossetti A Messina G Mori A Casucci M Tomasini B Treatment of patent ductus arteriosus (PDA) using ibuprofen: renal side-effects in VLBW and ELBW newborns J Matern Fetal Neonatal Med 2013 26 4 423 429 23057804 9 Lee GY Sohn YB Kim MJ Outcome following surgical closure of patent ductus arteriosus in very low birth weight infants in neonatal intensive care unit Yonsei Med J 2008 49 2 265 271 18452264 10 Mosalli R AlFaleh K Paes B Role of prophylactic surgical ligation of patent ductus arteriosus in extremely low birth weight infants: systematic review and implications for clinical practice Ann Pediatr Cardiol 2009 2 2 120 126 20808624	25512700	PMC4251059	NO-CC CODE	2021-01-05 02:18:56	yes	Open J Cardiovasc Surg. 2014 Aug 17; 7:1-4
==== Front PancreasPancreasMPAPancreas0885-31771536-4828Lippincott Williams & Wilkins MPA1366710.1097/MPA.000000000000021700017Original ArticlesRandomized Trial Comparing the Flexible 19G and 25G Needles for Endoscopic Ultrasound-Guided Fine Needle Aspiration of Solid Pancreatic Mass Lesions Ramesh Jayapal MD, FRCP (UK)Bang Ji Young MBBS, MPHHebert-Magee Shantel MD†Trevino Jessica MDEltoum Isam MD, MBA†Frost Andra MD†Hasan Muhammad K. MD‡Logue Amy MSN‡Hawes Robert MD‡Varadarajulu Shyam MD‡From the Division of Gastroenterology and Hepatology and †Department of Pathology, University of Alabama at Birmingham, Birmingham, AL; and ‡Center for Interventional Endoscopy, Florida Hospital, Orlando, FL.Reprints: Shyam Varadarajulu, MD, Center for Interventional Endoscopy, Florida Hospital, 601 East Rollins Street, Orlando, FL 32803 (e-mail: [email protected]).1 2015 11 12 2014 44 1 128 133 7 12 2013 25 6 2014 Copyright © 2014 by Lippincott Williams & Wilkins2014Lippincott Williams & WilkinsThis is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License, where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially.Supplemental digital content is available in the text. Objectives Although a large gauge needle can procure more tissue at endoscopic ultrasound-guided fine needle aspiration (EUS-FNA), its advantage over smaller needles is unclear. This study compared flexible 19G and 25G needles for EUS-FNA of solid pancreatic masses. Methods This was a randomized trial of patients undergoing EUS-FNA of pancreatic masses using flexible 19G or 25G needle. Main outcome measure was to compare median number of passes for on-site diagnosis. Secondary measures were to compare specimen bloodiness, complications, technical failures, and histological core tissue procurement. Results One hundred patients were randomized to EUS-FNA using flexible 19G or 25G needle. Median of 1 pass was required to achieve on-site diagnosis of 96% and 92% (P = 0.68) in 19G and 25G cohorts. There was no significant difference in technical failure (0% vs 2%, P = 0.99) or adverse events (2% vs 0%, P = 0.99) between 19G and 25G cohorts. Although histological core tissue procurement was significantly better with flexible 19G needle (88% vs 44%, P < 0.001), specimens were bloodier (severe bloodiness, 36% vs 4%; P < 0.001). Conclusions As there is no significant difference in the performance of flexible 19G and 25G needles, needle choice for sampling pancreatic masses should be based on endoscopist preference and need for histology. Key Words EUS-FNAsolid pancreatic masspancreatic cancerpancreatic biopsyflexible 19G needlerandomized trialOPEN-ACCESSTRUESDCT ==== Body Endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) is an integral part of the diagnostic algorithm for the evaluation of solid pancreatic mass lesions. Randomized trials have compared the 25G, 22G, and 19G needles in an attempt to identify the optimal needle for tissue acquisition.1–3 Of the 3 studies that compared the 22G and 25G needles,1–3 whereas there was no significant difference in diagnostic accuracy, there was a trend toward better performance with the 25G needle for the FNA of pancreatic head masses. These findings were confirmed in a recent meta-analysis that revealed higher sensitivity of 25G over 22G needles for the EUS-FNA of pancreatic mass lesions.4 A limitation of FNA procedures, particularly when using smaller gauge (22G or 25G) needles, is the limited ability to procure adequate histological samples. Tissue architecture and morphology are essential for accurate pathological assessment of certain lesions, such as lymphomas and gastrointestinal stromal tumors, for which histological core is preferred over a cytological aspirate.5 Although a larger 19G needle can procure histological samples, published data are limited.6–8 In a study of 120 patients who underwent EUS-FNA using the 19G needle, the procedure was technically successful in 98.9%, and adequate histological sample was obtained in 97.5%.6 However, patients with pancreatic head or uncinate masses were excluded in this study. A major limitation of the 19G needle has been its rigidity that makes transduodenal sampling of pancreatic masses difficult because of the stiffness induced by the needle assembly on the echoendoscope shaft. Only 1 randomized trial has compared the performance of 19G and 22G needles for sampling of solid pancreatic mass lesions.7 In that study, although the diagnostic accuracy of the 19G needle was superior, the technical failure rate associated with its use was significantly higher than that for the 22G needle. In a recent study of 548 patients who underwent EUS-FNA, the 25G needle had superior technical performance over the 19G and 22G needles for the sampling of pancreatic head and uncinate lesions.9 An inherent advantage of the 25G needle is its thin caliber that makes transduodenal sampling relatively easier. To circumvent the technical problems encountered with 19G needles, a new nitinol-based platform has recently been developed with the objective of enhancing its flexibility. In a preliminary study of 50 patients with 32 pancreatic head/uncinate mass lesions who underwent transduodenal tissue acquisition with this nitinol-based flexible 19G needle, EUS-FNA was diagnostic in 92.1%, histological core tissue was procured successfully in 94.7%, and the combined diagnostic accuracy was 100%.8 Given the increased use of 25G needles for the FNA of pancreatic masses, particularly head lesions, and recent advancements in the 19G platform, we conducted a randomized trial to compare the technical performance of both needle systems for the sampling of solid pancreatic mass lesions. MATERIALS AND METHODS Patients and Settings This was a multicenter randomized trial conducted at Florida Hospital and the University of Alabama at Birmingham (ClinicalTrials.gov identifier: NCT01677312; registration date: August 28, 2012). All patients 19 years and older with suspected solid pancreatic mass lesions that were identified on computed tomogram scan and referred for EUS-FNA were eligible for participation in this study. Patients were excluded if a pancreatic mass lesion was not seen at EUS, the mass had a cystic component, or if the coagulation parameters were abnormal. The respective institutional review boards of Florida Hospital and the University of Alabama at Birmingham approved the study on August 28, 2012. Written informed consents were obtained from all patients for participation in the trial. The full study protocol can be accessed from the ClinicalTrials.gov Web site, and the CONSORT checklist for randomized trials has been included as Supplemental Digital Content 1 (http://links.lww.com/MPA/A329). All authors had access to the study data and have reviewed and approved the final manuscript. Randomization and Masking Computer-generated randomization assignments were obtained before study enrollment using the block randomization method by the statistician. These were then placed in sequentially numbered sealed opaque envelopes and opened by the endoscopy nurse during the procedure when patients met criteria for study inclusion. Patients were randomized equally to the 2 needle types (1:1 allocation). Procedural Technique Patients underwent EUS-FNA with either the flexible 19G or 25G needle (Expect; Boston Scientific Corporation, Natick, MA) by 1 of the 5 experienced endosonographers (all with advanced endoscopy training and >500 EUS procedures/year) at either medical center. All pancreatic head and uncinate masses were accessed via the duodenum, whereas all pancreatic body and tail masses were sampled via the stomach.9 All procedures were performed using a linear array echoendoscope (Olympus UCT140; Olympus America Corp, Center Valley, PA) with patients in the left lateral decubitus position under moderate sedation or after administration of propofol. At EUS, during individual FNA passes, after puncturing the pancreatic mass, the stylet was removed, and the needle was moved to-and-fro, 12 to 16 times, at different areas within the lesion using the fanning technique.10 As described in a previous report, suction was not applied, and the stylet was not reintroduced into the needle after the first pass in any patient.11 Specimen Handling Tissue material was expressed onto slides by advancing the stylet within the needle assembly. After the initial pass, which was collected in cell block (Hank balanced salt solution; Invitrogen, Grand Island, NY), an attending pathologist who was blinded to the type of needle being used processed the subsequent specimens on-site. A maximum of 6 passes (excluding the cell block) were performed using the original needle type, and if there was diagnostic or technical failure, the patient underwent crossover to the alternate needle. However, if a definitive diagnosis was established during the initial attempt, the procedure was terminated, and the number of passes performed was documented. Preparation of Specimen for On-Site Analysis Air-dried and alcohol-stained smears were prepared on-site after individual passes. Air-dried smears were stained with Three-Step Stain (Richard-Allan Scientific, Kalamazoo, MI) and immediately reviewed by a cytopathologist who was blinded to the needle type used, to establish the on-site diagnosis and bloodiness of the specimen. Alcohol-stained smears were prepared off-site in the pathology laboratory using the Papanicolaou stain. Preparation of Cell Block for Histological Analysis In the laboratory, a 10-mL vial of Hank balanced salt solution containing the collected specimen was placed into the centrifuge, counter-balanced, and spun for 5 minutes. If the specimen quantity was sufficient, the supernatant was removed, and 3 drops of plasma and thrombin were added to the sediment. Upon formation of a clot, the cell button was removed intact, enclosed in a Tissue-Loc HistoScreen cassette (Microm International, Walldorf, Germany), and fixed in formalin. The cassette was processed, embedded in paraffin, and then prepared in hematoxylin and eosin to be evaluated for the presence of a histological core. Core tissue was quantified using cellSens (Olympus America Inc, Center Valley, PA), a specially designed software that measures dimensions of core tissue ex-vivo under microscopy in millimeters.2 When required, immunohistochemical or special staining was performed for the differentiation of morphologically challenging lesions. Outcome Measures and Follow-Up The primary objective was to compare the median number of passes required to establish on-site diagnosis. The secondary outcome measure was to compare the bloodiness of specimen, adverse events, technical failure, and the ability to procure histological core. Immediate complications were documented at the time of the procedure, and late complications were documented via telephone follow-up at 24 hours and 10 days after the procedure. Statistical Analysis In line with our primary objective, a 2-tailed sample size calculation was performed with type I error rate (α) set at 0.05 to attain 90% of power for detecting a difference of 1 pass in the number of passes needed to acquire an on-site diagnosis. On the basis of previous studies,1,8 SDs of 0.79 for the 19G needle group and 2.0 for the 25G needle group were used, which produced target sample sizes of 49 for each group and was set at 50 patients per group to allow for possible dropouts. Baseline characteristics of the patient population, pancreatic mass lesions, technical details, and procedure outcomes were summarized as means (with SD) or medians (with interquartile range [IQR] and range) for continuous data and as frequencies and proportions for categorical data. For the comparison of categorical data, χ2 or Fisher exact test was used as indicated, whereas the 2-sample t test or the Wilcoxon rank-sum test was used as appropriate for the comparison of continuous data. Statistical significance was determined as P < 0.05. Data sets were compiled using Microsoft Excel, and all statistical analyses were performed using Stata 13 (Stata Corp, College Station, TX). Definitions On-site diagnosis was defined as the proportion of patients in whom the tissue was diagnostically sufficient to render a preliminary on-site diagnosis using the original needle. Diagnostic failure (nondiagnostic) was defined as an inability to obtain sufficient diagnostic material for on-site diagnosis despite 6 passes with the original needle. Technical failure was defined as malfunction of the needle before an on-site assessment could be rendered. Core tissue was defined as a continuous string of material that retains its histological tissue architecture on microscopic examination. Specimen bloodiness was categorized based on the percentage of blood in the microscopic field: mild, less than 33%; moderate, 33% to 66%; and severe, greater than 66%. RESULTS Of the 130 patients who were screened for participation in this study between August 30, 2012, and January 25, 2013, 30 were excluded as a pancreatic lesion was not visualized at EUS in 19 patients, the lesion was cystic in 10 patients, and 1 patient refused participation. A total of 100 patients with solid pancreatic mass lesions constituted the study cohort and were randomized equally to the 2 needle groups. Patient Demographics and Tumor Characteristics Except for tumor size, which was larger in patients randomized to the flexible 19G needle (median size, 37.5 vs 35 mm; P = 0.03), there was no difference in patient demographics or tumor characteristics between the 2 cohorts (Table 1). More than 80% of lesions in both cohorts were neoplastic, and 61% of masses were located in the pancreatic head/uncinate region requiring transduodenal sampling. Vascular invasion was observed at EUS in 61% of patients. The final diagnosis was pancreatic adenocarcinoma or other type of malignant tumor in 76 patients (76%), neuroendocrine tumor in 7 patients (7%), chronic pancreatitis in 13 patients (13%), primary pancreatic lymphoma in 2 patients (2%), and intrapancreatic accessory spleen in 2 patients (2%). TABLE 1 Patient and Pancreatic Mass Characteristics Outcome Measures There was no significant difference in the median number of passes required to establish on-site diagnosis (1 [IQR, 1–1] vs 1 [IQR, 1–1], P = 0.41), rates of technical failure (0% vs 2%, P = 0.99), or procedural complications (2% vs 0%, P = 0.99) between the flexible 19G and 25G cohorts, respectively (Table 2). On-site diagnosis could not be established in 2 patients with pancreatic head masses in the flexible 19G cohort: 1 patient had a nondiagnostic aspirate, was crossed over to the 25G cohort, and was diagnosed to have pancreatic adenocarcinoma. Another patient developed bleeding (adverse event) at the sixth pass, and the procedure was terminated without crossover. However, at cell block, this patient was diagnosed to have pancreatic adenocarcinoma. On-site diagnosis could not be established in 4 patients in the 25G cohort, because of 3 nondiagnostic aspirates and 1 technical failure. Of the 3 patients with nondiagnostic aspirates who underwent crossover to the flexible 19G needle, one was diagnosed with pancreatic spindle cell cancer located in the pancreatic body, and two had chronic pancreatitis (1 lesion located in the head and the other in the tail of the pancreas). Technical failure was encountered in 1 patient who underwent transgastric FNA of a pancreatic body mass: the bent 25G needle could not be released out of the sheath after the third pass (all of which were nondiagnostic), and the patient was crossed over to the flexible 19G needle with FNA revealing a lymphoma. A flow diagram of the study results is shown in Figure 1. FIGURE 1 Flow diagram of the study results. TABLE 2 Technical Details and Outcomes Intraprocedural bleeding was observed at the sixth pass in a male patient who underwent transduodenal FNA of a pancreatic head mass using the flexible 19G needle. The bleeding resolved spontaneously, but the patient also developed postprocedure fever that was managed by administration of intravenous antibiotics. As stated earlier, the cell block in this patient eventually revealed a pancreatic adenocarcinoma. At the on-site assessment, severe bloodiness was observed in significantly more patients randomized to the flexible 19G than to the 25G needle (36% vs 4%, P < 0.001). Histological core tissue was also present in significantly greater proportion of patients randomized to the flexible 19G than to the 25G needle (88% vs 44%, P < 0.001), with a significant difference in the area of diagnostic core tissue between the 2 needles (median area, 1.79 mm2 for flexible 19G needle vs 0.37 mm2 for 25G needle; P = 0.007). Follow-Up At a median follow-up of 7 months (range, 2–12 months), 37 patients with neoplastic disease were undergoing chemoradiation, 13 had undergone definitive surgery, 12 elected for palliative care, and 22 were deceased. Fifteen patients with benign disease and one with neuroendocrine tumor on follow-up had no clinical or radiological disease progression. All patients who underwent surgical resection were found to have malignant disease, and there were no false-positive diagnoses in any patient. Likewise, on follow-up, none of the patients diagnosed with chronic pancreatitis had disease progression either clinically or on radiological imaging and were doing well. In addition, none of the patients who underwent chemoradiation had subsequent surgical resection as there was documented disease progression on follow-up computed tomogram scan with deterioration in clinical well-being. DISCUSSION In this study, the first randomized trial to compare the flexible 19G and 25G needles for sampling solid pancreatic mass lesions, the technical performance of the flexible 19G needle was comparable with that of the 25G needle. The major advantage of the flexible 19G needle was the ability to procure histological core tissue. The EUS-FNA of pancreatic lesions with the large bore 19G needle has been fraught with technical challenges. In a comparative study that evaluated the 19G Trucut, 19G, and 22G FNA needles for sampling solid pancreatic mass lesions, the technical success rates for sampling uncinate lesions were 0% versus 0% versus 100% and, for head lesions, were 60% versus 60% versus 100%, respectively.12 In a prospective randomized trial that compared the 19 and 22G needles, the technical success rate for sampling pancreatic head masses was significantly lower for the 19G needle than for the 22G needle (80.8% vs 100%).7 In another study that evaluated the 19G needle, although FNA of pancreatic uncinate lesions was not feasible in any patient, the success rate for FNA of head lesions was only 33.3%.13 In contrast, the present study shows that, when using the flexible 19G needle, mass lesions located anywhere in the pancreas can be successfully sampled, including patients crossed over from the 25G cohort because of technical failure. The diagnostic sensitivity of EUS-FNA is incumbent on the presence of an on-site cytopathologist, and the 25G needle is considered the most optimal for on-site tissue acquisition.14,15 There are limited data on the ability to provide rapid on-site interpretation when using the 19G needle.8 In the present study, although there was no difference in the ability to render on-site diagnosis between the flexible 19G and 25G needles, specimens procured using the flexible 19G needle were bloodier. Therefore, when using the flexible 19G needle, to minimize specimen bloodiness, it is important not to “jab” the same area in the mass repeatedly but rather “fan” the needle at different locations within the mass to procure a better quality sample. In the present study, although the specimens procured using the flexible 19G needles were bloodier, it did not impede on-site assessment in most patients because of the presence of adequate diagnostic tissue. The ability to render immediate diagnosis is particularly relevant for centers that have access to on-site cytopathology services so that patient care can be expedited. Several types of biopsy needles have been developed to procure histological core specimens, which sometimes are important for the diagnosis of challenging diseases such as well-differentiated pancreatic cancer or lymphoma and for the assessment of molecular markers in metastatic lung or breast cancer to facilitate receptor-specific chemotherapy.16,17 Although the relevance of core tissue procurement for such indications is still being debated and is outside the scope of this study, herein, we demonstrate that the flexible 19G FNA needle can procure a histological core tissue, in 1 pass, in more than 85% of patients. In addition, two of the patients randomized to the 25G needle were crossed over to the flexible 19G needle (because of nondiagnostic FNA in 1 patient and needle dysfunction in the other), after which the diagnoses of pancreatic lymphoma and spindle cell neoplasm were established. In rare instances, a core biopsy needle or the large caliber 19G needle is required for better morphological characterization of tumors that may not be always feasible with standard FNA needles.5 On the other hand, a diagnostic core was procured in only 44% of the patients when using the 25G needle. It is likely that, if multiple passes are performed for cell block using a 25G needle, then histological assessment may be possible in a higher proportion of patients. However, the number of passes required for an adequate cell block when using the 25G needle is unclear. Although not proven by this trial, an indirect implication of these findings is that, in centers that do not have access to on-site cytopathology services, the 19G needle can be used to procure histological specimens, which can then be processed off-site more easily using standard techniques. There are limited data on the safety profile of the 19G FNA needle for tissue procurement.14 In this study, 1 patient experienced intraprocedural bleeding and postprocedure fever after transduodenal EUS-FNA using a flexible 19G needle. Although we encountered only 1 adverse event when using the flexible 19G needle, the study was not adequately powered to compare the rate of adverse events between the 2 cohorts. Other studies that have evaluated the 19G needle for tissue acquisition report no adverse events associated with its use.6–8,12 In a recent meta-analysis that compared the 19G versus 22G/25G needles for EUS-FNA of solid pancreatic mass lesions, there was no difference in the rates of complications between the 2 cohorts.18 There are several limitations to our study. First, only solid pancreatic masses were evaluated, and the performance of both needles in other organs was not compared. Second, the median size of the pancreatic mass lesions was more than 30 mm, which may have made tissue acquisition less challenging with the flexible 19G needle. Third, per protocol, only 1 dedicated pass was obtained for histological core tissue assessment (cell block). It is therefore possible that more passes could have significantly improved the diagnostic yield in cell block. Fourth, the endoscopists were not blinded to the type of needle being used, and the possibility of bias cannot be excluded. However, the pathologists were blinded to the procedural details, and the influence of bias on the study findings is likely minimal. Fifth, all procedures were performed by expert endosonographers in tertiary referral centers with experienced on-site cytopathology support, and these results may not be applicable to all units. Sixth, although we recommend the fanning technique and not puncturing the same area repeatedly, the optimal technique for tissue procurement using the 19G needle is not standardized. Prospective, comparative studies are therefore needed to identify the optimal maneuver for tissue procurement when using a large caliber 19G needle. In addition, comparative studies evaluating the different 19G platforms are required to identify the most suitable needle for transduodenal sampling. Finally, the diagnostic accuracy of the cytological samples obtained using EUS-FNA was not compared with the final histological diagnosis (gold standard). However, with the exception of 1 patient in the flexible 19G cohort who experienced bleeding and subsequently had a nondiagnostic on-site aspirate that was later proven to be adenocarcinoma on cell block, there was no difference in diagnostic assessment between on-site versus final cytopathological reporting. These findings are in line with a previous study from our institution that revealed an excellent agreement (kappa, 84%) between on-site and final cytologic evaluation.19 In conclusion, the current platform of 19G needles enables tissue acquisition irrespective of the location of the mass in the pancreas. As there is no significant difference in the performance of flexible 19G and 25G needles, the choice of a needle should be based on endoscopist preference and the need for histological core tissue procurement. Supplementary Material SUPPLEMENTARY MATERIAL Presented at the ASGE Topic Forum, Digestive Diseases Week 2013, Orlando, Florida. Shyam Varadarajulu and Robert Hawes are consultants for Boston Scientific Corporation and Olympus America Inc. The remaining authors have no relevant disclosures. Supplemental digital contents are available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal’s Web site (www.pancreasjournal.com). ==== Refs REFERENCES 1 Camellini L Carlinfante G Azzolini F A randomized clinical trial comparing 22G and 25G needles in endoscopic ultrasound-guided fine-needle aspiration of solid lesions . Endoscopy . 2011 ; 43 : 709 – 715 .21611946 2 Fabbri C Polifemo AM Luigiano C Endoscopic ultrasound-guided fine needle aspiration with 22- and 25-gauge needles in solid pancreatic masses: a prospective comparative study with randomisation of needle sequence . 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==== Front Environ Sci TechnolEnviron. Sci. TechnolesesthagEnvironmental Science & Technology0013-936X1520-5851American Chemical Society 10.1021/es503827yArticlePolycyclic Aromatic Hydrocarbon (PAH) and Oxygenated PAH (OPAH) Air–Water Exchange during the Deepwater Horizon Oil Spill Tidwell Lane G. Allan Sarah E. O’Connell Steven G. Hobbie Kevin A. Smith Brian W. Anderson Kim A. Environmental and Molecular Toxicology Department, Oregon State University, ALS 1007, 2750 SW Campus Way, Corvallis, Oregon 97331, United States E-mail: [email protected]. Phone: 541-737-8501. Fax: 541-737-0497.20 11 2015 20 11 2014 06 01 2015 49 1 141 149 06 08 2014 20 11 2014 17 11 2014 Copyright © 2014 American Chemical Society2014American Chemical SocietyThis is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes. Passive sampling devices were used to measure air vapor and water dissolved phase concentrations of 33 polycyclic aromatic hydrocarbons (PAHs) and 22 oxygenated PAHs (OPAHs) at four Gulf of Mexico coastal sites prior to, during, and after shoreline oiling from the Deepwater Horizon oil spill (DWH). Measurements were taken at each site over a 13 month period, and flux across the water–air boundary was determined. This is the first report of vapor phase and flux of both PAHs and OPAHs during the DWH. Vapor phase sum PAH and OPAH concentrations ranged between 1 and 24 ng/m3 and 0.3 and 27 ng/m3, respectively. PAH and OPAH concentrations in air exhibited different spatial and temporal trends than in water, and air–water flux of 13 individual PAHs were strongly associated with the DWH incident. The largest PAH volatilizations occurred at the sites in Alabama and Mississippi in the summer, each nominally 10 000 ng/m2/day. Acenaphthene was the PAH with the highest observed volatilization rate of 6800 ng/m2/day in September 2010. This work represents additional evidence of the DWH incident contributing to air contamination, and provides one of the first quantitative air–water chemical flux determinations with passive sampling technology. National Institutes of Health, United Statesdocument-id-old-9es503827ydocument-id-new-14es-2014-03827yccc-price ==== Body Introduction The explosion of the Deepwater Horizon (DWH) oil rig on April 20, 2010 led to the release of approximately 4.1 million gallons of oil into the Gulf of Mexico.1 Between April 28 and July 19, 411 in situ burns were undertaken to remove oil from the water surface.2 An additional nonmechanical response included the application of 2.1 million gallons of chemical dispersants at the wellhead and to off-shore surface waters which likely increased the freely dissolved fraction of the oil constituents.3,4 Crude oil released from the Macondo 252 well during the DWH incident contained an estimated 3.9% of polycyclic aromatic hydrocarbons (PAHs) by weight.5 PAHs are chemicals of concern in oil, and their fate and transport in the environment is an important component of understanding potential impacts from spills. Whereas PAHs have been widely studied for many decades, oxygenated polycyclic aromatic hydrocarbons (OPAHs) are an emerging contaminant of concern. Interest in OPAHs has increased in the past decade due to their presence in the environment coupled with the toxicity of some OPAHs.6,7 Individual OPAHs consist of one or more oxygen atoms attached to an aromatic ring structure that may also contain other chemical groups.8 Formation of these compounds through both biotic and abiotic mechanisms could be expected during the DWH incident, especially through photo-oxidation of PAHs in air and waters.6,9 Low-density polyethylene (LDPE) passive sampling devices (PSDs) have been used in water and air to assess time-integrated environmental concentrations of many dissolved and vapor phase contaminants, respectively, including PAHs and OPAHs.10−12 Air sampling is often focused on determining the concentration of particulate-bound chemicals; however, exposure to the PAH vapor phase has been shown to account for up to 86% of the cancer risk from inhalation exposure.13−15 Vapor-phase PAHs are by definition unbound to particulates which means this atmospheric fraction is respirable and bioavailable. Similarly, the dissolved fraction of contaminants in water is bioavailable for passive uptake by organisms.10,16 In addition to being biologically relevant, the gas-phase and dissolved concentrations of chemicals are the fractions that flux from one environmental compartment to another.17−19 PSDs are ideally suited for flux measurements since they specifically sequester the dissolved-water phase and air-vapor phase fractions. Generally, great effort is required to acquire the dissolved water fraction including multiple filtration steps and solid phase cleanup(s).20 Additionally, the filtered water is only operationally defined as dissolved, as any particles smaller than the filter are also extracted. The PSDs employed are not subject to filter bias due to their lipophilic carbon polymer design and average pore size of 10 Å that characterize diffusion samplers.11 Until recently, investigation of flux using PSDs has only assessed the overall direction of flux based on the air–water partition compound coefficients (Kaw), or through concentration gradients measured in both air and water samples.21,22 To the authors’ knowledge there are only two reports of measuring the actual magnitude of PAH flux using PSDs, and a separate investigation of flux targeting a different chemical class.23−25 The air–water flux of PAHs is an important factor in understanding the fate of spilled oil.26 Though air and water quality monitoring were conducted during the DWH oil spill, no studies to the authors’ knowledge have reported on flux of PAHs and OPAHs across the air–water boundary. In this study, we present the air-vapor phase PAH and OPAHs at coastal sites in Louisiana, Mississippi, Alabama, and Florida prior to, during, and after shoreline oiling. Spatial and temporal trends for individual PAHs and OPAHs were examined during 13 months. We also address the data gap of aromatic hydrocarbon flux during episodic events. Using PSDs for quantitative flux assessment is a developing technological advancement in environmental chemistry, and this work is the first to quantitatively measure flux during an environmental disaster. Materials and Methods Sample Collection Sampling was performed at four coastal sites: Grand Isle, Louisiana (LA); Gulfport, Mississippi (MS); Gulf Shores, Alabama (AL); and Gulf Breeze, Florida (FL) (Figure 1A and B). Air and water samples were collected concurrently during 12 sampling events from May 2010 to June 2011; sampling durations for flux assessment ranged from 3 to 41 days (see Supporting Information (SI) Table 1 for specific dates).16 Figure 1 (A) Sampling locations along the Gulf of Mexico. (B) Samplers deployed off piers at each sampling site. (C) Air sampling cage affixed to pier in Gulf Breeze, FL. Stainless steel air sampler cages that allowed for air circulation while minimizing sampler exposure to water, particulate depositions, and UV (Figure 1C) were deployed concurrently with water cages described previously.16 A total of five PSDs were deployed in each air or water cage. Air samplers were located between 1 and 5 m above the water surface and were directly above water samplers.16 Approximately 1-m-long PSDs were constructed from LDPE tubing, and were fortified with deuterated PAH performance reference compounds (PRCs) for water or air sampling rate calculations. A list of PRCs can be found in SI List 1. PRCs spanned a range of Koa/Kows similar to the target analyte PAHs and OPAHs, and the most similar PRC was used for quantification (SI List 1). OPAHs were quantified using PAH PRCs; any biases generated from this approach are conservative since PAHs have slightly higher Koa/Kows than the analogous OPAHs.27−30 Detailed PSD conditioning, construction, cleanup, and extraction is described in Anderson et al. 2008.31 PSDs were transported in sealed polytetrafluoroethylene (PTFE) airtight bags. Samples were stored in the laboratory at 4 °C until extraction within 2 weeks of receipt. Sample Processing and Chemical Analysis All solvents used were Optima grade or better (Fisher Scientific, Pittsburgh, PA), and standards were purchased at purities ≥97%. All five PSDs from each cage were extracted as a composite representing a single sample in order to increase analytic sensitivity. PAH and OPAH in PSDs were extracted by dialysis with n-hexane detailed in Anderson et al.;31 use of n-hexane for extraction of OPAH is explained in O’Connell et al..32 Extracts were stored in amber glass vials at −20 °C until instrumental analysis. PAH and OPAH analysis used an Agilent 5975B gas chromatograph–mass spectrometer (GC-MS) with an Agilent DB-5MS column (30 m × 0.25 mm × 0.25 μm) in electron impact mode (70 eV) using selective ion monitoring (SIM). PAH GC parameters are detailed in Allan et al.,16 and OPAH parameters are detailed in O’Connell et al.12 Six and nine point calibration curves for PAHs and OPAHs, respectively, had correlation coefficients >0.98 for all target analytes. A list of measured analytes is provided in SI Lists 1 and 2. Quality Control Quality control (QC) samples accounted for over 30% of the total number of samples analyzed and included the following: PSD construction blanks, field and trip blanks for each deployment and retrieval, postdeployment cleaning blanks, and laboratory reagent blanks. Extraction surrogates were added to all samples immediately prior to extraction, and concentrations were surrogate corrected. All compounds were below detection limits in all blank QC samples. Mean extraction surrogate recoveries were 52.5% (range 37–113) for naphthalene-D8, 67.8% (range 53–116) for acenaphthylene-D8, 80.1% (range 77–113) phenanthrene-D10, 97.8% (84–118) for fluoranthene-D10, 105% (86–139) for chrysene-D12, 80.5%(68–90) for benzo(a)pyrene-D12, 66.7% (50–85) for dibenzo(g,h,i)perylene-D12, 66% (44–80) for 1,4-naphthalenequnione-D8, 104% (80–140) for 9-flourenone-D8, and 96% (60–150) for 9,10-anthraquinone-D8. Air–Water Flux Calculation Environmental vapor concentrations were determined using an empirical uptake model with sampling rates derived by measuring PRC loss as described by Huckins et al. and others.11,27,29,33 Details and formulas are presented in the SI. Previously published water concentrations were used for calculation of PAH flux and are described in detail in Allan et al.16 The exchange of chemicals between air and water at the interface can be described as the movement of a chemical from the bulk phase, followed by transport across the thin films of each phase into the receiving compartment. The Whitman two film model is used to calculate this movement: 1 where F is the flux (ng/m2 day–1), the total mass-transfer rate coefficient is Kol (m/day), and Cw and Ca are the dissolved and vapor phase concentrations in the water and air, respectively.17,18H′, in this case, is a compound-specific temperature-corrected Henry’s law value, and can be calculated using eq 2: 2 where R is the ideal gas constant (8.2057 × 10–5 m3 atm K–1 mol–1) and T is the temperature in Kelvin. Air and water temperatures were collected hourly using temperature loggers co-located with PSDs at each sampling site. The average temperature over each deployment was calculated and used for assessment of the temperature-corrected Henry’s law values. The total mass transfer coefficient in eq 1 can be calculated according to eq 3: 3 where ka is the air side mass transfer coefficient and kw is the water side mass transfer coefficient. Average wind speed over the course of the deployment was calculated from NOAA data published on the tides and currents web interface.34 Published diffusivity values for 13 PAHs were used to calculate Schmidt values as inputs for mass transfer coefficients.35 An estimate of OPAH flux was performed on 7 OPAHs, using PAH analogue diffusivity values, and are considered semiquantitative as a result. Details of the calculations are further described in Johnson and Bamford et al.36,18 Flux was only assessed when the compound was detected in both environmental compartments. PSD concentrations represent a time-weighted average concentration, therefore the net flux for each sampling period is the time-weighted average flux over the sampling duration. Using PSDs to assess the time-weighted flux provides an alternative new way to characterize movement of chemicals over a time period. PSD flux is especially well suited to applications where episodic changes and releases are important to capture and characterize. Assigning additional uncertainties to mass transfer coefficients derived from average values was determined to be an overly conservative approach. As a result, the error bars present on the flux figures in Figures 2 and 4 represent the pooled variance of the flux from an n = 12 replication study performed in the Gulf of Mexico during this study. Figure 2 (A) Σ33PAH vapor phase concentrations in air. (B) Σ33PAH dissolved concentrations in water.16 (C) Σ13PAH net flux. (D) Phenanthrene flux. (E) Naphthalene flux. (F) Fluoranthene flux. Error bars represent the calculated 95% confidence interval based on pooled variance from a replication study. Data Modeling Differences between sites and between sampling times were assessed using Wilcoxon rank-sum tests, and differences were considered significant at a probability value of p ≤ 0.05. Confidence intervals were calculated from a Gulf of Mexico air and water replication study performed using n = 12 PSDs and represents the pooled variance.37 The average percent difference between SUM PAH replicates in water and air at each site were 18 and 41, respectively. Principal component analysis (PCA) was used to explore changes in chemical profiles of samples; a specific description can be found in the SI. Analytes in the PCA below detection limits were assigned a value of one-half the limit of detection, information on detection limits can be found in SI Table 3. Results and Discussion Vapor PAHs in Coastal Air of Four Gulf Coast States Prior to shoreline oiling at LA, the measured ∑33airPAH at this site was 16 (±5) ng/m3. This increased to 23 (±7) ng/m3 the following month when there was visible shoreline oiling (Figure 2A). The June-1 sampling event was significantly greater (p ≤ 0.04) than sampling periods later in summer 2010. ∑33airPAH concentrations tended to increase earlier than PAH concentrations in water (Figure 2B from Allan et al.16), which could be due to faster atmospheric transport, as well as contributions from in situ burn events.38,39 At MS, the May 2010 and June-1 ∑33airPAH were significantly above all other sampling times (p < 0.05). The May 2010 and June-1 maximum concentrations observed in air are similar to the LA site. Although Middlebrook et al. did not measure PAHs (except naphthalene) their bulk organic carbon measurements, taken at concurrent time points with the June-1 sampling, are consistent with our high ∑33airPAH, providing converging lines of evidence that the DWH incident had tangible impacts on near-shore Gulf of Mexico air.40 The temporal trend of bioavailable PAHs at the AL site was different from that of LA and MS sites (Figure 2A). The ∑33airPAH concentrations were generally ≤2 ng/m3. The highest observed ∑33waterPAH was in September at 25 (±2) ng/m3; the highest measured ∑33airPAH concentration was during the winter.16 High wind events and continued near-shore cleanup activities were observed during those sampling periods (SI Table 2) and the air PAH trend observed is consistent with a recirculation/suspension of contaminated waters/sediments and some volatilization. Other possible explanations include increased local inputs such as marine traffic or other oil sources. The coastal air at FL had an initial ∑33airPAH concentration of 4 (±1) ng/m3. A trend of decreasing ∑33airPAH from May 2010 through August was observed, but was not statistically different from other sampling periods (p = 0.7). The FL air ∑33airPAH are about 8-fold less than those observed in LA or MS in May 2010 and June-1. All sites taken together displayed a temporal pattern of increases in the maximum air PAH concentrations occurring earlier at the western sites and later in the eastern locations. This could be explained by the distance of the sites from the wellhead, in addition to in situ burns and air currents in the Gulf of Mexico.40,41 The sites at LA and MS were most heavily impacted in May and June 2010. A similar trend was observed in water samples.16 Dispersion and aging of oil and oil chemicals could also explain this trend; if DWH were a primary source of PAHs during this time period, then a decrease of vapor phase PAH would be expected.42,43 Comparing Gulf of Mexico Air PAHs to Literature Values The vapor-phase ∑33airPAH concentrations in this study ranged between 2 and 23 ng/m3, and are similar to vapor-phase Σ13PAH concentrations of 3.06 and 24.1 ng/m3 recorded in the coastal metropolitan region of Kozani and the rural region of Petrana Greece, respectively.44 A 2006 study near a petroleum industry harbor in Belgium found Σ16PAH vapor phase concentrations to range between 15 and 135 ng/m3 during different seasons, overlapping with the measured PAHs in this study.45 Conversely, very high vapor phase concentrations were observed in the inland metropolitan region of Alexandria Egypt, with Σ42PAH concentrations ranging between 390 and 990 ng/m3.46 The highest individual PAH contributions to the total PAH load in this study were phenanthrene and 2-methyl phenanthrene (SI Figures S1 and S2), which are similar to other studies of vapor-phase PAHs at petroleum impacted sites or areas of moderate urbanization.44,45 PAH Air–Water Exchange Predicting the fate of PAHs during environmental disasters includes characterizing the exchange of PAHs across the air–water boundary.18,20 Whereas many fate models for the DWH oil spill assumed volatilization was an important transfer and fate mechanism, this pathway has not been directly quantified.26 Air–water exchange (flux) of 13 PAHs was determined at the four sites over a 13-month period that spanned the DWH incident (Figure 2C–E). Σ13PAH net flux was positive; meaning volatilization of PAHs from the water to the air occurred at all sampling sites and all time points during this investigation. The greatest ∑13PAH net flux to air occurred at MS and AL, observed during June-2 at 9570 (±5000) ng/m2/day, and September at 11 200 (±6000) ng/m2/day, respectively. The ∑13PAH flux to air peaked at LA in June-1, at 7600 (±4000) ng/m2/day. After the DWH in situ burns stopped and the well head was capped, the ∑13PAH flux generally decreased at those sites. Interestingly, ten months after the peak (i.e., June-1) ∑13PAH flux volatilization was about 6-fold lower at the LA site, but flux of PAHs from water to air was still 2.5-fold greater than observed in May 2010 prior to shoreline oiling. This may be due to the continuing influence of DWH oil in this area. The FL ∑13PAH net flux, volatilization, was significantly less (p = 0.05) than that at the three other sites. Individual PAHs showed more variability in flux direction and magnitude than the net PAH flux. The greatest volatilization at MS and AL was naphthalene at 9370 (±600) and 4850 (±300) ng/m2/day during the June-2 sampling event. The largest individual PAH volatilization at LA was phenanthrene at 7390 (±5000) ng/m2/day in June-1 and the largest deposition was −665 (±400) ng/m2/day in May 2010. The shift of flux from deposition to volatilization for phenanthrene is an important indicator of increased dissolved PAH levels in water rather than decreased vapor-phase PAH levels in air. In 1999, Bamford et al. found that local inputs from an urban setting to surface waters resulted in similar degassing events, indicating that local sources such as industrial activities, or an oil spill in this case, may strongly influence the flux of PAHs.18 Phenanthrene and fluoranthene underwent the largest observed single PAH deposition events at MS and AL at a rates of −905 (±600) ng/m2/day in May 2010 and −45.5 (±20) ng/m2/day in June-1, respectively (Figure 2D and 2F). Few other relevant studies are available for comparison of individual PAHs, but a study in a heavily industrialized harbor in Taiwan showed phenanthrene to be undergoing deposition during 19 of the 22 sampling time points.19 Additionally, another study reported the observed mean annual flux of phenanthrene along the southern coastline of Singapore to be −457 (±490) ng/m2/day.47 A third investigation shows phenanthrene in deposition phase for all but one observation in Lake Erie and Lake Ontario,23 and a fourth investigation of PAH flux found phenanthrene to be in or near equilibrium for all sites with detectable levels of phenanthrene in both air and water.25 All of these studies illustrate that the typical trend for phenanthrene is deposition under many environmental conditions, however, we found during the DWH incident along the coast of the Gulf of Mexico phenanthrene was volatizing. The change from deposition to volatilization of phenanthrene at LA, MS, and FL sites give strength to the supposition that the influx of hydrocarbons from the DWH incident changed the direction of phenanthrene flux well after visible oil was gone. The AL site did not shift from deposition to volatilization as observed at the other three sites and does not exhibit the characteristic phenanthrene deposition observed in other flux investigations.19,23,47 However, there was an increase in volatilization later in the study showing that a perturbation of the steady-state flux at this site occurred. Continuous volatilization of phenanthrene at AL might be explained by the local marine and residential activities which were in close proximity. The proximity of residential and marine activity at AL may have introduced phenanthrene directly to the water through runoff or marine engine use and maintenance. Sources such as local residences and other anthropogenic activities have been shown to affect PAH air–water dynamics.18,23 PAH Chemical Profiles and Source Modeling Principle component analysis (PCA) using air data in profile form was used to produce score and loading plots, shown in Figure 3A. The two score plots differ only by the choice of deployment time or state labels. PCA was also performed on individual state air data, as seen in Figure 3B. The score plots show good delineation between precap and postcap and also give a clear time trajectory for the first five sampling events, where the first samples taken in each state have the majority of the variability explained by PC1. As the sampling progressed after the DWH incident, p12 (1-methylphenathrene) and p14 (2-methylphenathrene) variability and percent contribution decreased until sampling events 6–10 (September–May 2011) show little to no difference between site or deployment in terms of PAH profile (Figure 3). Furthermore, postcap sampling events 4–10 (July–May 2011) show less intrasample variation than precap observations, suggesting the homogeneity of postcap samples consistent with a single episodic event (Figure 3). Postcap sampling periods of July and August (labeled as 4 and 5) show a transitional behavior which is most apparent when looking at PCA graphs for individual states (Figure 3B). The loading plot in Figure 3A shows precap samples have relatively high percentages of the alkylated PAHs compared to the parent PAHs, labeled as p12 (1-methylphenanthrene) and p14 (2-methylphenanthrene) and are consistent with a petrogenic source (see SI Figure S2).48,49 In contrast, the postcap samples tend to have high percentages of the parent PAHs consistent with pyrogenic sources, such as pyrene labeled as p20.48,49 Figure 3 (A) Principal component analysis (PCA) plots. Green and red triangles represent samples prior to, and after, the well head was capped, respectively. A1 is labeled by state, A2 is labeled by events numbered 1–10 (representing May 2010–May 2011), and A3 are PAH vectors (p20 = pyrene, p17 = fluoranthene, p14 = 1-methylphenanthrene, p12 = 2methylphenanthrene, p10 = phenanthrene). (B) Individual state PCA plots. (C) Ratio of 2–3 ring/4–6 ring PAHs for each site during each sampling event. Figure 3C shows source ratios enriched in 2- and 3-ring PAHs compared to 4- to 6-ring PAHs in May and June-1 at all sites, consistent with a petrogenic source.50,51 Ratios with values greater than 1 are indicative of a petrogenic source.51 The alkylated-PAH versus parent PAH profiles are dominantly also petrogenic in June-1 at LA. In urban environments, no single source was expected; this was consistent with our observations of mixed alkylated profiles observed later in 2010 and 2011. Oxygenated Polycyclic Aromatic Hydrocarbons in Air and Water Few OPAH concentrations in air and water have been quantified using PSDs.12 Oxygenated hydrocarbons were reported during the DWH incident using active sampling techniques; however, the specific oxygenated analytes were not identified.52 OPAHs detected in DWH crude oil were identified in both water and air samples (SI Table 4). During this study 11 OPAHs were quantified, five of those OPAHs were detected in most of the samples (SI Figure S3). The most abundant OPAHs in air during the DWH incident were acenaphthenequinone, benzofluorenone, 9,10-anthraquinone, and 9-fluorenone (Figures 2A–C and SI S3). Over the course of the study, the OPAHs with the highest concentrations in water were phenanthrene-1,4-dione, and acenaphthenequinone in LA, MS, and AL, while 1,4-anthraquinone was the largest contribution in water at FL (SI Figure S3). Sum OPAHs at LA peaked in June-1 in air and water, ∑22airOPAH 15.1 (±1) ng/m3 and ∑22waterOPAH 635 (±60) ng/L. Unlike PAHs, the ∑22airOPAH remained elevated in the June-2 sample, after which a decrease of 10–15 fold was observed. However, water concentrations of phenanthrene-1,4-dione 257 (±40) ng/L, and acenaphthenequinone 185 (±30) ng/L remained elevated at LA through May 2011. ∑22waterOPAH concentrations at the MS site were less than 25 ng/L for the first six sampling periods (Figure 2C). The most frequently observed was benzofluorenone (SI Figure S3). ∑22waterOPAH concentrations were elevated in February, April, and May 2011 at concentrations of 369 (±60), 262 (±40), and 112 (±20) ng/L respectively, p < 0.05, when compared to the other 5 sampling events for each of these observations. In each instance, acenaphthenequinone had the greatest contribution to the∑22waterOPAH. Conversely, ∑22airOPAH at MS was significantly higher in May 2010 at 20 (±1) ng/m3 than in all subsequent sampling times (p = 0.01). Similar to LA, the OPAH that contributed the most to MS ∑22airOPAH was acenaphthenequinone. The high concentrations of OPAHs in air in May 2010 suggest that air quality may have been impacted by the DWH before shoreline oiling was observed. Also, given the proximity of this site to urban and industrial activities, it is also important to consider possible impacts from local sources. Increased levels of OPAHs in Gulf of Mexico waters during later sampling in 2011 may be evidence of the continuing transformation of PAHs in the system into OPAHs. The ∑22airOPAH levels at AL were approximately 20-fold less than the highest concentrations observed at LA or MS. The highest ∑22airOPAH at AL was during June-1 sampling at 1.5 (±1) ng/m3, and concentrations gradually decreased throughout the study to a minimum of 0.19 (±0.1) ng/m3 in May 2011 (Figure 4A). OPAHs in water were minimal at the onset of sampling and peaked during the last sampling event. The lowest ∑22waterOPAH concentration at AL, 2.2 (±0.5), was observed during May 2010 and the highest ∑22waterOPAH, 617 (±100) ng/L, was recorded during May 2011. Figure 4 (A) Σ22OPAH vapor phase concentrations in air. (B) Acenaphthenequinone vapor phase concentrations in air. (C) Σ22OPAH dissolved concentrations in water. (D) Σ7OPAH net flux. (E) Benzofluorenone flux. (F) Acenaphthenequinone flux. Error bars represent the calculated 95% confidence interval based on pooled variance from a replication study. The highest concentration ∑22airOPAH observed during the study was measured at FL in May 2010 at level of 26 (±2) ng/m3; this observation was significantly different from all other samples (p < 0.05) (Figure 4A). The high concentration is largely due to acenaphthenequinone and 1,4-anthraquinone (Figure 4B, SI Figure S3). Due to short atmospheric half-lives and multiple formation pathways, positively identifying sources of OPAH is an ongoing research question. The potential for May 2010 ∑22airOPAH at this site to be affected by factors other than DWH is likely. ∑22waterOPAH concentrations in FL reached a peak concentration of 92 (±50) in June-1, a value nominally six times lower than the highest observed concentration at the LA site (Figure 4C). Unlike the other three sites, waterborne OPAH in Gulf Breeze, FL showed little temporal variation during the course of this research. In a study in southern France, the highest combined gas and particle OPAH values observed were nominally 50 to 65% less than the highest concentrations observed in FL and MS, respectively.53 The high concentrations of vapor-phase OPAHs observed during May 2010 are much higher than what has been previously characterized in typical urban settings.53 A study in Texas showed elevated vapor phase levels of benz[a]anthracene-7,12-dione during the summer, which was proposed to be a result of temperature-dependent partitioning between the particle-bound and vapor-phase OPAHs.54 Although similar meteorological conditions occurred during this study, the OPAH levels actually decreased dramatically during the summer. This observation lends support to the idea that elevated levels were due to a specific input(s) disrupting typical environmental conditions, and not simply a shift in partitioning between the vapor and aerosol phase.55−57 A study performed in Oregon on the Willamette River in the Portland Harbor Superfund site found dissolved OPAH concentrations in water ranging from 6 to 50 ng/L.32 This observation by O’Connell et al. is nominally 12 times lower than the highest water value reported here.32 The May 2010 event yielded OPAHs 10–20 fold higher at the LA, MS, and FL sites compared to sampling events later in the study. The consistently low levels of OPAHs in air after June 2010 are different from the PAH temporal profile where increases were observed in April 2011 in both LA and MS. Although toxicity of OPAHs is not thoroughly known, early evidence suggests development toxicity may be the same or higher for some OPAHs than the parent PAH.7 Therefore, OPAHs appear to be an important consideration as part of the transport, weathering, and ecosystem health during environmental disasters.9,52 OPAH Air–Water Exchange To the authors’ knowledge this is the first report of OPAH flux. Direction and approximate magnitude for the OPAH flux is show in Figure 4D–F. Unlike PAHs, where the net flux was consistently volatilization during the study, Σ7OPAH flux underwent repeated periods of deposition and volatilization at both MS and FL. LA and MS had the largest magnitude of volatilization in June-1 and August, respectively. Large Σ7OPAH volatilization at all four sites was primarily driven by the movement of acenaphthenequinone from water to air. MS underwent a change from net deposition to net volatilization between May 2010 and June-1, primarily attributable to acenaphthenequinone. Benzofluorenone flux at each site was of significantly lower magnitude than acenaphthenequinone; however, benzofluorenone was found to be of a very transient nature, undergoing volatilization in LA, deposition in FL, and changed from deposition to volatilization in MS. The nature of the dynamic movement between environmental compartments and the observed magnitudes indicate further investigation is warranted. The extremely high magnitude of OPAH flux to the atmosphere concurrent with the DWH incident shows that OPAHs as well as PAHs need to be assessed for environmental fate and transport when assessing the long-term impacts of an environmental disaster. Supporting Information Available Figures that present the full characterization of PAH and OPAH chemical profiles and PAH flux for all samples. This material is available free of charge via the Internet at http://pubs.acs.org/. Supplementary Material es503827y_si_001.pdf The authors declare no competing financial interest. Acknowledgments This project was supported in part by awards P42 ES016465 and the associated Analytical Chemistry Facility Core, P30 ES000210, and R21 ES020120 from the National Institute of Environmental Health Sciences. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIEHS or the National Institutes of Health. We appreciate valuable help from Alan Bergmann, Ricky Scott, Gary Points, and Glenn Wilson. We thank Grand Isle State Park, Gulfport Harbor, Bon Secour National Wildlife Refuge, and Gulf Shores National Seashore. Gulf Shores National Seashore permits: GUIS-2010-SCI-0022, GUIS-2011-SCI-0042. Bon Secour National Wildlife Refuge permits: 10-011, 11-002. 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==== Front Med Sci MonitMed. Sci. MonitMedical Science MonitorMedical Science Monitor : International Medical Journal of Experimental and Clinical Research1234-10101643-3750International Scientific Literature, Inc. 2536626610.12659/MSM.890654890654Diagnostic TechniquesAtomic Absorption Spectrometry Analysis of Trace Elements in Degenerated Intervertebral Disc Tissue Kubaszewski Łukasz 1ABCDEFGZioła-Frankowska Anetta 2BCDEFGFrankowski Marcin 2ABCDEFGNowakowski Andrzej 3DEFCzabak-Garbacz Róża 4AEFKaczmarczyk Jacek 5ADFGGasik Robert 6ACDEG1 Department of Orthopaedic and Traumatology, W. Dega University Hospital, University of Medical Sciences, Poznań, Poland2 Department of Water and Soil Analysis, Faculty of Chemistry, Adam Mickiewicz University in Poznań, Poznań, Poland3 Department of Spine Surgery, Oncologic Orthopaedics and Traumatology, W. Dega University Hospital, University of Medical Sciences, Poznań, Poland4 Department of Human Physiology, Medical University of Lublin, Lublin, Poland5 Department of Orthopaedic and Traumatology, W. Dega University Hospital, University of Medical Sciences, Poznań, Poland6 Clinic and Polyclinic of Neuro-orthopedic and Neurology, Institute of Rheumatology, Warsaw, PolandCorresponding Author: Łukasz Kubaszewski, e-mail: [email protected] Study Design B Data Collection C Statistical Analysis D Data Interpretation E Manuscript Preparation F Literature Search G Funds Collection 2014 04 11 2014 20 2157 2164 08 3 2014 14 5 2014 © Med Sci Monit, 20142014This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported LicenseBackground Few studies have investigated trace elements (TE) in human intervertebral disc (IVD) tissue. Trace element presence can have diverse meanings: essential TE show the metabolic modalities of the tissue, while environmentally-related TE indicate pollution and tissue-specific absorption and accumulation. IVD is a highly specific compartment with impaired communication with adjacent bone. Analysis of TE in IVD provides new insights regarding tissue metabolism and IVD communication with other tissues. Material/Methods Thirty intervertebral discs were acquired from 22 patients during surgical treatment for degenerative disease. Atomic absorption spectrometry was used to evaluate the concentrations of Al, Cd, Pb, Cu, Ni, Mo, Mg, and Zn. Results Al, Pb, Cu, Mg, and Zn were detected in all samples. Pb was significantly positively correlated with age, and Ni concentration was weakly correlated with population count in the patient’s place of residence. Only Cu was observed in higher concentrations in IVD compared to in other tissues. Significant positive correlations were observed between the following pairs: Mg/Zn, Mg/Al, Mg/Pb, Zn/Al, Zn/Pb, and Al/Pb. Negative correlations were observed between Mg/Cd, Zn/Cd, Mg/Mo, and Mo/Pb. Conclusions This study is one of few to profile the elements in intervertebral discs in patients with degenerative changes. We report significant differences between trace element concentrations in intervertebral discs compared to in other tissues. Knowledge of the TE accumulation pattern is vital for better understanding intervertebral disc nutrition and metabolism. MeSH Keywords Intervertebral Disc DegenerationSpineTrace Elements ==== Body Background Trace elements (TE) are of growing interest in medical studies, with the majority of publications focused on element deficiencies and related syndromes. Advancements in biochemical knowledge are leading to a better understanding of the roles of various elements in metabolic processes and tissue accumulation. The definition of TE in biology is somewhat flexible, with the term used to refer to dietary minerals needed in small quantities for proper physiology and development of an organism (including active elements of metabolic processes, such as copper, magnesium, and zinc) as well as elements that accumulate due to environmental pollution (lead and cadmium). There is not yet a comprehensive biological definition or complete list of trace elements found in humans. TE content is most easily analyzed in bone tissue because it is the trace elements repository, reflecting the turnover for the whole organism [1]. TE content can also be examined in more unstable compartments, such as serum, urine, or cerebrospinal fluid, which show more rapid changes secondary to exposure or specific response of the organism [2]. One of the most highly specific tissues is human intervertebral disc (IVD). In the first decade of life, this tissue becomes avascular, such that all metabolism relies on passive nutrient transport through the end-plates, which are cartilage-resembling tissue that act as barriers, separating IVD from the bone of the vertebrae. Confined in the center of the IVD, the nucleus pulposus environment is rich in lactic acid and has a lower oxygen concentration and higher pH compared to other tissues. These conditions result in a low cell concentration, with slow metabolism and abundant extracellular matrix (ECM). Degenerative disc disease (DDD) involves changes related to ECM dysfunction. The causes of DDD are complex and not fully understood, but appear to mainly involve aging, and genetic or environmental factors. The mainstream concept of degeneration assumes disproportionate levels of anabolic and catabolic activity in the ECM. These processes involve enzymes such as cathepsins, aggrecanases, and matrix metalloproteinases (MMPs) [3]. MMPs are zinc-dependent endopeptidases that are active in protein degradation [4]. Other TE are also involved in enzyme activity – for example, copper is present in cytochrome oxidase or ceruloplasmin – or are involved in collagen synthesis and connective tissue development [5]. Recent studies have analyzed the correlation between DDD and TE concentration in serum [6]. However, few papers have examined the role of TE in human IVD tissue. Tohno et al. [7] evaluated post-mortem Ca, P, S, Mg, and Na concentrations. Minami et al. [8] analyzed platinum levels in bone and IVD from cisplatin-treated ovarian cancer patients, and found that intradiscal metal accumulation after exposure exceeded bone accumulation by up to 4.3-fold. Other observations have also shown that in some cases TE concentrations can be higher in disc or bone tissue than in internal organs, such as liver and kidneys [9]. The present study aimed to analyze the selected TE profile (Al, Cd, Pb, Cu, Ni, Mo, Mg, and Zn) in human degenerated intervertebral disc tissue, and to compare the concentrations with those found in other tissues. Material and Methods The study material included 30 intervertebral discs obtained from 22 patients undergoing surgical discectomy or spine fusion due to degenerative disc disease. Twelve specimens were from the cervical spines of 6 patients, and 18 specimens were harvested from the lumbar spines of 16 patients. Preoperative magnetic resonance images were used to evaluate the degeneration status of the operated disc according to Pfirrmann score [10]. Atomic absorption spectrometry (AAS) was used to determine the concentrations of the trace elements Al, Cd, Pb, Cu, Ni, Mo, Mg, and Zn, which were calculated using the dry weight (dw) of the disc. The use of tissue in this study was approved by the appropriate bioethics committee, and written consent was obtained from all patients. The mean age of the patients at the time of operation was 47.6 (range, 28–64 years). All patients were interviewed using a questionnaire to collect data on demography, health status, and occupational heavy metals exposure. None of the patients had knowledge of being inadvertently exposed to heavy metal pollution. Based on postal zip codes, we obtained information regarding each patient’s places of residence and work over the last 5 years. Places of residence and employment were stratified according to population count as follows: >500 k, 200–499 k, 100–199 k, 20–99 k, <20 k, and village inhabitants. Analytical procedure First, the frozen intervertebral disc samples were freeze-dried using a Lyovac lyophilizer GT2e (Steris, Germany) for 24 hours. The samples were weighed after drying. Then 65% nitric acid (Merck, Germany) was added to obtain a dilution factor (DF) of 10, with amounts in the ranges of 0.2–0.6 g of sample and 2.0–6.0 ml of nitric acid. The prepared samples were allowed to stand overnight to slow mineralization. Then the samples were mineralized in a microwave oven (Mars Xpress 5, CEM USA). Trace element concentrations in the mineralized samples were determined using an AAS 7000 spectrometer (Shimadzu, Japan) with graphite furnace atomization (GF-AAS) for Al, Cu, Cd, Mo, Ni, and Pb, or with flame atomization (F-AAS) for Mg and Zn. All analyses were run in 3 replicates. The percent RSD did not exceed 5% for GF-AAS analysis, and did not exceed 7% for F-AAS analysis. Table 1 presents the basic optimized parameters for the determination of Al, Cu, Cd, Mo, Ni, Pb, Zn, and Mg using AAS. Statistical analysis The results are presented as means and ranges. Where applicable, standard deviation (SD) was also calculated. Spearman’s rank-order correlation analysis was used to determine the relationships among different parameters. P<0.05 was accepted as indicating statistical significance. When an element was not detected in the sample, the ½ limit of detection (LOD) value was left out of calculations. Statistica (Statsoft, Tulsa, USA) software was used for statistical analysis. Results The elements Al, Pb, Cu, Mg, and Zn were detected in every tested sample. Ni was detected in 97% of samples, Mo in 83%, and Cd in 57% (Table 2). The concentrations of Mg, Zn, and Cu were 1 order of magnitude higher than the concentrations of the other examined elements (mg·kg−1 dw vs. μg·kg−1 dw). The measured concentrations were as follows (range values with mean value and standard deviation, respectively): Al, mean of 663.71, range of 165.7–1271, SD of 288.84 (in μg·kg−1 dw); Cu, mean of 3.41, range of 0.97–23.64, SD of 4.045 (in mg·kg−1 dw); Mg, mean of 800.1, range of 182.6–2132, SD of 525.5 (in mg·kg−1 dw); Zn, mean of 39.60, range of 10.56–184.5, SD of 35.95 (in mg·kg−1 dw); Pb, mean of 8.435, range of 0.562–24.76, SD of 5.596 (in μg·kg−1 dw); Cd, mean of 8.435, range of 0.562–24.76 (in μg·kg−1 dw); Ni mean of 251.38, range of 25.48–444.2 (in μg·kg−1 dw); and Mo, mean of 54.33, range of 20.02–143.2 (in μg·kg−1 dw) (Table 2). Among the elements that were found in only some of the tested samples, the LOD values were: 0.2 μg·kg−1 for Cd and 1 μg·kg−1 for Ni and Mo. Correlation analysis showed significant positive correlation of Pb concentration with age (Table 3). Ni concentration showed a weak positive correlation with the population size of the place of residence. A strong positive correlation was observed between the pair Mg/Zn, both of which also showed a positive correlation with the non-essential element Al and the toxic element Pb. There was also a significant positive correlation between Al and Pb. Mg and Zn each also showed a weak negative correlation with Cd. Mo showed negative weak correlations with both Mg and Pb. Discussion Our present analysis detected Al, Pb, Cu, Mg, and Zn in all tested samples. Of these 5 elements, Cu, Mg, and Zn are considered essential in human metabolism, Pb is considered non-essential and toxic, and the role of Al is not yet fully understood. The other studied elements were only found in some of the tested samples. Of these, Cd is not essential and is considered toxic, but Mo and Ni are considered potentially essential. The present study had possible methodological flaws; however, considering the differences between LOD and mean levels in the rest of the samples, we can likely regard Cd, Mo, and Ni as non-essential in IVD metabolism and as unrelated to degenerative changes. Except for Pb, the majority of non-essential elements tend to concentrate in the tissue up to some critical point, and once this threshold is exceeded, the element is excreted through a variety of methods. For some TE, the accumulation capacity has only been determined for bone tissue; thus, complete analysis regarding bioaccumulation was not possible in our present study. The trace elements present in intervertebral disc tissue have not yet been extensively studied. Tohno et al. [7] investigated the elements abundantly present in IVD tissue, but their report did not include degeneration criteria. Due to the lack of relevant data from this particular biological compartment, here, we primarily compare our present findings to previous results from other tissues – the most relevant being cartilage and tendons, due to their metabolism, morphology, and biomechanical role [11,12]. Mg Magnesium content has been reported for a variety of tissues, including intervertebral disc and temporomandibular joint disc (TMJD) [13–15]. Tohno el al [7] reported the average Mg content of IVD to be 1.196 mg·g−1 (range, 0.6–2.2 mg·g−1) from a study of 9 specimens that were acquired post-mortem with no definition of degeneration stage. Our present results showed a lower mean Mg concentration of 800.1 mg·kg−1, with a range of 182.6–2132 mg·kg−1. This discrepancy could be due to the different methods of tissue acquisition between the 2 studies, and our present results may be grounds to question the use of cadavers as a control group. In another study of cadavers, Takano el al. [15] reported the average Mg concentration in TMJD, which was substantially lower than our present results (mean value 524.74 mg·kg−1 vs. 800.1 mg·kg−1 dw), and showed a narrower concentration range (393.4–764.9 mg·kg−1 dw). Another study shows Mg concentrations of 445 mg·kg−1 in cervical spine posterior longitudinal ligaments (PLL) [14], which are lower than those presently found in IVD and comparable to concentrations reported in TMJD. The standard deviation reported for cervical spine PLL Mg concentrations (161 mg·kg−1) was smaller than that in our study (525.5 mg·kg−1). The Mg concentration in bone has been reported as 1792.9 mg·kg−1 [13], which is more than 2 times higher than that found for IVD. Magnesium has many biological functions, and is the fourth most abundant cation in the body and the second most abundant intracellular cation [16]. Soft tissue accumulates only 19.3% of Mg ions. The majority of Mg ions are found in bone and muscle, with up to 60% of magnesium located within the bone, where it most probably forms the hydroxyapatite surface. In cases of deficiency, Mg from bone can be readily exchanged with serum, although this exchangeable form can decline with increasing age [17]. Our results showed that Mg concentrations in IVD of living patients with DDD were higher than those in ligamentous tissue and TMJD, and smaller than the concentrations found in cadaver IVD. The difference in Mg concentration between bone and IVD tissues was less pronounced than we expected based on the cellularity of each tissue. Since the cellularity of bone is substantially higher than that of IVD, we can conclude that Mg in IVD is mainly extracellular. The intracellular range of Mg concentration is maintained within strict limits, except under severe hypoxic stress or magnesium depletion. Up to 5% of intracellular magnesium is present in the form of free ions, with the rest bound to ionic compounds, such as ATP, ADP, citrate, proteins, RNA, and DNA, or stored in the mitochondria and endoplasmic reticulum. It has been shown that Mg can decrease MMP secretion to the extracellular matrix after stimulation with growth factors [18]. Migration of cells, such as macrophages and fibroblasts, in the early stages of skin wound repair is associated with Mg2+ increase [19]. Mg is connected with migratory phenotype activation and maintenance through modifications of integrins and E-cadherin. It has further been observed that magnesium deficiency results in loss of replicative capacity, and increases the expression of senescence-associated biomarkers in human fibroblasts [20]. Overall, Mg is predominantly located in ECM and its activity is related to repair process, which is supported by the presently observed higher concentration in DDD tissue compared to in ligaments. Cu Cu concentration is best documented in bone tissue, with average reported concentrations of 0.62 mg·kg−1 dw [13] and 0.8 mg·kg−1 dw [21]. Similar Cu concentrations have also been found in cartilage, with an average value of 0.79 mg·kg−1 dw and range of 0.20–1.78 mg·kg−1. Our results showed that the copper concentration in IVD (mean 3.41, range 0.97–23.64 mg·kg−1 dw) was more than 3 times higher than those previously observed in bone and cartilage. Copper and zinc are both predominantly associated with animal proteins, and are known for their antagonism, in that they tend to nullify each other’s actions. Our results did not confirm a negative correlation between these 2 elements. Copper is a redox-active metal that is predominantly used by organisms living in oxygen-rich environments, and that fluctuates between the oxidized (Cu2+) and reduced (Cu+) states [22]. It is also a compound of the XIAP protein, which acts as an apoptosis inhibitor. Cu ions are a key element of cytochrome oxidase, which is responsible for mitochondrial phosphorylation and ATP production. Thus, Cu deficiency may lead to respiratory disturbance. The integral membrane protein Ctr1 functions as a major copper importer at the plasma membrane. Cu+ is the only known physiological substrate for Ctr1. Recent findings in yeast and mammals strongly suggest that Ctr1 facilitates uptake of the metal-based anti-neoplastic drug cisplatin. This may explain the higher Pt concentration in cisplatin-treated patients reported by Minami et al. [8]. On the other hand, Ctr1 knock-out mice exhibit growth retardation. There may be a connection between high Cu concentration, low oxygen environment, and the Warburg effect [22]. The Warburg effect includes a down-regulation of cell respiratory capacity that is observed in many cancer cell types, with a corresponding shift to glycolysis for cell energy generation. This process can be related to p53 protein mutation, which is a common genetic change found in a broad range of cancer cells. P53-inactive mutation cells show significantly reduced oxygen consumption, while generating increased levels of lactate. It has been proposed that a link between p53 and SCO2 gene expression levels may hamper copper acquisition and promotion of the Warburg effect. The high lactic acid concentration due to glycolysis is the natural environment for IVD tissue. It is possible that glycolysis may be related to Cu concentration other than in the Warburg effect, which, in turn, may also be related to the p53 gene. However, IVD tissue is virtually free from mutagenesis, except for chordoma that arises from the remnants of the notochord, suggesting a low risk of p53 mutations in IVD cells. Another hypothesis is that higher Cu concentration may be a tissue-specific countermeasure to primary oxygen deficiency or promotion of oxygen-based energy acquisition. A possible third explanation of higher Cu concentration in disc tissue may relate to the element’s role in the repair process. Higher copper concentrations have been observed in wounds during the healing process, and the element has been observed to induce vascular endothelial growth factor expression [23]. However, this explanation is less likely because the process is bound with vascular ingrowth, which is not observed in disc tissue except for outer parts of the annulus fibrosus. Zn The presently determined intervertebral disc zinc concentration (39.9 mg·kg−1 dw) was similar to the levels in PLL (36 mg·kg−1 dw) reported by Kumai et al. [24]. On the other hand, the presently reported IVD concentrations are less than half that of the average concentrations reported in cartilage (88.3 mg·kg−1; range, 54.3–163.8 mg·kg−1 dw) [13] and bone (84.58 mg·kg−1; SD 17.68 mg·kg−1) [21]. Zn plays a well-recognized role in the immune system, is an antioxidant, and has anti-inflammatory actions [25]. Zinc-deficient cells display decreased gene expressions of interleukin-2 and IL-2 receptor alpha [26], suggesting that immune reaction would be less likely during the disc degeneration process. Zinc supplementation has been shown to decrease oxidative stress markers and inflammatory cytokine generation. It also stabilizes the molecular structure of cellular membranes and organelles, contributing to cell and organ integrity. Zinc also plays an essential role in DNA transcription and genetic expression [27]. MMPs are Zn-dependent compounds involved in ECM turnover, and we thus expected Zn concentration to be higher in IVD than in ligaments. The similar levels of Zn in ligaments and IVD raise questions regarding to what degree Zn level reflects the MMP concentration and the timing of MMP activation in IVD degenerative processes. The strong correlation between Zn and Mg confirms their major metabolic role in IVD tissue. This correlation was also observed in bone tissue of the Cracow, Poland population (correlation, 0.68) by Jurkiewicz et al. [13]. Further research is required to link metabolic background with TE content. Al It was very interesting that our results showed aluminum in all tested samples. Aluminum is the third most common element on earth, but is not considered essential in humans. Exogenic sources of aluminum may include oral antacids and antiperspirants applied to the skin. Aluminum cookware is considered safe, since only uncoated cookware can lead to leaching of particles into food. Aluminum can be part of highly reactive biomolecules. Although its role is not entirely known, several studies have shown its influence on neurodegenerative disorders, including Alzheimer’s disease, and brain tissue is considered the sink for systemic aluminum. The average concentration in the human tissue is at the level of 2.6 mg·kg−1 [28]. Our present data cannot exclude a metabolic role of the element, or the possibility that it plays a role in degeneration induction or the process itself. The levels observed in our study were smaller than reference values, suggesting that Al content is environment-related and depends on end-plate permeability. Answering these questions will require further analysis of Al compounds in IVD. Pb Lead accumulates in bone, and is thereafter difficult to excrete and is retained by the tissue, such that its concentration tends to increase over time. The Pb accumulation presently observed in IVD resembles that in bone, as Pb was the only element that was positively correlated with age. Compared to that in IVD, the Pb concentration in bone is up to 2 times higher, with an average of 1.35 mg·kg−1 dw [13]. The cartilage Pb concentration is lower, with an average of 0.41 mg·kg−1 and range of 0.21–0.65 mg·kg−1 [21], which is comparable to our results in IVD tissue. Viewing cartilage as a reference, we may conclude that Pb accumulation is not related to end-plate permeability, in contrast to Al. Cd Compared to the presently determined Cd concentration in IVD (8.4 μg·kg−1 dw), the concentration in bone is reportedly up to 3 times higher (30 μg·kg−1 dw) [13]. Łanocha et al. [29] reported a slightly lower Cd concentration in bone tissue (average, 22 μg·kg−1; range, 13–34 μg·kg−1). In a population that is occupationally exposed to Cd, the bone concentration can be higher, ranging from 0.11–1.2 mg·kg−1 dw. Cartilage Cd concentrations are congruous with levels observed in bone (average, 0.031 mg·kg−1; range, 0.001–0.151 mg·kg−1 dw) [21]. Interestingly, in our study of IVD, cadmium was detected in only 57% of the samples. The lower concentration of this element in IVD compared to cartilage, and its detection in only some of the tested samples, suggest that the end-plate acts as a selective barrier in Cd transport. Ni Nickel is predominantly associated with vegetable food sources, and has no potential biological activity in human metabolism [30]. Brodziak et al. [31] reported a mean Ni concentration in bone of 4.82 mg·kg−1 (SD, 10.74 mg·kg−1; range, 0.03–71.49 mg·kg−1). The same study found a similar cartilage content (mean, 4.40 mg·kg−1; SD, 7.38 mg·kg−1), and a lower concentration in the joint capsule (mean, 1.38 mg·kg−1; SD, 2.47 mg·kg−1). The Ni concentration in IVD found in our study (mean, 251.38 μg·kg−1; range, 25.48–444.2 μg·kg−1) was only 18% of the previously reported concentration in joint capsule and only 5% of the bone concentration. Although Ni presence was confirmed in 97% of tested samples, the results suggest impaired transport of this element into the disc. Mo Like zinc and copper, molybdenum is predominantly bound with animal protein, and is related to animal food intake. Mo concentration in human tissues ranges from 1 to 400 μg·kg−1, with the skeletal concentration being in the upper part of this range [30]. In our study, the mean Mo concentration in IVD was 54.33 μg·kg−1 dw, which is more than half of the reference value from the literature (75 μg·kg−1). Conclusions The present analysis is one of the few to address trace element concentrations in IVD tissue. The results add to a preliminary picture of the IVD chemical environment, and possible dependencies between TE in IVD tissue. This study showed significant differences between the trace element concentrations in intervertebral disc and in other tissues, especially bone. TE concentration may be influenced by dietary components rich in particular elements, as well as by biogenic factors, such as pregnancy and lactation. The major consideration in IVD tissue is selective end-plate permeability, which can be suspected in the cases of Ni, Cd, and Al, but not Pb. Biochemical, environmental, and metabolic functions could also favor the accumulation of some elements in higher quantities, as is the case for Cu. Further cross-referencing against other tissues and a better understanding of the biochemistry of the IVD are essential to answer the questions arising in this study. Source of support: Departmental sources Table 1 Presents the basic optimized parameters for the determination of Al, Cu, Cd, Mo, Ni, Pb, Zn, and Mg using AAS analytical technique. Parameter Al Cd Cu Mo Ni Pb Zn* Mg* Wavelength [nm] 309.3 228.8 324.8 313.3 232 283.3 213.9 285.2 Slit [nm] 0.7 0.7 0.7 0.7 0.2 0.7 0.7 0.7 Lamp current [mA] 10 8 8 10 12 10 5 8 Lamp mode BGC SR D2 D2 D2 D2 D2 D2 D2 Drying [°C] 150 150 150 150 120 150 – – Drying time [s] 45 30 30 30 25 30 – – Ashing [°C] 1000 500 800 1000 900 800 – – Ashing time [s] 25 20 20 20 20 20 – – Atomization [°C] 2600 2200 2300 2600 2600 2400 – – Atomization time [s] 5 5 5 5 6 5 – – Cleaning [°C] 2700 2400 2500 2700 2700 2600 – – Clearing time [s] 2 2 2 2 2 2 – – LOD [μg·kg−1] <20 <0.4 <30 <20 <15 <5 <0.1 <0.03 * Air-C2H2 atomisation mode; ** LOD in [mg·kg−1]. Table 2 The contents of elements in intervertebral disc. Sample No. Case No. Al Cd Pb Ni Mo Cu Mg Zn [μg/kg dw] [mg/kg dw] 1 1 539.5 4.921 170.1 222.9 111.2 5.602 413.9 18.29 2 2 1203 – 1264 134.8 54.35 2.492 1195 61.26 3 2 915.1 9.171 209.7 387.2 62.67 2.793 489.5 16.88 4 3 992.1 – 1252 369.9 29.38 2.092 1599 71.22 5 3 1084 – 1343 62.23 35.55 1.844 854.7 36.12 6 3 1021 – 1417 444.2 33.41 1.889 2132 121.75 7 4 572.7 16.02 867.3 143.4 77.23 1.459 672.1 25.72 8 5 1003 – 1825 43.22 – 0.9701 1622 29.38 9 6 1271 – 1599 373.4 38.89 2.317 1475 64.62 10 7 451.2 4.751 191.2 131.3 72.12 1.079 607.7 18.29 11 8 539.5 – 61.47 53.58 – 1.326 634.8 12.65 12 9 552.2 – 566.9 245.4 42.56 1.542 1008 38.97 13 10 470.1 6.444 167.3 213.4 69.82 1.616 371.4 14.06 14 11 520.6 0.562 2233 165.9 21.24 1.664 1032 55.76 15 11 697.3 9.292 314.2 312.9 35.89 1.799 516.7 34.35 16 12 490.6 7.640 665.9 421.2 43.58 2.046 977.9 43.84 17 13 545.9 – 525.9 117.5 – 2.519 999.4 18.53 18 14 440.1 1.625 266.3 162.5 55.21 1.417 482.8 36.78 19 15 444.7 – 589.2 48.15 73.16 6.089 856.2 41.18 20 16 240.1 5.634 495.9 181.2 62.38 2.715 368.3 48.51 21 17 165.7 7.595 198.3 103.3 53.91 4.562 246.5 11.35 22 18 192.6 8.520 186.6 169.9 34.65 4.101 182.6 10.56 23 19 546.6 13.209 478.4 106.2 35.42 3.827 371.5 22.19 24 20 748.9 24.764 274.2 308.6 143.2 3.073 291.1 15.44 25 20 389.2 6.218 338.3 427.6 61.61 3.356 276.4 16.11 26 21 461.1 8.026 204.2 400.7 43.13 3.695 299.4 17.56 27 21 743.2 – 1050 311.5 – 23.64 2016 184.5 28 22 632.9 – 773.2 – – 2.517 512.5 37.32 29 22 776.8 – 717.6 158.5 47.75 4.968 614.3 35.46 30 22 360.6 9.016 355.8 25.48 20.02 3.356 884.3 29.62 Table 3 Spearman’s rank correlation between elements, place of living, work and age. Pfirrmann grade Al Cd Pb Ni Mo Cu Mg Zn Age Place of living −0.17 0.02 0.24 −0.16 0.36* −0.03 0.29 −0.18 −0.14 −0.27 Place of work 0.18 0.03 0.37 −0.07 0.26 0.05 0.21 −0.15 −0.16 −0.12 Pfirrmann grade −0.03 0.32 0.03 −0.29 0.16 −0.07 0.00 −0.19 0.20 Al −0.21 0.61* 0.17 −0.12 −0.21 0.60* 0.44* 0.26 Cd −0.36 0.00 0.24 0.28 −0.48* −0.58* −0.14 Pb 0.02 −0.43* −0.15 0.76* 0.79* 0.50* Ni −0.02 0.09 −0.02 0.19 0.10 Mo 0.05 −0.40* −0.38 0.18 Cu −0.31 −0.08 −0.02 Mg 0.75* 0.21 Zn 0.28 * Significant p<0.05. ==== Refs References 1 Ericson JE Smith DR Flegal AR Skeletal concentrations of lead, cadmium, zinc, and silver in ancient. North American Pecos Indians Environ Health Perspect 1991 93 217 24 1773793 2 Carpenter WE Lam D Toney GM Zinc, copper, and blood pressure: Human population studies Med Sci Monit 2013 19 1 8 23291705 3 Longo UG Maffulli N Denaro V Growth Factors and Anticatabolic Substances for Prevention and Management of Intervertebral Disc Degeneration Stem Cells International 2012 ID 897183 4 Birkedal-Hansen H Moore WG Bodden MK Matrix metalloproteinases: a review Crit Rev Oral Biol Med 1993 4 197 250 8435466 5 Cai L Li XK Song Y Cherian MG Essentiality, toxicology and chelation therapy of zinc and copper Curr Med Chem 2005 12 2753 63 16305470 6 Zhao B Wang K Zhao J Luo Y Serum calcium concentration as an indicator of intervertebral disk degeneration prognosis Biol Trace Elem Res 2013 154 333 37 23892694 7 Tohno S Tohno Y Minami T Difference of mineral contents in human intervertebral disks and its age-related change Biol Trace Elem Res 1996 52 117 24 8773752 8 Minami T Hashii K Tateyama I Accumulation of platinum in the intervertebral discs and vertebrae of ovarian tumor-bearing patients treated with cisplatin Biol Trace Elem Res 1994 42 253 57 7826818 9 Minami T Tohno Y Tohno S Tissue platinum after clinical treatment with cisplatin or carboplatin in tumor-bearing patients Biol Trace Elem Res 1997 58 77 83 9363322 10 Pfirrmann CW Metzdorf A Zanetti M Magnetic resonance classification of lumbar intervertebral disc degeneration Spine 2001 26 1873 78 11568697 11 Arana CJ Diamandis EP Kandel RA Cartilage tissue enhances proteoglycan retention by nucleus pulposus cells in vitro Arthritis Rheum 2010 62 3395 403 20662071 12 Roughley PJ The structure and function of cartilage proteoglycans Eur Cell Mater 2006 12 92 101 17136680 13 Jurkiewicz A Wiechuła D Nowak R Metal content in femoral head spongious bone of people living in regions of different degrees of environmental pollution in Southern and Middle Poland Ecotoxicol Environ Saf 2004 59 95 101 15261729 14 Yamada M Tohno Y Tohno S Age-related changes of elements and relationships among elements in human tendons and ligaments Biol Trace Elem Res 2004 98 129 42 15073411 15 Takano Y Moriwake Y Tohno Y Age-related changes of elements in the human articular disk of the temporomandibular joint Biol Trace Elem Res 1999 67 269 76 10201333 16 Swaminathan R Magnesium metabolism and its disorders Clin Biochem Rev 2003 24 47 66 18568054 17 Magnesium in Report of a joint FAO/WHO expert consultation Bangkok 2002 http://www.fao.org/DoCREP/004/Y2809E/y2809e0k.htm 18 Yue H Lee JD Shimizu H Effects of magnesium on the production of extracellular matrix metalloproteinases in cultured rat vascular smooth muscle cells Atherosclerosis 2003 166 271 77 12535739 19 Grzesiak JJ Pierschbacher MD Shifts in the concentrations of magnesium and calcium in early porcine and rat wound fluids activate the cell migratory response J Clin Invest 1995 95 227 33 7814620 20 Killilea DW Ames BN Magnesium deficiency accelerates cellular senescence in cultured human fibroblasts Proc Natl Acad Sci USA 2008 105 5768 73 18391207 21 Lanocha N Kalisinska E Kosik-Bogacka DI Comparison of metal concentrations in bones of long-living mammals Biol Trace Elem Res 2013 152 195 203 23377610 22 Turski ML Thiele DJ New roles for copper metabolism in cell proliferation, signaling, and disease J Biol Chem 2009 284 717 21 18757361 23 Sen CK Khanna S Venojarvi M Copper-induced vascular endothelial growth factor expression and wound healing Am J Physiol Heart Circ Physiol 2002 282 1821 27 24 Kumai T Yamada G Takakura Y Trace elements in human tendons and ligaments Biol Trace Elem Res 2006 114 151 61 17205998 25 Shankar AH Prasad AS Zinc and immune function: the biological basis of altered resistance to infection Am J Clin Nutr 1998 68 447S 63S 9701160 26 Prasad AS Zinc in human health: effect of zinc on immune cells Mol Med 2008 14 353 57 18385818 27 Zinc in Report of a joint FAO/WHO expert consultation Bangkok 2002 http://www.fao.org/docrep/004/y2809e/y2809e0m.htm 28 Exley Ch The coordination chemistry of aluminium in neurodegenerative disease Coord Chem Rev 2012 256 2142 46 29 Lanocha N Kalisińska E Kosik-Bogacka D Comparison of concentrations of lead and cadmium in various parts of the femur head in patients after arthroplasty of the hip joint in Northwest Poland Biomed-Environ Sci 2012 25 577 82 23122316 30 Kabata-Pendias A Mukherjee AB Trace Elements from Soil to Human Berlin Heidelberg Springer-Verlag 2007 31 Brodziak-Dopierała B Kwapuliński J Sobczyk K Kowol J The occurrence of nickel and other elements in tissues of the hip joint Ecotoxicol Environ Saf 2011 74 630 35 20932577	25366266	PMC4301216	NO-CC CODE	2021-01-05 02:33:59	yes	Med Sci Monit. 2014 Nov 4; 20:2157-2164
==== Front Asian-Australas J Anim SciAsian-australas. J. Anim. SciAsian-Australasian Journal of Animal Sciences1011-23671976-5517Asian-Australasian Association of Animal Production Societies (AAAP) and Korean Society of Animal Science and Technology (KSAST) 10.5713/ajas.14.0761ajas-28-3-420ArticleCellular Uptake and Cytotoxicity of β-Lactoglobulin Nanoparticles: The Effects of Particle Size and Surface Charge Ha Ho-Kyung Kim Jin Wook Lee Mee-Ryung 1Jun Woojin 2Lee Won-Jae 1 Department of Food and Nutrition, Daegu University, Gyeongsan 712-714, Korea.2 Division of Food and Nutrition, Chonnam National University, Gwangju 550-757, Korea. Corresponding Author: Won-Jae Lee. Tel: +82-55-772-1884, Fax: +82-55-772-1889, E-mail: [email protected] of Animal Bioscience (Institute of Agriculture and Life Science), Gyeongsang National University, Jinju 660-701, Korea 3 2015 28 3 420 427 30 9 2014 16 11 2014 20 11 2014 Copyright © 2015 by Asian-Australasian Journal of Animal Sciences2015It is necessary to understand the cellular uptake and cytotoxicity of food-grade delivery systems, such as β-lactoglobulin (β-lg) nanoparticles, for the application of bioactive compounds to functional foods. The objectives of this study were to investigate the relationships between the physicochemical properties of β-lg nanoparticles, such as particle size and zeta-potential value, and their cellular uptakes and cytotoxicity in Caco-2 cells. Physicochemical properties of β-lg nanoparticles were evaluated using particle size analyzer. Flow cytometry and confocal laser scanning microscopy were used to investigate cellular uptake and cytotoxicity of β-lg nanoparticles. The β-lg nanoparticles with various particle sizes (98 to 192 nm) and zeta-potential values (−14.8 to −17.6 mV) were successfully formed. A decrease in heating temperature from 70°C to 60°C resulted in a decrease in the particle size and an increase in the zeta-potential value of β-lg nanoparticles. Non-cytotoxicity was observed in Caco-2 cells treated with β-lg nanoparticles. There was an increase in cellular uptake of β-lg nanoparticles with a decrease in particle size and an increase in zeta-potential value. Cellular uptake β-lg nanoparticles was negatively correlated with particle size and positively correlated with zeta-potential value. Therefore, these results suggest that the particle size and zeta-potential value of β-lg nanoparticles play an important role in the cellular uptake. The β-lg nanoparticles can be used as a delivery system in foods due to its high cellular uptake and non-cytotoxicity. β-LactoglobulinNanoparticleParticle SizeZeta-potentialCellular UptakeCytotoxicity ==== Body INTRODUCTION Bioactive compounds, such as plant polyphenols and carotenoids, have been used in functional foods providing many health benefits since they may enhance antioxidant, anticancer, and antiviral activities and decrease the risks of diseases (Chen and Subirade, 2005; Chen et al., 2006; Hu et al., 2012). However, these bioactive compounds often have poor bioavailability due to their low aqueous solubility, low permeability across the intestinal cells, and low stability during food processing and storage (Chen and Subirade, 2005; Chen et al., 2006; Hu et al., 2012). To overcome these problems, various delivery systems have been developed to entrap, protect, and deliver bioactive compounds to small intestine enhancing the bioavailability of compounds (Chen and Subirade, 2005; Chen et al., 2006; Zimet and Livney, 2009; Kumari and Yadav, 2011). Although many delivery systems have been successfully made using synthetic polymers, the production of synthetic polymer-based delivery systems may need the use of organic solvent and relatively hasher formulation condition. Therefore, food-grade materials should be used for the manufacture of delivery systems for food applications (Chen and Subirade, 2005; Chen et al., 2006). In this study, beta-lactoglobulin (β-lg), the major component of whey proteins, was selected as a food-grade delivery material. The β-lg has great potentials for the preparation of delivery systems, which is applicable to foods due to its generally regarded as safe status, high nutritional level, biodegradable nature and gel forming capability (Chen and subirade, 2005; Livney, 2010). In our previous study, modulating manufacturing variables including heat treatment resulted in the formation of whey protein nano delivery systems with various sizes and surface charges (Lee et al., 2008; Ha et al., 2013; Lee et al., 2013). It is known that reducing the particle size of delivery systems may enhance the bioavailability of bioactive compounds due to an increase in gastrointestinal transit time, adhesion to and interaction with biological cells, and cellular uptake of delivery systems (Win and Feng, 2005; Zhang et al., 2008; Iversen et al., 2011; Kumari and Yadav, 2011). Moreover, a decrease in the particle size from macroscopic scale to nanoscopic leads to changes in physico-chemical properties of the delivery materials and thus particles with smaller size may elicit different biological responses including toxicity (Yin et al., 2005; Napierska et al., 2009; Powell et al., 2010). On the other hand, the surface charge of nanoparticles can be considered as an important factor to affect the cellular uptake of nanoparticles because of the electrostatic interactions between the surface of nanoparticles and charged cell surface (Mansouri et al., 2006; Cho et al., 2009; Verma and Stellacci, 2010). Although the cellular uptake and cytotoxicity of several food-grade nanoparticles, such as chitosan-based (Zhang et al., 2008) and chitosan-caseinophosphopeptide (Hu et al., 2012) nanoparticles has been examined, there is a lack of knowledge on the delivery of food-grade nanoparticles, especially β-lg nanoparticles, to intestinal cells and cytotoxicity. Therefore, more studies on the cellular uptake and cytotoxicity of β-lg nanoparticles are needed to apply β-lg nanoparticles with bioactive compounds to functional foods. In this study, we hypothesize that the particle size and surface charges of β-lg nanoparticles modulated by heat treatments can be a major factor to affect their cellular uptakes in Caco-2 cells. The objectives of this study were to manufacture β-lg nanoparticles with various sizes and surface charges by changing heating temperature, to determine the relationships between the particle size and surface charge of β-lg nanoparticles and their cellular uptakes in Caco-2 cells, and to evaluate the cytotoxicity of β-lg nanoparticles. MATERIALS AND METHODS Chemicals and reagents The β-lg was generously supplied by Davisco International, Inc. (Le Sueur, MN, USA). Calcium chloride (CaCl2), fluorescein isothiocyanate (FITC), and propidium iodide (PI) were purchased from Sigma-Aldirich Inc. (St. Louis, MO, USA). Sodium hydroxide (NaOH) was purchased from Yakuri Pure Chemicals Co. (Osaka, Japan). Preparation of fluorescein isothiocynate-conjugated beta-lactoglobulin nanoparticles The FITC-conjugated β-lg nanoparticles were manufactured by the internal gelation method of Ha et al. (2013) and Hu et al. (2012). The β-lg solutions (1%, w/v) were treated with 4 mM CaCl2 and adjusted to pH 9.5 using 0.1 M NaOH. To investigate the cellular uptake of β-lg nanoparticles, FITC, a fluorescent marker, was used (Hu et al., 2012). To manufacture FITC-conjugated β-lg, 0.1 mL of FITC solution (40 mg/mL in ethanol) was added to 10 mL of β-lg solution in 0.1 M carbonate buffer at pH 9.0 and then FITC/β-lg mixtures reacted for 2 h in a dark room. To remove unreacted FITC, FITC/β-lg mixtures were dialyzed against distilled water for 48 h using a dialysis membrane (3.5 kDa molecular weight cut-off, Thermo Scientific, Rockford, USA) in a dark room. Those dialyzed mixtures were freeze dried to from FITC-conjugated β-lg. To manufacture FITC-conjugated β-lg nanoparticles, 1% (w/v) FITC-conjugated β-lg solutions were treated with 4 mM CaCl2 and adjusted to pH 9.5 using 0.1 M NaOH. The FITC-conjugated β-lg solutions were heated at 60°C, 65°C, or 70°C for 10 min using a water bath (Wisecircu, Daihan Scientific, Inc., Seoul, Korea) and then cooled to room temperature in ice water. Particle size and zeta-potential measurements The particle size (mean particle diameter, Z-average) and zeta-potential value of β-lg nanoparticles were assessed by the use of Zetasizer Nano ZS (Malvern Instruments, UK). Nanoparticles were diluted (1:10) with deionized water prior to analysis. The particle size and zeta-potential value of nanoparticles were measured at room temperature with scattering angles of 90° and 20°, respectively. Cell culture condition Caco-2 cells, which are human epithelial colorectal adenocarcinoma cells, were used as in vitro model to investigate the cellular uptake and cytotoxicity of β-lg nanoparticles. Caco-2 cells (ATCC HTB37) were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). Caco-2 cells were cultured in Dulbecco-Vogt modified Eagle medium (DMEM, Lonza BioWhittaker, Walkersvile, MD, USA) including 20% fetal bovine serum and 1% penicillin-streptomycin and maintained in 5% CO2 incubator at 37°C. Cytotoxicity and cellular uptake of beta-lactoglobulin nanoparticles: Quantitative studies Caco-2 cells were seeded in 6-well plates and incubated at 37°C, 5% CO2 in CO2 incubator for 24 h to form a confluent monolayer. Culture medium was replaced by serum-free DMEM and incubated at 37°C for 30 min. FITC-conjugated β-lg nanoparticles dissolved in serum-free DMEM were added to Caco-2 cells and then incubated at 37°C for 0.5, 1, 2, 3, or 4 h to initiate the cellular uptake of nanoparticles. Final concentration of β-lg nanoparticles in serum-free DMEM was 100, 250, or 500 μg/mL. To remove free nanoparticles in serum-free DMEM, cells were washed three times with phosphate-buffered saline (PBS, pH 7.4) and collected with trypsin treatment. The FITC-conjugated β-lg nanoparticles were used as a marker to quantify the cellular uptake of β-lg nanoparticles. Cytotoxicity and cellular uptake of nanoparticles were evaluated by FACSCalibur flow cytometry and CellQuest Pro software (Becton Dickenson, Mississauga, CA, USA). In nanoparticle treated cells, cell viability was evaluated with PI staining followed by flow cytometric analysis (Russell et al., 2004; Chen et al., 2009). Excitation and emission wavelengths were 488 nm and 590 nm, respectively. The percentage of live and dead cells was determined by measuring the PI-negative cells and PI-positive cells, respectively. The cell survival ratio was expressed as percent of PI-negative cells per control cells which were cultured in nanoparticle-free medium. The cellular uptake of FITC-conjugated β-lg nanoparticles in Caco-2 cells was also analyzed using flow cytometry at 488 nm excitation wavelength and 530 nm emission wavelength (Pushpanathan et al., 2012). The FITC-positive cells indicate the number of Caco-2 cells that have uptaken β-lg nanoparticles. Cellular uptake efficiency was expressed as percent of FITC-positive cells per total cells. Cellular uptake of beta-lactoglobulin nanoparticles: Qualitative studies Confocal laser scanning microscopy (CLSM) was used to observe the cellular uptake of FITC-conjugated β-lg nanoparticle in Caco-2 cells. To observe Caco-2 cells, cells were grown on a glass microscope cover-slip. The nuclei of Caco-2 cells were stained with PI (0.1 mg/mL). Cover-slips were then inverted and mounted onto microscope slides and cellular uptake of nanoparticles were observed using a CLSM (FV1000, Olympus, Tokyo, Japan) equipped with a ×40 (numerical aperture, 1.0) oil immersion lens. Fluorescent images were analyzed using a confocal microscopy (Fluoview FV1000, Olympus, Tokyo, Japan). Fluorescence signals were detected at excitation/emission wavelengths of 535/617 nm (PI, red) and 488/530 nm (FITC, green), respectively. Statistical analysis All data were expressed as mean±standard errors. The significance of the effects of FITC conjugation, heating temperature, and their interaction was analyzed by a two-way analysis of variance (ANOVA). One-way ANOVA with the Fisher’s least significant difference (LSD) test was used to analyze the effects of heating temperature on the zeta-potential value, cytotoxicity, and cellular uptake of β-lg nanoparticles. Pearson’s correlation coefficient analysis was used to determine the relationships between dependent variables (heating temperature, particle size, and zeta-potential of β-lg nanoparticles). The level of significance was set at the 5% level (p<0.05). All statistical analyses were used with SAS software package (2003). RESULTS AND DISCUSSION Physicochemical properties of fluorescein isothiocyanate- conjugated beta-lactoglobulin nanoparticles Effects of heating temperature on the physicochemical properties of β-lg nanoparticles, such as particle size and zeta-potential, were shown in Figures 1 and 2. An increase in heating temperature from 60°C to 70°C resulted in a significant (p<0.05) increase in the size of β-lg nanoparticles from 97 to 176 nm (Figure 1). It was reported that the heat treatment of β-lg from 60°C to 70°C may lead to increased surface hydrophobicity and free sulfhydryl groups exposed to aqueous environment determined by ANS probe method and DTNB method, respectively (Lee et al., 2008; Lee et al., 2013). An increase in surface hydrophobicity and exposed free sulfhydryl residues can be a driving force to increase intermolecular hydrophobic attractions and thiol-disulfide interactions, which may lead an increase in the size of β-lg nanoparticles. Similar results were reported that the size of whey protein particles was increased from 70 to 200 nm as heating temperature was increased from 58°C to 77°C (Gracia-Julia et al., 2008). Conjugation of FITC with β-lg did not significantly (p<0.05) affect the size of nanoparticles (Figure 1). Negative zeta-potential values (−14.8 to −17.6 mV) were observed in β-lg nanoparticles, which indicates that β-lg nanoparticle had negative surface charges. Since the isoelectric point of β-lg is about 5.3, β-lg nanoparticles manufactured at pH 9.5 had negative surface charges. Zeta-potential value of β-lg nanoparticles was significantly (p<0.05) decreased from −14.8 to −17.6 mV when heating temperature was increased from 60°C to 70°C (Figure 2). An increase in heating temperature may lead to partial unfolding of β-lg. And thus, negatively charged amino acids buried inside of β-lg could be exposed to the surface of β-lg, which could result in a decrease in the zeta-potential value of β-lg nanoparticles. Similar results were observed for β-lg dispersions heated at 85°C for 10 min (Schmitt et al., 2009). Cytotoxicity of beta-lactoglobulin nanoparticles There were growing interests in the safety and toxicity of nanoparticles since the small size of nanoparticles may cause toxicity problem (Yin et al., 2005; Napierska et al., 2009). A reduction in the size of nanoparticle led to an increase in the surface area, which can enhance interactions between nanoparticles and cell membranes (Zhang et al., 2008). Interactions between charged nanoparticles and cell membranes may induce the transient poration of cell membranes, which can be related to the cytotoxicity (Cockburn et al., 2012). The cytotoxicity of β-lg nanoparticles in Caco-2 cells were determined by measuring the cell survival ratio using flow cytometry prior to cellular uptake study (Figure 3). The measurement of cell survival ratio is the common method to investigate the in vitro cytotoxicity of biomaterials (Hu et al., 2012). Nanoparticle concentration (100 to 500 μg/mL) and incubation time (0.5 to 4 h) did not significantly (p<0.05) affect the cell survival ratio. When Caco-2 cells were treated with FITC-conjugated β-lg nanoparticles at concentration of 250 μg/mL for 2 h, no significant differences in cell survival ratio were observed indicating that β-lg nanoparticles had no cytotoxicity to Caco-2 cells (Figure 3). Although β-lg nanoparticles had very small size ranging from 98 to 192 nm, the size of β-lg nanoparticles was not an important determinant in cytotoxicity. Similar results were reported for food-grade nanoparticles. Zhang et al. (2008) reported that oleyl-chitosan nanoparticles exhibited no cytotoxicity when nanoparticles were treated with concentration up to 400 μg/mL on A549 cells. Chitosan-caseinophosphopeptide nanoparticles with concentration below 500 μg/mL were also non-toxic to Caco-2 cells (Hu et al., 2012). Cellular uptake of beta-lactoglobulin nanoparticles: Quantitative studies The in vitro or in vivo cellular uptake of nanoparticles was investigated by the use of fluorescent-based method (Win and Feng, 2005). In this study, Caco-2 cells were selected as in vitro model to investigate the cellular uptake of β-lg nanoparticles in small intestine since there are similar morphological and functional properties between Caco-2 cell lines and small intestinal enterocytes (Win and Feng, 2005; Powell et al., 2010). Fluorescein isothiocynate, was used as a fluorescent marker to determine the cellular uptake of β-lg nanoparticles in Caco-2 cells (Hu et al., 2012). Since the concentration and incubation time of nanoparticle could be key factors to affect the in vitro cellular uptake of nanoparticles (Zhang et al., 2008), the effects of concentration and incubation time of nanoparticles on the cellular uptake of β-lg nanoparticles were examined (Figure 4). As the concentration of β-lg nanoparticles was increased from 100 to 250 μg/mL, the cellular uptake of β-lg nanoparticles was significantly (p<0.05) increased (Figure 4A). However, a further increase in the concentration of nanoparticles from 250 to 500 μg/mL did not significantly (p<0.05) affect the cellular uptake of nanoparticles indicating that a saturation limit of nanoparticle was 250 μg/mL. An increase in incubation time from 0.5 to 2 h resulted in a significant (p<0.05) increase in the cellular uptake of β-lg nanoparticles while no significant (p<0.05) differences in the cellular uptake of β-lg nanoparticles after 2 h incubation were observed (Figure 4B). Therefore, FITC-conjugated β-lg nanoparticle suspensions with 250 μg/mL concentration and 2 h incubation time were used for further experiments. It is believed that the cellular uptake of nanoparticles was affected by various morphological- and physicochemical properties of nanoparticles, such as their size and surface charge (Mansouri et al., 2006; Kumari and Yadav, 2011; Yoo et al., 2011). Effect of heating temperature on the cellular uptake of β-lg nanoparticles was displayed in Figure 5. A decrease in heating temperature from 70°C to 60°C led to a decrease in the size of β-lg nanoparticles from 192 to 98 nm (Figure 1). The cellular uptake of β-lg nanoparticles was significantly (p<0.05) increased as heating temperature decreased from 70°C to 60°C (Figure 5). Smaller β-lg nanoparticles prepared at lower heating temperature had higher uptake efficiency of Caco-2 cell compared to larger β-lg nanoparticles treated at higher heating temperature. There were significantly negative correlations between the particle size and the cellular uptake of β-lg nanoparticles with r values of −0.76 (p<0.001) (Table 1). Since nanoparticles with smaller size had larger surface areas available to adhere and interact with cell membranes, a decrease in the size of nanoparticles may lead to an increase in the interactions and binding with cell membranes, which can result in enhanced cellular uptake of nanoparticles (Win and Feng, 2005; Chen et al., 2006; Zhang et al., 2008; Kumari and Yadav, 2011; Iversen et al., 2011; Ha et al., 2013). Similar results were reported for oleyl chitosan and PLGA nanoparticles (Win and Feng, 2005; Zhang et al., 2008). The cellular uptake of oleyl chitosan nanoparticles was increased from 7.6% to 8.8% when the size of nanoparticle was decreased from 307 to 199 nm (Zhang et al., 2008). Win and Feng (2005) reported that a reduction in the size of PLGA nanoparticles from 1,000 to 100 nm resulted in an increase in the cellular uptake of nanoparticles from 9% to 14% in Caco-2 cells. On the other hand, an increase in the cellular uptake of β-lg nanoparticles was observed when β-lg nanoparticles treated at lower heating temperature had higher zeta-potential value (surface charge) (Figure 5). Cellular uptake of β-lg nanoparticles was positively correlated with zeta-potential of β-lg nanoparticles (r = 0.75, p<0.01) (Table 1). Similar results was reported by He et al. (2010), who found that an increase in zeta-potential values of carboxymethyl chitosan grafted methyl methacrylate nanoparticles from −40 to −15 mV resulted in a significant increase in the cellular uptake of nanoparticles in murine macrophage cells. An increase in heating temperature may lead to an increase in the zeta-potential value of β-lg nanoparticles, which could result in stronger electrostatic repulsion between the β-lg nanoparticles and the cell membranes (He et al., 2010). This may cause a reduction in the adhesion and interaction of β-lg nanoparticles with Caco-2 cell surface and hence a decrease in cellular uptake of nanoparticles. Cellular uptake of beta-lactoglobulin nanoparticles: Qualitative studies Qualitative study on the cellular uptake of FITC-conjugated β-lg nanoparticles were carried out using a CLSM. The cellular uptake of β-lg nanoparticles treated at various heating temperatures was shown in Figure 6. Propidium iodide (red fluorescence) was used to visualize the nuclei of Caco-2 cells while FITC (green fluorescence) was conjugated to β-lg to observe the cellular uptake of nanoparticles. Confocal microscopic images of Caco-2 cells treated with FITC-conjugated β-lg nanoparticles were examined with PI channel (Figure 6, left panel), FITC channel (Figure 6, middle panel), and combined PI channel and FITC channel (Figure 6, right panel). The nuclei of Caco-2 cells were stained with PI (red) and displayed in Figure 6 (left panel). In Figure 6 (middle panel), the green color exhibited FITC-conjugated β-lg nanoparticles prepared at 60°C, 65°C, or 70°C heating temperature. The overlaying images of Caco-2 cell nuclei (PI, red) and β-lg nanoparticles (FITC, green) were shown in Figure 6 (right panel). In Figure 6 (right panel), β-lg nanoparticles were observed around the nuclei of Caco-2 cells (in the cell membrane and inside the cytosol) indicating that β-lg nanoparticles were successfully taken up by Caco-2 cells. No fluorescence was observed in the confocal microscopic image of control cells (data not shown) indicating that Caco-2 cell or β-lg do not have autofluorescence. In conclusion, food-grade β-lg nanoparticles with various particle size and surface charge were successfully manufactured by the internal gelation method. The β-lg nanoparticles were non-cytotoxic to Caco-2 cells and can be taken up by Caco-2 cells. Heating temperature can modulate the size and surface charge of β-lg nanoparticles. The size and surface charge of β-lg nanoparticles were crucial factors affecting the cellular uptake of β-lg nanoparticles in Caco-2 cells. A better understanding of the factors contributing to the cellular uptake of nanoparticles could allow dairy processors to apply β-lg nanoparticlesas a delivery system in functional foods. ACKNOWLEDGMENTS This research was carried out with the support of “Cooperative Research Program for Agriculture Science & Technology Development (PJ009659032013)” Rural Development Administration and Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2011-0013978; 2012R1A1A4A01013793), Republic of Korea. Figure 1 Particle size of β-lactoglobulin (β-lg) and fluorescein isothiocyanate-conjugated β-lg nanoparticles prepared at various heating temperature (60°C, 65°C, and 70°C). NPs, nanoparticles; HT, heating temperature; ns, not significant (p>0.05). Figure 2 Zeta-potential of fluorescein isothiocyanate-conjugated β-lactoglobulin nanoparticles prepared at various heating temperature (60°C, 65°C, and 70°C). Different letters on a column indicate significant differences (p<0.05). Figure 3 Viability of Caco-2 cells treated with β-lactoglobulin (β-lg) nanoparticles prepared at various heating temperature (60°C, 65°C, and 70°C). The β-lg nanoparticles (250 μg/mL) were added to Caco-2 cells and incubated at 37°C for 2 h. Different letters on a column indicate significant differences (p<0.05). Figure 4 Effects of nanoparticle concentration and incubation time on the cellular uptake of β-lactoglobulin (β-lg) nanoparticles in Caco-2 cells. (A) Various concentration of fluorescein isothiocyanate (FITC)-conjugated β-lg nanoparticles (100, 250, and 500 μg/mL) were added to Caco-2 cells and incubated at 37°C for 2 h. (B) FITC-conjugated β-lg nanoparticles (250 μg/mL) were added to Caco-2 cells and incubated at 37°C for 0, 0.5, 1, 2, 3, 4 h. Different letters on a column indicate significant differences (p<0.05). Figure 5 Effect of heating temperature on the cellular uptake of fluorescein isothiocyanate (FITC)-conjugated β-lactoglobulin (β-lg) nanoparticles in Caco-2 cells. FITC-conjugated β-lg nanoparticles (250 μg/mL) were added to Caco-2 cells and incubated at 37°C for 2 h. Different letters on a column indicate significant differences (p<0.05). Figure 6 Confocal laser scanning microscopy images of Caco-2 cells after 2 h incubation at 37°C with fluorescein isothiocyanate (FITC)-conjugated β-lactoglobulin (β-lg) nanoparticles (250 μg/mL) prepared at (A) 60°C, (B) 65°C, and (C) 70°C heating temperatures. To observe the cellular uptake of β-lg nanoparticles, cells were stained with propidium iodide (PI) and FITC-conjugated β-lg nanoparticles were used. Cellular uptake of FITC-conjugated β-lg nanoparticles was imaged by PI channel (left panel), FITC channel (middle panel), and combined PI channel and FITC channel (right panel), respectively. Table 1 Pearson’s correlation coefficients (r) between dependent variables Particle size Zeta-potential Cellular uptake Particle size −0.83* −0.76* Zeta-potential −0.83* 0.75 Cellular uptake −0.76* 0.75 , * Significantly different at p<0.01 and p<0.001, respectively. ==== Refs REFERENCES Chen L Subirade M 2005 Chitosan/β -lactoglobulin core–shell nanoparticles as nutraceutical carriers Biomaterials 26 6041 6053 15885766 Chen L Remondetto GE Subirade M 2006 Food protein-based materials as nutraceutical delivery systems Trends Food Sci Technol 17 272 283 Cho EC Xie J Wurm PA Xia Y 2009 Understanding the role of surface charges in cellular adsorption versus internalization by selectively removing gold nanoparticles on the cell surface with a I2 /KI etchant Nano Lett 9 1080 1084 19199477 Cockburn A Bradford R Buck N Constable A Edwards G Haber B Hepburn P Howlett J Kampers F Klein C Radomski M Stamm H Wijnhoven S Wildemann T 2012 Approaches to the safety assessment of engineered nanomaterials (ENM) in food Food Chem Toxicol 50 2224 2242 22245376 Ha HK Kim JW Lee M-R Lee W-J 2013 Formation and characterization of quercetin-loaded chitosan oligosaccharide/β -lactogloublin nanoparticle Food Res Int 52 82 90 He C Hu Y Yin L Tang C Yin C 2010 Effects of particle size and surface charge on cellular uptake and biodistribution of polymeric nanoparticles Biomaterials 31 3657 3666 20138662 Hu B Ting Y Zeng X Huang Q 2012 Cellular uptake and cytotoxicity of chitosan–caseinophosphopeptides nanocomplexes loaded with epigallocatechin gallate Carbohydr Polym 89 362 370 24750731 Iversen TG Skotland T Sandvig K 2011 Endocytosis and intracellular transport of nanoparticles: Present knowledge and need for future studies Nano Today 6 176 185 Kumari A Yadav SK 2011 Cellular interactions of therapeutically delivered nanoparticles Expert Opin Drug Deliv 8 141 151 21219249 Lee MR Nam G-W Choi H-N Yun H-S Kim S-H You S-K Park D-J Lee W-J 2008 Structure and chemical properties of beta-lactoglobulin nanoparticles J Agric Life Sci 42 31 36 Lee MR Choi H-N Ha H-K Lee W-J 2013 Production and characterization of beta-lactoglobulin/alginate nanoemulsion containing coenzyme Q10 : Impact of heat treatment and alginate concentrate Korean J Food Sci Anim 33 67 74 Livney YD 2010 Milk proteins as vehicles for bioactives Curr Opin Colloid Interface Sci 15 73 83 Mansouri S Cuie Y Winnik F Shi Q Lavigne P Benderdour M Beaumont E Fernandes JC 2006 Characterization of folate-chitosan-DNA nanoparticles for gene therapy Biomaterials 27 2060 2065 16202449 Napierska D Thomassen LCJ Rabolli V Lison D Gonzalez L Kirsch-Volders M Martens JA Hoet PH 2009 Size-dependent cytotoxicity of monodisperse silica nanoparticles in human endothelial cells Small 5 846 853 19288475 Powell JJ Faria N Thomas-McKay E Pele LC 2010 Origin and fate of dietary nanoparticles and microparticles in the gastrointestinal tract J Autoimmun 34 J226 J233 20096538 Pushpanathan M Rajendhran J Jayashree S Sundarakrishnan B Jayachandran S Gunasekaran P 2012 Direct cell penetration of the antifungal peptide, MMGP1, in Canadida albicans J Pept Sci 18 657 660 23080316 Russell P Hewish D Carter T Sterling-Levis K Ow K Hattarki M Doughty L Guthrie R Shapira D Molloy PL Werkmeister JA Kortt AA 2004 Cytotoxic properties of immunoconjugates containing melittin-like peptide 101 against prostate cancer: in vitro and in vivo studies Cancer Immunol Immunother 53 411 421 14722668 SAS Institute Inc 2003 SAS User’s Guide: version 9.1 Cary, NC, USA Schmitt C Bovay C Vuilliomenet AM Rouvet M Bovetto L Barbar R Sanchez C 2009 Multiscale characterization of individualized β-lactoglobulin microgels formed upon heat treatment under narrow pH range conditions Langmuir 25 7899 7909 19594178 Verma A Stellacci F 2010 Effect of surface properties on nanoparticle–cell interactions Small 6 12 21 19844908 Win KY Feng S-S 2005 Effects of particle size and surface coating on cellular uptake of polymeric nanoparticles for oral delivery of anticancer drugs Biomaterials 26 2713 2722 15585275 Yin H Too HP Chow GM 2005 The effects of particle size and surface coating on the cytotoxicity of nickel ferrite Biomaterials 26 5818 5826 15949547 Yoo JW Doshi N Mitragotri S 2011 Adaptive micro and nanoparticles: Temporal control over carrier properties to facilitate drug delivery Adv Drug Deliv Rev 63 1247 1256 21605607 Zhang J Chen XG Peng WB Liu CS 2008 Uptake of oleoyl-chitosan nanoparticles by A549 cells Nanomedicine 4 208 214 18508414 Zimet P Livney YD 2009 Beta-lactoglobulin and its nanocomplexes with pectin as vehicles for ω-3 polyunsaturated fatty acids Food Hydrocoll 23 1120 1126	25656189	PMC4341088	NO-CC CODE	2021-01-05 02:53:14	yes	Asian-Australas J Anim Sci. 2015 Mar; 28(3):420-427
==== Front Proc Natl Acad Sci U S AProc. Natl. Acad. Sci. U.S.ApnaspnasPNASProceedings of the National Academy of Sciences of the United States of America0027-84241091-6490National Academy of Sciences 2577553320141727310.1073/pnas.1417273112Biological SciencesMedical SciencesAtrial natriuretic peptide prevents cancer metastasis through vascular endothelial cells ANP-mediated prevention of cancer metastasisNojiri Takashi abHosoda Hiroshi cTokudome Takeshi aMiura Koichi cIshikane Shin aOtani Kentaro cKishimoto Ichiro dShintani Yasushi bInoue Masayoshi bKimura Toru abSawabata Noriyoshi bMinami Masato bNakagiri Tomoyuki bFunaki Soichiro bTakeuchi Yukiyasu eMaeda Hajime eKidoya Hiroyasu fKiyonari Hiroshi gShioi Go gArai Yuji hHasegawa Takeshi iTakakura Nobuyuki fHori Megumi jOhno Yuko jMiyazato Mikiya aMochizuki Naoki kOkumura Meinoshin bKangawa Kenji a1aDepartment of Biochemistry,cDepartment of Regenerative Medicine and Tissue Engineering,hDepartment of Molecular Biology, andkJST-CREST/Department of Cell Biology, National Cerebral and Cardiovascular Center Research Institute, Suita-city, Osaka 565-8565, Japan;bDepartment of General Thoracic Surgery andjDepartment of Mathematical Health Science, Osaka University Graduate School of Medicine, Suita-city, Osaka, 565-0871, Japan;dDepartment of Endocrinology and Metabolism, National Cerebral and Cardiovascular Center Hospital, Suita-city, Osaka 565-8565, Japan;eDepartment of General Thoracic Surgery, National Hospital Organization Toneyama Hospital, Toyonaka-city, Osaka, 560-8552, Japan;fDepartment of Signal Transduction, Research Institute for Microbial Diseases, Suita-city, Osaka 565-0871, Japan;gLaboratory for Animal Resources and Genetic Engineering, RIKEN Center for Developmental Biology, Kobe-city, Hyogo 650-0047, Japan; andiDrug Research Section II, Fukushima Research Laboratories, TOA EIYO Ltd., Fukushima-city, Fukushima 960-0280, Japan1To whom correspondence should be addressed. Email: [email protected]* by Masashi Yanagisawa, University of Texas Southwestern Medical Center, Dallas, TX, and approved February 18, 2015 (received for review September 7, 2014) Author contributions: T. Nojiri, H.H., T.T., N.T., M. Miyazato, N.M., M.O., and K.K. designed research; T. Nojiri, H.H., T.T., K.M., S.I., K.O., I.K., Y.S., M.I., T.K., N.S., M. Minami, T. Nakagiri, S.F., Y.T., H.M., H. Kidoya, and N.M. performed research; T. Nojiri, H. Kiyonari, G.S., and Y.A. contributed new reagents/analytic tools; T. Nojiri, T.H., M.H., and Y.O. analyzed data; and T. Nojiri, N.M., and K.K. wrote the paper. 31 3 2015 16 3 2015 16 3 2015 112 13 4086 4091 Freely available online through the PNAS open access option.Significance Postoperative cancer recurrence is a major problem following curative cancer surgery. Perioperative systemic inflammation induces the adhesion of circulating tumor cells released from the primary tumor to the vascular endothelium of distant organs, which is the first step in hematogenous metastasis. We have previously reported that administration of atrial natriuretic peptide (ANP) during the perioperative period reduces inflammatory response and has a prophylactic effect on postoperative cardiopulmonary complications in lung cancer surgery. Here, we demonstrate that cancer recurrence after lung cancer surgery was significantly lower in ANP-treated patients than in control patients (surgery alone). We show that ANP prevents cancer metastasis by suppressing the inflammatory reaction of endothelial cells, thereby inhibiting cancer cell adhesion to vascular endothelial cells. Most patients suffering from cancer die of metastatic disease. Surgical removal of solid tumors is performed as an initial attempt to cure patients; however, surgery is often accompanied with trauma, which can promote early recurrence by provoking detachment of tumor cells into the blood stream or inducing systemic inflammation or both. We have previously reported that administration of atrial natriuretic peptide (ANP) during the perioperative period reduces inflammatory response and has a prophylactic effect on postoperative cardiopulmonary complications in lung cancer surgery. Here we demonstrate that cancer recurrence after curative surgery was significantly lower in ANP-treated patients than in control patients (surgery alone). ANP is known to bind specifically to NPR1 [also called guanylyl cyclase-A (GC-A) receptor]. In mouse models, we found that metastasis of GC-A–nonexpressing tumor cells (i.e., B16 mouse melanoma cells) to the lung was increased in vascular endothelium-specific GC-A knockout mice and decreased in vascular endothelium-specific GC-A transgenic mice compared with control mice. We examined the effect of ANP on tumor metastasis in mice treated with lipopolysaccharide, which mimics systemic inflammation induced by surgical stress. ANP inhibited the adhesion of cancer cells to pulmonary arterial and micro-vascular endothelial cells by suppressing the E-selectin expression that is promoted by inflammation. These results suggest that ANP prevents cancer metastasis by inhibiting the adhesion of tumor cells to inflamed endothelial cells. cardiac peptidecancer metastasisvascular endothelial cellinflammationsurgery ==== Body The majority of cancer patients die from tumor metastasis. Despite substantial advances in our understanding of the mechanisms of tumor metastasis, effective prevention of metastasis has not been well established. Surgical removal of solid tumors is performed to cure patients if the primary tumor meets surgical indications; however, postoperative cancer recurrence is a major problem. Surgical trauma itself influences the development of early recurrence (1, 2). First, the procedure during tumor removal might provoke detachment of tumor cells; consistently, the number of circulating tumor cells is increased during primary tumor resection (3, 4). We previously reported that the presence of circulating tumor cells in pulmonary veins during lung cancer surgery could be a prognostic indicator for early cancer recurrence (4). Second, surgical trauma provokes a severe systemic inflammatory reaction. Emerging evidence suggests that systemic inflammation can accelerate the adhesion of circulating tumor cells to the vascular endothelium of distant organs, which is the first step of extravasation in hematogenous metastasis (5, 6). We identified human atrial natriuretic peptide (ANP) as a diuretic, natriuretic, and vasodilating hormone from the human heart in 1984 (7). ANP binds specifically to the guanylyl cyclase-A (GC-A) receptor to exhibit biological functions, including promotion of diuresis, antifibrotic action, and inhibition of renin-angiotensin-aldosterone (8, 9). Thus, ANP has been used clinically for the treatment of heart failure since 1995 in Japan. We previously reported that administration of human ANP during the perioperative period reduces inflammatory responses and has a prophylactic effect on postoperative cardiopulmonary complications in lung cancer surgery (10–12). In those studies, ANP was used to promote diuresis during perioperative right-side heart failure caused by lung damage. Here, we further analyzed the effect of ANP on prevention of cancer recurrence after surgery and found that ANP might have antitumor metastatic activity. We explored the antimetastatic action of ANP by using tissue-specific GC-A transgenic and knockout mice of tumor metastasis models. Our results suggest that ANP could be useful as an antimetastasis peptide to prevent cancer recurrence after surgery. Results Clinical Impacts of ANP Therapy on Cancer Recurrence After Lung Cancer Surgery. We performed a retrospective study of the incidence of cancer recurrence in lung cancer patients after curative surgery, comparing patients who underwent perioperative ANP treatment with those who were subjected to surgery alone (control patients). The 2-y relapse-free survival (RFS) after surgery was significantly greater in ANP-treated patients than in control patients (91% vs. 75%, P = 0.018) (Fig. 1A). To eliminate bias, we reanalyzed the data by using propensity score matching. The 2-y RFS in the propensity score-matched analysis was also significantly greater in ANP-treated patients than in control patients (91% vs. 67%, P = 0.0013) (Fig. 1B and SI Appendix, Table S1). We hypothesized from these retrospective observations that ANP may prevent recurrence of lung cancer. Fig. 1. Effect of ANP treatment on RFS in patients with surgically resected nonsmall cell lung cancer. (A) Kaplan–Meier curves of the ANP group and control group (surgery alone) in all patients (P = 0.018, log-rank test). (B) Kaplan–Meier curves of the above groups in propensity score-matched patients (P = 0.0013, log-rank test). RFS was measured from the day of surgery to cancer recurrence. Antimetastatic Effects of ANP in Hematogenous Pulmonary Metastatic Models. Vascular inflammation is considered to render the endothelium adhesive to circulating tumor cells, thereby allowing the metastasis of tumor cells (5, 6). We previously reported that postoperative complications induced by inflammation are reduced by ANP (10–12). Therefore, to investigate whether ANP inhibits the metastasis of cancer cells to inflamed organs, we examined the effect of ANP on tumor metastases in mice injected with LPS, which mimics systemic inflammation induced by surgical stress (6, 13). The LPS-treated mice showed numerous hematogenous pulmonary metastases of intravenously injected A549 lung cancer cells expressing EGFP (A549-EGFP) cells (Fig. 2 A and B and SI Appendix, Fig. S1A). In contrast, the mice pretreated with ANP exhibited a large and significant reduction of LPS-induced pulmonary metastasis of A549-EGFP cells (Fig. 2 A and B and SI Appendix, Fig. S1A). ANP also significantly inhibited the pulmonary hematogenous metastasis of B16/F10 melanoma cells, which do not express GC-A (Fig. 2 C and D and SI Appendix, Fig. S1 B and C). Furthermore, we confirmed that ANP significantly inhibited the pulmonary hematogenous metastasis of A549-EGFP (SI Appendix, Fig. S2 A and B) and B16/F10 (SI Appendix, Fig. S2 C and D) cells even without LPS, suggesting that ANP inhibits tumor metastasis both under LPS-induced general inflammation and under non–LPS-induced massive inflammation. More importantly, these data indicate that ANP acts through GC-A expressed in nontumor mouse cells. Fig. 2. ANP inhibits the LPS-augmented metastasis of A549-EGFP lung cancer cells and B16/F10 mice melanoma cells to the lung. (A) Representative EGFP images of the lungs of mice that were pretreated with or without LPS and then injected with A549-EGFP cells (1 × 106 cells per mouse) and continuously treated with or without ANP for 4 wk. The mice were killed 6 wk after the injection of tumor cells. (B) Bar graph showing the number of nodules representing pulmonary metastasis of A549-EGFP cells in mice grouped as in A. Data are means ± SD (n = 6, each group). *P < 0.001, unpaired two-tailed t test. (C) Representative images of the lungs of mice that were pretreated with or without LPS and then injected with B16/F10 cells (2 × 105 cells per mouse) and continuously treated with or without ANP for 2 wk. The mice were killed 2 wk after the injection of the tumor cells. (D) Bar graph showing the number of nodules representing pulmonary metastasis of B16/F10 cells in mice grouped as in C. Data are means ± SD (n = 6, each group). P < 0.001, unpaired two-tailed t test. (E) Representative images of the lungs and hearts (Top and Middle, respectively) and histological cross-sections of the hearts (H&E staining, Bottom) of the GC-Aflox/flox mice and EC GC-A-KO mice after injection of B16/F10 cells (2 × 105 cells per mouse). The mice were killed 2 wk after the injection of the tumor cells. (Scale bars, 500 μm.) Red arrows indicate metastasis in the heart. (F) Bar graph showing the number of nodules representing pulmonary metastasis of B16/F10 cells in mice grouped as in E. Data are means ± SD (n = 9, 7, each group). P < 0.01, unpaired two-tailed t test. (G) Kaplan–Meier curves comparing survival times between GC-Aflox/flox and EC GC-A-KO mice after injection of B16/F10 cells (2 × 105 cells per mouse). n = 12, 11 (each group), P < 0.05, log-rank test. (H) Representative images of the lungs of WT and EC GC-A-Tg mice after injection of B16/F10 cells (5 × 105 cells per mouse). The mice were killed 2 wk after the injection of tumor cells. (I) Bar graph showing the number of nodules representing pulmonary metastasis of B16/F10 cells in mice grouped as in H. Data are means ± SD (n = 10, 8, each group). *P < 0.01, unpaired two-tailed t test. (J) Kaplan–Meier curves comparing survival times between WT and EC GC-A-Tg mice after injection of B16/F10 (5 × 105 cells per mouse). n = 15 (each group), P < 0.05, log-rank test. Whole images of lungs were shown in SI Appendix, Fig. S1. To eliminate the direct effect of ANP on tumor cell proliferation, we first examined the direct effects of ANP on the growth of cancer cells and found that GC-A was expressed in A549 and H460 human lung cancer cells (SI Appendix, Fig. S1B). Even though GC-A was expressed on A549 and H460 cells, ANP did not induce the proliferation of these tumor cells (SI Appendix, Fig. S3 A–C). Natriuretic peptide receptor-C, which is also known as a receptor of ANP, was expressed in A549, H460, and B16/F10 cells (SI Appendix, Fig. S3A); however, there were no significant effects of ANP on the growth of A549, H460, or B16/F10 cells (SI Appendix, Fig. S3 B–D). These results suggest that the inhibitory effect of ANP on tumor metastasis is dependent upon GC-A expressed on cells other than tumor cells. We considered that GC-A expressed on endothelial cells might be responsible for the antimetastatic effect of ANP because cancer cell attachment to endothelial cells is the initial step in metastasis (5, 6). Vascular endothelial cells abundantly express GC-A, which exhibits a protective role in the cardiovascular system (8, 9). Therefore, to clearly show that the antimetastatic effect of ANP does not depend on GC-A expression in tumor cells, we examined the hematogenous pulmonary metastasis of B16/F10 cells in both endothelium-specific GC-A knockout mice (termed EC GC-A-KO mice) and GC-A transgenic mice (termed EC GC-A-Tg mice) (SI Appendix, Fig. S4). EC GC-A-KO mice exhibited a significant elevation of blood pressure and cardiac hypertrophy compared with GC-Aflox/flox mice. These phenotypic data were consistent with the previous report (14). The number of pulmonary metastases was significantly higher in EC GC-A-KO mice than in GC-Aflox/flox mice (Fig. 2 E and F). Furthermore, cardiac metastases were found in one-third of EC GC-A-KO mice, whereas no cardiac metastasis was found in GC-Aflox/flox mice (Fig. 2E). Overall survival was significantly shorter in EC GC-A-KO mice compared with GC-Aflox/flox mice (Fig. 2G). In contrast, the number of pulmonary metastases was significantly lower in EC GC-A-Tg mice than in WT mice (Fig. 2 H and I), and EC GC-A-Tg mice survived significantly longer than WT mice (Fig. 2J). Collectively, these data suggest that endothelial GC-A activated by ANP prevents hematogenous pulmonary metastasis of cancer cells in mice. Mechanism of the Effect of ANP on Cancer Cell Adhesion to Vascular Endothelial Cells. We next attempted to uncover the molecular mechanism behind ANP-mediated inhibition of tumor metastasis through vascular endothelial cells. The attachment of A549-EGFP and H460 lung cancer cells expressing EGFP (H460-EGFP) cells to cultured human pulmonary artery endothelial cells (HPAECs) stimulated with LPS was dependent on the dose of LPS (Fig. 3A). ANP significantly inhibited LPS-induced tumor cell attachment to HPAECs (Fig. 3 B and C) and human lung microvascular endothelial cells (HMVEC-L) (SI Appendix, Fig. S5 A–C). LPS induces the expression of cell adhesion molecules, including E-selectin, vascular cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1) (15), which in turn promote the infiltration of inflammatory cells, thereby increasing inflammation. Among these cell adhesion molecules, E-selectin is considered to play a central role in hematogenous metastasis (16–18). We therefore examined the adhesion molecule-dependent attachment of tumor cells to vascular endothelial cells. The attachment of A549-EGFP cells to LPS-induced HPAECs was significantly inhibited by knockdown of E-selectin, but not by knockdown of VCAM-1 or ICAM-1 (Fig. 3 D and E and SI Appendix, Fig. S6A). Fig. 3. ANP inhibits LPS-regulated E-selectin–dependent adhesion of cancer cells to vascular endothelial cells. (A) Representative images of the adhesion of tumor cells (A549-EGFP, Upper; H460-EGFP, Lower) to monolayer-cultured HPAECs pretreated with or without ANP. (B and C) Bar graphs showing the number of A549-EGFP cells (B) or H460-EGFP cells (C) attached to monolayer-cultured HPAECs pretreated with or without ANP. Data are means ± SEM (n = 3, each group). P < 0.05, P < 0.01, *P < 0.001, unpaired two-tailed t test. (D) Representative images of A549-EGFP cells attached to HPAECs depleted of the indicated molecules by siRNA treatment and treated with LPS. (E) Bar graph showing the number of A549-EGFP cells attached to HPAECs treated as in (D). Data are means ± SEM (n = 4–5, each group). P < 0.05, one-way ANOVA. (Scale bars, 500 μm.) To search for genes that could be responsible for the ANP-mediated inhibition of tumor cell attachment to vascular endothelial cells, we performed microarray analyses of human umbilical vein endothelial cells (HUVECs) stimulated by ANP. E-selectin expression was markedly reduced in ANP-treated HUVECs compared with those treated with vehicle alone (SI Appendix, Table S2). Consistently, the expression of E-selectin induced by LPS in HPAECs was inhibited by ANP, whereas that of neither VCAM-1 nor ICAM-1 was affected (Fig. 4A). ANP also significantly inhibited the expression of E-selectin induced by LPS in HMVEC-L (SI Appendix, Fig. S5D). Furthermore, ANP-mediated suppression of LPS-induced E-selectin expression was not observed in HPAECs depleted of GC-A (Fig. 4B and SI Appendix, Fig. S6B). These data indicate that ANP suppresses E-selectin expression through GC-A. Because LPS induces inflammation by inducing nuclear translocation of NF-κB, which in turn promotes E-selectin expression (19), we examined the effect of ANP on nuclear translocation induced by LPS. ANP significantly inhibited the accumulation of NF-κB in the nucleus of LPS-induced HPAECs (Fig. 4 C and D). Fig. 4. ANP–GC-A signaling attenuates LPS-induced E-selectin expression. (A) Immunoblot analysis of the lysates of HPAECs pretreated with or without ANP followed by LPS stimulation; antibodies used are indicated on the left. Each blot is representative of six independent experiments. (B) E-selectin expression assessed by immunoblot analysis of the lysates of HPAECs transfected with the indicated siRNAs and stimulated with LPS. The result shown is representative of six independent experiments. (C) Bright field images (Left) and NF-κB immunofluorescence images (Right) of HPAECs that were unstimulated (control, Top), stimulated with LPS alone (Middle), or pretreated with ANP followed by LPS stimulation (Bottom). Each image is representative of five independent experiments. (Scale bars, 100 μm.) (D) Quantitative analyses of C. Each column shows the percentage of HPAECs with nuclear NF-κB expression in the indicated group. Data are means ± SEM (n = 5, each group); *P < 0.01, unpaired two-tailed t test. (E) Quantitative reverse transcriptase PCR analysis of E-selectin mRNA levels in the lungs of mice pretreated with ANP or vehicle (control) and treated with LPS. Data are normalized relative to 36B4 mRNA levels. Data are means ± SEM (n = 6, each group); P < 0.05, unpaired two-tailed t test. (F) Immunoblot analysis of E-selectin levels in lung lysates of mice pretreated with or without ANP followed by LPS stimulation (1.0 mg/kg) for 5 h. Each blot is representative of six independent experiments. (G) E-selectin images (Left), CD31 images (Center), and merged images with DAPI staining (Right) of the lungs of mice pretreated with or without ANP followed by LPS stimulation (1.0 mg/kg) for 5 h. Each image is representative of six independent experiments. Nuclei are stained with DAPI (blue). (Scale bars, 100 μm.) Finally, we performed an in vivo study to evaluate the effects on LPS-induced E-selectin expression in the lung. Five hours after injection of LPS, E-selectin expression in the lung increased dose-dependently, whereas pretreatment with ANP attenuated this increase at both the gene and protein levels (Fig. 4 E and F). Consistently, immunohistochemical analysis showed that pretreatment with ANP inhibited LPS-induced E-selectin expression in the CD31+ vascular endothelium (Fig. 4G). Furthermore, mice pretreated with E-selectin–neutralizing antibody exhibited a significant reduction of LPS-induced pulmonary metastasis of B16/F10 cells (SI Appendix, Fig. S7). Collectively, these data suggest that ANP inhibits E-selectin expression and reduces the E-selectin–mediated adhesion of tumor cells to vascular endothelium of the lung upon LPS-induced inflammation. Discussion Although many clinical trials aimed at preventing cancer recurrence during the perioperative period have been conducted, no prophylactic treatments have been established. The failure of these trials might be ascribed to the risk of surgery alone, and the side effects of the chemicals used in the trials (20, 21). We previously showed that ANP prevents the incidence of postoperative complications after lung cancer surgery (10–12). Here we demonstrate that cancer recurrence after curative surgery was significantly lower in ANP-treated patients than in control patients, suggesting that ANP could potentially be used to prevent cancer recurrence after surgery. We assumed two possibilities as to how ANP inhibited tumor metastases; one was that ANP directly inhibited the tumor cell proliferation and the other was that ANP indirectly inhibited tumor cell metastases by acting on nontumor cells. In previous studies of the direct effects of ANP on cancer cells, both inhibitory and stimulatory effects of ANP on the growth of cancer cells have been reported (22, 23); therefore, the direct effects of ANP on cancer cells remain controversial. In the present study, we focused on the possibility that ANP indirectly inhibits tumor cell metastases through effects on nontumor cells. Our discovery that mice pretreated with ANP exhibited a dramatic reduction of LPS-induced pulmonary metastasis of introduced cancer cells provides direct evidence that ANP can prevent tumor metastasis in mice. This notion is supported by our finding that mice that specifically overexpress or lack expression of the receptor of ANP (i.e., GC-A) in the vascular endothelium have reduced or enhanced numbers of metastases, respectively, compared with the appropriate control mice. These results suggest that ANP prevents early relapse in patients at least in part by preventing metastasis through the vascular endothelium. Surgical procedures induce postoperative complications and early recurrence after surgery by releasing inflammatory cytokines, such as IL-1β and TNF-α (1, 2). Recent studies indicate that postoperative complications, including severe inflammatory reaction and infection, after various types of cancer surgery are associated with poor cancer-specific survival (24–26). Endothelial cells that become inflamed during surgery are considered to be prone to adhering to circulating tumor cells, thereby allowing the initiation of metastasis (5, 6). Although most circulating tumor cells undergo rapid cell death by apoptosis (27, 28), it is possible that surgical inflammation promotes the adherence of residual cancer cells to inflamed endothelial cells (5, 6). ANP has anti-inflammatory and anti-infectious activity on endothelial cells. ANP pretreatment reduces serum TNF-α levels and NF-κB activation by inhibiting IκB-phosphorylation in mice injected with LPS (29) and has a protective role against LPS-induced lung injury and endothelial barrier dysfunction (30). Our finding that ANP has anti-inflammatory action (i.e., suppression of LPS-induced E-selectin) in vascular endothelial cells in mice is consistent with these studies. Taken together, our results suggest that ANP-mediated inhibition of metastasis occurs through inhibition of the inflammatory response. Among the vascular adhesion molecules, E-selectin is essential for recruitment of inflammatory cells to damaged tissues (31), and it enables circulating tumor cells to roll and tether on the endothelium. Recent studies have shown that cross-talk between E-selectin and integrins could facilitate the movement of not only inflammatory cells but also tumor cells through the endothelium to inflammatory foci (16–18). In fact, tumor metastasis is increased in the lungs of E-selectin–overexpressing mice and reduced in E-selectin knockout mice (18). Therefore, E-selectin is considered to play a central role in hematogenous metastasis (16–18). In a clinical study, Gogali et al. reported that serum levels of soluble E-selectin in lung cancer patients were significantly elevated compared with those in control subjects (32). However, we assume that the antimetastasis activity of ANP does not solely depend upon the suppression of E-selectin, because extravasation of cancer cells in the metastatic process is regulated by many other steps. Recent experimental reports demonstrated that inflammatory chemokines including chemokine ligand (CCL) 2 and CCL5 contributed to not only leukocyte recruitment but also tumor cell homing to activated endothelial cells (33, 34). Because we focused on only E-selectin expressions in this study, further studies are necessary to elucidate the detailed mechanism and role of the ANP–GC-A system in cancer metastasis. Because most current chemotherapeutic agents are cytotoxic and cause many side effects, chemotherapy cannot be used during surgical resection to prevent cancer recurrence. In contrast, ANP is an endogenous and physiological peptide and has been proved not to cause severe adverse effects when used in patients with heart failure (35). Because the target of ANP is considered to be vascular endothelium in all organs that express the GC-A receptor, including lung, liver, and brain, ANP might inhibit hematogenous cancer metastasis to all organs expressing GC-A receptor and could be used for all kinds of malignant tumors. Materials and Methods Clinical Study. We retrospectively evaluated 552 consecutive patients who underwent curative surgery for nonsmall cell lung cancer at Osaka University Hospital and National Hospital Organization Toneyama Hospital from August 2007 to December 2011. Patients with carcinoma in situ and those undergoing a limited resection, including wedge resection, were excluded. Patients with incomplete postoperative follow up (n = 8) were also excluded. Segmentectomy for curative surgery was not excluded. Finally, 467 patients who underwent curative surgery were included in the present study. RFS, defined as the time from the day of surgery to cancer recurrence, was compared between patients who received ANP during the perioperative period and those that received surgery only. In the ANP group, the subjects received ANP intravenously at 0.025 μg⋅kg⋅min (Daiichi-Sankyo Pharmaceutical) without a bolus for 3 d continuously, starting just before the induction of general anesthesia. We previously reported that ANP has a prophylactic effect against postoperative cardiopulmonary complications for patients with elevated preoperative brain natriuretic peptide levels (10, 12, 36). Therefore, we performed a propensity score-matched analysis to reduce the treatment selection bias for each group. The propensity score was estimated by using a logistic regression model adjusted for age, sex, pathological staging [lung cancer tumor, node, metastases (TMN) staging seventh edition], cancer histology, and preoperative brain natriuretic peptide levels. These variables were chosen for potential associations with the outcome of interest. An independent statistician selected the patients by matching propensity scores without access to clinical outcome information. Patient characteristics for the full and propensity score-matched cohorts are listed in SI Appendix, Table S1. In our matching algorithm, one patient who received ANP was matched to one patient who did not receive ANP by using nearest-neighbor matching without replacement. To measure covariate balance, we used the standardized difference. Estimation of propensity scores and matching were performed by using MATLAB r2011b software (Mathworks). Lung cancer-specific RFS was compared by using Kaplan–Meier estimates and the log-rank test for equality of survival curves. Calculations were conducted by using JMP statistical software (SAS Institute). All patients received predefined treatment including chemotherapy according to the clinical guidelines for lung cancer in Japan. ANP treatment was performed just to prevent postoperative complications; therefore, there were no differences in the treatments including chemotherapy between control patients and ANP patients. We obtained complete pathological and follow-up data from all subjects. The study protocol was approved by the Institutional Review Boards of both institutions, and all patients gave written informed consent to participate in the study (Trial registration ID: JPRN-UMIN4880). The median follow-up duration was 36 mo (18–60 mo). All subjects underwent follow-up examinations at 3-mo intervals postoperatively: each evaluation included a physical examination, chest X-ray and blood tests including tumor markers. Thoraco-abdominal CT scans were generally performed at 6-mo intervals and additional bone scintigraphy and MRI of the brain for the detection of cancer recurrence were performed every year. Cell Lines. The human lung cancer cell lines, A549-EGFP and H460-EGFP, were obtained from Wako and maintained in RPMI-1640 medium supplemented with 10% (vol/vol) FBS. The mice melanoma cell line, B16/F10, and the A549, H460, MCF-7, OVCAR3, CaCo2, GCIY, HepG2, ME-180, PANC1, and PC3 cell lines were obtained from the American Type Culture Collection and maintained in DMEM supplemented with 10% FBS at 37 °C under 5% CO2. HPAECs, HMVEC-L, and HUVECs were purchased from Lonza, maintained in EGM-2 according to the manufacturer’s instructions, and used within passages three to five. Experimental Lung Metastasis Model. All animal experiments were performed according to the protocol approved by the Animal Care Ethics Committee of the National Cerebral and Cardiovascular Center Research Institute. Six-week-old BALB/c nu/nu and C57BL/6 mice were purchased from Japan SLC. BALB/c nu/nu mice for A549-EGFP cells (1 × 106 cells per mouse) or C57BL/6 mice for B16/F10 cells (2–5 × 105 cells per mouse) were used in this study. In the experiments with LPS (Fig. 2 A–D), mice were divided into three groups: vehicle alone (control), vehicle/LPS, and ANP/LPS group with two kinds of cancer cells (either A549-EGFP or B16/F10 cells). ANP in 0.9% saline or vehicle was continuously injected by using osmotic pumps (Alzet Model 1002 or 2004, DURECT) implanted subcutaneously in the upper back of the mice 1 d before LPS injection. On the next day, the mice were intravenously injected with or without 1.0 mg/kg LPS (Wako). Five hours after LPS injection, cancer cells (either A549-EGFP or B16/F10 cells) were injected into the tail vein. In the experiments without LPS (SI Appendix, Fig. S2), mice were divided into two groups: vehicle and ANP groups with two kinds of cancer cells (either A549-EGFP or B16/F10 cells). ANP or vehicle alone was implanted 1 d before cancer cells injection. On the next day, cancer cells (either A549-EGFP or B16/F10 cells) were injected into the tail vein. To ascertain the efficiency of ANP administration, we measured the blood levels of cGMP. However, the data did not reach statistical significance when ANP (0.5 μg⋅kg⋅min) was infused subcutaneously in the mice (SI Appendix, Fig. S8), although the blood cGMP levels showed a tendency of increase after ANP administration. ANP (0.5 μg⋅kg⋅min) or vehicle infusion was started 1 d before the injection of cancer cells. At this dose, ANP did not change the blood pressure or heart rate of the mice (SI Appendix, Table S3). ANP or vehicle infusion was continued until the mice were euthanized. Six or 8 wk (A549-EGFP) or 2 wk (B16/F10) after tumor cell injection, the mice were killed for evaluation of pulmonary metastases. The number of nodules reflecting pulmonary metastasis of A549-EGFP or B16/F10 cells was counted by using images obtained with a fluorescent microscope (OV100, Olympus) or a camera (CX6, Ricoh). Adhesion Assay. To quantify tumor cell adhesion to HPAECs or HMVEC-L, a standardized cell adhesion assay was performed by using a modification of the method of van Rossen et al. (37). Briefly, HPAEC or HMVEC-L monolayers were established in 35-mm collagen-coated dishes (IWAKI). Before coculture with tumor cells, HPAECs or HMVEC-L were either pretreated with 0.1 μM ANP for 15 min or untreated and subsequently treated with LPS (0–50 pg/mL) for 30 min, then washed three times with fresh M199 medium (Gibco; Invitrogen) containing 1% BSA (Sigma-Aldrich). A549-EGFP or H460-EGFP tumor cells (2 × 105 cells per dish) were added to the confluent monolayer-cultured HPAECs or HMVEC-L and cocultured for 3 h. The dishes were then washed three times with PBS to remove nonadherent tumor cells, and the cells were fixed with 4% (wt/vol) paraformaldehyde. The number of remaining EGFP+ cells in the fixed dishes was counted by using images obtained with a fluorescence microscope (FSX100, Olympus) and a computer-aided manipulator program (Cell-sense, Olympus). In addition, the adhesion of the tumor cells to HPAECs depleted of either E-selectin, VCAM-1, or ICAM-1, and stimulated with LPS, was similarly analyzed. Statistics. Data are presented as means ± SEM and were analyzed by using a two-tailed Student's t-test for paired samples or one-way ANOVA for multiple groups. P values less than 0.05 were considered statistically significant. Supplementary Material Supplementary File We thank T. N. Sato (Nara Institute of Science and Technology) and M. Yanagisawa (University of Texas Southwestern Medical Center) for their gift of Tie2-Cre mice; M. M. Taketo and M. Sonoshita (Kyoto University) for giving valuable advice; T. Mabuchi, H. Mondo, Y. Nakamura, and AntiCancer Japan Inc. for their technical assistance; and K. Shioya for helping us to care for the mice. This work was supported by research grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan; Osaka Cancer Society, Japan Research Foundation for Clinical Pharmacology, Kobayashi Foundation for Cancer Research, Takeda Science Foundation, and Mochida Memorial Foundation for Medical and Pharmaceutical Research (to T. Nojiri); and grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan and Takeda Science Foundation (to K.K.). Conflict of interest statement: K.K., T. Nojiri, H.H., and M.O. have filed the patent related to atrial natriuretic peptide for the treatment of cancer metastasis with Daiichi-Sankyo Pharmaceutical Inc. (PCT/JP2012/054841). *This Direct Submission article had a prearranged editor. Data deposition: The data reported in this paper have been deposited in the Gene Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE56976). 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==== Front Med Sci MonitMed. Sci. MonitMedical Science MonitorMedical Science Monitor : International Medical Journal of Experimental and Clinical Research1234-10101643-3750International Scientific Literature, Inc. 2596166410.12659/MSM.893553893553Meta-AnalysisEffects of Bariatric Surgery on Incidence of Obesity-Related Cancers: A Meta-Analysis Yang Xiang-wu ABCLi Peng-zhou ACDZhu Li-yong CDEFZhu Shai-hong AEFGThird Xiangya Hospital, Centre South University, Changsha, Hunan, P.R. ChinaCorresponding Author: Shai-hong Zhu, e-mail: [email protected] Study Design B Data Collection C Statistical Analysis D Data Interpretation E Manuscript Preparation F Literature Search G Funds Collection 2015 11 5 2015 21 1350 1357 13 1 2015 23 2 2015 © Med Sci Monit, 20152015This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported LicenseBackground The aim of this meta-analysis was to investigate possible relationships between bariatric surgery and incidence of obesity-related cancers. Obesity is an established risk factor for obesity-related cancers but the effects of bariatric surgery on incidence of obesity-related cancers are uncertain. Material/Methods We searched 4 electronic databases to identify eligible studies: PubMed, Embase, Web of Science, and Google Scholar. Five observational studies were eligible and included in this meta-analysis. Random-effects or fixed-effects odds ratio (OR) and its corresponding 95% confidence interval (CI) were pooled. Results Meta-analysis of these 5 observational studies revealed that bariatric surgery was associated with a significantly (p=0.0004) reduced incidence of obesity-related cancers (OR=0.43, 95%CI, 0.27–0.69) when compared with control individuals. Pooled estimated data showed that bariatric surgery is associated with a 24% lower colorectal cancer (CRC) risk. No publication bias was detected by Egger’s or Begg’s tests. Conclusions Although bariatric surgery may significantly reduce incidence of obesity-related cancers, considering the limitations of these included studies, these findings should be confirmed by further well-designed studies. MeSH Keywords Bariatric SurgeryColorectal NeoplasmsObesity ==== Body Background Colorectal cancer (CRC) is among the most common cancers in the world. Obesity is a well-established risk factor for obesity-related cancers, especially for CRC [1,2]. The prevalence of obesity has doubled world-wide 1980 and at least 500 million people are classified as obese (body mass index [BMI] ≥30 kg/m2). Lifestyle-based behavioral and pharmacological interventions for weight loss remain the major approaches to obesity prevention and management [3–5], but with limited success. Surgical treatment for obesity (bariatric surgery) should be considered in patients with BMI >40 kg/m2 or for those have other significant obesity-related comorbidities but BMI 35–40 kg/m2. Bariatric surgery has been shown to be successful in achieving significant sustained weight loss with low operative mortality and proven safety in older (>55 years) obese patients [6–8]. Weight loss after bariatric surgery yields important health benefits, including resolution of type 2 diabetes in most treated patients and lower total mortality, attributed mainly to reduced incidence of major cardiovascular events and cancer overall [9,10]. A meta-analysis of 21 observational studies from Ma et al. [11] indicated that obesity is associated with increased thyroid cancer risk, except for medullary thyroid cancer. Given the role of overweight and obesity in increasing obesity-related cancers risk, one might expect that weight loss achieved through bariatric surgery would result in reduced risk of obesity-related cancers. We performed a systematic review and meta-analysis aiming to summarize the relationship between bariatric surgery and incidence of obesity-related cancers. Material and Methods Our systematic review was conducted according to Cochrane and the Centre for Reviews and Dissemination guidelines and is reported according to PRISMA guidelines [12–14]. Search strategy and study selection PubMed, Embase, Web of Science, and Google Scholar were independently searched by 2 investigators (Xiang-wu Yang and Shai-hong Zhu) to identify potential eligible studies. The original searches were performed in October 2014 and updated in January 2015. In addition, the reference lists of relevant reviews and studies retrieved were manually searched to identify additional eligible studies. The above databases were searched using a combination of indexed terms and text word searches of title, abstract, and keywords. The following index words were used: “overweight or obese,” “behavioral or lifestyle-based or pharmacological or bariatric surgery or weight loss” and “obesity-related cancer or cancer.” This search strategy was adapted for use with other databases, and further details are available on request. The search was restricted to human studies with abstracts published in English. Databases were searched from inception and thus no date limits were applied. Two reviewers (Peng-zhou Li and Li-yong Zhu) performed the initial screening of titles and abstracts against the inclusion and exclusion criteria to identify potentially relevant papers. Full-text versions of potentially relevant papers identified from the initial screening were retrieved. In cases of disagreement in the initial screening stage, full text of the articles involved was retrieved. Where multiple articles from the same study were found, only the report with the longest follow-up period was included. Both reviewers screened all full-text articles to generate the final list of articles to be included in the systematic review and meta-analysis. Data extraction and quality assessment The following data were extracted using a standardized form: study design, country of origin, period of study, follow-up period, baseline characteristics of population, inclusion/exclusion criteria, description of the intervention, and any relevant outcome measures (as described above). Data extraction was performed by 1 reviewer and was verified by another reviewer. Disagreements were resolved by discussion. Quality assessment of non-randomized studies was performed using the Newcastle-Ottawa scale (NOS) [15]. Study quality was not an exclusion criterion. Statistical analysis We combined studies reporting obesity-related cancers incidence using a random- or fixed-effects meta-analysis. Heterogeneity levels in these studies were quantified using the I2 statistic, and the 95% confidence interval (CI) for I2 was calculated using the Higgins et al. method [16,17]. Statistical analyses were performed using the Review Manager (RevMan) computer program, version 5.3. Copenhagen: the Nordic Cochrane Centre, the Cochrane Collaboration, 2014. Results The searches generated a total of 142 publications, of which titles and abstracts were screened. Five studies that met our inclusion criteria were included in the present review and meta-analysis [18–22]. No studies reporting on obesity-related cancers outcomes after bariatric surgery were excluded on the basis of study design or quality. A flow diagram of the article selection process is shown in Figure 1. Study characteristics The included studies were all registry-based, retrospective studies. They reported on obesity-related cancers incidence in a total study population of 26 331 individuals after bariatric surgery and 82 903 obese controls (Table 1) [18–22]. All studies had a predominance of female subjects (mean 79% and 66.8% in the bariatric surgery and control groups, respectively). McCawley et al. [21] included female subjects only. Adams et al. [18] identified controls with a self reported BMI ≥35 kg/m2 on their driver’s license identification application. The other 4 included studies identified the control population using the diagnosis of morbid obesity as recorded on respective data registries [18–22]. The study by Adams et al. [18] was unique in reporting baseline BMI data and using BMI to match the bariatric surgery and control groups. This study and only 1 other used age- and sex-matched controls [18]. None of the studies specified the treatment (if any) given to the control groups. Gastric bypass was either the sole or the most commonly employed surgical procedure used in the included studies (Table 1). Follow-up BMI data was reported in only 1 study and this was for the bariatric surgery group only (mean BMI reduction 31.9%; 95% CI, 31.1–32.2) [18]. The other 4 studies did not report on any weight loss measure outcomes. Christou et al. [19] reported a lower CRC risk in the bariatric surgery group compared with the non-surgically treated controls (unadjusted RR 0.32; 95% CI, 0.076–1.313, p=0.063). Adams et al. [18] also reported reduced CRC risk in the bariatric surgery group (HR 0.70; 95% CI, 0.43–1.15, p=0.15). Study quality and publication bias Quality assessment scores using the NOS tool are summarized in Table 2 [15]. Four of the studies had high scores (7–9, max score=9) using the NOS tool, whereas the other had a lower score (4). There were too few studies to perform a funnel plot analysis of potential publication bias. Quantitative results (meta-analysis) Data from the 5 bariatric surgery studies were included in a meta-analysis to estimate the overall effect of surgery on obesity-related cancers diagnosis using a random-effects model (Figure 2). Subgroup analysis was carried out to explore the potential influence of different cancer types and found that the estimated data were significantly altered by colorectal cancer (p=0.57) (Figure 3). The meta-analysis revealed that weight loss after surgery was associated with significantly (p=0.0004) lower risk of subsequent obesity-related cancers diagnosis (OR 0.43; 95% CI, 0.27–0.69) (Figure 2). The meta-analysis also showed that weight loss after surgery was associated with significantly (p=0.02) lower risk of subsequent CRC diagnosis (OR 0.76; 95% CI, 0.61–0.95) (Figure 4). Discussion To the best of our knowledge, this is the most complete systematic assessment and meta-analysis of the effects of bariatric surgery on the subsequent risk of obesity-related cancers. Our meta-analysis of data from 5 observational studies involving 109 234 individuals followed for 5–12.3 years (where reported) revealed that bariatric surgery is associated with a 57% lower (p=0.0004) subsequent risk of obesity-related cancers diagnosis. This association was consistent across the 5 included studies. No studies reporting on obesity-related cancers outcomes after bariatric surgery were excluded on the basis of study design or quality; therefore, these results summarize the evidence currently available. Our meta-analysis also revealed that bariatric surgery is associated with a 24% lower (p=0.02) subsequent risk of CRC diagnosis. This association was consistent across the 4 included studies. No studies reporting on CRC-related outcomes after bariatric surgery were excluded on the basis of study design or quality; therefore, these results summarize the evidence currently available. Obesity is a complex multi-system health problem and it is acknowledged that a “one system fits all” mechanism is unlikely [23]. Given that bariatric surgery reduces inflammatory markers, reduces genomic damage, and/or enhances antineoplastic responses, one would expect a reduction in obesity-related cancers risk after bariatric surgery [24–26]. The major limitation of this review is the small number of studies that met our inclusion criteria. The studies reviewed here, all on bariatric surgery, were observational and results from meta-analyses of observational studies should be treated with caution [27]. There were no RCTs that addressed our proposed questions directly. Such RCTs would be difficult to conduct due to requiring many participants with lengthy follow-up to achieve sufficient power to detect any effect. The studies included in our meta-analysis had different lengths of follow-up. Because of insufficient data in the primary studies, we were unable to perform regression analysis to investigate the effect of length of follow-up. However, the consistency in outcomes across the 5 studies suggests that the heterogeneity in duration of follow-up is unlikely to have biased the outcome. Overweight or obese patients undergoing bariatric surgery are more likely to be motivated to lead a healthier lifestyle than untreated obese controls. In addition, 4 of the 5 studies included in our meta-analysis identified controls using the diagnosis of morbid obesity, which is an approach that could have selected less healthy obese individuals as controls [19,20,28]. Many environmental and lifestyle factors influence the risk of obesity-related cancers; therefore, it is possible that factors other than weight loss following bariatric surgery are responsible for the apparently protective effect against obesity-related cancers observed in the present study [1]. Despite attempts to adjust for some confounders by design in some of the included studies (e.g., use of age- and sex-matched controls), potential confounding factors such as cigarette smoking and alcohol drinking were not adjusted for in any of the included studies. Despite the inability to adjust for smoking or alcohol use because of a lack of direct data, Derogar et al. attempted to examine possible effects by performing a sensitivity analysis in which those with smoking- and alcohol-related diagnoses were excluded from the analysis [20]. A lower proportion of individuals had such a diagnosis in the bariatric surgery than in the control group (9.7% vs. 15.0%, respectively), but this exclusion did not change their findings. A study by the same group has shown a hyperproliferative state in rectal mucosal biopsies 6 months after RYGB when compared to obese controls [29]. A similar increase in proliferation status was not seen after a sleeve gastrectomy [30]. The hypothesis states that the predominantly “malabsorptive” bariatric procedures, such as RYGB, may expose the colorectal mucosa to harmful luminal contents and, given the latency in CRC carcinogenesis, this effect becomes more apparent with time after surgery. Conclusions There is a lack of high-quality evidence about the effects of bariatric surgery on the subsequent risk of obesity-related cancers. To date, all relevant data are from non-randomized observational studies. Our meta-analysis of observational studies has shown that bariatric surgery (predominately using Roux-en-Y gastric bypass) was associated with 57% lower obesity-related cancers risk and 24% lower CRC risk. Well-designed prospective clinical studies of the long-term effects of weight loss interventions, including bariatric surgery, on obesity-related cancers risk are required. Conflict of interest All the authors declare that they have no conflicts of interest. Source of support: Departmental sources Figure 1 Flow diagram of study identification. Figure 2 Forest plot of new obesity-related cancers diagnosis rates in the bariatric surgery and no surgery groups. Figure 3 Forest plot of new diagnosis rates for different cancer types in the bariatric surgery and no surgery groups. Figure 4 Forest plot of new CRC diagnosis rates in the bariatric surgery and no surgery groups. Table 1 Study characteristics. Author Year Country Type of study Participants (n) Females (%) Age Baseline BMIa Type of bariatric surgery Follow-up (years) Adams et al. 2009 USA Retrospective S: 6,709 S: 86% S: 38.9 (10.3) S: 44.9 (7.6) All RYGB S: 12.3 (5.7)c Two cohort study C: 9,609 C: 86% C: 39.1 (10.7) C: 47.4 (6.5) – C: 11.8 (5.6)c Christou et al. 2008 Canada Retrospective S: 1,035 S: 66% S: 45.1 (11.6) S: 50.0 (8.2) 81.3% RYGB; S: 5c Two cohort study C: 5,746 C: 64% C: 46.7 (13.1) C: no data 18.7% VBG C: 5c Derogar et al. 2013 Sweden Retrospective S: 15,095 S: 77% S: 39.0 S: no data 51% RYGB; 25% VBG; S: 10 (1–30)b Two cohort study C: 62,016 C: 63% C: 49.0 C: no data 24% GB; 12% >1 procedure C: 7 (1–30)b McCawley et al. 2009 USA Retrospective S: 1,482 S: 100% S: 41.7 S: 51.6 93.5% gastric bypass; No data Two cohort study C: 3,495 C: 100% C: 46.9 C: no data 3.8% GB; 1.8% VBG No data Sjöström et al. 2009 Sweden Retrospective S: 2,010 S: 70.6% S: 47.2(5.9) S: 41.7 68.1% VBG;18.7% GB; S: 10.8c Two cohort study C: 2,037 C: 71% C: 48.7(6.3) C: 40.9 13.2% gastric bypass C: 10.9c S – surgery group; C – control group; RCT – randomised control trial; RYGB – Roux-en-Y gastric bypass; VBG – vertical banded gastroplasty; GB – gastric band; a mean (±standard deviation) (kg/m2, where reported); b median (range); c mean (±standard deviation) (where reported). Table 2 Quality assessment of the studies using the NOS. Author Selection (max. 4) Comparability (max. 2) Exposure (max. 3) Total (max. 3) Adams 2009 4 2 3 9 Christou 2008 4 1 3 8 Derogar 2013 4 0 3 7 McCawley 2009 3 0 1 4 Sjöström 2009 4 2 3 9 NOS – Newcastle-Ottawa criteria. ==== Refs References 1 Wiseman M The second World Cancer Research Fund/American Institute for Cancer Research expert report. 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==== Front 10126486032902Obesity (Silver Spring)Obesity (Silver Spring)Obesity (Silver Spring, Md.)1930-73811930-739X2596014610.1002/oby.21077nihpa666172ArticleLA Sprouts Randomized Controlled Nutrition, Cooking and Gardening Program Reduces Obesity and Metabolic Risk in Latino Youth Gatto Nicole M. MPH, PhD1Martinez Lauren C. 2Spruijt-Metz Donna MA, PhD234Davis Jaimie N. RD, PhD51 Center for Nutrition, Healthy Lifestyles & Disease Prevention, School of Public Health, Loma Linda University2 Department of Preventive Medicine, University of Southern California3 Center for Economic and Social Research, University of Southern California4 Department of Psychology, University of Southern California5 Department of Nutritional Sciences, University of Texas at AustinCorresponding Author: Nicole M. Gatto, MPH, PhD, Associate Professor of Epidemiology, Center for Nutrition, Healthy Lifestyles & Disease Prevention, Loma Linda University \| School of Public Health, 24951 North Circle Drive, Nichol Hall 2025, Loma Linda, California 92350, Office: (909) 558-7597 Cell: (323) 244-6039, [email protected] 3 2015 09 5 2015 6 2015 18 5 2016 23 6 1244 1251 Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:http://www.nature.com/authors/editorial_policies/license.html#termsObjective To assess the effects of a 12-week gardening, nutrition, and cooking intervention (“LA Sprouts”) on dietary intake, obesity parameters and metabolic disease risk among low-income, primarily Hispanic/Latino youth in Los Angeles. Methods Randomized control trial involving four elementary schools [2 schools randomized to intervention (172, 3rd–5th grade students); 2 schools randomized to control (147, 3rd–5th grade students)]. Classes were taught in 90-minute sessions once a week to each grade level for 12 weeks. Data collected at pre- and post-intervention included dietary intake via food frequency questionnaire (FFQ), anthropometric measures [BMI, waist circumference (WC)], body fat, and fasting blood samples. Results LA Sprouts participants had significantly greater reductions in BMI z-scores (0.1 versus 0.04 point decrease, respectively; p=0.01) and WC (−1.2 cm vs. no change; p<0.001). Fewer LA Sprouts participants had the metabolic syndrome (MetSyn) after the intervention than before, while the number of controls with MetSyn increased. LA Sprouts participants had improvements in dietary fiber intake (+3.5% vs. −15.5%; p=0.04) and less decreases in vegetable intake (−3.6% vs. −26.4%; p=0.04). Change in fruit intake before and after the intervention did not significantly differ between LAS and control subjects. Conclusions LA Sprouts was effective in reducing obesity and metabolic risk. LatinoHispanicchildrenchildhood obesityBMIwaist circumferencefruit and vegetable consumptionfibermetabolic syndrome ==== Body INTRODUCTION The increased prevalence of childhood obesity in the US is concerning, and has led to projections that one in three male and two in five female children born in the year 2000 will develop diabetes in their lifetime [1]. Nearly one-third (31.8%) of US children and adolescents aged 2–19 years were either overweight or obese in 2011–2012, including 16.9% who were obese [2]. Pediatric obesity is associated with an increase in cardiovascular disease (CVD) risk factors, asthma, and psychological problems during childhood [3, 4]. Significant disparities in obesity prevalence exist by racial/ethnic group, with adolescent Hispanic/Latinos having higher rates of obesity than their Caucasian counterparts [2]. Socioeconomic status (SES) is an important determinant of access to healthy and affordable, high-quality fresh fruits, vegetables and other foods [5]. Low income residents of “food desert” neighborhoods in urban areas are less likely to have access to fresh and healthy foods than residents of higher income neighborhoods [6]. Low intakes of dietary fiber, specifically from fruits and vegetables, coupled with high consumption of refined grains and added sugar are linked to obesity and related disorders in Hispanic/Latino youth aged 8–18 years in Los Angeles (LA) [7, 8]. Interventions that target these dietary habits could be effective in reducing obesity risk in Hispanic/Latino youth [8]. It is known that food preferences are shaped when children are young [9], and children’s preferences for vegetables are strong predictors of vegetable consumption [10]. Studies suggest that having a direct experience with growing food enhances children’s understanding of foods and their relationship to health [11]. While many programs for children that involve both gardening and nutrition components exist, none have included experimental designs that would allow more rigorous evaluation of their impact on obesity and metabolic risk factors [11–22]. In 2010, we developed and pilot-tested a non-randomized 12-week gardening, nutrition/cooking intervention called “LA Sprouts” in predominantly low-income Hispanic/Latino elementary school children in LA. The LA Sprouts intervention was demonstrated to be effective in reducing body mass index (BMI) and systolic blood pressure (SBP) [13]. LA Sprouts also increased dietary fiber intake and preferences for target fruits and vegetables, and changed carbohydrate composition [23, 24]. Preliminary findings from our pilot program led us to conduct a larger randomized control experimental study of LA Sprouts in this population. This is the first experimental, randomized controlled study to date to examine the effects of an afterschool gardening, nutrition, and cooking program on dietary intake, obesity parameters and associated metabolic disease risk in Hispanic/Latino youth. We hypothesize that students participating in the LA Sprouts program compared to controls would experience a reduction in adiposity and metabolic risk factors and an increase in intake of dietary fiber, fruit, and vegetables. METHODS AND PROCEDURES Participants A full description of our LA Sprouts randomized controlled trial (RCT) study design and sample is provided elsewhere [25]. Briefly, during 2011–13elementary schools within LA Unified School District (LAUSD) were eligible if they: 1) offered the LA’s BEST after-school care program, 2) had a student body ≥75% Hispanic/Latino, 3) had ≥75% of students in the free/reduced cost school meal program, 4) were located within 10 miles of University of Southern California (USC) Health Science Campus, 5) had expressed interest by school personnel in having a garden/hosting our program, and 6) could make an administrative commitment (including assisting with securing LAUSD approval and building parent support). Four elementary schools were identified, and all 3rd–5th grade students at the schools who were enrolled in LA’s BEST(n=409) were invited to participate; 375 (92%) agreed. Two schools (n=204 students) were randomized to receive the LA Sprouts intervention and two other schools were randomized to controls (n=171 students) receiving a delayed intervention. At least partial obesity/metabolic measures and questionnaire data were collected on 364 participants (n=198 intervention, n=164 controls) at baseline (pre-intervention). Follow-up (post-intervention) data was missing on 44 participants who changed schools, left the parent LA’s BEST program, or were absent on data collection days. Main analyses herein are based on 319 (88% of those with baseline data; n=172 intervention, n=147 controls; n= 130 3rd, n=103 4th, n=86 5th grade students) children for whom both baseline and follow-up data were available on our primary outcome, BMI(Figure 1). One hundred and sixty-nine children (46% of the total sample) participated in blood draws at baseline; 113 of these (67%; n=67 intervention, n=46 controls) returned for follow-up draws are included in analyses of blood measures. Six percent more children who provided a sample reported having internet access at home than those who did not provide a sample, otherwise that subset did not differ from the total sample in demographic or socioeconomic characteristics or BMI parameters. Institutional Review Boards of USC, the University of Texas at Austin, Loma Linda University, and LAUSD approved the study. Informed written consent from parents and assent from children were obtained. ClinicalTrials.gov identifier NCT02291146. Description of the Intervention The LA Sprouts intervention was taught on campus at school gardens specifically constructed for the program. Each school garden design took into consideration the specific needs and challenges of the individual schools, and the design and planning process involved key stakeholders including school principals and teachers, afterschool staff, and LAUSD personnel. All gardens utilized raised bed garden planter boxes placed either on unpaved, grassy areas of the school yard or on areas where asphalt was removed. An area near the garden was designated as teaching space. Each school garden was outfitted with a minimum number of tools needed for gardening and cooking supplies for an outdoor kitchen. Intervention classes were taught in 90-minute sessions once a week to each grade level for 12 weeks during either a fall or winter/spring school semester. Sessions consisted of a 45-minute interactive cooking/nutrition lesson and a 45-minute gardening lesson taught by an educator with a nutrition or gardening background who was employed specifically for this intervention. Our program’s curriculum and theoretical framework is described in greater detail [13, 25, 26]. Students worked in small teams led by an educator to cook/prepare a recipe each week, which emphasized fruit and/or vegetable ingredients. The snack was eaten in a “family-style” manner, i.e., together at a table, with a tablecloth, non-disposable plates and silverware. The gardening activities also used a “hands-on” approach, where children participated in planting, growing and harvesting organic fruits and vegetables. A teacher to student ratio of 1: 3–6 was maintained. Description of Control Group Third, fourth and fifth grade students at the two control schools did not receive any nutritional/cooking or gardening information from investigators between pre- and post-testing, and schools were asked to refrain from augmenting their curriculum with similar lessons during the study period. After post-testing was completed, students at the control schools received the full LA Sprouts program (“delayed intervention”), including a school garden being built. Data Collection LA Sprouts and control participants completed questionnaires and had obesity and metabolic data collected at baseline and post intervention. Data collection occurred during the week prior to instruction being initiated (for baseline measures) or 7–14 days after the final day of instruction (for post-intervention measures), and took place during after-school sessions, in the morning before school or on weekends. Study personnel who were not blinded to group assignment were trained to perform data collection using standardized protocols. All staff were directed to review the protocols; participated in demonstrations of procedures by the principal investigator (PI) and or project manager (PM); and were observed to ensure a proper technique. A PI or PM was present to supervise data collection. We strove, whenever feasible, to schedule a given staff member to collect the same measurement at baseline and post-intervention. Anthropometric and Metabolic Disease Risk Data Height was measured with a free-standing stadiometer (Seca, Birmingham, UK); weight and percent body fat were measured via bioelectrical impedance (Tanita TBF 300A, Arlington Heights, IL). BMI z-scores and percentiles were determined using CDC cut-points for age and sex [27]. Blood pressure (BP) was measured with an automated monitor with appropriate child cuffs (Omron, Schaumberg, IL), and waist circumference (WC) measures followed NHANES protocol [28]. Child Questionnaires The child questionnaire included items on demographics and socioeconomic status. Dietary intake was measured using the Block Kids Food Screener (“last week” version). This 41-item screener was developed and adapted from the Block Kids 2004 Food Frequency Questionnaire [29], and has been validated in youth living in a metropolitan area [30]. The screener was designed to assess intake by food group, and includes questions used to estimate intake of fruit and fruit juices, vegetables, potatoes, whole grains, meat/poultry/fish, dairy and added sugars. National dietary surveys such as NHANES were used to inform the selection of foods to query, and to apply appropriate portion sizes and nutrient composition. Fasting Blood Sample Optional fasting blood draws were collected during non-academic hours and off-campus from participants by bilingual, licensed phlebotomists with experience drawing blood in overweight children. Samples were processed and stored at USC until they were shipped for assays. Glucose was assayed using a Yellow Springs Instruments analyzer (Yellow Springs, OH). Total cholesterol, high-density lipoprotein cholesterol (HDL), and triglyceride levels were measured using the enzymatic methods [31] on a Stanbio Sirrus analyzer (Stanbio Laboratory, Boerne, TX); Low-density lipoprotein (LDL) was calculated using the Friedwald equation. Insulin was quantified using an ELISA kit (EMD Millipore, St. Charles, MO). Homeostatic model assessment (HOMA-IR) was calculated as a measure of insulin resistance [32]. Metabolic Syndrome Participants were identified as having the metabolic syndrome (MetSyn) following the work of Cook et al. [33], which provides recommendations for reference values to define cut-offs for component MetSyn factors. Statistical Analysis Anthropometric and metabolic data were screened for plausibility by conducting residual analyses examining how the baseline value of a given variable predicted the value of that variable at follow-up. Original data was checked to resolve possible measurement errors for participants with standardized residuals > \|3\|, otherwise, that observation was removed from analyses. For Block data, we selected for analysis variables for individual food questions as well as estimates of consumption of nutrients and food groups that were pertinent to our study hypotheses. We examined and excluded as implausible or outlying observations for which the change in reported intake between pre- and post-intervention was at or below the 1st, or at or above the 99th percentile. While the number of observations set to missing varied by analysis, fewer than a total of 2.2% of all observations were excluded. All variables were examined for normality and data transformations were attempted for SBP, HDL cholesterol and fasting insulin, but improvements were not substantial. Thus, analyses used the original untransformed data for these variables. Frequencies were tabulated for categorical socio-demographic variables at baseline; mean ± standard errors (SE) for continuous variables at baseline and follow-up were calculated, adjusting for age (continuous), sex, Hispanic/Latino (yes, no), English spoken at home (yes, no), school (Monte Vista, Loreto, Sierra Park, Euclid Elementary). Means for nutrients and foods/food groups were additionally adjusted for energy intake (kcal). Repeated measures analysis of variance (ANOVA) was used to assess whether mean changes in anthropometric, clinical and dietary (continuous) variables over the 12-week intervention period differed between intervention and control groups. Models were adjusted for the covariates as above. We did not include season (Fall, Winter/Spring) in models because it was explained by school. In sensitivity analyses, we examined the effect of additional adjustment for baseline BMI in models where dietary variables and clinical variables (other than BMI) were the dependent variable. We also restricted analyses to the overweight/obese subsample to examine whether results were similar to the total sample. All analyses used SAS version 9.4 (SAS Institute Inc., Cary, NC, USA.). RESULTS By design, the study population was ~89% Hispanic/Latino and ~90% eligible for free lunch at school (Table 1). The majority (>50%) were overweight (BMI ≥85th percentile), and more than one-third were obese (BMI ≥95th percentile). There were no differences at baseline between LA Sprouts participants and controls in age, sex, ethnicity, BMI and most sociodemographic factors examined. LA Sprouts participants were less likely to speak English at home than controls (p=0.06). After the 12-week program, LA Sprouts participants had significantly greater reductions in BMI than controls (0.1 versus 0.04 decrease in BMI z-score, respectively; p=0.01). LA Sprouts participants had a 1.2 cm (1.7%) reduction in WC, while controls had no change after the intervention (p<0.001) (Table 2). The number of students overall who fit criteria for the MetSyn was small. However, there were fewer LA Sprouts participants with the MetSyn after (n=1) the intervention than before (n=7), while the number of controls with the MetSyn increased between pre- (n=3) and post-intervention (n=4). For percent body fat, SBP and DBP, and other blood measures, the change between pre- and post- intervention was not statistically different between LA Sprouts participants and controls. Adjustment for BMI did not appreciably alter the change estimates or impact our conclusions about the effect of the intervention on obesity or metabolic measures (data not shown). Results in the overweight/obese strata were similar to those in the total sample (data not shown). LA Sprouts participants increased dietary fiber consumption by 0.4 g/d (3.5%), compared to a 2.0 g/d (15.5%) decrease in controls (p=0.04) (Table 3). LA Sprouts participants compared to controls had smaller decreases in vegetable intake per day (−0.03 CE/d or a 3.6% decrease, versus −0.2 CE/d, or a 26.4% decrease; p=0.04). LA Sprouts had increases in consumption of whole grains and green beans and peas, while controls decreased their intake of these foods (p ≤ 0.10). Change in fruit intake overall and intake of apples, bananas and oranges before and after the intervention did not significantly differ between LAS and control subjects. DISCUSSION LA Sprouts is the first randomized school gardening, nutrition, and cooking intervention to demonstrate effectiveness in reducing obesity (measured by BMI and WC) in predominantly Hispanic/Latino elementary school aged children. While the reductions were relatively small in magnitude (decreases of 0.1 in BMI z-score and 1.2 cm in WC), it is nonetheless notable that the study was able to show changes over a 12-week period. By extension, our findings suggest that similar interventions implemented over a longer term may expect greater change in reduction of these obesity parameters. Additional studies would be needed to evaluate this. For comparison, other more intensive RCTs such as those that included dietary modification, rigorous nutrition education, intense and monitored physical activity sessions, or a clinic-based component with or without healthcare professionals have demonstrated inconsistent success in achieving reductions in BMI [34, 35]. A 12-week behavioral modification program for Hispanic/Latino children aged 7–15 years and their families that provided alternative foods to substitute for those with high glycemic index, dietary prescription plans and physical activity sessions found a 0.156 point reduction in BMI z-score after 3 months [36]. The modest reduction in BMI associated with our educational program may be interpreted relative to the magnitude of those observed under more intense dietary and/or physical activity conditions. Furthermore, as the prevalence of overweight and obesity in our study population was higher than national averages for Hispanic youth [2] (which are higher than those for non-Hispanic Whites nationally), we believe this underscores the need to address disparities in obesity risk, and even small risk reductions in this high-risk population represent progress in tackling the problem. LA Sprouts was also effective in changing dietary intake, with an observed increase in dietary fiber intake among participants, which was an intention of the intervention design. LA Sprouts participants increased intake of whole grains and green beans/peas while controls decreased intake of these foods. Some [13, 15, 16, 18–20, 22], but not all [12, 14, 17, 19, 21] previous non-randomized studies of school-garden based educational programs have demonstrated an effect on increasing fruit or vegetable intake in children. It should be noted that with over 90% of students in the study eligible for free or reduced cost breakfast and lunch served at school, this likely implies that 2/3 of their daily dietary intake was determined by school availability, which was the same between LA Sprouts participants and controls. This further suggests that children had little control over food options for two of three of their meals, which further contextualizes an interpretation of the magnitude of change in dietary intakes associated with the intervention. While the number of students overall who fit the definition of the MetSyn was small, we did observe a decrease in MetSyn among LA Sprouts participants and an increase in controls from pre- to post-intervention. While this finding should be interpreted with caution, it may suggest that LA Sprouts had an effect on the biochemical processes associated with this clustering of metabolic risk factors. While mean differences between pre- and post-intervention in the anthropometric and lipid variables that comprise the MetSyn were statistically significant only for WC, fewer LA Sprouts participants than controls met individual metabolic syndrome criteria after the intervention. The importance of our WC finding is further supported by the emphasis placed on the role of abdominal obesity for metabolic syndrome risk by the International Diabetes Foundation, who advocates that WC be used to identify children aged < 10 years to target weight reduction and be a required criterion to diagnose the MetSyn for children aged 10– <16 years [37]. Our intervention was designed to be culturally tailored, for example, by including recipes that targeted foods familiar to our study population such as salsas and vegetable quesadillas. We did not observe an effect of the intervention on fruit consumption, which is not in line with our study hypothesis but is concordant with some other non-randomized school-garden based educational interventions targeting fruit intake [14, 16, 17, 19]. The food screener used did not provide a broad assessment of vegetables and fruits, and particular fruits and vegetables which may be more commonly consumed by cultures reflected in our study population (i.e., papayas, nopales). Thus it is possible that our null findings for fruits may reflect inadequate sensitivity of our selected data collection instrument. Furthermore, FFQs are not able to precisely quantify intake of nutrient consumption or differences among varieties of foods that may be captured in a single food item question [38]. Funding, time and sample size limitations prevented collection of 24-hour dietary recalls, which would have provided a more accurate assessment of dietary intake [39]. Nevertheless, the Block screener demonstrated good validity against 3, 24-hour dietary recalls in 99 youth in a metropolitan area, with de-attenuated correlations between the two dietary assessment instruments ranging from 0.526 for vegetables to 0.878 for potatoes [30]. Furthermore, given that our analysis focused on comparisons between groups of participants and not individual assessments, we feel that the associated efficiency and cost savings afforded by automated data entry and analysis made the FFQ instrument an appropriate choice. Our intervention was developed to take place during the after school hours because this time is ideal for implementing such health programs. Students who remain on campus after school dismissal and prior to parent pick up are captive audiences for three to four hours. Local and national data suggest that 50% of school children in kindergarten through eighth grade aged 5–13 years are regularly in non-parental care before and after school [40], a statistic that likely differs by geographic region and sociodemographic factors. The afterschool hours are an opportunity to enhance students’ academic achievement as an extension of instructional time, while engaging students in topics or activities otherwise not part of the academic school day such as gardening or nutrition. Many after-school care providers include scheduled time for enrichment in their programming and seek activities that fulfill their needs. It is possible to incorporate “fun” “hands on” activities such as cooking or gardening that may not be feasible in a classroom setting. After school programs do not compete with required school day instruction and are not restricted by a requirement that they meet school standards. Nevertheless, our curriculum has mapped on school standards (i.e., math, science, language arts, and health) and could be utilized during the academic school day. Garden-based programs are multi-faceted, can be utilized during both school and after-school hours, and integrate academic subjects and other subjects such as health. There were several limitations of our study. We do not have data on long-term sustainability of the program or maintenance of our results beyond the 12-week study period. Additional longer-term studies are needed to understand these issues and long-term health benefits of a garden-based intervention. We provided trained educators to teach our program, and it is uncertain whether similar results can be expected when the program is taught by afterschool staff. However, after the intervention we held several train-the-trainer workshops and provided all educational resources and supplies associated with our lessons to the schools to help sustain the program. We had a smaller sample size for blood measures, as these were optional, which could explain our lack of findings for these variables. While we recognized the importance of involving parents and offered parallel classes to them on mornings, evenings and weekends, these classes were poorly attended. Future efforts should be directed to increasing parental support for such programs, and should obtain evaluation measures on parents, as it is recognized that the home food environment reinforces material taught to children. While gardening is a source of physical activity for children, and the imbalance between energy intake and expenditure is at the root of obesity, future garden-based studies may want to supplement the exercise component of their programs to include more high intensity gardening activities such as digging and weeding, which were not as emphasized in our intervention. In conclusion, LA Sprouts is the first school garden-based, nutrition, cooking and gardening experimental intervention developed, and which resulted in a decreased risk of obesity and metabolic disease and improvements in dietary intake in high-risk Hispanic/Latino youth. Our findings suggest that teaching children to grow, prepare, and eat fruits and vegetables is an efficacious approach to reducing disease risk. However, longer RCTs are warranted to understand the long-term effects of garden-based programs. In addition, more studies are needed to examine how to sustain such garden-based programs in school settings. Programs with a school garden component provide children access to healthy foods in otherwise food desert neighborhoods. CONFLICTS OF INTEREST No author has any financial interest or conflict of interest to disclose. ACKNOWLEDGEMENTS Authors NMG and JND designed and conducted the research and have primary responsibility for final content. NMG and LCM analyzed data and performed statistical analysis. NMG, JND, LCM and DSM wrote the paper. This study was supported by funding from the NIH (grant number 5R21DK094066). A grant from the Keck Foundation provided funding to build the school gardens. Figure 1 Flow of participants through the LA Sprouts study. Table 1 Demographic characteristics of LA Sprouts and control participants at baseline Characteristic, n (%) or mean ± SD LA Sprouts (n=172) Controls (n=147) p-valuea Pre Pre Male 82 (47.7) 71 (48.3) 0.91 Hispanic/Latino 153 (89.0) 127 (88.8) 0.97 Age, years 9.3 ± 0.9 9.3 ± 0.9 0.9 Height, cm 135.0 ± 8.5 135.0 ± 8.5 0.96 Weight, kg 36.9 ± 10.6 38.1 ± 12.6 0.30 BMI, kg/m2 19.8 ± 4.1 20.6 ± 4.6 0.13 Overweight (≥85th percentile) 82 (51.3) 73 (53.3) 0.73 Obese (≥95th percentile) 54 (33.8) 54 (39.4) 0.31 Socioeconomic factors No English spoken at home 48 (28.7) 27 (19.6) 0.06 No computer at home 42 (26.1) 32 (23.2) 0.56 No internet at home 39 (23.2) 32 (23.2) 0.99 Mother does not have own car 57 (34.3) 38 (27.1) 0.17 Eligible for free lunch at school 152 (90.5) 125 (89.3) 0.73 a p-value for difference between groups from chi-square tests (categorical variables) or independent t-tests (continuous variables). Table 2 Adjusted mean ± SEa anthropometric and clinical characteristics of LA Sprouts participants and controls before and after intervention, and adjusted mean (percent) change between pre- and post-intervention Characteristic, mean ± SE LA Sprouts (n=172) Controls (n=147) p-valueb Pre Post Absolute change Percent change Pre Post Absolute change Percent change Anthropometrics BMI Percentile 75.6 ± 2.3 73.7 ± 2.3 −1.9 −2.5 74.8 ± 2.6 73.8 ± 2.6 −1.0 −1.3 0.13 BMI z-score 0.95 ± 0.09 0.85 ± 0.09 −0.1 −10.5 1.01 ± 0.10 0.97 ± 0.10 −0.04 −4.0 0.01 Waist circumference (WC), cm 70.3 ± 0.5 69.1 ± 0.5 −1.2 −1.7 71.6 ± 0.6 71.6 ± 0.6 0.0 0.0 <0.001 Body fat, % 24.7 ± 0.4 24.2 ± 0.4 −0.5 −2.0 25.2 ± 0.4 24.6 ± 0.4 −0.6 −2.4 0.82 Clinical characteristics Systolic blood pressure, mmHg 109.4 ± 1.0 108.9 ± 1.0 −0.5 −0.5 112.0 ± 1.1 111.7 ± 1.1 −0.3 −0.3 0.87 Diastolic blood pressure, mmHg 64.7 ± 0.9 64.0 ± 0.9 −0.7 −1.1 66.2 ± 1.0 63.6 ± 1.0 −2.6 −3.9 0.28 Cholesterol Total 156.8 ± 3.3 162.5 ± 3.5 5.7 3.6 158.5 ± 4.2 160.4 ± 4.6 1.9 1.2 0.32 LDL-C 84.6 ± 2.7 85.9 ± 2.9 1.3 1.5 86.9 ± 3.5 86.6 ± 3.8 −0.3 −0.3 0.62 HDL-C 58.0 ± 1.3 60.0 ± 1.4 2.0 3.4 57.3 ± 1.7 58.6 ± 1.8 1.3 2.3 0.42 Triglycerides 69.5 ± 3.5 73.8 ± 3.8 4.3 6.2 70.6 ± 4.5 72.6 ± 4.9 2.0 2.8 0.63 Insulin, µU/mL 10.7 ± 0.8 11.3 ± 0.9 0.6 5.6 10.9 ± 1.1 11.4 ± 1.2 0.5 4.6 0.90 HOMA-IR 2.4 ± 0.2 2.6 ± 0.2 0.2 8.3 2.5 ± 0.3 2.6 ± 0.3 0.1 4.0 0.85 Glucose, mg/dL 91.5 ± 0.8 93.6 ± 0.8 2.1 2.3 91.4 ± 0.9 92.8 ± 1.1 1.4 1.5 0.56 Metabolic Syndrome 7 (4.2) 1 (0.6) −6 85.7 3 (2.1) 4 (2.72) 1 WC ≥90th percentile, age-and sex-specific 45 (27.4) 41 (24.4) −4 −8.9 47 (34.1) 49 (34.0) 2 4.2 Fasting glucose ≥110mg/dL 0 0 - - 0 1 (2.3) 1 100 Triglycerides ≥110mg/dL, age-specific 9 (10.5) 8 (12.9) −1 −11.1 12 (19.4) 8 (18.2) −4 −33.3 HDL-C ≤40 mg/dL 3 (3.5) 2 (3.2) −1 −33.3 6 (9.7) 5 (11.4) −1 −16.7 BP ≥90th percentile, age-,sex-and height-specific 66 (39.8) 56 (32.9) −10 −15.2 72 (52.2) 68 (47.2) −4 −5.6 a Means are adjusted for age (continuous), sex, ethnicity (hispanic/latino versus not), english spoken at home (yes, no), school (Monte Vista, Loreto, Sierra Park, Euclid Elementary) b p-value for multiplicative interaction term indicating change from pre to post for each measure between groups from mixed effects regression models Table 3 Adjusteda mean intake of select foods and nutrients of LA Sprouts and control participants before and after intervention, absolute and percent change between pre- and post-intervention LA Sprouts Controls p-valuec Nutrient or Food, mean ± SE Pre Post Absolute change Percent change Pre Post Absolute change Percent change Nutrient Energy, kcalb 1265.1 ± 95.6 1261.3 ± 95.6 −3.8 −0.3 1389.0 ± 109.5 1239.3 ± 109.5 −149.7 −10.8 0.25 Protein, g/d 55.2 ± 2.2 54.1 ± 2.2 −1.1 −2.0 56.3 ± 2.5 47.1 ± 2.5 −9.2 −16.3 0.19 Fat, g/d 54.1 ± 2.1 52.1 ± 2.1 −2.0 −3.7 55.1 ± 2.4 47.3 ± 2.4 −7.8 −14.2 0.33 Carbohydrates, g/d 149.9 ± 5.2 154.3 ± 5.2 4.4 2.9 165.3 ± 5.9 153.9 ± 5.9 −11.4 −6.9 0.27 Added sugar, tsp/d 7.2 ± 0.5 8.0 ± 0.5 0.8 11.1 8.5 ± 0.5 7.8 ± 0.5 −0.7 −8.2 0.11 Dietary fiber, g/d 11.5 ± 0.5 11.9 ± 0.5 0.4 3.5 12.9 ± 0.6 10.9 ± 0.6 −2.0 −15.5 0.04 Food or Food Group Meat, OEd 2.8 ± 0.2 2.7 ± 0.2 −0.1 −3.6 2.8 ± 0.2 2.2 ± 0.2 −0.6 −21.4 0.18 Dairy, CEe 1.4 ± 0.1 1.3 ± 0.1 −0.1 −7.1 1.5 ± 0.1 1.3 ± 0.1 −0.2 −13.3 0.56 Whole grains, OE 0.49 ± 0.03 0.53 ± 0.03 0.04 8.2 0.56 ± 0.04 0.48 ± 0.04 −0.1 −14.3 0.10 Vegetables, CE 0.83 ± 0.05 0.80 ± 0.05 −0.03 −3.6 0.91 ± 0.06 0.67 ± 0.06 −0.2 −26.4 0.04 Fruit, fruit juice, CE 1.3 ± 0.09 1.3 ± 0.09 0.0 0.0 1.5 ± 0.1 1.4 ± 0.1 −0.1 −6.7 0.56 Apples, bananas, oranges, CE 0.43 ± 0.04 0.45 ± 0.04 0.02 4.7 0.5 ± 0.04 0.42 ± 0.04 −0.1 −16.0 0.12 Lettuce salad, CE 0.18 ± 0.02 0.18 ± 0.02 0.0 0.0 0.20 ± 0.02 0.16 ± 0.02 −0.04 −20.0 0.28 Green beans, peas, CE 0.03 ± 0.01 0.04 ± 0.01 0.01 33.3 0.03 ± 0.01 0.02 ± 0.01 −0.01 −33.3 0.08 Tomatoes, CE 0.05 ± 0.01 0.04 ± 0.01 −0.01 −20.0 0.05 ± 0.01 0.03 ± 0.01 −0.02 −40.0 0.21 a Means are adjusted for age (continuous), sex, ethnicity (hispanic/latino versus not), english spoken at home (yes, no), school (Monte Vista, Loreto, Sierra Park, Euclid Elementary) b not adjusted for energy(kcal) c p-value for multiplicative interaction term indicating change from pre to post for each measure between groups from mixed effects regression models d OE: ounce equivalent e CE: cup equivalent WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT Food preferences are shaped when children are young Having a direct experience with growing food enhances children’s understanding of foods Existing programs for children that involve gardening and nutrition components have been effective at shaping attitudes and preferences WHAT THIS STUDY ADDS The first randomized controlled trial of a gardening and nutrition education program on obesity risk A specific focus on a high-risk Latino youth population LA Sprouts was effective in reducing obesity measured by BMI and waist circumference ==== Refs REFERENCES 1 Narayan KMV Boyle JP Thompson TJ Sorenson SW W DF Lifetime risk for diabetes mellitus in the United States Journal of American Medical Association 2003 290 1994 1890 2 Ogden CL Carroll MD Kit BK Flegal KM Prevalence of childhood and adult obesity in the United States, 2011–2012 Jama 2014 311 8 806 814 24570244 3 Daniels SR Arnett DK Eckel RH Gidding SS Hayman LL Kumanyika S Overweight in children and adolescents: pathophysiology, consequences, prevention, and treatment Circulation 2005 111 15 1999 2012 15837955 4 Reilly JJ Methven E McDowell ZC Hacking B Alexander D Stewart L Health consequences of obesity Arch Dis Child 2003 88 9 748 752 12937090 5 Sobal J Stunkard A Wadden Stunkard Influence of the home environment on the development of obesity in children. 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==== Front EXCLI JEXCLI JEXCLI JEXCLI Journal1611-2156Leibniz Research Centre for Working Environment and Human Factors 2014-239Doc461Review ArticleNon-alcoholic fatty liver disease, diet and gut microbiota Finelli Carmine 1Tarantino Giovanni 231 Center of Obesity and Eating Disorders, Stella Maris Mediterraneum Foundation, Potenza, Italy2 Department of Clinical Medicine and Surgery, Federico II University Medical School of Naples, Italy3 National Cancer Institute "Foundation G. Pascale" -IRCS- 83013 Mercogliano (Av), ItalyTo whom correspondence should be addressed: Giovanni Tarantino, Department of Clinical Medicine and Surgery, Federico II University Medical School of Naples, Via Sergio Pansini, 5, 80131 Naples, Italy, E-mail: [email protected] 5 2014 2014 13 461 490 12 3 2013 31 3 2014 Copyright © 2014 Finelli et al.2014This is an Open Access article distributed under the following Assignment of Rights http://www.excli.de/documents/assignment_of_rights.pdf. You are free to copy, distribute and transmit the work, provided the original author and source are credited.This article is available from http://www.excli.de/vol13/Tarantino_07052014_proof.pdfNon-alcoholic fatty liver disease (NAFLD) is a severe liver disease that is increasing in prevalence with the worldwide epidemic of obesity and its related insulin-resistance state. Evidence for the role of the gut microbiota in energy storage and the subsequent development of obesity and some of its related diseases is now well established. More recently, a new role of gut microbiota has emerged in NAFLD. The gut microbiota is involved in gut permeability, low-grade inflammation and immune balance, it modulates dietary choline metabolism, regulates bile acid metabolism and produces endogenous ethanol. All of these factors are molecular mechanisms by which the microbiota can induce NAFLD or its progression toward overt non-alcoholic steatohepatitis. Modification of the gut microbiota composition and/or its biochemical capacity by specific dietary or pharmacological interventions may advantageously affect host metabolism. Large-scale intervention trials, investigating the potential benefit of prebiotics and probiotics in improving cardiometabolic health in high-risk populations, are fervently awaited. Gut microbiomeNAFLDinterventions ==== Body Introduction The rising incidence of obesity in today’s environment is associated with many obesity-related health complications, including cardiovascular disease, type 2 diabetes (T2D), hyperlipidemia, hypertension, and non-alcoholic fatty liver disease (NAFLD) (Tarantino et al., 2007[180]; 2012[179]; 2013[174]; Finelli and Tarantino, 2013[56]). This constellation is also recognized as the metabolic syndrome and is characterized by underlying insulin resistance (IR). NAFLD or generally speaking hepatic steatosis (HS) is defined as the accumulation of lipid, primarily in the form of triacylglycerols in individuals who do not consume significant amounts of alcohol (< 20 g ethanol/d) and in whom other known causes of steatosis, such as certain drugs and toxins, have been excluded (Vuppalanchi and Chalasani, 2009[194]). The spectrum of NAFLD includes simple fatty liver, non-alcoholic steatohepatitis (NASH) - characterized by inflammation, apoptosis, ballooning degeneration, Mallory hyaline, fibrosis-, cirrhosis post NASH, hepatocellular carcinoma and advanced liver disease, which leads to liver-related death (Vuppalanchi and Chalasani, 2009[194]; Sorrentino et al., 2004[164]; Tarantino and Finelli, 2013[178]; Tarantino, 2007[172]; Tarantino et al., 2011[176]; Finelli and Tarantino, 2013[55]). Given the close relations between obesity, the metabolic syndrome, and the development of NAFLD, it is not surprising that many NAFLD patients have multiple components of the metabolic syndrome, whether or not they are overweight or obese. IR is present in and is a significant predictor of NAFLD and NASH in most patients (Clark, 2006[36]), even the ~ 10–15 % of patients who are not overweight (Chiang et al., 2011[32]; Hamaguchi et al., 2012[72]). NAFLD is a multifactorial disease that involves a complex interaction of genetics, diet, and lifestyle, all of which combine to form the NAFLD phenotype. A cornerstone of the management strategy in such patients with fatty liver is the use of diet to decrease body weight, and improve glycemic control, dyslipidemia and cardiovascular risks as well (Finelli and Tarantino, 2012[53]). Gut microbiota are thought to play a role in the pathogenesis of NASH for several reasons. First, gut microbiota are known to have a large effect on the digestion and absorption of nutrients (van der Hoeven-Hangoor et al., 2013[191]). Microbiota transplantation experiments in mice suggested that certain microbiota is capable of inducing obesity independent of other environmental factors (Kallus and Brandt, 2012[86]). Second, gut microbiota participate in the development and homeostasis of the overall immunity of the host (Gigante et al., 2011[63]). Therefore, certain microbiota may influence the development of liver inflammation. The links between gut microbiota and the host immune system include TLRs and short-chain fatty acids (Vinolo et al., 2011[193]). In fact, the innate immune system might influence the metabolic syndrome and obesity, as mice deficient in Toll-like receptor 5 develop hyperphagia, become obese and insulin resistant (Tilg, 2010[184]). Third, gut microbiota may influence the production of gut hormones, such as glucagon-like peptide 1, and, subsequently, have an effect on the overall metabolism of the host (Flint, 2011[58]). The liver appears as the first point of contact for (and produces the initial immunological response to) bacteria and microbial components, as well as other endogenous and exogenous toxins present in the portal blood. Given the capacity of the liver to regulate metabolism in a form that can affect the entire organism, to distribute numerous substances to the gut through bile and the entero hepatic circulation, and to regulate numerous hormonal and immunological responses, the potential for the liver to influence gut function can be quickly appreciated. Interactions between the gut, the diet and the liver are, naturally, bidirectional; hormones, inflammatory mediators and the products of digestion and absorption all unequivocally influence liver function. The focus of this review is on gut–liver-diet interactions that contribute to the pathogenesis of a common liver disorder, NAFLD. Interactions between the intestinal microbiota and liver The interactions of the gut microbiota and the liver have only recently been investigated in detail. Receiving approximately 70 % of its blood supply from the intestinal venous outflow, the liver represents the first line of defense against gut-derived antigens, food antigens, toxins, microbial-derived products, and microorganisms (Henao-Mejia et al., 2013[76]). The liver, therefore, is equipped with a broad array of immune cells (i.e., macrophages, lymphocytes, natural killer cells, and dendritic cells) to accomplish this function (Henao-Mejia et al., 2013[76]). Small intestinal bacterial overgrowth (SIBO) is common in patients with cirrhosis (Wiest et al., 2014[200]; Savarino et al., 2011[152]; Bellot et al., 2013[17]; Gupta et al., 2010[69]) and its prevalence correlates directly with the severity of liver disease (Pande et al., 2009[119]). A physiological basis for SIBO in liver disease was added by reports of impaired small bowel motility and prolonged orocecal transit time in patients with cirrhosis (Seo and Shah, 2012[158]; Gunnarsdottir et al., 2003[68]). Some studies reinforced the concept that disturbed motility predisposes to SIBO, and suggested a link between alterations in intestinal motility and the development of both hepatic encephalopathy and spontaneous bacterial peritonitis (Romeiro et al., 2013[143]; Ancel et al., 2006[8]; Bouin et al., 2004[22]; Garcovich et al., 2012[61]). Bacterial translocation (passage of viable bacteria resident in the gastrointestinal tract to generally sterile tissues) in the context of SIBO is facilitated by augmented intestinal permeability, another feature of liver cirrhosis (Hada et al., 2010[70]; Cariello et al., 2010[26]). Indeed, increased intestinal permeability is associated with an increased risk of spontaneous bacterial peritonitis (Benjamin et al., 2013[18]; Assimakopoulos et al., 2012[10]). Having identified bacterial products as hepatotoxins (Lamontagne et al., 2013[97]; Kirsch et al., 2006[92]) and having figured out that multiple metabolic activities of the gut microbiota could affect the liver function, the potential for the gut microbiota to induce or maintain various liver diseases, mainly at the light of its immunological interactions with the host has been clearly reckoned. NAFLD, intestinal microbiota and metabolic changes NAFLD is a multifactorial disease. Numerous genetic, metabolic, inflammatory and environmental factors are consideration to contribute to its pathogenesis (Figure 1(Fig. 1)). In the middle of the environmental factors, diet is the most important, though its role may be more complex than one of simply inducing fat accumulation in the liver, and may involve interactions with the microbiota. Indeed, the microbiota may contribute to NAFLD in a number of ways, some of which are quite unexpected. Obesity Obesity, especially visceral obesity, is a major risk factor for NAFLD in humans (Finelli and Tarantino, 2012[54]). The gut microbiota is partly responsible for body fat deposition in mice (Flint, 2011[58]). Usually colonized animals have a higher body fat content than do germfree animals; the inoculation of germfree mice with micro biota obtained from usually colonized adult animals resulted in a 57 % increase in total body fat (Wolf, 2006[201]). Organisms in the Bacteroidetes and Firmicutes phyla collectively represent about 90 % of the microbiota composition in mice, as they do in humans (Murphy, 2010[111]). However, their relative contributions vary considerably according to body composition. In a murine model, it was shown that the relative abundance of these two phyla differs among lean and obese mice; the obese mouse had a higher proportion of Firmicutes to Bacteroidetes (50 % greater) than the lean mouse (Kallus and Brandt, 2012[86]). The same results were appreciated in obese humans compared to lean subjects, as reported by Kallus and Brandt (2012[86]). The postulated explanation for this finding is that Firmicutes produce more complete metabolism of a given energy source than do Bacteroidetes, thus promoting more efficient absorption of calories and subsequent weight gain (Kallus and Brandt, 2012[86]). These findings indicate a possible role for components of the microbiota in determining the efficiency of calorie extraction from the diet and, thereby, influencing body weight (Blaut and Klaus, 2012[20]). It showed that the significant increase in mannitol excretion rate, in patients undergoing Roux-en-Y Gastric Bypass (RYGB), from the first postoperative month to the sixth postoperative month most likely reflects the occurrence of intestinal adaptation (mucosal hyperplasia), which would tend to minimize the malabsorption of macronutrients (Savassi-Rocha et al., 2014[153]). A subgroup of patients who undergo RYGB exhibit pronounced increase in their intestinal permeability (assessed by the lactulose/mannitol ratio and the lactulose excretion rate) at the sixth postoperative month (Savassi-Rocha et al., 2014[153]). Obese humans harbour considerably fewer Bacteroidetes and more Firmicutes than lean controls (Krznaric et al., 2012[96]). Changes in the gut microbiota have also been documented among obese individuals after gastric bypass surgery (Osto et al., 2013[118]). Osto et al. (2013[118]) showed that RYGB surgery might differently modify the gut microbiota composition in the three distinct anatomical sections of the small intestine compared to sham surgery. RYGB induced changes in the microbiota of the alimentary limb and the common channel resembling those seen after prebiotic treatment or weight loss by dieting, as reported by Osto et al. (2013[118]). These changes may be associated with altered production of intestinal hormones known to control energy balance (Osto et al., 2013[118]). Postsurgical modulation of gut microbiota may significantly contribute to the beneficial metabolic effects of RYGB surgery (Osto et al., 2013[118]), not excluding those on NAFLD. Santacruz et al. (2009[150]) indicated that calorie restriction and physical activity have an impact on gut microbiota composition related to body weight loss, which also seem to be influenced by the individual's microbiota. Limited evidence suggests a role for increased microbial extraction of calories among obese humans. DiBaise et al. (2008[45]), in their review, suggests that the gut microbiota affects nutrient acquisition and energy regulation and its composition has also been shown to differ in lean vs obese animals and humans. Some evidence suggests that the metabolic activities of the gut microbiota facilitate the extraction of calories from ingested dietary substances and help to store these calories in host adipose tissue for later use (DiBaise et al., 2008[45]). Furthermore, the gut bacterial flora of obese mice and humans include fewer Bacteroidetes and correspondingly more Firmicutes than that of their lean counterparts, suggesting that differences in caloric extraction of ingested food substances may be due to the composition of the gut microbiota (DiBaise et al., 2008[45]). Bacterial lipopolysaccharide derived from the intestinal microbiota may act as a triggering factor linking inflammation to high-fat diet-induced metabolic syndrome (DiBaise et al., 2008[45]). DiBaise et al. (2008[45]) concluded that existing evidence warrants further investigation of the microbial ecology of the human gut and points to modification of the gut microbiota as one means to treat people who are over-weight or obese. Several studies reveal how the microbiota might influence body weight and composition. Gut microbiota could precisely affect the proportion of calories obtained from the intestinal contents (caloric salvage). For example, Bacteroides thetaiotamicron is able to break most glycosidic linkages of numerous constituents of our diet. This Gram-negative anaerobic bacterium can degrade indigestible poly saccharides from plant, representing a quota part of calorie needs, nearly 10–15 % (Zocco et al., 2007[210]). Furthermore, microbes are predominantly found in surface-attached and spatially structured polymicrobial communities (Estrela and Brown, 2013[49]). Within these communities, microbial cells excrete a wide range of metabolites, setting the stage for interspecific metabolic interactions (Estrela and Brown, 2013[49]). The links, however, between metabolic and ecological interactions (functional relationships), and species spatial organization (structural relationships) are still poorly understood, as reported by Estrela et al. (2013[49]). It showed that strong metabolic interdependence drives the emergence of mutualism, robust interspecific mixing, and increased community productivity. These emergent community properties are driven by demographic feedbacks, such that aid from neighboring cells directly enhances focal cell growth, which in turn feeds back to neighbour fecundity. In contrast, weak metabolic interdependence drives conflict (exploitation or competition), and in turn greater interspecific segregation. Together, these results support the idea that species structural and functional relationships represent the net balance of metabolic interdependencies (Estrela and Brown, 2013[49]). The caloric restore is furnished by the presence of genes encoding enzymes that split vegetable and dietary polysaccharides in the microbiota of obese mouse. Administration of a conventional microbiota to germ free mice induced a rapid increase in body fat associated with increased hepatic triglyceride production related to increased activity of two crucial enzymes involved in de novo fatty acid synthesis—acetyl coenzyme a carboxylase and fatty acid synthetase (Wolf, 2006[201]). The microbiota inhibits angiopoietin related protein 4, which suppresses lipoprotein lipase, a key regulator of fatty acid release from triglyceride rich chylomicrons. Inhibition of angiopoietin related protein 4 causes hyper-expression of lipoprotein lipase, giving place to an augmented uptake of fatty acids and storage of triglycerides in adipocytes (Wolf, 2006[201]; Fleissner et al., 2010[57]) and liver (Fleissner et al., 2010[57]). Gut microbes have a pivotal role in the intra luminal metabolism of bile acids (Ruiz et al., 2013[146]). Given that bile acids are fundamental for the absorption and emulsification of dietary fats and lipid soluble vitamins in the small intestine, disorganized bile acid physiology could result in an altered energy balance. The role of bile acids in maintaining the intestinal barrier function and the luminal environment must too be called to mind, including their capacity to prevent SIBO and bacterial translocation (Hagey and Krasowski, 2013[71]; Karatepete al., 2010[87]). Additionally, bile acids are involved in energy and lipid metabolism, being capable of lowering triglyceride levels, for example (Trauner et al., 2010[187]). Therefore, the microbiota could, because to effects on bile acid metabolism in the gut lumen, influence signalling pathways involved in energy and lipid metabolism. The consequences of this involvement include the regulation of secondary bile acid metabolism, the inhibition of bile acid synthesis, modifications to lipid peroxidation and the storage of fatty acids in the liver (Sayin et al. 2013[154]). Insulin resistance Insulin resistance is crucial in the pathogenesis of the metabolic syndrome, of which NAFLD is considered as the hepatic component. Insulin resistance appears to have a crucial role in the pathogenesis of NAFLD and NASH (Finelli and Tarantino, 2013[56]). Besides the suggested role of insulin resistance in the development of steatosis, hepatic insulin resistance could promote hepatocyte injury and inflammation (Tarantino et al., 2009[177]). Gut flora and gut derived endotoxemia are considered main factors in developing insulin resistance. The key link is represented by the lipopolysaccharide–toll like receptor 4 (TLR4)–monocyte differentiation antigen CD14 system (Penas-Steinhardt et al., 2012[122]; Ma et al., 2013[105]; Belforte et al., 2013[16]; Krautbauer et al., 2014[95]). Even if this evidence has been largely gleaned from animal models, one study documented elevated plasma levels of lipopolysaccharide among patients with obesity and type 2 diabetes mellitus (Hawkesworth et al., 2013[75]). Confirmation of these findings and elucidation of the role of the microbiota, gut damage and the pathways for translocation of bacterial debris could open new avenues for prevention and treatment of type 2 diabetes, as reported by Hawkesworth et al. (2013[75]). Suppression or modification of SIBO, by leading to reduced proinflammatory cytokine production, results in a fall in fasting insulin concentrations and decreased insulin resistance (Penas-Steinhardt et al., 2012[122]; Rodríguez-Hernández et al., 2013[140]). Moreover, Carvalho and Saad (2013[28]) suggested that several strategies focusing on modulation of the gut microbiota (antibiotics, probiotics, and prebiotics) are being experimentally employed in metabolic derangement in order to reduce intestinal permeability, increase the production of short chain fatty acids and anorectic gut hormones, and promote insulin sensitivity to counteract the inflammatory status and insulin resistance found in obese individuals. In another study, it hypothesized that ampicillin improve glucose tolerance in mice only if treatment is initiated prior to weaning and that it disappears when treatment is terminated (Rune et al., 2013[147]). The results supported the hypothesis that a "window" exists early in life in which an alteration of the gut microbiota affects glucose tolerance as well as development of gut immunity and that this window may disappear after weaning (Rune et al., 2013[147]). The gut microbiota has also been implicated, though in a very different manner, in the pathogenesis of type 1 diabetes mellitus and is an exceedingly complex microenvironment that is intimately linked with the immune system, including the regulation of immune responses (Atkinson and Chervonsky, 2012[11]). Murri et al. (2013[112]) hypothesized that type 1 diabetes in humans could also be linked to a specific gut microbiota Their aim was to quantify and evaluate the difference in the composition of gut microbiota between children with type 1 diabetes and healthy children and to determine the possible relationship of the gut microbiota of children with type 1 diabetes with the glycemic level. This is the first study showing that type 1 diabetes is associated with compositional changes in gut microbiota. The significant differences in the number of Bifidobacterium, Lactobacillus and Clostridium and in the Firmicutes to Bacteroidetes ratio observed between the two groups could be related to the glycemic level in the group with diabetes (Murri et al., 2013[112]). Moreover, the quantity of bacteria essential to maintain gut integrity was significantly lower in the children with diabetes than the healthy children. Therefore, Murri et al. (2013[112]) suggested that these findings could be useful for developing strategies to control the development of type 1 diabetes by modifying the gut microbiota. There is increasing evidence that environmental factors acting at the intestinal level, with a special regard to the diverse bacterial species that constitute the microbiota, influence the course of autoimmune diseases in tissues outside the intestine both in humans and in preclinical models (Sorini and Falcone, 2013[163]). These observations suggest factors in the modern environment promote pancreatic islet autoimmunity and destruction of insulin-producing beta cells (Penno et al., 2013[123]). The Environmental Determinants of Islet Autoimmunity (ENDIA) Study is investigating candidate environmental exposures and gene-environment interactions that may contribute to the development of islet autoimmunity and type 1 diabetes (Penno et al., 2013[123]). ENDIA evaluated the microbiome, nutrition, bodyweight/composition, metabolome-lipidome, insulin resistance, innate and adaptive immune function and viral infections (Penno et al., 2013[123]). Therefore, Penno et al. (2013[123]) suggested that defining gene-environment interactions that initiate and/or promote destruction of the insulin-producing beta cells in early life will inform approaches to primary prevention of type 1 diabetes. Altered choline metabolism Diets deficient in both methionine and choline have been consistently associated with the development and progression of hepatic steatosis, and have been indicated that synergistic effects of protein restriction and choline deficiency influence integrated metabolism and hepatic pathology in mice when nutritional fat content is very high, and support the consideration of dietary choline content in ketogenic diet studies in rodents to limit hepatic mitochondrial dysfunction and fat accumulation (Schugar et al., 2013[157]). Decreased choline intake is significantly associated with increased fibrosis in postmenopausal women with NAFLD (Guerrerio et al., 2012[67]). Wattacheril et al. (2013[197]) suggest that phospholipid zonation may be associated with the presence of an intrahepatic proinflammatory phenotype and thus have broad implications in the etiopathogenesis of. Enzymes produced by the gut microbiota catalyze the first step in the conversion of dietary choline to dimethylamine and trimethylamine (Craciun and Balskus, 2012[39]). These metabolites (Rezzi et al., 2007[138]) are absorbed through the microvilli and reach the liver via the portal vein (Tang et al., 2013[170]) where trimethylamine is largely cleared by hepatic first-pass metabolism before it enters the systemic circulation. Germ free mice do not excrete trimethylamine, supporting an essential role for the gut microbiota in the conversion of choline to this compound (Bain et al., 2005[13]). Létoffé et al. (2014[99]) showed that exposure to trimethylamine increases the pH of the growth medium of exposed bacteria, resulting in modifications in antibiotic uptake and transient alteration of antibiotic resistance. This study therefore presents a new mechanism by which volatile compounds, during food transformation and fermentation, can affect community behavior and structure in physically separated bacteria, and it illustrates how airborne chemical interactions between bacteria contribute to the development of bacterial communities (Létoffé et al., 2014[99]). Diet and mutations of the gut microbiota and host metabolism Modification of gut microbiota and/or its biochemical ability by specific dietary or pharmacological interventions, may conveniently affect host metabolism. However, in humans to date it is unclear, whether the diet-induced effects depend on pre-existent gut microbial composition, in interaction with the host phenotype, whether oral co-administration of specific bacterial species together with the dietary substrate is required, and which mechanisms are involved. Crucially, it is unknown whether the observations made in rodents can be extrapolated to humans and ultimately harnessed for clinical purposes. In association with the tools to modulate the gut microbiota, prebiotics (i.e. ‘non-digestible food ingredients that beneficially affect the host by selectively stimulating the growth and/or activity of one or a limited number of the bacteria in the guts’) (Gibson et al., 2004[62]), and probiotics (i.e. ‘live microorganisms which, when given orally in quantities adequate to allow colonization of the colon, confer a health benefit to the host’) (Bertazzoni et al., 2013[19]), are the most important. Supplementation with inulin-type fructooligosaccharides (FOS), stimulated growth of Bifidobacterium spp. and in some cases Lactobacillus spp. in humans (Dewulf et al., 2013[42]; Bedani et al., 2013[15]; O'Connell Motherway et al., 2013[116]). These groups of bacteria, often administrated as probiotics, were associated with reduction of intestinal endotoxin levels and improvement of mucosal barrier function (Pinzone et al., 2012[124]; Rao and Samak, 2013[134]; Fouhy et al., 2013[59]). The Bifidobacteria count at baseline is strictly related to the increased count after treatment, clearly showing that pre-existent composition of the gut microbiota is central to the response of the intervention (Dewulf et al., 2013[42]). Van Bearlen et al. (2009[190]) showed that expression profiles of human mucosa displayed striking differences in modulation of NF-kappaB-dependent pathways, notably after consumption of living Lactobacillus plantarum bacteria in different growth phases. In a randomized, double-blind, placebo-controlled trial, independently of other lifestyle changes, oligofructose supplementation has the potential to promote weight loss and improve glucose regulation in overweight adults (Parnell and Reimer, 2009[120]). Also, Pedersen et al. showed that oligofructose dose-dependently increased peptide YY, decreased pancreatic polypeptide and tended to decrease ghrelin, but did not significantly affect appetite profile, energy intake, glucose, insulin, or glucagon-like peptide 1 concentrations during appetite study sessions (Pedersen et al., 2013[121]). Pedersen et al. concluded that oligo-fructose supplementation at ≥ 35 g/day increased peptide YY and suppressed pancreatic polypeptide and hunger; however, energy intake did not change significantly (Pedersen et al., 2013[121]). It has demonstrated that a single gene (encoding linoleic acid isomerase) expressed in an intestinal microbe can influence the fatty acid composition of host fat (Rosberg-Cody et al., 2011[144]). Fava et al. (2013[51]) suggested a new evidence from a large-scale dietary intervention study that high carbohydrate diets, irrespective of glycemic index, can modulate human faecal saccharolytic bacteria, including bacteroides and bifidobacteria Conversely, high fat diets reduced bacterial numbers, and in the high saturated fat diet, increased excretion of short-chain fatty acids (SCFAs), which may suggest a compensatory mechanism to eliminate excess dietary energy (Fava et al., 2013[51]). In contrast, supplementation of non-fermentable carbohydrates such as FOS, which lead to an increase in SFCA formation, had beneficial effects on the host metabolic phenotype, including increased satiety, body weight and -fat loss and improvement in insulin sensitivity and glucose tolerance, with several mechanisms involved (Fouhyet al., 2013[59]; Pourghassem Gargari et al., 2013[126]; Schroeder et al., 2013[156]; Whelan, 2013[199]). Of note, butyrate shows an obvious function of anti-obesity, and can alleviate the metabolic stress, maintain the ß-cell function and protect them from inflammatory response in pregnant obese mouse without obvious fetus toxicity (Li et al., 2013[100]). Evidence supporting that dietary inulin alone was effective to prevent the development of hepatic steatosis, ameliorate nutritional effects, and alleviate the hepatic change in the expression of hepatic cytochrome P450 (CYP) mRNA, while co-treatment with statin did not have additive or synergistic effects and statin may cause adverse effects in rats fed the high-fat and high-sucrose diet (Sugatani et al., 2012[169]). Reimer et al. (2012[136]) reported that novel polysaccharide (NPS) PolyGlycopleX (PGX) and Sitagliptin improve several metabolic outcomes in Zucher diabetic fatty rats, but combined, their ability to markedly reduce glycemia suggests they may be a promising dietary/pharmacological co-therapy for type 2 diabetes management. Probably, the SCFA-induced physiological effects on colonic functions might be attributable to the activation of SCFA receptors on epithelial cells in the colon (Tazoe et al., 2008[183]). However, highly viscous, non-fermentable fibers may limit weight gain and reduce adiposity and non-fermentable fibers, regardless of viscosity, may promote meal termination (Schroeder et al., 2013[156]). Another, fermentable indigestible carbohydrate increases the number of free fatty acid receptor 2 -positive L-cells in the proximal colon (Schroeder et al., 2013[156]). Free fatty acid receptor 2 activation by SCFAs might be an important trigger for produce and release GLP-1 by enteroendocrine L-cells in the lower intestine (Schroeder et al., 2013[156]). Also, FOS in mice increased the number of intestinal bifidobacteria and reduced the impact of high-fat diet-induced endotoxaemia and inflammation (Pourghassem Gargari et al., 2013[126]; Schroeder et al., 2013[156]). Several studies in humans already support interest in FOS in the control of satiety, triglyceridemia, or steatohepatitis (Delzenne et la., 2007[40]). Moran-Ramos et al. (2012[110]) suggested that the potential for diet interventions as a promising strategy for modulating gut hormone responses to food ingestion and, ultimately, preventing or treating metabolic diseases is being emphasized considering that these diseases are currently a public health burden. The link with gut peptides production in humans remains to be proven. Therefore, we hypothesize that the effects of dietary factors on gut microbiota and host metabolism, particularly in humans, are as yet widely unknown. These may depend on both the dietary intervention and the pre-existent gut microbial composition, in relation to the host phenotype. Gut microflora's composition and non-dietary factors Gut microflora composition in an individual’s colon likely is influenced by a combination of dietary habits and other host- and non-host-associated factors. For example, the exposure of individuals to microbes capable of establishing residence in the gut might depend on geographic location, with large differences expected between individuals living in areas with different levels of drinking water purity and food quality; different levels of hygiene; or with different climates. Other factors that may contribute to the progression of the intestinal microflora include initial colonization after birth, driven by the presence of selective nutrients in the mother’s milk; host genetic factors that influence the secretion of substances that facilitate selection for specific bacteria; immune control that favors growth of some groups of bacteria; and random chance that results in a colonization cascade. Another factor that can alter an established microflora is antibiotic treatment. Antibiotics interfere with the existing microflora by selecting in contrast to vulnerable bacteria and, even after treatment, the re-establishment of the full complexity of the microflora might result in a changed composition. Buccigrossi et al. (2013[23]) sustained that a relationship exists between eubiosis and functions and conversely between dysbiosis and dysfunctions or even diseases. Abnormalities in microflora composition may trigger or contribute to specific diseases. This raises the hypothesis to target microflora in order to restore eubiosis through the use of antibiotics, probiotics or nutrients (Buccigrossi et al., 2013[23]). Differences between physical activity levels might too change gut microflora composition. Even if moderate exercise has not been shown to reduce transit time through the intestinal tract, elevated activity levels might change other aspects of intestinal physiology and, in this manner, the conditions for microbial growth (Kim, 2012[91]; Cho et al., 2013[34]). Valdés-Ramos et al. (2010[189]) suggest that high-fat diets combined with exercise are able to induce an increase in CD3+ lymphocytes due to increased CD8+ cells and a decrease in B-cells and the authors concluded that explanations and consequences of the effects of diet and exercise on the gut mucosal immunity are still being explored (Valdés-Ramos et al., 2010[189]). Another, it observed substantial taxonomic changes in the microbiome, changes in copies of key genes involved in the metabolism of carbohydrates to short-chain fatty acids, increases in colonic short-chain fatty acid levels, and alterations in the regulation of hepatic metabolism of lipids and cholesterol (Cho et al., 2012[33]). Therefore, Cho et al. (2012[33]) demonstrated the alteration of early-life murine metabolic homeostasis through antibiotic manipulation. For these findings, we hypothesized that as-yet-undiscovered factors, or random chance, too is partly responsible for the establishment and maintenance of the intestinal microbiota. In a human dietary intervention study reporting beneficial effects of green and black tea drinking on serum lipids (Hartley et al., 2013[73]), Henning et al. (2013[77]) observed that the consumption of both, green tea and black tea, was associated with a significant increase in urinary and serum phenolic acids. Tea polyphenols are metabolized by the colonic microflora yielding phenolic metabolites, which may contribute to the health benefits of tea (Henning et al., 2013[77]). We hypothesize that, at least for short study intervals, the constituted microflora had an overwhelming effect on the flora’s final composition, thus indicating the need for a longer follow-up in dietary studies. Due to substrate availability, water content, and other physiologic conditions, the highest microbial activity is found in the proximal colon (Tannock, 2002[171]; Macfarlane and Macfarlane, 2012[106]). In that regard, although the microflora’s composition appears to be affected by factors that are primarily associated with diet (i.e., changes in substrate availability, pH, and reduction potential), it might also be influenced by genetic and other as-yet-undiscovered factors. Small intestinal bacterial overgrowth (SIBO) The progression of simple steatosis to steatohepatitis is essentially an inflammatory rather than a metabolic process; risk factors associated with this change include obesity and a high BMI (Tarantino et al., 2010[181]; Greene et al., 2014[66]; Alkhouri et al., 2014[6]). Several lines of evidence, detailed below, have suggested that SIBO might play an important part in progression of NAFLD to NASH. Intestinal failure and total parenteral nutrition (TPN) are associated with NAFLD and progression to NASH (Corbin and Zeisel, 2012[38]; Rollins et al., 2013[142]). SIBO, related probably to intestinal hypomotility as well as other factors, such as suppressed secretion of gastric acid and intestinal enzymes and reduced bile flow, has been considered as a causative factor (Corbin and Zeisel, 2012[38]; Rollins et al., 2013[142]). The use of TPN in the treatment of critically ill patients has been the subject of debate because it has been associated with alterations in intestinal homeostasis (Hodin et al., 2012[78]). Important factors in maintaining intestinal homeostasis are the intestinal microbiota and Paneth cells, which exist in a mutually amendable relationship. Hodin et al. (2012[78]) showed a shift in intestinal microbiota in TPN-fed rats that correlated with changes in Paneth cell lysozyme expression. Further studies that include interventions with microbiota or nutrients that modulate them may yield information on the involvement of the microbiota and Paneth cells in TPN-associated intestinal compromise (Hodin et al., 2012[78]). However, the contribution of the intestinal microbiome to liver disease goes beyond simple translocation of bacterial products that promote hepatic injury and inflammation (Schnabl and Brenner, 2014[155]). Microbial metabolites produced in a dysbiotic intestinal environment and host factors are equally important in the pathogenesis of liver disease (Schnabl and Brenner, 2014[155]). Therefore, we hypothesize that the combination of liver insult and disruptions in intestinal homeostasis contribute to liver disease. The increased abundance of alcohol-producing bacteria in NASH microbiomes, elevated blood-ethanol concentration in NASH patients, and the well-established role of alcohol metabolism in oxidative stress and, thus, liver inflammation suggest a role for alcohol-producing microbiota in the pathogenesis of NASH (Zhu et al., 2013[209]). Zhu et al. (2013[209]) postulated that the distinct composition of the gut microbiota among NASH, obese, and healthy controls could offer a target for intervention or a marker for disease. In addition, several experimental studies and clinical trials revealed promising effects of probiotics in improving NAFLD; however given the limited experience in this field, generalization of probiotics as treatment of NAFLD needs substantiation through more trials with a larger sample sizes and with longer-term follow up (Kelishadi et al., 2013[90]). Younossi et al. (2014[205]) suggested that NASH associated with metabolic syndrome can progress advanced fibrosis and cirrhosis. Weight loss and lifestyle modification have been shown to improve NASH. Other medications used for weight loss and metabolic syndrome have been evaluated, such as orlistat, metformin and thiazolidinediones, as reported by Younossi et al. (Younossi et al., 2014[205]). Alternative regimens using ursodeoxycholic acid, statins and probiotics as well as bariatric surgery have been evaluated, but have not been recommended as first-line treatment for NASH (Younossi et al., 2014[205]). Vitamin E for NASH patients without diabetes seems to be promising (Younossi et al., 2014[205]). The lack of effective treatment for NASH suggests the heterogeneity of patients presenting with the NASH phenotype (Younossi et al., 2014[205]). The best treatment strategy for these patients may be to identify their pathogenic target and develop personalised treatment protocols (Younossi et al., 2014[205]). Shanab et al. showed that NASH patients have a higher prevalence of SIBO which is associated with enhanced expression of TLR-4 and release of IL-8 (Shanab et al., 2011[159]). SIBO may have an important role in NASH through interactions with TLR-4 and induction of the pro-inflammatory cytokine, IL-8 (Shanab et al., 2011[159]). It showed that probiotic combination with metformin improves liver aminotransferases better than metformin alone in patients with NASH (Shavakhi et al., 2013[160]). Nevertheless, NASH recurs immediately after liver transplantation unless the jejunoileal bypass is removed (Charlton, 2013[29]). However, Wu et al. (2008[202]) suggested that SIBO may decrease small intestinal movement in NASH rats. Another, SIBO may be an important pathogenesis of NASH and treatment with cidomycin by mouth can alleviate the severity of NASH (Wu et al., 2008[202]). In addition, gut flora and bacterial translocation play important roles in the pathogenesis of chronic liver disease, including cirrhosis and its complications (Ilan, 2012[81]). Intestinal bacterial overgrowth and increased bacterial translocation of gut flora from the intestinal lumen predispose patients to bacterial infections, major complications and also play a role in the pathogenesis of chronic liver disorders (Ilan, 2012[81]). A better understanding of the cell-specific recognition and intracellular signaling events involved in sensing gut-derived microbes will help in the development of means to achieve an optimal balance in the gut-liver axis and ameliorate liver diseases (Ilan, 2012[81]). These may suggest new targets for potential therapeutic interventions for the treatment of NASH (Ilan, 2012[81]). Both obesity and diabetes, key factors in NASH progression, are also associated with intestinal dysmotility (Stenkamp-Strahm et al., 2013[166]), which could potentially lead to SIBO (Bures et al., 2010[25]; Jacobs et al., 2013[84]; Mushref and Srinivasan, 2013[113]). Ghrelin, a gastric hormone that regulates food intake, also exerts prokinetic effects (Strasser, 2012[168]; Queipo-Ortuño et al., 2013[129]). Patients with NASH show low ghrelin levels (Gonciarz et al., 2013[64]; Machado et al., 2012[107]), which could lead to reduced gut motility and encourage retrograde colonization of the small intestine by colonic bacteria and, probably, the progression of SIBO. Rana et al. (2014[133]) showed that increase in cytokines and decrease in anti-oxidants in ulcerative colitis patients would have resulted in oxidative stress causing delayed gastrointestinal motility leading to SIBO. Miele et al. (2009[108]) suggested that NAFLD in humans is associated with increased gut permeability and that this abnormality is related to the increased prevalence of SIBO in these patients. The increased permeability appears to be caused by disruption of intercellular tight junctions in the intestine, and it may play an important role in the pathogenesis of hepatic fat deposition (Miele et al., 2009[108]). Disruption of tight junctions between intestinal epithelial cells by bacterial toxins or other inflammatory mediators leads to translocation of intraluminal contents (and, notably, bacterial endotoxins) into the systemic circulation. Sachdev and Pimentel (2013[149]) suggested that quantitative culture of small bowel contents and a variety of indirect tests have been used over the years in an attempt to facilitate the diagnosis of SIBO. The indirect tests include breath tests and biochemical tests based on bacterial metabolism of a variety of substrates. Infact, Rana and Bhardwaj (2008[132]) suggested that SIBO can be diagnosed by: 1) culture of jejunum aspirate for bacterial counts, 2) 14C-D-xylose breath testing, 3) non-invasive hydrogen breath testing using glucose or lactulose or 4) 14C-glycocholic acid breath testing. Actually, there is no single valid test for SIBO, and the accuracy of all current tests remains limited due to the failure of culture to be a gold standard and the lack of standardization of the normal bowel flora in the small intestine (Sachdev and Pimentel, 2013[149]). Interestingly, in morbidly obese patients, bacterial overgrowth prevalence is higher than in healthy subjects and is associated with severe hepatic steatosis (Sabaté et al., 2008[148]). Therefore, the ideal approach to treat SIBO is to treat the underlying disease, eradicate overgrowth, and address nutritional deficiencies that may be associated with the development of SIBO (Sachdev and Pimentel, 2013[149]). The gut microbiota and hepatotoxic effects The gut microflora has been identified to have possible hepatotoxic effects for numerous years. Indeed, the intestinal microbiota produces a number of probably hepatotoxic compounds, such as ammonia, ethanol, acetaldehyde, phenols and benzodiazepines, which must be consequently metabolized in the liver. Bacterial endotoxins reaching the liver through the portal circulation activate the hepatic Kupffer cells and stimulate their production of nitric oxide and cytokines. Altered intestinal permeability might ease the delivery of these hepatotoxic factors to the liver. Bacterial endotoxin, such as lipopolysaccharide (LPS), plays an important role in the pathogenesis of NAFLD (Fukunishi et al., 2014[60]). In fact, Fukunishi et al. (2014[60]) suggest that LPS may accelerate the progression of hepatic steatosis. In association with the numerous bacterial products, lipopolysaccharide and ethanol appear to be the most important factors in NAFLD pathogenesis. Lipopolysaccharide Lipopolysaccharide (LPS), the active component of endotoxin, binds to lipopolysaccharide binding protein (LBP), CD14, TLR4 and lymphocyte antigen 96, among other receptors. Roh and Seki (2013[141]) suggested that gut microflora-derived bacterial products (i.e. LPS) and endogenous substances (i.e. high-mobility group protein B1 [HMGB1], free fatty acids) released from damaged cells activate hepatic TLRs that contribute to the development of alcoholic and NASH and liver fibrosis. The crucial role of TLR4, a receptor for LPS, has been implicated in the development of alcoholic steatohepatitis, NASH, liver fibrosis, and hepatocellular carcinoma (Roh and Seki, 2013[141]). In fact, LPS binds to LBP and the LBP–LPS complex binds to CD14 on Kupffer cells. Then TLR associates with CD14 on the cell surface, triggering an essential intracellular inflammatory cascade, including stress-activated and mitogen-activated protein kinases, c-Jun N-terminal kinase (JNK), p38 and the nuclear factor κB (NFκB) pathway. Activation of Inhibitor of NFκB kinase ß subunit kinase (IKK) leads to the phosphorylation and complete degradation of IKK-ß, an NFκB inhibitor. NFκB translocates to the nucleus, where it binds to the promoter region of a number of target genes involved in the inflammatory pathway, such as TNF and IL-1ß. Metabolic effects Endogenous lipopolysaccharide is a complex of polysaccharide components and lipids. The lipid moiety, termed lipid a, is thought to be relevant to the induction of metabolic effects. In mice, lipopolysaccharide infusion resulted in increased fasting levels of glucose and insulin, as well as weight gain; the effects of this treatment on total body fat, steatosis and adipose tissue were similar to those induced by a high-fat diet. In parallel with these changes, the numbers of macrophages in adipose tissue and levels of inflammatory markers and hepatic triglycerides increased. In addition, insulin sensitivity in the liver (but not in other body tissues) was modified in lipopolysaccharide infused mice. Visceral and subcutaneous fat deposition was likewise increased in both the high-fat diet and lipopolysaccharide infused groups of animals (Krautbauer et al., 2014[95]). Moreover, fat ingestion elevates the effectiveness of translocation of intestinal bacterial LPS (Lee, 2013[98]). A high-fat diet produces a considerable quantity of lipoprotein containing chylomicrons, which promote LPS translocation to extraintestinal tissues (Demignot et al., 2014[41]). For individuals on a high-fat diet, therefore, a primary factor in the induction of metabolic diseases could be activation of an inflammatory cascade, induced by lipopolysaccharide binding to the complex of lymphocyte antigen 96, CD14 and TRL4 on the surface of immune cells (Racioppi et al., 2012[130]). Another, Racioppi et al. (2012[130]) sustained that calcium/calmodulin-dependent kinase kinase 2 (CaMKK2) plays a key role in regulating food intake and energy expenditure at least in part by its actions in hypothalamic neurons. Lipopolysaccharide can stimulate monocytes and macrophages to produce the proinflammatory cytokines TNF, IL1 and IL6 (Li et al., 2014[101]). Accordingly, several studies have reported high levels of proinflammatory cytokines, notably TNF, in obese individuals (Miele et al., 2009[108]; Zhong et al., 2013[208]; Gonzalez-Quintela et al., 2013[65]; Zunino et al., 2013[211]). TNF can induce insulin resistance by dual effects on insulin sensitive tissues, and this cytokine rapidly abolishes insulin receptor signalling in adipocytes, hepatocytes and skeletal muscle cells in tissue culture (Lorenzo et al., 2008[103]; Di Renzo et al., 2013[44]; Carstensen et al., 2014[27]). Furthermore, TNF-α in male Wistar rats models showed improved glucose and insulin homeostasis (Ahmed et al., 2014[3]). CD14, which acts as a lipopolysaccharide co-receptor along with lymphocyte antigen 96 and TLR4, might be the main molecule mediating insulin resistance and, hence, the occurrence of obesity and diabetes. Obese rodents lacking CD14 were protected from obesity, diabetes, the development of steatosis and visceral fat mass accumulation after lipopolysaccharide administration (Krautbauer et al., 2014[95]). Proinflammatory effects In a murine model with NAFLD, hepatic fat accumulation induces the liver to further grave injury by hepatotoxins and/or infectious agents, leading to NASH and the eventual progression of cirrhosis (Vansaun et al., 2013[192]). Shen et al. (2005[161]) reported that addition of leptin to normal rats increased LPS-induced hepatic TNF-alpha production in vivo and leptin receptor-deficient Zucker rats showed reduced hepatic TNF-alpha production on addition of LPS in vivo. These findings indicate that P38 and JNK pathways are involved in the signal transduction of leptin enhancement of LPS-induced TNF-alpha production (Shen et al., 2005[161]). Furthermore, Imajo et al. demonstrated that up-regulation of CD14 by leptin-mediated signaling is critical to hyperreactivity against endotoxin during NASH progression (Imajo et al., 2012[82]). Up-regulation of CD14 in Kupffer cells and hyperreactivity against low-dose LPS were observed in high-fat diet (HFD)-induced steatosis mice, but not chow-fed-control mice (Imajo et al., 2012[82]). Hyperresponsivity against low-dose LPS led to accelerated NASH progression, including liver inflammation and fibrosis. Administering leptin in chow-fed mice caused increased hepatic expression of CD14 via STAT3 signaling, resulting in hyperreactivity against low-dose LPS without steatosis. In contrast, a marked decrease in hepatic CD14 expression was observed in leptin-deficient ob/ob mice, despite severe steatosis (Imajo et al., 2012[82]). Lipopolysaccharide induced production of cytokines is initiated by binding of lipopolysaccharide to LBP, followed by the attachment of this complex to CD14 on Kupffer cells. TLR4 associates with CD14 on the cell surface to initiate lipopolysaccharide induced signal transduction - notably, activation of nuclear factor κb (NFκB) and the subsequent production of proinflammatory cytokines, such as TNF and cyclooxygenase 2 (Imajo et al., 2012[82]; Ling et al., 2014[102]). Activation of TLR4 by lipopolysaccharide triggers an essential intracellular inflammatory cascade, including stress-activated and mitogen-activated protein kinases, c-Jun-N-terminal kinase, p38 and the nFκB pathway. Activation of inhibitor of NFκB kinase subunit ß (IKKß) kinase leads to the phosphorylation and complete degradation of IKKß, an NFκB inhibitor. Removal of IKKß allows NFκB to translocate to the nucleus, where it binds to the promoter region of a number of target genes involved in the inflammatory pathway, such as TNF and IL-1ß (Huang and Hung, 2013[80]). Thus, NFκB might be a key factor in the induction of pro-inflammatory cytokines. Evidence, mostly from animal models, shows that this pathway is activated in the presence of NASH. Ruiz et al. (2007[146]) showed that NAFLD patients have elevated plasma levels of LPS-binding and they are further increased in patients with NASH. This increase is related to a rise in TNF-alpha gene expression in the hepatic tissue which supports a role for endotoxemia in the development of steatohepatitis in obese patients, as reported by Ruiz et al. (2007[146]). Stankovic et al. (2014[165]) suggested that methionine-choline deficient (MCD) diet duration necessary for development of NAFLD and the dynamic of lipid profile and fatty acids are not completely established. Therefore, in their study examined dynamics and association between liver free fatty acids, serum lipid profile and liver morphological changes on MCD diet-induced NAFLD in mice. Stankovic et al. (2014[165]) concluded that supplementation with n-3 polyunsaturated acid, especially in the initial stage of fatty liver disease, may potentially have preventive effects and alleviate development of NAFLD/NASH and may also potentially reduce cardiovascular risk by moderating dyslipidemia (Stankovic et al., 2014[165]). Some studies have suggested that bacterial overgrowth and endotoxemia along with its receptor, TLR-4, play a role in the pathogenesis of NAFLD. Kiziltas et al. (2014[93]) reported that as the first-time-in-humans controlled study related to investigation of TLR4 gene polymorphism in NAFLD, their findings contribute to the available data that TLR-4 signaling is pivotal for the pathogenesis of NASH and indicate that the TLR4 codon 299 heterozygous gene mutation (Asp299Gly) in humans may have a preventive role against the genesis of NAFLD. Inflammatory cytokines, such as TNF-α and IFN-γ, induce, as reported by Kawaratani et al. (2013[89]), liver injury in the rat model of NASH. Another, hepatoprotective cytokines, such as IL-6, and anti-inflammatory cytokines, such as IL-10, are also associated with NASH (Kawaratani et al., 2013[89]). Besides, IL-6 improves NASH via activation of the signal transducer and activator of transcription 3 (STAT3) and the subsequent induction of a variety of hepatoprotective genes in hepatocytes (Kawaratani et al., 2013[89]). IL-10 inhibits alcoholic liver inflammation via activation of STAT3 in Kupffer cells and the subsequent inhibition of liver inflammation (Kawaratani et al., 2013[89]). Alcohol consumption promotes liver inflammation by increasing translocation of gut-derived endotoxins to the portal circulation and activating Kupffer cells through the LPS/TLR 4 pathways. Another, oxidative stress and microflora products are also associated with NASH (Kawaratani et al., 2013[89]). Therefore, interactions between pro- and anti-inflammatory cytokines and other cytokines and chemokines are likely to play important roles in the development of NASH (Kawaratani et al., 2013[89]). Hepatic stellate cells (HSCs) could play a main role in generating the liver inflammatory cascade associated with endotoxemia (Harvey et al., 2013[74]; Stewart et al., 2014[167]). HSCs are the major cell type involved in liver fibrosis. Lipopolysaccharide (LPS)-mediated signaling through TLR4 in HSCs has been identified as a key event in liver fibrosis, and as the molecular link between inflammation and liver fibrosis (Zhao et al., 2014[206]). Therefore, Zhao et al. investigated the effects of caffeic acid phenethyl ester (CAPE), one of the main medicinal components of propolis, on the pro-inflammatory and fibrogenic phenotypes of LPS-stimulated HSCs (Zhao et al., 2014[206]). HSCs from rats were isolated and cultured in Dulbecco's modified Eagle's medium (DMEM) (Zhao et al., 2014[206]). Following treatment with LPS, HSCs showed a strong pro-inflammatory phenotype with an up regulation of pro-inflammatory mediators, and a fibrogenic phenotype with enhanced collagen synthesis, mediated by transforming growth factor-ß1 (TGF-ß1) (Zhao et al., 2014[206]). CAPE significantly and dose-dependently reduced LPS-induced nitrite production, as well as the transcription and protein synthesis of monocyte chemoattractant protein-1 (MCP-1), interleukin-6 (IL-6) and inducible nitric oxide synthase (iNOS), as determined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR), western blotting and enzyme-linked immunosorbent assays (ELISA) (Zhao et al., 2014[206]). CAPE further reduced the TGF-ß1-induced transcription and translation (protein synthesis) of the gene coding for collagen type I α1 (col1A1), in LPS-stimulated HSCs (Zhao et al., 2014[206]). Following LPS stimulation, the phosphorylation of the nuclear factor-κB (NF-κB) inhibitor IκBα and consequently, the nuclear translocation of NF-κB, were markedly increased in the HSCs, and these changes were reversed by pre-treatment with CAPE (Zhao et al., 2014[206]). Zhao et al. (2014[206]) concluded that CAPE attenuates the pro-inflammatory phenotype of LPS-stimulated HSCs, as well as the LPS-induced sensitization of HSCs to fibrogenic cytokines by inhibiting NF-κB signaling. These results provide new insight into the treatment of hepatic fibrosis through regulation of the TLR4 signaling pathway (Zhao et al., 2014[206]). Thus, HSCs play an important role both in endotoxin-induced acute hepatocyte injury, with TNF-α and endothelin-1 as important mediators of these effects (Stewart et al., 2014[167]). Ethanol Acetaldehyde and acetate are two major metabolites of ethanol. Ethanol can increase production of acetate via inhibition of the tricarboxylic acid cycle. In turn, acetate is a substrate for fatty acid synthesis (Sato et al., 2014[151]). Acetaldehyde and its metabolites might lead to the formation of reactive oxygen species, which increase oxidative stress and, ultimately, induce liver injury (Tarantino et al., 2014[175]). Ye et al. (2013[203]) investigated the role of Cytochrome P4502E1 in sensitizing Kupffer cells to LPS-mediated inflammation after ethanol induction. As reported by Ye et al. (2013[203]), in cultured Kupffer cell, using chlormethiazole as inhibitor, ethanol-induced CYP2E1 overexpression was proved to contribute to the sensitization of Kupffer cells to LPS stimuli, with amplification of ROS production and activation of NF-κB, resulting in increased TNF-α production. Alkhouri et al. (2014[7]) showed that exhaled breath analysis is a promising non invasive method to detect fatty liver in children. Therefore, isoprene, acetone, trimethylamine, acetaldehyde, and pentane are novel biomarkers that may help to gain insight into pathophysiological processes leading to the development of NAFLD (Alkhouri et al., 2014[7]). Treating these animals with probiotics to modify the gut microbiota improved NAFLD histology and decreased serum levels of liver enzymes (Penas-Steinhardt et al., 2012[122]). Zhu et al. (2013[209]) showed that the increased abundance of alcohol-producing bacteria in NASH microbiomes, elevated blood-ethanol concentration in NASH patients, and the well-established role of alcohol metabolism in oxidative stress and, consequently, liver inflammation suggest a role for alcohol-producing microbiota in the pathogenesis of NASH. Ethanol is partly responsible for the physiological and morphological modifications in the intestinal barrier associated with SIBO, and thus enhances the passage of endotoxins from the gut lumen into the portal blood (Cariello et al., 2010[26]). Nair et al. (2001[114]) suggested that higher breath ethanol concentrations are observed in obese subjects than in leaner ones (Nair et al., 2001[114]). It is possible that intestinally derived ethanol may contribute to the pathogenesis of NASH. Probiotics and prebiotics In vitro studies, as reported by Druart et al. (2014[46]), have suggested that isolated gut bacteria are able to metabolize PUFA into CLA (conjugated linoleic acids) and CLnA (conjugated linolenic acids). However, the bioavailability of fatty acid metabolites produced in vivo by the gut microbes remains to be studied. Druart et al. (2014[46]) concluded that the accumulation of the main metabolites (CLA cis-9,trans-11-18:2 and CLnA cis-9, trans-11, cis-15-18:3) in the caecal tissue was not associated with their increase in the plasma, therefore suggesting that, if endogenously produced CLA and CLnA have any biological role in host metabolism regulation, their effect would be confined at the intestinal level, where the microbiota is abundant (Druart et al., 2014[46]). The effects of administering prebiotics have illustrated the ability of the gut microbiota to affect host metabolism by both reducing energy intake and protecting the host from weight gain; the latter effect might be mediated by altered release of gut peptides involved in appetite and weight control (Pyra et al., 2012[127]; Koleva et al., 2012[94]; Everard et al., 2013[50]; Bomhof et al., 2014[21]; Dewulf et al., 2013[42]; Closa-Monasterolo et al., 2013[37]). Rauch and Lynch (2012[135]) suggested that modulating microbial exposure through probiotic supplementation represents a long-held strategy towards ameliorating disease via intestinal microbial community restructuring. Therefore, this field has experienced somewhat of resurgence over the past few years, primarily due to the exponential increase in human microbiome studies and a growing appreciation of our dependence on resident microbiota to modulate human health (Rauch and Lynch, 2012[135]). Wang et al. (2013[196]) reported that the therapeutic effects of probiotic treatment in alcoholic liver disease have been studied in both patients and experimental animal models. Although the precise mechanisms of the pathogenesis of alcoholic liver disease are not fully understood, gut-derived endotoxin has been postulated to play a crucial role in hepatic inflammation (Wang et al., 2013[196]). Previous studies have demonstrated that probiotic therapy reduces circulating endotoxin derived from intestinal gram-negative bacteria in alcoholic liver disease. Wang et al. (2013[196]) concluded that probiotic Lactobacillus rhamnosus GG (LGG) treatment reduced alcohol-induced hepatic inflammation by attenuation of TNFα production via inhibition of TLR4- and TLR5-mediated endotoxin activation (Wang et al., 2013[196]). Another, early low volume oral synbiotic/prebiotic supplemented enteral stimulation of the gut seems to be a potentially valuable complement to the routine treatment protocol of severe acute pancreatitis, as reported by Plaudis et al. (Plaudis et al., 2012[125]). Therefore, the ethanol-induced pathogenic changes in the microbiome and the liver were prevented by LGG supplementation (Bull-Otterson et al., 2013[24]). Overall, significant alterations in the gut microbiome over time occur in response to chronic alcohol exposure and correspond to increases in intestinal barrier dysfunction and development of alcoholic liver disease (Bull-Otterson et al., 2013[24]). Furthermore, the altered bacterial communities of the gut may serve as significant therapeutic target for the prevention/treatment of chronic alcohol intake induced intestinal barrier dysfunction and liver disease (Bull-Otterson et al., 2013[24]). Dewulf et al showed that inulin-type fructans, which promote gut fermentation, paradoxically counteract GPR43 (a G protein-coupled receptor, potential link between gut fermentation processes and white adipose tissue development) overexpression induced in the adipose tissue by an high-fat diet, a phenomenon that correlates with a beneficial effect on adiposity and with potential decrease in PPARγ-activated processes (Dewulf et al., 2011[43]) Probiotics alter the intestinal microbiota with non-urease-producing organisms that reduce production of ammonia (Lunia et al., 2013[104]). In a prospective, randomized controlled trial conducted by Lunia et al., probiotics were found to be effective in preventing hepatic encephalopathy in patients with cirrhosis (Lunia et al., 2013[104]). Lactulose promotes equol production and changes the microbial community during in vitro fermentation of daidzein by fecal inocula of sows (Zheng et al., 2014[207]). Equol has higher biological effects than other isoflavones. However, only about 30-50 % of humans possess a microbiota capable of producing equol from dietary daidzein. In recent years, interest has grown in dietary applications to improve equol production in human and other animals. Zheng et al. (2014[207]) showed that lactulose was used as a potential equol-promoting prebiotic in vitro. The effect of lactulose on transformation of daidzein into equol by sows' fecal microbiota was investigated (Zheng et al., 2014[207]). Results showed that lactulose treatment improved bacteria growth parameters, changing the kinetics of fermentation in vitro. Lactulose significantly increased total gas production (Zheng et al., 2014[207]). Furthermore, lactulose altered the microflora composition, increased equol production associated with a reduction in the population of methanogen and increased the sulfate-reducing bacteria population during 24 h of incubation. Zheng et al. (2014[207]) reported for the first time that in a certain condition (sealing or high pressure), via a dihydrodaidzein pathway equol might be able to reform to daidzein by further metabolism using lactulose as a substrate. Zheng et al. (2014[207]), in this study, proposed that "hydrogen-producing prebiotic" might be a novel way to promote equol production in vivo or in vitro (Zheng et al., 2014[207]). Finally, experimental models, as reported by Imajo et al. (2014[83]), have highlighted several mechanisms connecting microbiota to the development of liver dysfunction in NASH such as increased energy harvesting from the diet, small intestine bacterial overgrowth, modulation of the intestinal barrier by glucagon-like peptide-2 secretions, activation of innate immunity through the lipopolysaccharide-CD14 axis caused by obesity-induced leptin, periodontitis, and sterile inflammation. The manipulation of microbiota through probiotics, prebiotics, antibiotics, and periodontitis treatment yields encouraging results for the treatment of obesity, diabetes, and NASH, but data in humans is scarce (Imajo et al., 2014[83]). Metabolic effects Tomaro-Duchesneau et al. (2014[185]) indicated that administration of the ferulic acid (a phenolic acid found in foods normally consumed by humans that has demonstrated antioxidant activity, cholesterol-lowering capabilities, and anti-tumorigenic properties) producing L. fermentum NCIMB 5221 has the potential to reduce insulin resistance, hyperinsulinemia, hypercholesterolemia, and other markers involved in the pathogenesis of metabolic syndrome. Certain probiotics, including Lactobacillus and Bifidobacterium spp., have the capacity to synthesize bile salt hydrolase (Ruiz et al., 2013[146]), a key enzyme in the deconjugation of bile acids. Deconjugated bile acids are less effective in micelle formation and the emulsification of ingested lipids than conjugated bile acids and, therefore, reduce the efficiency of fat absorption (Cirin et al., 2011[35]; Yokota et al., 2012[204]; Hagey and Krasowski, 2013[71]; Cherrington et al., 2013[31]; Chen et al., 2013[30]). Through cholesterol-lowering effects, Lactobacillus and Bifidobacterium spp. can ameliorate dyslipidemia (Banjoko et al., 2012[14]; Wang et al., 2013[195]). In obese and/or dyslipidemic patients, administration of the LAB probiotic mixture ameliorated the levels of total cholesterol and LDL-cholesterol (Jones et al., 2013[85]; Rai et al., 2013[131]; Tuohy et al., 2014[188]). The effects Lactobacillus reuteri GMNL-263 (Lr263), a new probiotic strain developed by Hsieh’s laboratory, on insulin resistance and the development of hepatic steatosis in high-fructose fed rats were explored (Hsieh et al., 2013[79]). The levels of serum glucose, insulin, leptin, C-peptide, glycated hemoglobin, GLP-1, liver injury markers, lipid profile in serum and liver were significantly increased in high-fructose-fed rats (Hsieh et al., 2013[79]). However, after Lr263 administration, the elevation of these parameters was significantly suppressed (Hsieh et al., 2013[79]). Therefore, the Hsieh’s study provided evidences clarifying the effectiveness of Lr263 on reducing insulin resistance as well as hepatic steatosis formation in high-fructose-fed rats and suggested that Lr263 may be a promising therapeutic agent in treating type 2 diabetes (Hsieh et al., 2013[79]). Experimental evidence revealed that obesity-associated NAFLD is linked to changes in intestinal permeability and translocation of bacterial products to the liver (Ritze et al., 2014[139]). Actually, no reliable therapy is available except for weight reduction. Ritze et al. (2014[139]) examined the possible effect of the probiotic bacterial strain Lactobacillus rhamnosus GG (LGG) as protective agent against experimental NAFLD in a mouse model. LGG increased beneficial bacteria in the distal small intestine. Moreover, LGG reduced duodenal IƙB protein levels and restored the duodenal tight junction protein concentration (Ritze et al., 2014[139]). Ritze et al. showed for the first time that LGG protects mice from NAFLD induced by a high-fructose diet. The underlying mechanisms of protection likely involve an increase of beneficial bacteria, restoration of gut barrier function and subsequent attenuation of liver inflammation and steatosis (Ritze et al., 2014[139]). Rosberg-Cody et al. (2011[144]) demonstrated that a single gene (encoding linoleic acid isomerase) expressed in an intestinal microbe can influence the fatty acid composition of host fat. Anti-inflammatory effects Probiotics have several anti-inflam-matory effects (Table 1(Tab. 1)) that could contribute to clinical benefit in NAFLD (Ritze et al., 2014[139]): competition with and displacement of pathogenic strains in SIBO, particularly those with limited adherence ability in vitro (Abedi et al., 2013[1]); alteration of inflammatory pathways produced by intestinal bacterial overgrowth via alteration of cytokine signalling (Audy et al., 2012[12]); amelioration of intestinal barrier function through modulation of cytoskeletal and tight-junction proteins (Miyauchi et al., 2013[109]; Noda et al., 2013[115]); enhancement of the integrity of the intestinal epithelium by providing essential nutrients, especially in the form of medium-chain fatty acids that inhibit apoptosis (Wen et al., 2012[198]); direct inhibition of the production of pro-inflam-matory mediators, such as TNF and induction of anti-inflammatory responses in intestinal-epithelial-cell–leukocyte co-cultures (Trapecar et al., 2014[186]); and stimulation of IgA release (Ashraf and Shah, 2014[9]). In conventional culture there was no Escherichia Coli bacterial translocation in control animals (Eizaguirre et al., 2011[48]). Polymerase chain reaction detected Escherichia Coli bacterial translocation showing higher sensitivity (Eizaguirre et al., 2011[48]). Administration of Lactobacillus johnsonii La1, without addition to antioxidants, not reduced bacterial translocation and not attenuated endotoxemia in a rat model of cirrhosis (Soriano et al., 2012[162]). Furthermore, mouse models of acute hepatitis have too showed reductions in the incidence of bacterial translocation and hepatic injury after the administration of several strains of Lactobacillus and Bifidobacterium (Osman et al., 2007[117]; Ahrne and Hagslat, 2011[4]). Future directions The importance of gut–liver interactions is also accentuated by the role of the intestinal microbiota in NAFLD. The gut microbiota and SIBO, in particular, are now considered to be crucial factors in the pathogenesis of NAFLD. Actually, evidence has been widely derived from a variety of animal models; the definition and diagnosis of SIBO in man continues to present a substantial challenge. Several bacterial components and products have been implicated in the pathogenesis of NAFLD and NASH; in animal models the role of lipopolysaccharide, through its capacity to regulate metabolic processes and activate proinflammatory cytokine production, has been particularly prominent. Since it is clear, from everything that has been described above, the possible important role of gut derived microbial factors in the development and/or progression of NAFLD, a logical proposition is that modifying the microbiota might have a beneficial effect on this pathological condition. Complications of liver disease could probably be reduced by altering the microbiota either qualitatively or quantitatively. For example, alteration of the gut microbiota by prebiotics or probiotics might be an important therapeutic strategy in the treatment of NAFLD. To understand the impact of gut microbes on human health and well-being it is crucial to assess their genetic potential (Qin et al., 2010[128]). The gene set, approximately 150 times larger than the human gene complement, contains an overwhelming majority of the prevalent (more frequent) microbial genes of the cohort and probably includes a large proportion of the prevalent human intestinal microbial genes (Qin et al., 2010[128]). Several issues remain to be determined: the exact prevalence of SIBO (defined using modern molecular techniques) in NAFLD and NASH, whether the translocation of bacterial products, such as LPs, across the gut wall is significant to these disorders in man and whether tailored interventions (with probiotics, prebiotics, antibiotics, or some combinations thereof) will exert meaningful benefits. Alcohol consumption increases the SIBO and intestinal permeability of endotoxin (Abhilash et al., 2014[2]). The endotoxin mediated inflammatory signaling plays a major role in alcoholic liver fibrosis (Abhilash et al., 2014[2]). The possible mechanism may be the inhibitory effect of acid ascorbic on SIBO, intestinal barrier defect and IKKß, which decreased the activation of NF-κB and synthesis of cytokines, as reported by Abhilash (Abhilash et al., 2014[2]). SIBO is also responsible for endotoxemia, systemic inflammation, and its consequences including obesity and NAFLD (Duseja and Chawla, 2014[47]). Relationship between gut microbiota and NAFLD is also dependent on altered choline and bile acid metabolism and endogenous alcohol production by gut bacteria (Duseja and Chawla, 2014[47]). Further evidence linking gut microbiota with obesity and NAFLD comes from studies showing usefulness of probiotics in animals and patients with NAFLD, as suggested by Duseja et al. (Duseja and Chawla, 2014[47]). Diet and nutritional status are among the most important, modifiable determinants of human health. The nutritional value of food is determined partially by a person’s gut microbial community (microbiota) and its component genes (microbiome). Separating the interactions between diet, the structure and operations of the gut microbiota, and nutrient and energy harvest is confounded by changes in human environmental exposures, microbial ecology and genotype. The human gut microbiota and microbial influences on lipid and glucose metabolism, satiety, and chronic low-grade inflammation are known to be involved in metabolic syndrome (Remely et al., 2014[137]). Fermentation end products, especially short chain fatty acids, are believed to engage the epigenetic regulation of inflammatory reactions via free fatty acid receptor and other short chain fatty acid receptors. Remely et al. (2014[137]) suggested that a different composition of gut microbiota in obesity and type 2 diabetes affect the epigenetic regulation of genes. Interactions between the microbiota and epigenetic regulation may involve not only short chain fatty acids binding to free fatty acid receptor. Therefore dietary interventions influencing microbial composition may be considered as an option in the engagement against metabolic syndrome (Remely et al., 2014[137]). We hypothesized that each step in the process to ultimately bring gut microbiota manipulation to the clinic, in particular to harness its potential for the prevention and treatment of dysmetabolic disorders, needs to be a small, careful and well-controlled one, to be taken in the setting of expert multidisciplinary collaborations (Karlsson et al., 2013[88]). The latter can only be answered by appropriately powered, randomized, controlled clinical trials. Further studies are required to investigate the human clinical potential of the probiotic formulation in affecting the markers and pathogenesis of metabolic syndrome (Tomaro-Duchesneau et al., 2014[185]), or associated morbidities, according to recent findings showing that modulation of gut microbiota by probiotics has beneficial effects on brain activity in stress conditions, displays anxiolytic-like activity and reduces apoptosis in the lymbic system in animal models of depression (Ait-Belgnaoui et al., 2013[5]). This long-distance effect of probiotics opens up a new field of research mainly at the light of the possible impact on the unbalance of apoptosis-antiapoptosis process , key mechanism inducing NAFLD/NASH (Tarantino et al., 2011[182]). The main approach to obesity is to unravel the mechanisms involved in nutrient absorption and then the role of gut flora. In conditions of over-nutrition, cells must cope with a multitude of extracellular signals generated by changes in nutrient load, hormonal milieu, adverse cytokine/adipokine profile, and apoptosis/anti-apoptosis processes. To date studies have demonstrate that among all nutrients, lipids and carbohydrates play a major regulatory role in the gene transcription of glycolytic and lipogenic enzymes (epigenetics), insulin, and adipokines. These nutrients mainly exert their effects through the gene expression of sterol responsive binding protein 1 and 2 (SREBP) and the mammalian target of rapamycin (mTOR) (Tarantino and Capone, 2013[173]). 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==== Front J Cell Mol MedJ. Cell. Mol. MedjcmmJournal of Cellular and Molecular Medicine1582-18381582-4934John Wiley & Sons, Ltd Chichester, UK 1868191010.1111/j.1582-4934.2008.00451.xThis Article Has Been RetractedIn vitro analysis of integrin expression in stem cells from bone marrow and cord blood during chondrogenic differentiation Goessler Ulrich Reinhart aBugert Peter bBieback Karen bStern-Straeter Jens aBran Gregor aSadick Haneen aHörmann Karl aRiedel Frank aa Department of Otolaryngology, Head and Neck Surgery, University Hospital Mannheim, University of HeidelbergMannheim, Germanyb Institute of Transfusion Medicine and Immunology, Red Cross Blood Service of Baden-Württemberg/ Hessen gGmbH, Ruprecht Karls-University Heidelberg, Faculty of Clinical Medicine MannheimGermany Correspondence to: Dr. Ulrich GOESSLER, Department of Otolaryngology, Head and Neck Surgery University Hospital Mannheim, D-68135 Mannheim, Germany. Tel.: +49 621 383 1600 Fax: +40 621 383 1972 E-Mail: [email protected] 2009 04 8 2008 13 6 1175 1184 30 9 2007 26 7 2008 © 2009 The Authors Journal compilation © 2009 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd2009The use of adult mesenchymal stem cells (MSC) in cartilage tissue engineering has been implemented in the field of regenerative medicine and offers new perspectives in the generation of transplants for reconstructive surgery. The extracellular matrix (ECM) plays a key role in modulating function and phenotype of the embedded cells and contains the integrins as adhesion receptors mediating cell–cell and cell–matrix interactions. In our study, characteristic changes in integrin expression during the course of chondrogenic differentiation of MSC from bone marrow and foetal cord blood were compared. MSC were isolated from bone marrow biopsies and cord blood. During cell culture, chondrogenic differentiation was performed. The expression of integrins and their signalling components were analysed with microarray and immunohistochemistry in freshly isolated MSC and after chondrogenic differentiation. The fibronectin-receptor (integrin a5b1) was expressed by undifferentiated MSC, expression rose during chondrogenic differentiation in both types of MSC. The components of the vitronectin/osteopontin-receptors (avb5) were not expressed by freshly isolated MSC, expression rose with ongoing differentiation. Receptors for collagens (a1b1, a2b1, a3b1) were weakly expressed by undifferentiated MSC and were activated during differentiation. As intracellular signalling components integrin linked kinase (ILK) and CD47 showed increasing expression with ongoing differentiation. For all integrins, no significant differences could be found in the two types of MSC. Integrin-mediated signalling seems to play an important role in the generation and maintenance of the chondrocytic phenotype during chondrogenic differentiation. Especially the receptors for fibronectin, vitronectin, osteopontin and collagens might be involved in the generation of the ECM. Intracellularly, their signals might be transduced by ILK and CD47. To fully harness the potential of these cells, future studies should be directed to ascertain their cellular and molecular characteristics for optimal identification, isolation and expansion. integrincartilagetissue engineeringdifferentiationextracellular matrixmesenchymal stem cellschondrogenic differentiation ==== Body Introduction Improved healthcare has resulted in dramatic demographic changes in developed countries, causing an increase in the prevalence of diseases associated with aging. Stem cell research and regenerative medicine offer unique opportunities for developing new therapeutic approaches to prevent and treat these debilitating and life-threatening diseases, and new ways to explore fundamental questions of biology. Still, each year, millions suffer from organ failure or tissue loss due to injury, disease or congenital malformation [1, 2]. With a progressively aging population, there is an increasing demand for therapies to regenerate or replace musculoskeletal tissues. More than three million musculoskeletal procedures are performed annually. The existing shortage of donor tissue and organs available for transplantation has driven a multidisciplinary effort to develop therapeutic solutions. The emerging field of tissue engineering promises to deliver improvements in the technologies and therapies for musculoskeletal disorders through the development of biological substitutes for tissue replacement. The creation of functional tissue addresses very complex biological problems, comprising a wide range of engineering, science and clinical disciplines. The next generation of engineered musculoskeletal tissues will be more complex and structurally organized to better mimic normal tissue structure and function. Tissue engineering as an interdisciplinary approach utilizes specific combinations of cells, scaffolds and bioactive factors to create, influence and maintain cellular phenotype and function [3]. Cartilage as a unique avascular, aneural and alymphatic loadbearing live tissue is unique in that the extracellular matrix is composed of a complex combination of type II collagen fibrils which are specifically arranged and have bonded to them by very large water-retaining molecules called aggrecan molecules [4]. The promise of tissue engineering is perhaps most relevant to chondrogenic defects because cartilage has little self-healing potential. Recently, the successful isolation of human stem cells from bone marrow (BM), periosteum and other newer sources was established by different groups [5–8]. These cells are highly proliferate and are capable of differentiating into different types of tissue such as bone, cartilage, tendon, muscle or fat. Human mes-enchymal stem cells are characterized by a specific pattern of cell surface markers, growth factors, cytokine receptors, integrins and other adhesion molecules [9, 10]. Although BM has been the main source for the isolation of multipotent MSCs and BM-MSCs are well characterized and safe in handling, the harvest of BM is a highly invasive procedure and the number, differentiation potential, and maximal life span of MSCs from BM decline with increasing age [11–13]. Therefore, alternative sources from which to isolate MSCs are subject to intensive investigation. As one alternative source umbilical cord blood (UCB) has proven to offer excellent potential for clinical scale allogenic transplantation. UCB can be obtained by a less invasive method, without harm for the mother or the infant [14, 15]. Essential preclinical studies proved a higher percentage of CD34+CD38− cells in UCB compared to BM, suggesting that more primitive progenitors may be abundant in neonatal blood [16]. The same might apply for the presence of MSCs or progenitor cells. However, previous attempts to isolate MSCs from UCB either failed [17–19] or have demonstrated a low frequency of mesenchymal progenitors [20, 21]. Controversy still exists whether full-term UCB can serve as a fully acknowledged source for isolating multipotent MSCs: although some groups did not succeed in isolating MSCs [18, 19], we and other groups succeeded in isolating MSCs from full-term UCB [20, 22, 23]. As cellular function and phenotype are influenced by intrinsic and extrinsic stimuli, the cell–cell and cell–matrix interactions are of special interest in understanding factors crucial to generation of a distinct cellular phenotype. The integrin family of cell surface receptors appears to play a major role in the mediation of the cell–ECM interactions associated with structural and functional changes in surrounding tissues [24–27]. The integrins are heterodimeric glycoproteins that are composed of an α- and a β-subunit, each of which has extracellular and cytoplasmic domains. The extracellular domains bind to a number of ECM-proteins, including collagen types II and VI, fibronectin and matrix Gla-protein. Several recent studies have provided evidence that chondrocytes express integrins [28–33]. Salter et al. used immunohistochemical staining in normal adult articular cartilage, and noted that integrin α5β1 was the most prominently expressed chondrocyte integrin [32]. A more recent study demonstrated that the chondrocyte expression of α1β1, α5β1 and αvβ5 were accompanied by weak expression of integrin α3β1 and αvβ3 [31]. Other integrins are known to have distinct functions in binding components of the ECM (Table 1). Table 1 Different integrins and their functions Integrin Receptor for Integrin α5β1 Fibronectin Integrin α 4 β 1 VCAM, Fibronectin Integrin α 6 β 1 and α 7 β 1 Laminin Integrin α 1 β 1 Collagen, Laminin, Tenascin Integrin α 2 β 1 Laminin, Collagen Integrin α v β 5 Osteopontin Integrin-mediated signalling is involved in a variety of cellular processes such as differentiation, adhesion and migration. Hannigan et al. found that integrin-linked kinase (ILK) coimmuno-precipitated with β1 integrin from cell lysates, and that overexpression of ILK disrupted cell architecture and inhibited adhesion to integrin substrates, suggesting that ILK regulates integrin-mediated signal transduction [34]. In addition to ILK, integrin cytoplasmic domain-associated protein 1 (ICAP1) interacts with the cytoplasmic domain of β1 integrin [35]. CD47 or integrin-associated protein (IAP) is a membrane protein that is involved in the increase in intracellular calcium concentration that occurs upon cell adhesion to extracellular matrix [36]. As the stem cell is responsible for modulating its environment and the chondrocyte phenotype is influenced by the diverse components of the extracellular matrix, the investigation of the molecular basis of distinct changes during developmental processes – for the generation of cartilage transplants especially the process of chondrogenic differentiation – might broaden the understanding of impediments in the field of tissue engineering. As BM-MSCs are best characterized, we asked whether MSCs derived from other sources share characteristic expression patterns of BM-MSCs. The aim of our study was to analyse MSCs isolated from BM and UCB under identical in vitro conditions and during chondrogenic differentiation with respect to integrin expression. Materials and methods Collection and isolation of MSC from BM BM was obtained from the femoral shaft of patients undergoing total hip replacement at the orthopaedic department of the University Hospital Mannheim. Cells were aspirated into a 5 ml syringe containing CPD anticoagulant. In total, six specimens from female patients were obtained, with the donor age ranging from 68 to 84 years. To isolate mononuclear cells (MNC), the BM aspirates were diluted 1:5 with PBS/2mM EDTA (Nexell, Baxter, Unterschlei?heim, Germany, and Merck, Darmstadt, Germany) and carefully loaded onto Ficoll-Hypaque solution (Amersham, Freiburg, Germany). After density gradient centrifu-gation at 435 × g for 30 min. at room temperature, MNC were removed from the interphase and washed two to three times with PBS/EDTA. Cell counts were performed using an automated cell analyzer (Cell-Dyn 3200, Abbott, Wiesbaden, Germany). BM-derived MNC were set in culture at a density of 1 × 105/cm2 into 75 cm2 tissue culture flasks (Nunc, Wiesbaden, Germany, http://www.nunc.de) in MSCGM medium (MSCGM BulletKit™, Cambrex, St. Katharinen, Germany). After overnight incubation at 37°C in humidified atmosphere containing 5% CO2, non-adherent cells were removed and fresh medium was added to the flasks. Cultures were maintained and remaining non-adherent cells were removed by complete exchange of culture medium every 3–4 days. The flasks were screened continuously to get hold of developing colonies of adherent cells. Fibroblastoid cells were recovered between day 7 and 10 after initial plating by using 0.04% Trypsin/0.03% EDTA (PromoCell, Heidelberg, Germany). Recovered cells were replated at a density of 4000–5000 cells/cm2 as passage 1 (P1) cells and thereafter. Collection of UCB UCB units (n= 59) were collected as previously described from the unborn placenta of full-term deliveries in a multiple bag system containing 17 ml of citrate phosphate dextrose buffer (Cord Blood Collection System; Eltest, Bonn, Germany) [22, 37] and processed within 24 hrs of collection. The collection was performed in accordance with the ethical standards of the local ethical committee. Isolation and culture of MNC from UCB The isolation of MSCs was performed as described for BM with a few exceptions. Prior to the isolation of MNC, the anticoagulated cord blood was diluted 1:1 with 2 mM EDTA-PBS. The MNC fraction was initially seeded at a density of 1 × 10 MNC/cm into foetal calf serum (FCS)-precoated culture plates (FCS batches S0113/1038E and S0113/892E; Biochrom, Berlin, Germany, http://www.biochrom.de) (Falcon, Becton Dickinson and Company, Franklin Lakes, NJ, USA, http://www.bd.com) [22]. Nonadherent cells were removed 12–18 hrs after initial plating. The same culture conditions and media were applied as described for BM-FACs. Adherent fibroblastoid cells only appeared as CFU-F and were harvested at subconfluence using Trypsin (PromoCell). Cells at the second passage and thereafter were replated at a mean density of 3.5 ? 4.8 × 103/cm2. Chondrogenic differentiation To promote chondrogenic differentiation, 2.5 × 105 cells were gently cen-trifuged (150 ×g, 5 min.) in a 15 ml polypropylene tube (Greiner) to form a pellet according to the protocol of Mackay et al.[38]. Without disturbing the pellet, the cells were cultured for 4 weeks in complete chondrogenic differentiation medium (Cambrex) including 10 ng/ml TGFβ3 (Strathmann Biotec AG, Hamburg, Germany) by feeding twice a week. After the culture period, cryosections were analysed by Safranin O staining. The sections were fixed with ice-cold acetone (Sigma) and stained with 0.1% aqueous Safranin O solution (Sigma). Cell nuclei were counterstained with Weigert’s iron haematoxylin (Sigma). For the RNA analysis we harvested and lysed the aggregates in RLT buffer (Qiagen, Hilden, Germany). The lysis was aggravated by freezing the pellet repeatedly in liquid nitrogen. RNA extraction and microarray hybridization Extraction of RNA was performed using RNA Mini Kit (Qiagen) according to the manufacturers’ protocol and as published before [39]. The RNA concentration was estimated from the absorbance at 260 nm. Approximately 1μg total RNA was used in each microarray experiment and for amplification and labelling of mRNA the SMART technique (SMART Fluorescent Probe Amplification Kit; BD Clontech, Heidelberg, Germany) was applied according to the manufacturers’ protocol. RNA samples from day 1 were labelled with Cy3 and day 6 or day 21 samples were labelled with Cy5 (Cy™3- and Cy™5-monoreactive dye; Amersham Pharmacia Biotech, Freiburg, Germany). Corresponding Cy3- and Cy5-labelled samples were mixed, vacuum dried and resuspended in 25 μl microarray hybridization buffer (MWG-Biotech; Ebersberg, Germany). Prior to hybridization the samples were heat denaturated at 95°C for 5 min. The human 10K (MWG-Biotech) oligo microarray systems on glass slides were used for mRNA profiling. Hybridization of Cy3/Cy5-cDNA was performed using cover slips and a hybridization chamber for 16 hrs at 42°C in a water bath. After stringent washing of the glass slides according to the manufacturers specifications the hybridization signals of the Cy3 and the Cy5 dyes were measured using a microarray laser scanner (GMS418; Affymetrix, MWG-Biotech). Microarray data analysis and statistics The ArrayVision (Imaging Research, Inc., St. Catharines, ON, Canada) software has been used for evaluation and calculation of signal intensities from the raw data images in 16-bit tagged-image-file (TIF) format as described previously [39]. In brief, for evaluation of hybridization results we defined a negative (<3.000), a grey area (3.000–4.999) and a positive range (≥5.000) of hybridization signal intensities. Signal-to-background (S/B) values were calculated by dividing the signal intensity for each spot with the background signal intensities of the hybridized glass slide. Computer-assisted evaluation of the raw data provides the mean signal intensity and the signal to background ration for each individual gene spot. For statistical evaluation the mean signal intensity and standard deviation (S.D.) was calculated for each spot from the values obtained in the 10 individual experiments. Functional grouping of genes was performed on the basis of the database supplied by the array manufacturer. Immunohistochemistry Immunohistochemistry for integrin αv, integrin (β1, integrin (β5, CD47 and the integrin-linked kinase (ILK) was performed by using a streptavidin-biotin complex procedure. Endogenous peroxidase was blocked with 0.3% hydrogen peroxide for 30 min. Sections were washed with phosphate-buffered saline (PBS) and incubated with normal rabbit serum in PBS for 30 min. at room temperature to block non-specific antibody reaction. The sections were then incubated over night at 4°C with the primary antibody (all from Santa Cruz Biotechnologies, Heidelberg, Germany). The slides were washed in several changes of PBS. The sections were then incubated with a peroxidase-conjugated secondary antibody (DAKO, Hamburg, Germany). After being washed twice in PBS, sections were then treated with a streptavidin-biotin-peroxidase complex and peroxidase reaction was performed using Diaminobenzidine DAB (DAKO, Hamburg, Germany) as chromogen. The different antibodies were diluted to the desired concentrations in PBS. Controls were carried out by omitting the primary antibody. Light microscopically investigation was performed using a Zeiss Axiophot microscope. Results Microarray analysis In MSC from bone marrow (BM-MSC, Table 2, Fig. 1), for the components of the fibronectin-receptor (integrin α5/β1) a constant expression for integrin α5 (day0: 11790, day20: 9144, Ratio day20/day0: 0,78) could be found, integrin β1 was inactivated (day0: 32134, day20: 15557, Ratio day20/day0: 0,48). The components of the receptor for VCAM and Fibronectin (integrin α4/β1) were not expressed (integrin α4, day0: 811, day20: 2548, Ratio day20/day0: 3,19) and an inactivation of the gene for integrin β1 (day0: 32134, day20: 15557, Ratio day20/day0: 0,48). The components of the receptor for laminin (integrin α/β1 and integrin α7/β1) showed a constant expression of integrin α7 (day0: 2872, day20: 4826, Ratio day20/day0: 1,68) and an inactivation of integrin β1 (day0: 32134, day 20: 15557, Ratio day20/day0: 0,64). The components of the receptor for collagen, laminin and tenascin (integrin α1/β1) were inactivated (integrin α1: day0: 7345, day20: 4639, Ratio day20/day0: 0,63; integrin β1: day0: 32134, day20: 15557, Ratio day20/day0: 0,48). The components of the receptor for laminin and collagen (integrin α2/β1) revealed a constant expression of integrin α2 (day0: 23892, day20: 30795, Ratio day20/day0: 1,29) and an inactivation of integrin β1 (day0: 32134, day20: 15557, Ratio day20/day0: 0,48). For the receptor for osteo-pontin (integrin αv/β5), an inactivation of integrin αv (day0: 4044, day20: 3306, Ratio day20/day0: 0,82) and an activation of integrin β5 (day0: 2958, day20: 15601, Ratio day20/day0: 5,27). The components of the intracellular signalling cascade revealed a constant expression of ILK (day0: 1931, day20: 2625, Ratio day20/day0: 1,36) and of CD47 (day0: 22758, day20: 23360, Ratio day20/day0: 1,03). ICAP-1 was not expressed (day0: 1041, day20: 2147, Ratio day20/day0: 2,06). Table 2 Signal intensities of hybridization signals as measured using the microarray laser scanner and calculated by The ArrayVision software in MSC from bone marrow Receptor for Day 0 Day 20 S.D. Day 0 S.D. Day 20 Ratio d20/d0 Fibronectin Integrin a5 11790 9144 4269 4093 0,78 Integrin b1 32134 15557 3040 3698 0,48 VCAM, Fibronectin Integrin a4 811 2584 307 821 3,19 Integrin b1 32134 15557 3040 3698 0,48 Laminin Integrin a6 1206 773 241 132 0,64 Integrin a7 2872 4826 1412 2412 1,68 Integrin b1 32134 15557 3040 3698 0,48 Collagen, Laminin, Tenascin Integrin a1 7345 4639 6079 4269 0,63 Integrin b1 32134 15557 3040 3698 0,48 Laminin, Collagen Integrin a2 23892 30795 9674 3988 1,29 Integrin b1 32134 15557 3040 3698 0,48 Osteopontin Integrin av 4044 3306 1112 1036 0,82 Integrin b5 2958 15601 804 8541 5,27 Signalling cascade CD47 1931 2625 1224 1921 1,36 ILK 22758 23360 11452 11496 1,03 ICAP-1 1041 2147 117 594 2,06 Signals were measured on day 1 and after chondrogenic differentiation on day 20. Signals are shown with standard deviation and the ratio day 20/day 1. Figure 1 Expression levels of genes for different integrins and integrin-associated proteins in MSC by microarray hybridization analysis. Results from mesenchymal stem cells from bone marrow (BM-MSC) during chondrogenic differentiation for the given genes in undifferentiated MSCs (white bars) and chondrocytes differentiated from the MSCs (black bars). In MSC from cord blood (Table 3, Fig. 2), the components of the fibronectin-receptor (integrin α5/β1) showed an inactivation for integrin α5 (day0: 7391, day20: 3358, Ratio day20/ day0: 0,45), and constant expression for integrin β1 (day0: 3474, day20: 3193, Ratio day20/ day0: 0,92). The components of the receptor for VCAM and fibronectin (integrin α4/β1) were not expressed (integrin β4: day0: 1018, day20: 2584, Ratio day20/ day0: 2,54) and constantly expressed (integrin p1: day0: 3474, day20: 3193, Ratio day20/ day0: 0,92). The components of the Receptor for Laminin (integrin α6/β1 and integrin α7/β1) revealed no expression of integrin α7 (day0: 1518, day20: 1936, Ratio day20/day0: 1,28) and constant expression of integrin β1 (day0: 681, day20: 1104, Ratio day20/ day0: 1,62). Integrin a6 (day0: 681, day20: 1104, Ratio day20/day0: 1,62) was not expressed. As for the receptor for collagen, laminin and tenascin (integrin α1/β1), a strong activation of integrin α1 (day0: 845, day20: 6783, Ratio day20/day0: 8,0) and constant expression of integrin β1 (day0: 3474, day20: 3193, Ratio day20/ day0: 0,92). The receptor for laminin and collagen (integrin a2/p1) revealed constant expression of integrin α2 (day0: 23892, day20: 30795, Ratio day20/day0: 1,29) and integrin β1 (day0: 3474, day20: 3193, Ratio day20/ day0: 0,92). For the receptor for osteopontin (integrin αv/β5), a constant expression of integrin αv (day0: 4259, day20: 5257, Ratio day20/day0: 1,23) and an inactivation of integrin β5 (day0: 7684, day20: 4344, Ratio day20/day0: 0,57) was found. The components of the intracellular signalling cascade showed a constant expression of ILK (day0: 6147, day20: 5222, Ratio day20/day0: 0,85) and inactivation of CD47 (day0: 21171, day20: 11173, Ratio day20/day0: 0,53), ICAP-1 was not expressed (day0: 1616, day20: 661, Ratio day20/day0: 0,41). Table 3 Signal intensities of hybridization signals as measured using the microarray laser scanner and calculated by The ArrayVision software in MSC from cord blood Receptor for Day 0 Day 20 S.D. Day 0 S.D. Day 20 Ratio d20/d0 Fibronectin Integrin a5 7391 3358 5283 1542 0,45 Integrin b1 3474 3193 2845 1496 0,92 VCAM, Fibronectin Integrin a4 1018 2584 322 821 2,54 Integrin b1 3474 3193 2845 1496 0,92 Integrin a6 681 1104 373 397 1,62 Integrin a7 1518 1936 369 1233 1,28 Integrin b1 3474 3193 2845 1496 0,92 Collagen, Laminin, Tenascin Integrin a1 845 6783 442 2854 8,02 Integrin b1 3474 3193 2845 1496 0,92 Laminin, Collagen Integrin a2 5060 9458 2954 4956 1,87 Integrin b1 3474 3193 2845 1496 0,92 Integrin av 4259 5257 1788 1911 1,23 Integrin b5 7684 4344 4811 2937 0,57 Signalling cascade CD47 6147 5222 3134 2497 0,85 ILK 21171 11173 6070 887 0,53 ICAP-1 1616 661 315 104 0,41 Signals were measured on day 1 and after chondrogenic differentiation on day 20. Signals are shown with standard deviation and the ratio day 20/day 1. Figure 2 Expression levels of genes for different integrins and integrin-associated proteins in MSC by microarray hybridization analysis. Results from mesenchymal stem cells from cord blood (CB-MSC) during chondrogenic differentiation for the given genes in undifferentiated MSCs (white bars) and chondrocytes differentiated from the MSCs (black bars). Immunohistochemistry The analysis of integrin-expression on protein-level was analysed with monoclonal antibodys against integrin av, integrin β1, integrin β5, CD47 and the integrin-linked kinase (ILK). For all markers, during the whole process of chondrogenic differentiation, a constant expression for all markers could be found (Fig. 3, Table 4). Figure 3 Immunohistochemical staining against different integrins in MSC during chondrogenic differentiation. Day 1 (left) and day 20 (right) of cell culture. A Integrin β1 (day 1) in CB MSC; B Integrin β1 (day 20) in CB MSC; C ILK (day 1) in CB MSC; D ILK (day 20) in CB MSC. Table 4 Immunohistochemical detection of integrins and integrin-associated proteins in freshly isolated MSC (day 1) and during chondrogenic differentiation (days 1, 10, 20 and 30) A Antibody specific for Staining pattern* BM MSC Day 0 Day 1 Day 10 Day 20 Day 30 Integrin av +++ + ++ +++ ++ Integrin b5 ++ + +++ +++ ++ Integrin b1 ++ + ++ ++ + CD47 +++ ++ +++ +++ ++ ILK +++ +++ +++ ++ ++ B Antibody specific for Staining pattern* CB MSC Day 0 Day 1 Day 10 Day 20 Day 30 Integrin av ++ ++ +++ +++ +++ Integrin b5 +++ ++ ++ ++ +++ Integrin b1 ++ ++ +++ ++ ++ CD47 +++ +++ +++ +++ ++ ILK +++ +++ +++ +++ +++ * Amount of cells stained by the monoclonal antibodies is symbolized by ++++ (80—100%), +++ (60–70%), +h—–h ++ (50–60%), ++ (40–50%), h—–h+ (30–40%), + (20–30%), ± (<20%) and – for no staining. Discussion The field of regenerative medicine encompasses various areas of technology, such as tissue engineering, stem cells and cloning. Tissue engineering, one of the major components of regenerative medicine, follows the principles of cell transplantation, materials science and engineering towards the development of biological substitutes that can restore and maintain normal function. BM-derived stem cells have been studied for decades, are well characterized and safe in even clinical settings. For clinical applications of stem cell transplantation therapy, direct manipulation of cells and their interactions would be desirable. Hitherto, establishing distinct protocols for precisely inducing and maintaining cellular differentiation with a defined phenotype and function has been extremely challenging. In addition, establishing knowledge about cell–cell and cell–matrix interactions would be desirable and the integrins as a family of adhesion receptors mediating these stimuli are a promising target for research. As mentioned above, the harvest of BM is a highly invasive procedure and the number, differentiation potential and maximal life span of MSCs from BM decline with increasing age [11, 12]. Therefore, alternative sources from which to isolate MSCs are subject to intensive investigation. UCB is an alternative source that can be obtained by a less invasive method and in larger quantities than BM. Expression of integrins was analysed in MSC from BM and cord blood during chondrogenic differentiation. The components of the fibronectin-receptor (integrin α5/β1) showed in both types of MSC with ongoing differentiation a diminished expression of integrin α5 in both MSC-types and inactivation of integrin β1 in BM-MSC and constant expression in CB-MSC, on protein-level, integrin β1 showed constant expression. With RT-PCR analysis it could be shown in previous studies that freshly isolated MSC express Collagen 2 and 10 [40]. These previous results suit the high expression of integrin β1α5 in undifferentiated MSC, as the interaction of MSC and components of the ECM (e.g. Collagen 2) viathis receptor is certain. So integrin (β1α5 might exert an influence on cellular phenotype in undifferentiated MSC, with ongoing differentiation this receptor seems to become less important, in both types of MSCs at the same time. The receptor for VCAM and fibronectin (integrin a4/(31) was not expressed by MSC from BM and CB in terms of integrin a4 and was inactivated during chondrogenic differentiation as for integrin (31, respectively. This receptor does not seem to be involved in the signalling during chondrogenic differentiation. The components of the receptor for laminin (integrin α6/β1 und α7β1) showed no expression in the two types of MSC of integrin α6 and α7 as well as a diminished expression of integrin β1 during chondrogenic differentiation. So this receptor does not seem to play an important role during chondrogenic diferentiation. The receptor for collagen, laminin and tenascin (integrin α1/β1) revealed adverse expression patterns in the two stem cell types: On RNA-level, inactivation of integrin α1 and β1 in BM MSC and activation of a1 with constant expression of integrin β1 in CB-MSC could be observed. However, on protein level, these diverging results could not be obtained, as integrin β1 showed constant expression in both types of MSC. These results do not allow a specific conclusion about the role of this receptor during chondrogenic differentiation. The osteopontin receptor (integrin αv/β5) showed constant expression for integrin av in both types of MSC. Integrin β5 was activated in BM-MSC and was inactivated in CB-MSC. Immunohistochemichal staining revealed constant expression of integrin β5 in both MSC-types. It has been established that integrin β5 plays a role in binding Vitronectin spielen könte [41]. In addition, it has been concluded that the osteopontin receptor might be involved in processes of cellular migration and proliferation, especially in smooth muscle cells during vascular trauma [42]. Furthermore, this receptor might assist in the cellular differentiation in vitro[43]. The expression of this receptor during chondrogenic differentiation might reflect the influence of this receptor during the generation of a distinct ECM. The adhesion to surrounding ECM molecules might guide the process of differentiation. The analysis of the components of the receptors for laminin and collagen (integrin α2/β1) resulted in an inactivation of integrin α2 in BM-MSC, in CB-MSC a rising expression during chondrogenic differentiation. As mentioned above, integrin (31 showed diminishing expression during chondrogenic differentiation in BM-MSC and constant expression in CB-MSC. For this receptor, a specific role during chondrogenic differentiation may not be established. A possible role might be in adhering to collagens to facilitate the synthesis of ECM. This process cannot be analysed in monolayer culture. In summary, different expression patterns were found only on RNA-level for 2 receptors if one compares the expression patterns in the two stem cell types. So one might conclude that there are no significant differences in chondrogenic differentiation capacity and the expression of integrins. Establishing new sources for MSCs might have a high impact on clinical usage these cells. Exploitation might be related to the abundance and expansion capacity of MSCs. Based on our results, both BM and CB are reliable sources for isolating and expanding MSCs in autologous settings. Advantages of CB-MSC include the less invasive harvesting. For the past decades, BM has been deployed as the main source for clinical application of MSCs, such as the treatment of osteoge-nesis imperfecta, graft versus host disease or acute myocardial infarction [44–46]. As an age-dependent decrease in number, frequency and differentiation capacity of BM-MSCs has been described, they could be clinically inefficient when derived from elderly patients. Taking into account all these factors, CB-MSC might provide a solid starting basis in reference to abundance, easy harvest and high MSC frequency. Conclusion In the present study, we analysed expression patterns of integrins and integrin-related signalling-proteins. One of the candidates for signal-transmission is the fibronectin receptor which might play a role in freshly isolated cells. Other receptors, e.g. for collagen, laminin and tenascin do not seem to be involved in signal transduction. The receptor for osteopontin seems to play a role during chondrogenic differentiation, in addition the receptor for laminin and collagen might assist the beginning chondrogenic differentiation. Intracellularly, ILK and CD47, but not ICAP1, might be involved in transduction of the integrin-dependent signals. Integrin-mediated signalling seems to play an important role in the generation and maintenance of the chondrocytic phenotype during chondrogenic differentiation. 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15498362	18681910	PMC4496112	NO-CC CODE	2021-01-05 03:54:10	yes	J Cell Mol Med. 2009 Jun 4; 13(6):1175-1184
==== Front J Cell Mol MedJ. Cell. Mol. MedjcmmJournal of Cellular and Molecular Medicine1582-18381582-4934John Wiley & Sons, Ltd Chichester, UK 1906776710.1111/j.1582-4934.2008.00601.xArticlesChemosensitization in non-small cell lung cancer cells by IKK inhibitor occurs via NF-κB and mitochondrial cytochrome c cascade Jin Xianqing aQiu Lin a#Zhang Dianliang ab#Zhang Mingman aWang Ziming cGuo Zhenhua aDeng Chun aGuo Chunbao aa Laboratory of Surgery, Children’s Hospital of Chongqing Medical UniversityChongqing, P.R. Chinab Department of Surgery, Affiliated Hospital of Qingdao UniversityQingdao, P.R. Chinac Department of Orthopaedics, Daping Hospital, Third Military Medical UniversityChongqing, P.R. China Correspondence to: Chunbao GUO, M.D., Ph.D., Laboratory of Surgery, Children’s Hospital of Chongqing Medical University, 136 Zhongshan 2nd Rd. Chongqing, 400014, P.R. China. Tel.: +86–23-63893006 Fax: +86-23-63893006 E-mail: [email protected]# Lin Qiu and Dianliang Zhang contributed equally to this work. Nov-Dec 2009 03 12 2008 13 11-12 4596 4607 11 8 2008 10 11 2008 © 2008 The Authors Journal compilation © 2009 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd2008In this study, we demonstrated with mechanistic evidence that parthenolide, a sesquiterpene lactone, could antagonize paclitaxel-mediated NF-κB nuclear translocation and activation by selectively targeting I-κB kinase (IKK) activity. We also found that parthenolide could target IKK activity and then inhibit NF-κB; this promoted cytochrome c release and activation of caspases 3 and 9. Inhibition of caspase activity blocked the activation of caspase cascade, implying that the observed synergy was related to caspases 3 and 9 activation of parthenolide. In contrast, paclitaxel individually induced apoptosis via a pathway independent of the mitochondrial cytochrome c cascade. Finally, exposure to parthenolide resulted in the inhibition of several NF-κB transcript anti-apoptotic proteins such as c-IAP1 and Bcl-xl. These data strengthen the rationale for using parthenolide to decrease the apoptotic threshold via caspase-dependent processes for treatment of non-small cell lung cancer with paclitaxel chemoresistance. chemosensitizationparthenolideIKKNF-κBcytochrome c ==== Body Introduction Systemic treatment, especially chemotherapy, is an indispensable step for curing or increasing survival in patients with lung cancer. However, resistance to chemotherapy is frequently observed; this can result in recurrence and metastasis [1, 2]. Therefore, exploration of molecular mechanisms involved in chemoresistance and further development of drugs and strategies that inhibit chemoresistance are a high priority for lung cancer therapy [3, 4]. The transcription factor NF-κB is known to be involved in the transcriptional regulation of various genes involved in cell proliferation, angiogenesis, metastasis and apoptosis [5, 6]. Anti-apoptotic signalling by NF-κB mediates resistance to chemotherapeutic drugs [7, 8] in ovarian cancer [9–11], pancreatic cancer [12], breast cancer [13, 14] and prostate cancer [15], implying that inhibition of NF-κB might be an effective treatment strategy for many resistant cancers. However, this has been challenged by evidence in other cell types; in neuroblastoma cells, NF-κB activation induced p53-mediated apoptotic signalling in response to the chemotherapy agent Dox [16]. It is presently unclear which signalling events ultimately determine whether NF-κB activation results in a pro- or anti-apoptotic response. The role of NF-κB might depend on the conditions of other oncogenes such as Myc, P53 and Bcl2. Specifically, aberrant expression of NF-κB has been related to lung cancer development and progression. Our recent work revealed that nuclear RelA and cytoplasmic pI-κBα were significantly associated with a poor prognosis [1]. Furthermore, activation of NF-κB by chemotherapy can blunt the efficacy of the chemotherapy itself to induce death in lung cancer cells [17, 18]. The finding that NF-κB is a key player in lung cancer survival has prompted researchers to explore drugs with NF-κB suppression activity in order to directly kill the cancer or render it more vulnerable to chemotherapeutic agents [19–21]. Additionally, the use of drug combinations with non-overlapping toxicity profiles could allow decreased doses of the individual agents, thereby reducing the severity of undesired side effects. NF-κB proteins are sequestered in the cytoplasm in an inactive state and in a complex with the inhibitor I-κB. Degradation of I-κB by I-κB kinase (IKK)-mediated phosphorylation can release NF-κB to translocate to the nucleus and promote target gene expression. NF-κB can be blocked by targeting elements of its various signalling cascades such as the IKK complex, the I-κB inhibitory protein, the p65 subunit of the transcriptionally active heterodimer and the proteasome. Thus, any molecular agents that inhibit NF-κB are potential therapeutic targets for cancers whose tumorigenicity depends on NF-κB activity [7, 22, 23]. A great number of such compounds have already been tested to suppress the growth of cancer cells [24–26]. The active compound of feverfew (Chrysanthemum parthenium), parthenolide, has shown potential as an anticancer agent [25, 27]. Parthenolide shows significant cancer suppression activity in vitro and in vivo [28, 29] through inhibition of NF-κB by direct inhibition of IKK [27], which can regulate the phosphorylation of I-κB at serine residues 32 and 36. Although the inhibitory effect of parthenolide on NF-κB activity has been reported previously in several cancer cell lines [28–31], its significance, detailed mechanism and potential use in treatment and as an adjuvant with chemotherapy have not yet been fully investigated in human non-small cell lung cancer (NSCLC). The aims of this study are to provide further information about the role of NF-κB in lung cancer cell biology and to further determine whether parthenolide could elicit anti-proliferative activity via inhibition of NF-κB activation, thereby increasing the efficacy of paclitaxel in human NSCLC cell lines. To our knowledge, the present study is the first to combine molecular and functional analyses of NF-κB and its related molecules in order to determine adjuvant effects of parthenolide and the synergistic combination of parthenolide and paclitaxel. Materials and methods Materials and antibodies Paclitaxel was purchased from Calbiochem (La Jolla, CA, USA) and dissolved in 100% dimethyl sulfoxide to make a stock solution of 1.0 mmol/l. The I-κB phosphorylation inhibitor (BAY 11–7082, Cat. #EI278–0010) and pancaspase inhibitor Z-Val-Ala-Asp-fluoromethyl ketone (Z-VAD-fmk) (Cat. #P416–0001) were purchased from Biomol (BIOMOL International L.P., PA, USA). Parthenolide was obtained from Alexis Biochemicals (San Diego, CA, USA). These agents were dissolved in DMSO at a concentration of 10 or 20 mmol/l and stored in the dark at −80°C. The entire stock solution was diluted to obtain the desired concentrations with growth medium. The concentration of dimethyl sulfoxide did not exceed 0.2% (vol/vol). Z-VAD-fmk was incubated at a desired concentration of 20 mol/l for 30 min. prior to induction of apoptosis (paclitaxel treatment). During prolonged experiments, supernumerary Z-VAD-fmk was further added for 12 to 16 hrs. All cell culture reagents, DNA-modifying enzymes and Lipofectamine reagent were purchased from Invitrogen (Carlsbad, CA, USA). Cell cultures The human lung adenocarcinoma cell line A549, squamous cell line NCI-H446 and A549 taxol-resistant derivative A549-T24 were selected for resistance to taxol in a stepwise manner and maintained in a final concentration of 24 nmol/l taxol. These cells were obtained from the cell bank of the Chinese Academy of Sciences and maintained at 37°C in a 5% CO2 atmosphere in air, in RPMI 1640 medium (Gibco, Gaithersburg, MD, USA) supplemented with 10% (vol/vol) foetal bovine serum and 100 units/ml each of penicillin and streptomycin. Small interfering RNA Small interfering RNA (siRNA) was applied in order to achieve gene silencing. High-purity SMARTpool™ siRNAs targeting IKK-α (Cat. # 60–055, accession no. NM_001278), IKK-β (Cat. # 60–025, accession no. NM_001556), p65 (Cat. # 60–076, accession no. NM_021975) and control siRNA were purchased from Upstate (Charlottesville, VA, USA). Lipid-encapsulated SMARTpool™ and control were used for transfection. At the time of transfection, the cell density was ∼70–90% confluent, or approximately 1 × 105 cells/ml. We transfected the cells using the siIMPORTER™ Transfection Reagent (Cat. # 64–101SP, Upstate) according to the manufacturer’s protocol after testing and optimizing the conditions best suited for each cell line or culture. To improve gene silencing, at 24 hrs after transfection the same siRNA reagents were added to the media for a second 24 hrs. At 24 hrs after transfection, the cells received paclitaxel as described above. Gene silencing effects were evaluated by Western blot analysis. Cell viability determination (MTT assay) Cells were exposed to different treatments in a medium containing 0.5% foetal bovine serum for 72 hrs, then 10 μl of 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyl-tetrazolium bromide (MTT) solution (10 mg/ml in PBS) were added followed by 100 μl of 10% sodium dodecyl sulphate (SDS) for formazan crystal formation. The absorption of the samples was determined using an ELISA reader (Anthos Mikrosysteme GmBH, Germany) at a wavelength of 570 nm. The optical density of untreated control cells was considered to represent 100% viability for calculation of the viability of treated cells. Terminal deoxynucleotidyltransferase-mediated dUTP nick end labelling (TUNEL) Control and treated cells collected at various time-points following treatment were subjected to terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick-end labelling (TUNEL) assay. The commercially available APO-BrdU™ TUNEL assay kit (with Alexa Fluor® 488 anti-BrdU, 60 assays; Cat. # A23210, Invitrogen) was utilized to label DNA fragmentation with terminal deoxynucleotide transferase following the instructions of the manufacturer. Quantification of the apoptotic cells by FACS analysis (Calibur, BD Biosciences, CA, USA) was achieved using the geo mean function in CellQuest Software (FACScan, BD Biosciences, CA, USA). All flow cytometry analyses were repeated three times. Nuclear extracts and electrophoretic mobility shift assay (EMSA) Nuclear proteins of the cultured control and treated cells were extracted using an NE-PER nuclear and cytoplasmic extract kit (Pierce Biotechnology, IL, USA), following the manufacturer’s instructions. EMSA was performed using a gel shift assay system kit (Promega, WI, USA) as described previously [1]. Briefly, double-stranded oligonucleotide probes targeting NF-κB (5′-AGT TGA GGG GAC TTT CCC AGG C-3′) and Oct-1 (5′-ATGCAAAT-3′) were labelled using T4 polynucleotide kinase and γ-32P ATP (Yahui Biotech Inc., Beijing, China). Ten micrograms of protein extract were incubated with 2 × 104 cpm of oligonucleotide probe in addition to 1 μg poly(dI-dC) (Sigma, St. Louis, MO, USA) in binding buffer for 15 min. at room temperature. Equal loading of nuclear extracts was monitored by Oct-1 binding. The resulting protein-DNA complexes were analysed on 4% non-denaturing polyacrylamide gels in 0.5× Tris-borate-ethylenediaminetetraacetic acid buffer and then subsequently dried and analysed by autoradiography. Western blot analysis Differently treated cells were lysed using protein extraction buffer and homogenized in 250 μl of lysing buffer. Cytoplasmic extracts were prepared to measure the activation of caspases 3 and 9. Ten micrograms of total protein were electrophoresed through SDS-PAGE on 4–20% gradient gels (Shanghai Sangon Biotech, Shanghai, China) and transferred to nylon membranes (Shanghai Sangon Biotech). The blots were probed with primary antibodies to recognize respective proteins; these antibodies included anti-RelA against the p65 (RelA) unit (p65; 1:150; sc-109, Santa Cruz Biotechnology, CA, USA), anti-phosphorylated I-κBα that recognizes the Ser32 epitope (1:40; sc-8404, Santa Cruz Biotechnology), anti-IκBα (1:100; sc-1643, Santa Cruz Biotechnology), anti-Phospho-IKK-α (Ser180)/IKK-β (Ser181) (1:100; #2681, Cell Signaling Technology, Beverly, MA, USA), anti-IKK-β (1:100; #2684, Cell Signaling Technology), anti-cleaved caspase-3 (Asp175) (1:100; #9661), anti-cleaved caspase-9 (Asp330) (1:100; #9501, Cell Signaling Technology), anti–X-linked inhibitor of apoptosis protein (XIAP) (1:100; #2404, Cell Signaling Technology), anti–Bcl-XL(1:100; #2762, Cell Signaling Technology), anti–c-IAP1 (1:100; #4952, Cell Signaling Technology), anti–c-IAP2 (1:100; #3130, Cell Signaling Technology), anti–Trail-R1 (DR4) (1:100; # sc-6823, Santa Cruz Biotechnology), anti–Trail-R2 (DR-5) (1:100; # sc-65314, Santa Cruz Biotechnology), anti-Fas (1:100; # sc-52395, Santa Cruz Biotechnology) and anti-β-actin (1:100; # sc-47778, Santa Cruz Biotechnology). The secondary antibody, horseradish peroxidase-coupled mouse anti-rabbit immunoglobulin (Jingmei Biotech Co., Ltd. Shenzhen, China), was then incubated for 1 hr at room temperature. The subsequent analysis was performed with chemiluminescence using an ECL Western blotting kit (Amersham Biosciences, IL, USA) following the manufacturer’s recommendations. Immunofluorescence assays For immunostaining, cells were fixed in 3.8% paraformaldehyde for 5 min. at room temperature, permeabilized in 0.1% saponin for 5 min., and stained with anti-NF-κB (p65) (1:200; Santa Cruz Biotechnology) and anti-cytochrome c (1:200; #4272, Cell Signaling Technology, MA, USA) antibodies for 30 min. at room temperature. After washing with PBS, cells were incubated with affinity-purified, rhodamine-conjugated mouse anti-rabbit IgG (1:4000; Jackson Immuno Research, West Grove, PA, USA). The stained cells were analysed using a confocal microscope (Leica Microsystems Heidelberg GmbH, Heidelberg, Germany) with excitation at 488 nm and emission at 525 nm. Cytochrome c release assay Isolated tumour cells (5 × 107) were collected by centrifugation at 600 ×g for 5 min. at 4°C and washed with ice-cold PBS. The cells were assayed with the Cytochrome c apoptosis assay kit (Cat. #K257–100, Biovision, CA, USA). Briefly, cells were homogenized with the cytosol extraction buffer provided in the kit and then centrifuged at 700 ×g for 10 min. at 4°C to remove debris. The supernatant was then centrifuged at 10,000 ×g for 30 min. at 4°C. The pellet contained the mitochondrial fraction, and the supernatant was collected as the cytosolic fraction. These fractions were analysed for cytochrome c content by Western blotting using the cytochrome c antibody provided in the kit. Statistical analysis Statistical analysis was carried out using one-way ANOVA followed by Fisher’s least significant difference test, and the level of significance was set at P < 0.05. Data are expressed as the mean ± S.E.M. Statistical comparisons were carried out using SPSS software for Windows (SPSS, Inc., Chicago, IL, USA). Results Paclitaxel treatment induces NF-κB activation and up-regulates its regulatory target Bcl-xl We first compared NF-κB DNA-binding activities among the human lung cancer cell lines A549, NCI-H446 and A549-T24, which had demonstrated resistance to taxol treatment, in order to detect possible effects of paclitaxel on NF-κB activity. As shown in Fig. 1, the specificity of NF-κB was first evaluated by performing EMSA gel supershift and competition assays (Fig. 1A). Basal NF-κB activity in A549 and NCI-H446 cells was not detectable. After exposure to 100 nmol/l paclitaxel for 12, 24 and 48 hrs, NF-κB activity in A549 and NCI-H446 cells increased notably in comparison with the basal level. Although the basal level of NF-κB activity in A549-T24 cells was higher than in A549 and NCI-H446 cells, NF-κB activity in A549-T24 cells also increased after 24 hrs of paclitaxel treatment, albeit to a lesser degree (Fig. 1B). Figure 1 Effect of paclitaxel on NF-κB activation in A549, NCI-H446 and A549-T24 cell lines. (A) Supershift analysis and competitive study were performed to confirm the specificity of NF-κB DNA binding in A549-T24 cells stimulated with 100 nmol/l paclitaxel for 48 hrs, with antibodies specific for RelA (p65) (recognizes RelA/p50) (line 3), NF-κB1 (p50) (recognizes p50/p50 and RelA/p50) (line 4) and a 50-fold excess of unlabelled oligonucleotide (diminished both RelA/p50 and p50/p50) (line 5) and another oligonucleotide AP-1 (line 6). Data from one representative experiment are shown. (B) The three cell lines were exposed to 100 nmol/l paclitaxel for sequential durations. Nuclear extracts were prepared and examined for NF-κB DNA binding activities (RelA/p50) by electrophoretic mobility shift assay. Oct-1 was selected as a constitutively expressed control. NF-κB activation-involved molecules such as pI-κB, I-κB and Bcl-xl (cytoplastic extracts) were subjected to Western blotting with β-actin as a loading control. Representative figures of at least three independent experiments with similar results are shown at the indicated hours after treatment. The positions of protein weight markers are noted on the right. As expected, paclitaxel treatment transiently increased the amount of phosphorylated I-κBα in A549 and NCI-H446 cells as detected by Western blotting. This was linked to a change in NF-κB DNA binding activity; however the total I-κBα was maintained at a high level similar to that without treatment, suggesting that paclitaxel did not affect the degradation of I-κBα in A549 or NCI-H446 cells. Consistent with the NF-κB DNA binding activity, the anti-apoptotic gene Bcl-xl was also up-regulated in response to paclitaxel treatment. Similar effects were also observed in the A549-T24 cells. IKK kinase complex mediates paclitaxel-induced NF-κB activation To determine the involvement of IKK in the response to paclitaxel, phospho-IKK-α /β was detected by immunoblotting with specific antibodies. Levels of phosphorylated (active) IKK increased 24 hrs after exposure to paclitaxel. Specifically, IKK-β and not IKK-α was phosphorylated in response to paclitaxel treatment, consistent with the changes in NF-κB and I-kB (Fig. 2A). Figure 2 BAY 11–7082-mediated inhibition of IKK resulted in reduced induction of NF-κB by paclitaxel. A549 cells with or without pre-treatment of 5 μmol/l BAY 11–7082 were treated with 100 nmol/l paclitaxel for 24 hrs. (A) NF-κB DNA binding activities in the nuclear extract and p-IKK-α/β, IKK-β, phospho-I-κBα, I-κBα, Bcl-xl in the cytosolic extract were compared. The experiment was repeated with identical outcomes for at least three times, and a representative result is shown. B. TUNEL analysis with flow cytometry was carried out to evaluate induction of apoptosis. Columns, average values of at least three independent experiments performed in triplicate; bars, ±S.E.M. #, P < 0.01 versus paclitaxel + BAY 11–7082. Next, we tested whether inhibition of IKK using an IKK inhibitor (BAY 11–7082) was sufficient to block paclitaxel-induced NF-κB activation. IKK activity (indicated as phospho-IKK-α/β) was induced in human NSCLC cell lines by paclitaxel treatment and inhibited by BAY 11–7082, whereas levels of IKK proteins (indicated as IKK-β) remained at the same level. As shown in Fig. 2A, paclitaxel-induced NF-κB activity measured with EMSA was abrogated by BAY 11–7082. Paclitaxel-induced Bcl-xl expression was also reduced by BAY 11–7082 (Fig. 2A), whereas the amount of total I-kBα was not increased by paclitaxel treatment (Fig. 2A). We further performed a TUNEL assay to examine whether apoptosis could account for the cell growth inhibition in this system. As seen in Fig. 2B, paclitaxel treatment alone resulted in an apoptosis rate of 25%. BAY 11–7082 at a concentration of 5 μmol/l did not show significant growth inhibition after 24 hrs treatment in NCI-H446 cell lines, and still less apoptotic induction in A549 cell lines. Co-treatment with paclitaxel and BAY 11–7082, resulted in a further 20% and 30% enhancement of the apoptotic response in A549 and NCI-H446 cells, respectively (Fig. 2B), suggesting that interference with NF-κB transcriptional activity could sensitize the paclitaxel response. Parthenolide inhibits paclitaxel-mediated activation of IKK Earlier studies reported that parthenolide could inhibit activation of IKK in pancreatic carcinoma cell lines [30]. Here we examined if it also had an effect in human NSCLC lines. As expected, after exposure to parthenolide prior to paclitaxel stimulation, paclitaxel-induced NF-κB activation was potently inhibited in A549 cells as measured by EMSA (Fig. 3A). Incubation with 5 μmol/l parthenolide for 24 hrs completely inhibited paclitaxel–induced activation of IKK activity (Fig. 3B). The activation of IKK was concurrent with degradation of I-κB that showed similar kinetics in both cells types and was prevented by increasing the time of incubation with parthenolide (data not shown). Figure 3 Regulation of NF-κB activation by parthenolide occurs through IKK inhibition. (A) A549 cells were pre-treated with 5 μmol/l parthenolide for various times (left) and for 24 hrs at various concentrations (right) courses, then incubated with 100 nmol/l paclitaxel for 24 hrs. Equal amounts of nuclear and cytosolic extracts were subjected to electrophoretic mobility shift assay (EMSA) for NF-κB binding and Western blotting for Bcl-xl, respectively. (B) A549 cells were alternatively pre-treated with either 5 μmol/l parthenolide or 5 μmol/l BAY 11–7082 for 24 hrs and then incubated with paclitaxel for 24 hrs. EMSA for NF-κB binding and Western blotting for p-IKK-α/β and Bcl-xl were performed. (C) Nuclear translocation of p65 was determined by indirect immunofluorescence. Representative fields show the exclusive cytoplasmic location of NF-κB in untreated control (a) and parthenolide-treated cells (b). Nuclear accumulation of p65 was observed in paclitaxel-treated cells (c) but not in paclitaxel + BAY 11–7082 pre-treated (d) or paclitaxel + parthenolide pre-treated cells (e). All the experiments shown in this figure were carried out two to four times, producing similar outcomes, and one representative result is shown. Next, we performed immunoblotting and EMSAs to determine whether parthenolide inhibited the nuclear translocation of NF-κB and/or NF-κB binding activity. As shown in Fig. 3C, strong p65 translocation to the nucleus was observed in A549 cells stimulated with paclitaxel. This increased NF-κB translocation was completely inhibited when cells were incubated with 10 μmol/l parthenolide prior to paclitaxel stimulation; the effect was similar to or stronger than that seen with BAY 11–7082. This effect was also concentration and time dependent, and concentrations that did not affect apoptosis induction also inhibited the DNA binding activity of NF-κB (5 μmol/l). Moreover, NF-κB DNA binding activity in nuclear extracts of A549 cells stimulated with paclitaxel were completely abolished by 10 μmol/l parthenolide (Fig. 3A). Parthenolide sensitizes to paclitaxel-induced apoptosis We further examined the involvement of parthenolide in sensitization to paclitaxel-induced apoptosis. Parthenolide induced cell growth inhibition in a concentration- and time-dependent manner, but at a concentration of 5 μmol/l for 12 hrs, parthenolide did not significantly inhibit growth (Fig. 4A). Treatment with paclitaxel for 24 hrs induced cell apoptosis by about 30%; co-treatment with paclitaxel and parthenolide further enhanced apoptosis by almost 55%. The inhibitory effect of parthenolide and paclitaxel administered together was more than that of BAY 11–7082 and paclitaxel (Fig. 4B). Similarly, a DNA fragmentation assay demonstrated that cell death induced by paclitaxel and parthenolide occurred via apoptotis (data not shown). Figure 4 Inhibition of the IKK/NF-κB pathway enhanced paclitaxel-mediated cytotoxicity. (A) Induction of apoptosis by parthenolide in concentrations (1, 3, 5,15 20 μmol/l) (a) and times (0, 12, 24, 48h) (b) courses in A549 (left), NCI-H446(middle) and A549-T24 (right) cells; TUNEL was assessed using flow cytometry, all of the columns depicted represent a mean of at least three independent experiments in triplicate; bars, ±S.E.M. (B) The apoptotic rate was analysed via TUNEL assay in A549 cells that were pre-treated with or without 5 μmol/l parthenolide for 24 hrs, then incubated with 100 nmol/l paclitaxel for another 24 hrs; bars, ±S.E.M. Significant differences are indicated by asterisks. #, P < 0.01 versus paclitaxel + parthenolide incubation with the same time. (C) Western blots of IKK-α and IKK-β protein levels in A549 cells 48 hrs after transfection with the indicated siRNAs (a). The experiments of Western blot were carried out with similar outcomes for at least three times, and a representative result is shown. A549 cells were transfected with the indicated siRNAs for 48 hrs then treated with 5 μmol/l parthenolide and/or 100 nmol/l paclitaxel in succession; the cell extracts were then subjected to electrophoretic mobility shift assay (EMSA) (NF-κB) (b) and TUNEL analysis (c). EMSA analyses were performed with identical outcomes for three times, and a representative result is shown. TUNEL analysis was carried out two independent siRNA experiments in triplicate, columns, mean of two independent experiments; bars, ±S.E.M. Significant differences are indicated as *, P < 0.01 versus the same cells treated with paclitaxel plus parthenolide and #, P < 0.01 versus cells treated with IKK-α or IKK-β siRNA as well as parthenolide. Although our results suggest that parthenolide potentiated paclitaxel-mediated apoptosis by inhibiting IKK/NF-κB, it remains possible that parthenolide might sensitize cells to pro-apoptotic processes via IKK-independent pathways. To further investigate this, we transfected cells with siRNA-targeting IKK-α or IKK-β to evaluate whether inhibition of IKK could abolish the effect of parthenolide. As seen in Fig. 4Cb, siRNA-IKK-α and siRNA-IKK-β both blocked the IKK/NF-κB signalling cascade as measured by the DNA binding activity of NF-κB. Blockade with the expression of IKK-α or IKK-β in A549/siRNA-IKK-α/β cells resulted in enhancement of apoptosis induced by paclitaxel. Interestingly, parthenolide treatment in this system did not further enhance paclitaxel-induced apoptosis (Fig. 4Cc). These results are consistent with the EMSA data shown in Fig. 4Cb, and with phosphorylated (active) IKK immunoblotting (data not shown). Biological NF-κB blockade contributes to parthenolide sensitization to paclitaxel To determine whether IKK-mediated parthenolide sensitization to paclitaxel relies on NF-κB regulation, gene silencing targeting the p65 RelA subunit was carried out, and cell lines were then co-treated with paclitaxel and parthenolide or BAY 11 7082. Compared to A549 cells, the expression of Bcl-xl was abolished in cells transfected with p65-targeting siRNA. This was accompanied by the decreased RelA DNA binding activity in the nucleus (Fig 5A), confirming that the siRNA worked. As expected, inhibition of NF-kB significantly augmented the response of A549 cells to paclitaxel as assessed by MTT assay, whereas it abolished the inhibitory effects of parthenolide or BAY 11 7082 even when used at high concentrations (Fig. 5B). Figure 5 Biological NF-κB blockade with p65 RelA siRNA enhanced paclitaxel-induced cytotoxicity. A549 cells were transfected with p65 RelA siRNA for 24 hrs and exposed to paclitaxel (100 nmol/l) for another 24 hrs. The scramble sequence siRNA control showed no effects on cell viability or NF-κB DNA binding activity (data not shown here). (A) NF-κB DNA binding activity and Bcl-xl expression were measured with electrophoretic mobility shift assay and Western blot analysis respectively at least three times with similar outcomes. (B) Cell viability was assessed by the MTT reduction assay with increasing concentrations of paclitaxel (0–200 nmol/l). All of the results represent the mean of at least three independent experiments performed in triplicate; bars, ±S.E.M. Significant differences are indicated by asterisks. #, P < 0.01 versus p65 siRNA-transfected cells treated with the same doses of paclitaxel. (C) TUNEL analysis with flow cytometry was carried out to evaluate induction of apoptosis. Untreated cells (none) served as the control (100%). The experiment was carried out with similar outcomes for at least three replications, and a representative result is shown. The apoptosis rates were 24.8% for A549 cells and 76.89% for A549/p65siRNA as indicated below the box. (D) A549 cells were transfected with p65 RelA siRNA and exposed to parthenolide (5 μmol/l)-paclitaxel (100 nmol/l) and BAY 11 7082 (5 μmol/l)-paclitaxel (100 nmol/l) combination treatments. TUNEL analysis with flow cytometry was carried out to evaluate induction of apoptosis. The data represent the average of two independent experiments performed in triplicate; Bars, ±S.E.M. #, P < 0.01 versus p65 siRNA-transfected cells treated with the same concentrations of paclitaxel. We next used the TUNEL assay to examine whether induction of apoptosis could account for the cell growth inhibition observed in p65-silenced A549 cells. These cells showed strong induction of DNA fragmentation, indicating apoptosis (Fig. 5C). These results suggest that specific inhibition of NF-κB activity, which regulates expression of the anti-apoptotic gene Bcl-xl, resulted in the induction of apoptosis. In order to further verify that parthenolide could sensitize the effects of paclitaxel via NF-κB regulation, cells genetically silenced for NF-κB were treated with parthenolide and paclitaxel, or with BAY 11 7082 and paclitaxel. As expected, blockade of p65 resulted in the enhancement of paclitaxel-mediated apoptosis comparable to the one obtained with parthenolide or BAY 7082 pre-treatments. But also these pre-treatments do not further enhance paclitaxel-induced apoptosis when p65 is already blocked by siRNA (Fig. 5D). Effect of IKK inhibition on NF-κB transcript target genes To determine whether NF-κB downstream target genes are up-regulated by parthenolide, we evaluated the expression of some endogenous genes known to be regulated by NF-κB. These included pro-apoptotic genes such as like Fas, DR4 and DR5 as well as anti-apoptotic genes such as c-IAP1, c-IAP2, XIAP and Bcl-xl. As indicated in Fig. 6, incubation with parthenolide somewhat increased Fas, DR4 and DR5 protein levels. In contrast to the previous finding that exposure to paclitaxel induced up-regulation of the anti-apoptotic molecule Bcl-xl, incubation of A549 cells with parthenolide resulted in a marked decrease of Bcl-xl, c-IAP1 and c-IAP2 expression but XIAP expression had no obvious change (Fig. 6). The inhibition of anti-apoptotic molecules in A549 cells by paclitaxel was also abolished by transfection with IKK-α or IKK-β siRNA, single or in combination (Fig. 6). These results indicate that parthenolide can antagonize paclitaxel to diminish NF-κB-activated anti-apoptotic molecule expression in A549 cells. Figure 6 Effect of parthenolide and/or silencing of IKK-β on apoptosis-related gene expression. IKK-β siRNA and control scrambled siRNA (data not shown)-transfected cells were treated with parthenolide (5 μmol/l) alone or in combination with paclitaxel (100 nmol/l). Equal amounts (50 μg/lane) of cytoplastic extracts were subjected to Western blotting at least three times with similar outcomes. The positions of molecular weight markers are noted on the right. Mitochondrial apoptosis signalling cascade and effects of parthenolide It has been reported that mitochondria play a minor role in paclitaxel-induced cell death of lung cancer cells [32, 33]. However, mitochondrial cytochrome c release during NF-κB inhibition was described previously [34]. Therefore, we next evaluated the possible contribution of cytochrome c release and caspase activation to the chemosensitization effect of parthenolide in A549 cells. A549 cells treated with paclitaxel only displayed a punctate staining pattern for cytochrome c (Fig. 7A); this pattern became diffuse upon parthenolide pre-stimulation, indicating release from the mitochondria. This was further verified by measurement of cytochrome c in the cytoplasm. A concentration of at least 5 μmol/l of parthenolide was necessary to induce cytochrome c release; this mirrored the concentration required to inhibit IKK and suggested a link between IKK inhibition and cytochrome c release. Figure 7 The IKK inhibitor parthenolide induced mitochondrial cytochrome c release, which precedes cell death. (A) A549 cells treated with 100 nmol/l paclitaxel and 5 μmol/l parthenolide either single or in combination were stained with monoclonal antibodies against cytochrome c and analysed using a confocal microscope (upper panel). The cytosolic extract was prepared and subjected to Western blotting with a cytochrome c antibody (lower panel). (B) A549 cells were incubated with Z-VAD-fmk, and subsequently either 100 nmol/l paclitaxel or 5 μmol/l parthenolide were added. Cytosolic proteins were extracted, separated and incubated with antibodies recognizing active caspases 3 and 9 as indicated. (C) A total of 106 cells were treated with paclitaxel or paclitaxel + parthenolide in the absence or presence of 20 μmol/l Z-VAD-fmk. Cells were labelled with a fluorescein TUNEL kit and analysed by flow cytometry. The percentage of apoptotic cells was 28% (a, paclitaxel), 17% (b, paclitaxel + Z-VAD-fmk), 48% (c, paclitaxel + parthenolide), and 32% (d, paclitaxel + parthenolide + Z-VAD-fmk). This experiment was performed at least five times with similar outcomes. All the experiments shown in this figure were performed two to four times, producing identical outcomes, and one representative result is shown. We next assessed by immunoblotting whether parthenolide induction of cytosol cytochrome c could lead to the activation of caspases 9 and 3. Activity of both caspases was influenced by parthenolide induction of cytochrome c leakage and associated with parthenolide-induced apoptosis. These effects were completely prevented in the presence of the pan-caspase inhibitor Z-VAD-fmk (Fig. 7B and C), further confirming that parthenolide regulates paclitaxel sensitivity by attenuating NF-κB activation and its downstream mitochondrial apoptosis signalling cascade. Discussion In the present study, activation of NF-κB was significantly induced by paclitaxel in chemotherapy-resistant cancer cell lines, supporting a role of NF-κB activation in the cellular mechanism of apoptosis resistance. This is consistent with previous reports in ovarian cancer, prostate cancer, pancreatic cancer and breast cancer. Our results also indicate that the phosphorylation of IKK-β was strongly increased by paclitaxel treatment, whereas p-IKK-α was essentially not affected. This result is in agreement with reports that IKK-β, and not IKK-α, is responsible for cytokineinduced activation of NF-κB [35, 36]. We also found that I-κB phosphorylation, which is catalysed by IKK (including IKK-α and IKK-β) [37, 38], was induced by paclitaxel treatment in parallel with NF-κB activation. Therefore, it is conceivable that a drug that inhibits NF-kB could inhibit or circumvent resistance to chemotherapeutic agents such as paclitaxel. We further investigated an adjuvant approach to increase the efficacy of paclitaxel through inhibition of NF-κB. We demonstrated that parthenolide, a promising new multifunctional anti-cancer drug previously proven to inhibit NF-κB activity [39, 40], could contribute to the sensitization of A549 cells to paclitaxel-induced apoptosis. We observed that the cytotoxic effects of paclitaxel were potentiated by pre-treatment with 5 μmol/l parthenolide. At this concentration, we did not see noticeable induction of apoptosis by parthenolide alone. These data are consistent with studies that have inhibited NF-κB signalling by NF-κB antisense oligonucleotides [48], ectopically expressed I-κBαM [49–51] or used the proteasome inhibitor PS-341[52, 53] or I-κB inhibitor BAY 11–7085 [54, 55] to block I-κB degradation in different cancer cell lines. Targeting of the NF-κB pathway by proteasome inhibitors and I-κB kinase inhibitors has been used for both preclinical models and clinical trials involving patients with various types of tumours [56–58]. It has been suggested that pre-treatment is a prerequisite for enhanced cell sensitization to chemotherapy agents [46, 47]. Similarly, we found that pre-treatment, but not simultaneous administration, had a synergic effect on paclitaxel cell sensitivity. Many researchers have shown that parthenolide can reversibly bind IKK and block its activity in vitro and in vivo[41, 42]. We demonstrated that parthenolide inhibited constitutive and paclitaxel-induced RelA/NF-κB activity by interfering with the effect of paclitaxel on IKK-β, thereby functionally inhibiting phosphorylation of I-κBα and subsequently leading to inhibition of NF-κB activity. However, whether paclitaxel-induced up-regulation and parthenolide-induced down-regulation of p65 are only regulated through IKK-β remains unclear. Some reports indicate that paclitaxel can directly induce phosphorylation of I-κBα[43]. Several other studies have also shown that paclitaxel directly activates survival pathway-related proteins such as Bcl-2, Akt, Cox-2 and mitogen-activated protein kinase independent of NF-κB [44, 45]. In the present study, transfection with siRNA-targeting IKK-α or IKK-β abolished parthenolide activity, indicating that when co-treated with paclitaxel, parthenolide might affect the NF-κB/I-κB cascade via IKK-β inhibition; this has not previously been indicated. It has been verified that this synergetic effect of parthenolide and chemotherapeutic agents is specific to tubulin-modifying agents and cannot be observed with a variety of other cytotoxic agents [46]. Mechanistically, paclitaxel is tubulin-modifying agent that is different from DNA-damaging agents such as cisplatin, doxorubicin or camptothecin. Although apoptosis triggered by DNA-damaging agents is largely dependent on mitochondrial pathways, activation of caspases and mitochondria appears to be a secondary effect of paclitaxel treatment of NSCLC cells [33, 59], even though paclitaxel-induced NF-κB activity mediates inhibition of caspases [60, 61]. Our results also indicate that mitochondrial apoptosis pathways did not play an essential role in paclitaxel-induced cell death because inhibition of these pathways by Z-VAD-fmk resulted only in a temporary protection. Because parthenolide-induced apoptosis has been shown to be dependent on caspase activity [27–30], we next explored the possibility that the synergistic effect of paclitaxel and parthenolide might involve both cascade-dependent (parthenolide-induced) and -independent (paclitaxel-induced) mechanisms. If so, the induction of NF-kB and subsequent apoptotic resistance should be abolished by parthenolide. The cytotoxic effect of paclitaxel was affected by Z-VAD-fmk treatment. However, caspase activation induced paclitaxel plus parthenolide at a concentration of 5 μmol/l was blocked by the caspase inhibitor Z-VAD-fmk, indicating that the effects of parthenolide occur mainly through a mitochondrial pathway. Treatment with paclitaxel plus parthenolide significantly increased cytochrome c release. Furthermore, the level of phosphorylated I-κBα, which was also reduced by paclitaxel administered in combination with parthenolide, showed an identical time course to the level of cytochrome c. These effects were blocked by siRNA-targeting IKK-β, indicating that cytochrome c and NF-κB might act in concert in response to the combined treatment. This result is consistent with previous studies in other systems [62, 63]. NF-κB activation can result in the transcription of some anti-apoptotic genes such as Bcl-xl [5, 60, 61, 64]. In the present study expression of Bcl-xl was reduced by parthenolide administered in combination with paclitaxel through down-regulation of NF-κB signalling; these effects paralleled the observed synergistic toxicity. Collectively, these observations indicate that the synergistic effect of paclitaxel and parthenolide is derived from reduced Bcl-xl induction via inhibition of NF-κB activity by parthenolide; thus synergistic cellular toxicity occurs at the level of NF-κB and downstream anti-apoptotic factors. Several ongoing clinical trials are currently evaluating whether NF-κB inhibitors can enhance or restore the response to a wide range of therapeutics against both solid and haematological tumours [65–68]. Parthenolide, which can activate the IKK pathway and subsequently inhibit NF-κB and its downstream anti-apoptotic proteins, was also tested in cell lines derived from solid tumours [65, 68, 69]. The present study therefore suggests that parthenolide might be useful in combination therapy with paclitaxel; such a combination might limit adverse side effects. We thank Prof. Wang Wei, Prof. Mingman Zhang and Prof. Mingqing Peng for providing technical assistance and insightful discussions during the preparation of the manuscript. We thank Dr Xiaoyong Zhang, at the Wistar Institute, USA, for help with the linguistic revision of the manuscript. 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==== Front J Insect SciJ. Insect ScijisjisJournal of Insect Science1536-2442Oxford University Press 2588163410.1093/jisesa/iev011iev011ResearchThe Effects of Colony Structure and Resource Abundance on Food Dispersal in Tapinoma sessile (Hymenoptera: Formicidae) Van Weelden M. T. 1Bennett G. Buczkowski G. Department of Entomology, Purdue University, West Lafayette, IN 479061Corresponding author, e-mail: [email protected] Editor: Paulo Oliveira 2015 15 4 2015 15 1 46The odorous house ant, Tapinoma sessile (Say) (Hymenoptera: Formicidae), exhibits a high degree of variation in colony spatial structure, which may have direct and indirect effects on foraging. Protein marking and mark-release-recapture techniques were utilized to examine the effect of colony spatial structure on food dispersal. Sucrose water spiked with rabbit IgG protein was presented to colonies with varying spatial configurations in laboratory and field experiments. In monodomous laboratory colonies, the rate and extent of food dispersal was rapid due to a decrease in foraging area. In polydomous colonies, food dispersal was slower because conspecifics were forced to forage and share food over longer distances. However, over time, food was present in all extremities of the colony. Experiments conducted in the field produced similar results, with nests in close proximity to food yielding higher percentages of workers scoring positive for the marker. However, the percentage of workers possessing the marker decreased over time. Results from this study provide experimental data on mechanisms of food dispersal in monodomous and polydomous colonies of ants and may be important for increasing the efficacy of management strategies against T. sessile and other pest ant species. foragingodorous house antimmunomarkingpolydomyfood dispersal	25881634	PMC4535483	NO-CC CODE	2021-01-05 04:04:48	yes	J Insect Sci. 2015 Apr 15; 15(1):46
==== Front Med Sci MonitMed. Sci. MonitMedical Science MonitorMedical Science Monitor : International Medical Journal of Experimental and Clinical Research1234-10101643-3750International Scientific Literature, Inc. 2643922410.12659/MSM.894640894640Lab/In Vitro ResearchExpression of TP53, BCL-2, and VEGFA Genes in Esophagus Carcinoma and its Biological Significance Wei Wei 1BCWang Yanqin 2CDYu Xiaoming 1DEYe Lan 1EFJiang Yuhua 1FGCheng Yufeng 3ABC1 Cancer Center, The Second Hospital of Shandong University, Jinan, Shandong, P.R. China2 Department of Rehabilitation Medicine, Qilu Hospital of Shandong University, Jinan, Shandong, P.R. China3 Department of Radiotherapy, Qilu Hospital of Shandong University, Jinan, Shandong, P.R. ChinaCorresponding Author: Yufeng Cheng, e-mail: [email protected] Study Design B Data Collection C Statistical Analysis D Data Interpretation E Manuscript Preparation F Literature Search G Funds Collection * These authors contributed equally to this work 2015 06 10 2015 21 3016 3022 13 5 2015 08 6 2015 © Med Sci Monit, 20152015This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported LicenseBackground The pathogenesis of esophagus carcinoma involves a cascade process consisting of multiple factors and accumulation of gene mutations. It is known that vascular endothelial growth factor (VEGF) mainly regulates de novo vascular formation while B-cell lymphoma-2 (BCL-2) gene exerts a tumor-suppressing effect. The prominent expression of VEGFA and BCL-2 genes, along with the most famous tumor-suppressor gene, TP53, raise the possibly of gene interaction. This study therefore investigated the effect and correlation of TP53, BCL-2, and VEGFA genes on cell proliferation and apoptosis of esophagus carcinoma. Material/Methods A total of 30 male rats were prepared by subcutaneous injection of methyl-benzyl-nitrosamine (MBNA) to induce esophagus cancer, along with 30 controlled rats which received saline instead. After 4, 10, 20, or 30 weeks, rats were sacrificed to observe the morphological changes of esophageal mucosa. Cell apoptosis was quantified by terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling (TUNEL) assay. Immunohistochemical (IHC) staining was employed to examine the expression of TP53, BCL-2 and VEGFA genes. Results With the progression of cancer, pathological damages of esophageal tissue aggravated while the cancer cell apoptosis gradually decreased compared to controlled animals. Protein levels of p53, Bcl-2, and VEGF in the model group were significantly elevated at each time point. Positive correlations existed between p53 and Bcl-2 or VEGF. Conclusions Abnormally elevated expression of TP53, BCL-2, and VEGFA genes may participate in the proliferation of esophagus cancer cells in a synergistic manner. MeSH Keywords Barrett EsophagusProto-Oncogene Proteins c-bcl-6Tumor Suppressor Protein p53 ==== Body Background As a common malignant tumor in the digestive tract, esophagus carcinoma derives from a cascade process which is dependent on multiple factors. Cells in the precancerous lesion are able to re-differentiate into normal cells or transform into primary carcinoma. Therefore, elucidation of the molecular mechanism of esophagus carcinoma pathogenesis and the establishment of drug targets are critically important for the prevention and treatment of disease [1,2]. Both the occurrence and progression of malignant tumors are correlated with cell proliferation/apoptosis [3,4], which is mainly regulated by tumor suppressor gene B-cell lymphoma-2 (BCL-2). The over-expression of Bcl-2 proteins in tumor cells may promote the growth of lesions, suggesting the correlation of BCL-2 and biological behavior of esophagus cancer cells [4]. Another tumor suppressor gene, TP53, may induce oncogenesis when it has been mutated. Vascular epithelial growth factor (VEGF) is also known to be related to the invasion and metastasis of malignant tumors. Studies have revealed the expression of TP53, BCL-2, and VEGFA genes in both esophagus tumor and adjacent tissues [5,6], suggesting the biological significance of those 3 factors in esophagus cancer. This study therefore investigated the effect of TP53, BCL-2, and VEGFA genes on the proliferation and apoptosis of esophagus carcinoma cells, via a methyl-benzyl-nitrosamine (MBNA)-induced rat esophagus cancer model, in an attempt to investigate the potential molecular mechanism underlying cancer pathogenesis. Material and Methods Animal model A cohort of 60 male Wistar rats (7~8 weeks old, body weight between 180 and 200 grams) were provided by the Animal Center of the Institute of Oncology, Shandong University. Animals were kept in a specific pathogen-free (SPF) grade facility with food and water provided ad libitum. The experimental protocol was pre-approved by the ethics committee of our hospital. After acclimation, all animals were randomly divided into control and model groups (N=30 each). Rats in the model group received bi-weekly subcutaneous injection of 0.15% MBNA (at 3.5 mg/kg, Sigma, USA) in 0.9% saline. Controlled animals received an equal volume of 0.9% saline. General condition of animals was daily observed during the whole experiment, which lasted 30 consecutive weeks. Morphological observation Animals from both groups were sacrificed at week 4, 10, 20, and 30 (N=7 each). Esophagus tissues were longitudinally dissected for examination of mucosa morphology. Tissue samples were then fixed in 10% neutral buffered formalin (NBF) solution and stained by HE method. Microscopic morphology was then examined under a light-field microscope. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling (TUNEL) assay We also quantified the condition of tumor cell apoptosis using a TUNEL kit (Boster Biotech, China) following the manual instruction. A total of 100 nuclei were observed from each of 5 randomly selected fields from 1 slide. The apoptosis index (AI) was calculated as: number of apoptotic cells/total cell number ×100%. Immunohistochemical (IHC) staining Esophagus tissues were fixed in NBF, embedded in paraffin, and sectioned. After dewaxing and rehydration, mouse anti-p53 monoclonal antibody (1:100, Boster Biotech, China), mouse anti-Bcl-2 monoclonal antibody (1:60, Boster Biotech, China), or mouse anti-VEGF monoclonal antibody (1:100, Boster Biotech, China) were added for overnight incubation. Goat anti-mouse IgG secondary antibody conjugated with horseradish peroxidase (Boster Biotech, China) was applied, followed by color development using DAB substrate. Positive signals were deduced as yellow/brown granules in the cytoplasm (for VEGF and Bcl-2) or the nucleus (for p53). The relative staining intensity was calculated by the averaged optical density (OD) value. The staining scoring is described in Table 1. Statistical analysis The SPSS 19.0 software package was used to analyze all collected data, of which measurement data with normal distribution are presented as mean ± standard deviation (SD). Between-group comparison was done by t-test or one-way analysis of variance (ANOVA). Significance of correlation between parameters was performed by Spearman correlation analysis. A statistical significance was defined when p<0.05. Results Morphological features of esophagus mucosa At each time point, control rats had smooth mucosa with no significant abnormality in the esophagus (Figure 1A). After 4 weeks, small granules occurred in the rough mucosa of rat esophagus in the model group (Figure 1B). Papillomas were observed from esophagus mucosa from 10 weeks, and were aggravated at 20 and 30 weeks. We further checked the morphological features of esophagus mucosa under the microscope. Control rats had a basal cell layer in a regular arrangement, with evenly distributed epithelial cells (Figure 2A). Model rats, however, had inflammation from 4 weeks, as shown by irregular arranged epithelial cells, hyperplasia nucleus containing granular chromatin, and unclear cell border (Figure 2B). Esophagus tissue at 10 weeks had atypical proliferation, loosely arranged cells, and enlarged nuclei containing granular chromatin (Figure 2C). At 20 weeks, atypical proliferation was aggravated, as shown by cells with discrete sizes, enlarged and darkened nuclei, and abnormal nucleus/cytoplasm ratio (Figure 2D). After 30 weeks, features of squamous carcinoma could be identified by irregular cell arrangement, abnormal cell shape, elevated chromatin, and nuclei with dark color and abnormal shape (Figure 2E). Expression of p53, Bcl-2 and VEGF proteins IHC staining results showed that Bcl-2 proteins were mainly identified in the cell membrane and cytoplasm, VEGF proteins existed in the cytoplasm, and p53 proteins occurred in the nucleus. In the control group, all those 3 proteins had lower expression levels (Figures 3A, 4A, and 5A). In the model group, after 4 weeks of MBNA-induction, expression levels of p53, Bcl-2, and VEGF proteins were all elevated (Figures 3B, 4B, and 5B). With the progression of the disease, expression levels of those 3 proteins were further elevated (Figures 3C–3E, 4C–4E, and 5C–5E) in a time-dependent manner. Cell apoptosis With the elongation of induction, model rats had decreased levels of esophagus carcinoma cells, as supported by the significantly suppressed AI compared to the control group (p<0.05, Figure 6). Correlation analysis We analyzed the correlation among p53, Bcl-2, and VEGF proteins in esophagus carcinoma tissues. Results showed a significant positive correlation between p53 and Bcl-2 or VEGF proteins (r=0.753 and 0.762, p<0.05 in both cases). The correlation between Bcl-2 and VEGF proteins was not significant (r=0.109, p>0.05). Discussion The pathogenesis of esophagus carcinoma is closely related with the imbalance of cell apoptosis. Under normal physiological conditions, a dynamic balance exists between cell proliferation and apoptosis. The dysregulation of apoptosis, therefore, may lead to various diseases, including malignant tumors. Actually, it has been well established that the inactivation of tumor-suppressing genes and activation of oncogenes are molecular mechanisms underlying the immortality of cancer cells. Tumor-suppressor genes inhibit the formation of tumors via its participation in cell cycle-related signal transduction, cell apoptosis, and differentiation regulation. It has been shown that inhibition of cell apoptosis play an important role in the pathogenesis of esophagus carcinoma [7,8]. The most widely studied tumor-related genes in esophagus cancer are TP53 and BCL-2 genes. As an anti-apoptotic gene, the BCL-2 gene locates at chromosome 14q18 and encodes a membrane protein to prolong the cell survival period. Bcl-2 protein is an oncogene that inhibits cell death due to various causes. The over-expression of BCL-2 gene in tumor cells can cause resistance to chemotherapy. The elevated expression of BCL-2 gene in esophagus carcinoma cells may sustain the cell cycle via its inhibition on intracellular Ca2+, blocking nucleus transportation and anti-oxidation [9,10]. The wild-type TP53 gene encodes p53 protein, which can suppress tumor formation by its surveillance on cell growth and genome, along with the induction of apoptosis of cells with mutated DNA. Bcl-2 protein expression can be down-regulated by wild-type TP53 gene to facilitate cell apoptosis, accompanied with elevated expression of bax proteins [11,12]. VEGF, on the other hand, directly induces the tumor angiogenesis by the elevated permeability of micro-vessels, thereby providing a favorable environment for endothelial cell proliferation. The highly specific induction of vascular endothelial cell mitosis by VEGF directly facilitates de novo formation of tumor vessels. It has been reported that both tumor proliferation and invasion are correlated with elevated VEGF expression, which can be regulated by the TP53 gene [13,14]. Various oncology studies all suggested the correlation between TP53 and BCL-2/VEGFA expression and the existence of a possibly shared transcriptional activation pathway. This study thus investigated the role and correlation of p53, Bcl-2, and VEGF in the apoptosis of esophagus carcinoma. Our results showed significantly lowered apoptosis level in the esophagus model group compared to control animals. With the disease progression, esophagus cell apoptosis decreased gradually, as shown by lowered AI, suggesting the potential involvement of apoptosis in esophagus cancer. Compared to those in the control group, model rats had elevated expression of Bcl-2, p53, and VEGF proteins in their esophagus tissues, in addition to the existence of inflammatory lesions and atypical hyperplasia. Expression of these proteins also increased with the progression of disease, in a time-dependent manner. All those results suggest the involvement of these proteins in the dysregulation of cell apoptosis and further tumor progression. In esophagus carcinoma tissues, TP53 gene expression level was positively correlated with Bcl-2 or VEGF proteins, suggesting the role of p53 in the directing apoptotic pathway in the occurrence of esophagus cancer. Because elevated Bcl-2 may keep cells in the active cycle and decrease apoptotic level, it can increase tumor cell proliferation. VEGF also facilitates tumor progression by regulating angiogenesis. Our results thus suggest the synergistic effect of TP53, BCL-2, and VEGFA genes in esophagus cancer occurrence. The possible cellular pathway among those proteins has been reported previously: Bcl-2 altered the redox status of mitochondrial and membrane potential, thus releasing apoptotic protein precursor Apaf-1, cytochrome C and activating caspase-9; p53 induced the death receptor via cascade activation of Fas, DR5 and DR4 to facilitate cell apoptosis [15,16]. p53 also regulated the endogenous apoptotic pathway as an upstream factor of the Bax/Bak pathway to mediate Bcl-2 protein expression [17,18]. As the downstream effector of p53 gene, both Bax and Bcl-2 can be selectively regulated by p53. The imbalance between Bcl-2 and Bax gene expression may favor the occurrence of esophagus cancer. Further studies supported the role of p53 in tumor angiogenesis via its regulation of VEGF expression [19,20]. Our study provided further evidence, as the expression of p53 was closely related with Bcl-2 in esophagus carcinoma tissues, suggesting potential synergistic effects. Previous study of genomic screening in esophagus cancer patients revealed the loss of chromosome 17p, where TP53 gene localizes in cancer patients, suggesting the genetic grounds of oncogenesis [21]. Moreover, the gene polymorphism of the TP53 gene may also reside in the tumor pathogenesis as the so-called unique tumor principle, in which each individual tumor may have various tumor suppressor gene polymorphisms, in addition to certain epigenetic regulations, such as methylation patterns [22]. Therefore, the expressional regulation of TP53 gene is of crucial importance in the development of esophagus tumor. Our study provides further evidence regarding the role of TP53 in BCL-2 gene expression regulation. Such selective modulation of TP53 on BAX and BCL-2 gene expression may be affected by environmental factors, leaving the detailed cellular pathway unclear. It is commonly believed that TP53 gene mutation and protein deposition occur early in the oncogenesis of esophagus cancer. The wild-type TP53 gene may inhibit tumor angiogenesis via its down-regulation of VEGF proteins; however, mutant p53 can facilitate the proliferation and metastasis of tumor cells. These phenomena suggest the existence of a common pathway that is regulated by both p53 and VEGF. The illustration of a synergistic effect between VEGF and p53 proteins requires further knowledge about the role of p53 in mediating tumor angiogenesis [20]. It has been postulated that p53 may regulate tumor vascular formation via the increase of protein kinase C activity and modulating VEGF expression. Wild-type TP53 gene may inhibit VEGF expression via negative feedback in its transcription, thereby inhibiting tumor vessel formation. Mutant TP53 gene, however, loses its ability to inhibit VEGF transcription, as does the inhibition of vascular formation and tumor invasiveness. Our results revealed that a time-dependent relationship between p53 and Bcl-2 or VEGF protein levels, suggesting their close relationship with tumor cell proliferation and apoptosis. Conclusions The abnormally elevated expression of TP53, BCL-2, and VEGFA genes in esophagus carcinoma suggest potential synergistic effects between these proteins, which may all participate in the proliferation and apoptosis of tumor cells. Source of support: Departmental sources Figure 1 Morphological changes of rat esophagus. Representative images of rat esophagus from control (A) and model (B) groups. Magnification, ×10. Figure 2 Pathological alternations of rat esophagus by HE staining. (A) Control group: (B) Model group at 4 weeks; (C) Model group at 10 weeks; (D) Model group at 20 weeks; (E) Model group at 30 weeks. Magnification, ×200. Figure 3 Expression of Bcl-2 proteins by IHC staining. (A) Control group: (B) Model group at 4 weeks; (C) Model group at 10 weeks; (D) Model group at 20 weeks; (E) Model group at 30 weeks. Magnification, ×200. Figure 4 Expression of VEGF proteins by IHC staining. (A) Control group: (B) Model group at 4 weeks; (C) Model group at 10 weeks; (D) Model group at 20 weeks; (E) Model group at 30 weeks. Magnification, ×200. Figure 5 Expression of p53 proteins by IHC staining. (A) Control group: (B) Model group at 4 weeks; (C) Model group at 10 weeks; (D) Model group at 20 weeks; (E) Model group at 30 weeks. Magnification, ×200. Figure 6 Apoptosis of esophagus carcinoma cells. The condition of cell apoptosis in both control and model rat esophagus tissues is presented as apoptosis index (AI). * p<0.05 compared to controlled animals at the same time point. Table 1 Standards and scores of IHC staining. Intensity score Percentage score Staining score=(1)×(2) Score Criteria Score Criteria Score Description 0 No staining 0 No positive cell 0 Negative (−) 1 Light yellow 1 0~25% positive cells 1~3 Weak positive (+) 2 Brown yellow 2 26~50% positive cells 4~5 Moderate (++) 3 Dark brown 3 51~75% positive cells 6~7 Strong (+++) 4 >75% positive cells ==== Refs References 1 Zhang J Zhu Z Liu Y Diagnostic value of multiple tumor markers for patients with esophageal carcinoma PLoS One 2015 10 2 e0116951 25693076 2 Baba Y Ishimoto T Harada K Molecular characteristics of basaloid squamous cell carcinoma of the esophagus: Analysis of KRAS, BRAF , and PIK3CA mutations and LINE-1 methylation Ann Surg Oncol 2015 [Epub ahead of print] 3 Ning Z Zhu H Li F Tumor suppression by miR-31 in esophageal carcinoma is p21-dependent Genes Cancer 2014 5 11–12 436 44 25568668 4 Lv H Liu R Fu J Epithelial cell-derived periostin functions as a tumor suppressor in gastric cancer through stabilizing p53 and E-cadherin proteins via the Rb/E2F1/p14ARF/Mdm2 signaling pathway Cell Cycle 2014 13 18 2962 74 25486483 5 Nason KS Predicting response to neoadjuvant therapy in esophageal cancer with p53 genotyping: a fortune-teller’s crystal ball or a viable prognostic tool? J Thorac Cardiovasc Surg 2014 148 5 2286 87 25444198 6 Chang Z Zhang W Chang Z Expression characteristics of FHIT , p53 , BRCA2 and MLH1 in families with a history of oesophageal cancer in a region with a high incidence of oesophageal cancer Oncol Lett 2015 9 1 430 36 25436004 7 Yao W Qin X Qi B Association of p53 expression with prognosis in patients with esophageal squamous cell carcinoma Int J Clin Exp Pathol 2014 7 10 7158 63 25400812 8 Zhang H Xia J Wang K Zhang J Serum autoantibodies in the early detection of esophageal cancer: a systematic review Tumour Biol 2015 36 1 95 109 25433500 9 Shang L Liu HJ Hao JJ A panel of overexpressed proteins for prognosis in esophageal squamous cell carcinoma PLoS One 2014 9 10 e111045 25337715 10 Buas MF Levine DM Makar KW Integrative post-genome-wide association analysis of CDKN2A and TP53 SNPs and risk of esophageal adenocarcinoma Carcinogenesis 2014 35 12 2740 47 25280564 11 Xu XL Zheng WH Tao KY p53 is an independent prognostic factor in operable esophageal squamous cell carcinoma: a large-scale study with a long follow-up Med Oncol 2014 31 11 257 25270283 12 Appelman HD Matejcic M Parker MI Progression of esophageal dysplasia to cancer Ann NY Acad Sci 2014 1325 96 107 25266019 13 Huang K Chen L Zhang J Elevated p53 expression levels correlate with tumor progression and poor prognosis in patients exhibiting esophageal squamous cell carcinoma Oncol Lett 2014 8 4 1441 46 25202347 14 Kandioler D Schoppmann SF Zwrtek R The biomarker TP53 divides patients with neoadjuvantly treated esophageal cancer into 2 subgroups with markedly different outcomes. A p53 Research Group study J Thorac Cardiovasc Surg 2014 148 5 2280 86 25135238 15 Yu JP Lu WB Wang JL Pathologic response during chemo-radiotherapy and variation of serum VEGF levels could predict effects of chemo-radiotherapy in patients with esophageal cancer Asian Pac J Cancer Prev 2015 16 3 1111 16 25735340 16 Xu YW Peng YH Chen B Autoantibodies as potential biomarkers for the early detection of esophageal squamous cell carcinoma Am J Gastroenterol 2014 109 1 36 45 24296751 17 Yang Z Wang YG Su K VEGF-C and VEGF-D expression and its correlation with lymph node metastasis in esophageal squamous cell cancer tissue Asian Pac J Cancer Prev 2015 16 1 271 74 25640364 18 Omoto I Matsumoto M Okumura H Expression of vascular endothelial growth factor-C and vascular endothelial growth factor receptor-3 in esophageal squamous cell carcinoma Oncol Lett 2014 7 4 1027 32 24944663 19 Yang PW Hsieh MS Huang YC Genetic variants of EGF and VEGF predict prognosis of patients with advanced esophageal squamous cell carcinoma PLoS One 2014 9 6 e100326 24945674 20 Song HY Deng XH Yuan GY Expression of bcl-2 and p53 in induction of esophageal cancer cell apoptosis by ECRG2 in combination with cisplatin Asian Pac J Cancer Prev 2014 15 3 1397 401 24606472 21 Dulak AM Schumacher SE van Lieshout J Gastrointestinal adenocarcinomas of the esophagus, stomach and colon exhibit distinct patterns of genome instability and oncogenesis Cancer Res 2012 72 17 4383 93 22751462 22 Ogino S Fuchs CS Giovannucci E How many molecular subtypes? Implications of the unique tumor principle in personalized medicine Expert Rev Mol Diagn 2012 12 6 621 28 22845482	26439224	PMC4601357	NO-CC CODE	2021-01-05 04:29:21	yes	Med Sci Monit. 2015 Oct 6; 21:3016-3022
==== Front Med Sci MonitMed. Sci. MonitMedical Science MonitorMedical Science Monitor : International Medical Journal of Experimental and Clinical Research1234-10101643-3750International Scientific Literature, Inc. 2647403110.12659/MSM.894485894485Clinical ResearchCorrelation Between High-Density Lipoprotein and Monocyte Subsets in Patients with Stable Coronary Heart Disease Jiang Shaoyan 1ABCLi Dan 2DELi Jian 2FGAn Yi 2EFG1 Department of Cardiology, The Affiliated Cardiovascular Hospital of Qingdao University, Qingdao, Shandong, P.R. China2 Department of Cardiology, The Affiliated Hospital of Qingdao University, Qingdao, Shandong, P.R. ChinaCorresponding Author: Yi An, e-mail: [email protected] Study Design B Data Collection C Statistical Analysis D Data Interpretation E Manuscript Preparation F Literature Search G Funds Collection 2015 16 10 2015 21 3129 3135 28 4 2015 08 6 2015 © Med Sci Monit, 20152015This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported LicenseBackground High-density lipoprotein (HDL) consists of heterogeneous particles with a variety of structures and functions. Its role in atherosclerosis has been gradually recognized. Studies have shown dysfunction of small HDL in patients with coronary artery disease (CAD). Monocytes play an important role in atherosclerosis, which can be divided into 3 subgroups based on the expression of surface markers CD14 and CD16. This study aimed to investigate the association between HDL and monocyte subsets in CAD patients. Material/Methods A total of 90 patients with stable CAD were selected in this study. Monocytes were divided into classical monocytes (CM, CD14++CD16−), intermediate monocytes (IM, CD14++CD16+), and non-classical monocytes (NCM, CD14+CD16++). HDL components in serum were determined by high-resolution polyacrylamide gel electrophoresis (detected by Quantimetrix HDL Lipoprint system, referring to HDL subfractions analysis: A new laboratory diagnostic assay for patients with cardiovascular diseases and dyslipoproteinemia). Results Serum level of small HDL was positively correlated with circulating proinflammatory NCM (r=0.30; p=0.004), negatively correlated with CM, and not correlated with IM. We also found that disease severity was not associated with diabetes mellitus, glycosylated hemoglobin, hypertension, smoking history, or statin dosage. Conclusions Our study confirmed that small HDL level is associated with an increase in NCM and a decrease in CM, suggesting the proinflammatory relationship between small HDL and intrinsic immune function during the progression of stable CAD. MeSH Keywords HypoalphalipoproteinemiasIntracranial ArteriosclerosisRibosome Subunits, Small, Archaeal ==== Body Background Cardiovascular disease remains the leading cause of death in elderly patients worldwide despite the rapid development of cardiovascular drugs. Several clinical and epidemiological researches have indicated that the level of high-density lipoprotein (HDL) is closely related to cardiovascular disease. Studies on the treatment of such cardiovascular disease have been focused on how to improve HDL level [1]. Nevertheless, increasing HDL level by cholesteryl ester transfer protein (CETP) inhibitor has not reduced the risk of coronary heart disease, but increased the morbidity and mortality rate in patients with such disease [2,3]. Some researchers have suggested that the function of HDL might have been damaged in this pathological environment; therefore, it is crucial to ensure its function. HDL consists of heterogeneous lipoprotein particles characterized by specific structures, metabolic functions, and atherosclerosis-resistance. Small HDL in healthy people has been confirmed to impact the progression of atherosclerosis by increasing the consumption of cholesterol, as well as antioxidant and anti-inflammatory responses. However, its function is abnormal in patients with atherosclerosis and dyslipidemia [4–6]. Clinical research shows that small HDL is associated with the incidence and severity of CAD, whereas large molecular HDL showed a negative correlation. Monocytes play an important role in the inflammatory reaction during atherosclerosis. Monocytes can be divided into 3 subgroups based on the expression of surface CD14 and CD16: classical monocytes (CM, CD14++CD16−), intermediate monocytes (IM, CD14++CD16+), and non-classical monocytes (NCM, CD14+CD16++). Both IM and NCM are proinflammatory cells, whose proportion is related to the occurrence of CAD, intima-media thickness, and plaque stability. Total cholesterol, low-density lipoprotein (LDL) cholesterol, and triglycerides are related to proinflammatory NCM, whereas HDL has a negative correlation. A previous study on 900 cases of CAD patients showed that IM has a predictive value for cardiovascular disease [7–11]. In the current study, we explored the correlation between different HDL components and monocytes subsets in CAD patients. Material and Methods Patients and study design All subjects were CAD patients treated in our hospital between September 2009 and August 2010. Inclusion criteria were: age above 18 years and, with stable CAD diagnosed by selective coronary angiography. Exclusion criteria were: recent occurrence of acute coronary syndrome with ST segment elevation myocardial infarction, non-ST elevation myocardial infarction, or unstable angina, a history of percutaneous coronary intervention (PCI), cardiac failure, cancer, and acute or chronic liver or renal failure. All selected patients provided signed informed consent. The study was approved by the Institutional Ethics Committee in our hospital. Blood sample Before the selective coronary arteriography, a blood sample was collected from the antecubital vein of each patient, using 3.8% sodium citrate tube, serum separation tube, and EDTA tube (Greiner Bio-One, Frickenhausen, Germany) and centrifuged at 3000 rpm at 4°C for 15 min. Laboratory detection The levels of granulocyte colony-stimulating factor (g-CSF), macrophage colony-stimulating factor (M-CSF), and interleukin-6 (IL-6) were determined by enzyme-linked immunosorbent assay (ELISA; R&D Systems, Minneapolis, MN). The levels of IL-10 and granulocytes-macrophage colony-stimulating factor (GM-CSF) were determined by Luminex assay (R&D Systems, catalog number FCST03). Flow cytometry White blood cells and monocyte subsets were detected by flow cytometry. The staining and gating of cells is shown in Figure 1. CD45-PerCP monoclonal antibody (BD Biosciences, catalog number 345809, San Diego, CA, USA), CD14-FITC monoclonal antibody (BD Biosciences, catalog number 345784), CD16-APC-H7 monoclonal antibody (BD Biosciences, catalog number 560195), CD3-APC monoclonal antibody (BD Biosciences, catalog number 345767), CD19 (BD Biosciences, catalog number 345791), CD56 (Beckton Dickinson, catalog number 341027), and isotype control were used for staining. The cells were incubated in the dark for 15 min, mixed with 1.5 ml of lysate (BD FACS lysing solution, BD Biosciences), incubated in the dark for another 15 min, and washed 3 times with PBS. Cells were resuspended in 3 ml of fixing solution and analyzed by flow cytometry. Data analyses were performed using FACS Canto II and FACS Diva software (BD Biosciences). CD45+CD3− and CD19–CD56− cells with specific forward scatter (FSC) and side scatter (SSC) were monocytes. The monocytes were divided into CM, IM, and NCM as previously described. The absolute number of monocytes was calculated based on the number of white blood cells and CD45+ cells detected in flow cytometry. Detection of lipid The serum levels of total cholesterol, HDL, LDL, and triglyceride were determined. HDL subgroup components were quantified using the Quantimetrix HDL Lipoprint System® (Quantimetrix Corporation, Redondo Beach, CA, USA). HDL was divided into 10 subgroups based on their locations on SDS-PAGE: 1–3, 4–7, and 8–10 represented large, medium, and small HDL particles, respectively (Figure 2). Statistical analysis All statistical analyses were performed using SPSS20.0 software (Chicago, IL, USA). Classified variables are expressed as count or percentage and compared by χ2 or Fisher exact tests. Numerical data are presented as mean±standard deviation (χ̄±S) and analyzed by one-way ANOVA. Deflection data were compared by ANOVA after logarithmic transformation. Relevance was determined by Pearson correlation analysis. Three subgroups (CM, IM, and NCM) were incorporated into the linear regression model. These subgroups were also incorporated into the model when the clinical features, statins usage, or lipid parameters were correlated with monocyte subgroups or small HDL level (p<0.2). P values smaller than 0.05 were considered significantly different. Results Patient information A total of 90 patients diagnosed as having stable CAD by angiography were enrolled in this study, including 72 males (80%) and 21 smokers (23%). The mean age of the patients was 64.1±10.0 years old. Among these patients, a total of 25, 36, and 29 cases suffered from single-, 2-, and 3-vessel coronary arterial disease, respectively; 31% and 52% patients received high- and low-dose statin treatment, respectively and 17% patients received no statin therapy. Correlation between monocytes subgroups and serum level of small HDL Monocyte subsets were analyzed by flow cytometry (Figure 1). The mean density of CM, NCM, and IM was 270.5±142.7 cells/μl (82.1±6.7%), 39.7±28.9 cells/μl (12.3±5.9%), and 18.7±15.1 cells/μl (5.6±3.3%), respectively. Serum level of small HDL was negatively correlated with circulating CM (r=−0.33, p=0.001; Figure 3A), positively correlated with NCM (r=0.30, p=0.004; Figure 3B), and not correlated with IM (r=0.14, p=0.20; Figure 3C). As shown in Table 1, no correlation was observed between monocyte subsets and medium HDL, large HDL, and total HDL. Linear regression analysis revealed that small HDL was independently correlated with proinflammatory NCM and circulating CM compared with other lipid parameters, risk factors, and statin. As shown in Table 2, IM was only correlated with total cholesterol and LDL. CM value was the lowest (79.3±7% vs. 83.7±6% and 83.9±6%; p=0.004; Figure 4A) with the serum level of small HDL at the high tertiles (13–20 mg/L) compared with that at medium (9–12 mg/L) and low tertiles (2–8 mg/L). Furthermore, the density of proinflammatory NCM was highest (14.7±7% vs. 10.7±5% and 10.8±5%; p=0.006; Figure 4B) in patients with small HDL at the high tertiles, whereas IM was not correlated with the tertiles of small HDL (5.9±3% vs. 5.6±3% vs. 5.3±3%; p=0.54; Figure 4C). Correlation between HDL subsets with lipid parameters and cardiovascular risk factors Small HDL level was significantly correlated with triglycerides, VLDL, LDL, and total cholesterol (Table 3), but no association was observed between small HDL level and total HDL level. Large HDL was negatively correlated with small HDL, VLDL, and triglycerides, and was highly associated with total cholesterol. Medium HDL was correlated with large HDL, LDL, and VLDL, but not with triglycerides. Medium HDL level (28.8±7.1 vs. 23.7±5.8 mg/dl; p=0.002) and large HDL level (19.3±11.1 vs. 10.7±5.7 mg/dl; p=0.005) in female patients was significantly higher compared with male patients, whereas there was no significant difference in small HDL between female and male patients (11.3±3.6 vs. 11.3±4.3 mg/dl; p=0.99). We also found that large HDL was negatively correlated with weight (r=−0.28; p=0.008). In addition, diabetes mellitus, glycosylated hemoglobin, hypertension, and smoking history were not related to HDL subgroup, statin dosage, or severity of disease. Association between HDL subgroups with colony-stimulating factor and inflammation markers Serum level of small HDL was significantly correlated with granulocyte colony-stimulating factor (G-CSF; r=0.22, p=0.05) but not with granulocyte macrophage colony-stimulating factor (GM-CSF; r=0.05, p=0.66) or macrophage colony-stimulating factor (M-CSF; r=−0.09, p=0.37). Medium, large, and total HDLs were not related to any of the 3 types of CSF. Serum level of small HDL was not associated with proinflammatory marker hsCRP (r=−0.05; p=0.64), IL-6 (r=−0.10; p=0.38) or IL-10 (r=0.06; p=0.62). In addition, intermediate-density lipoprotein, large HDL, and total HDL were not related to hsCRP, IL-6, or IL-10. Neither of the monocyte subsets were correlated with hsCRP (CM: R=−0.11, p=0.32; IM: R=0.14, p=0.19; NCM: R=0.04, p=0.68), IL-6 (CM: R=0.06, p=0.59; IM: R=0.13, p=0.24; NCM: R=−0.14, p=0.24), or IL-10 (CM: R=0.01,p=0.94; IM: R=−0.01, p=0.98; NCM: R=−0.01, p=0.94). Discussion Manyepidemiological and prospective studies have clearly shown that serum HDL level is negatively correlated with the risk of coronary heart disease. HDL exerts a variety of protective effects on arteries, including cholesterol outflow, antioxidation, anti-inflammation, cell protection, vasodilator, and antithrombosis [12]. Moreover, several studies have also confirmed that small HDL particles can potentially prevent atherosclerosis. During dyslipidemia, including elevation of triglycerides or total cholesterol, small HDL level was significantly increased, whereas the number of large HDL particles was substantially reduced, leading to significant change in HDL metabolism and distribution of subsets. This study confirmed that serum level of small HDL was correlated with lipid index, such as total cholesterol, LDL, VLDL, and triglyceride, but not with total HDL, lipoprotein, or statin usage in 90 patients with stable CAD diagnosed by angiography. The level of small HDL changes in patients with dyslipidemia or obesity and in patients with cardiovascular disease [13]. A study of 115 patients with CAD suggested that large HDL level was significantly increased, as revealed by coronary angiography. Another 10-year follow-up study covering 1000 patients revealed that small and large HDLs have prognostic value to the progression of ischemic heart disease [14–16]. The incidence of CAD in females is more closely related to smaller HDL particles. Furthermore, small HDL level in patients with acute ischemic shock is significantly higher than that in healthy populations. A clinical study of 60 patients has confirmed that small HDL is associated with non-calcified plaques by coronary artery CT and intravascular ultrasound. In another study, covering 102 patients with myocardial infarction and 200 healthy controls, large and middle HDL are negatively correlated with early-stage acute myocardial infarction, whereas small HDL level is up-regulated in young patients with acute myocardial infarction. Furthermore, small HDL level is also increased in patients with acute coronary syndrome, whereas large HDL level is reduced [17,18]. The heterogeneity of monocytes and its association with atherosclerosis has been confirmed by the detection of expression of surface markers CD14 and CD16. The number of CD16+ monocytes is increased in acute and chronic inflammation and atherosclerosis, which are rapidly activated by the stimulation of inflammation [19]. Our study demonstrated that the increase in small HDL level was associated with the distribution of proinflammatory monocyte subsets in patients with stable CAD. Specifically, serum level of small HDL was positively correlated with cell density of NCM (CD14+CD16++), negatively correlated with CM (CD14++CD16−), and not correlated with IM (CD14++CD16+). Several studies have shown that the correlation between total HDL or LDL and monocyte subsets disappears after adjusting for BMI. In this study, the correlation between small HDL and monocyte subsets was independent of age, sex, smoking history, BMI, diabetes, and statin usage. The patients were divided into 3 subgroups based on the level of small HDL. Monocyte subsets significantly promoted atherosclerosis and other inflammatory responses in the group with the highest level of small HDL. In addition, the proportion of NCM was increased while that of CM was decreased. However, monocyte subsets were not relevant to serum CRP, IL-6, or IL-10. Currently, the association between CD16+ monocytes and hsCRP level remains controversial. While some studies report that CD16+ monocytes were correlated with hsCRP level in patients with unstable angina, others have shown that CD16+ monocytes were related to TNF-α level instead of the level of hsCRP or IL-6 [20]. Furthermore, it has been confirmed that several colony-stimulating factors (CSFs) are expressed in various vascular cells, which can affect the progression of atherosclerosis by regulating macrophage phenotype and cholesterol intake [21]. In this study, the plasma level of G-CSFs was significantly correlated with small HDL. The association between HDL subgroups and monocyte subsets was only analyzed at a certain time point; therefore, their association with functional changes in the process of atherosclerosis cannot be determined. Conclusions Our study has demonstrated that small HDL level is associated with an elevation in IM and a reduction in CM, revealing the proinflammatory correlation between small HDL and intrinsic immune function in stable CAD. Source of support: Departmental sources Figure 1 Identification of monocyte subsets. Monocytes were defined as CD45+ cells (B) with typical FSC and SSC (A). CD3−, CD19−, and CD56− labeled cells (C) were excluded for possible T cell contamination. CD45+CD3/19/56− cells were monocytes (D). Figure 2 Quantimetrix HDL Lipoprint system separate and quantify HDL subgroup component. Figure 3 Correlation between serum level of small HDL and monocyte subsets. Figure 4 Correlation between level grade of small HDL and monocyte subsets. Table 1 Correlation between HDL subgroups and circulating monocyte subsets. Monocyte subgroup CM CD14++CD16− IM CD14++CD16+ NCM CD14+CD16++ R P value R P value R P value Total HDL −0.08 0.45 −0.06 0.60 0.12 0.25 Small HDL −0.33 0.001 0.14 0.20 0.30 0.004 Medium HDL −0.05 0.66 −0.32 0.76 0.07 0.50 Large HDL 0.06 0.55 −0.12 0.26 −0.01 0.96 The significant differences are indicated in bold. Table 2 Multivariable regression model for the correlation between small HDL and monocyte subsets. CM CD14++CD16− IM CD14++CD16+ NCM CD14+CD16++ Univariate P value β P value Univariate P value β P value Univariate P value β P value Small HDL 0.001 −0.33 0.006 0.20 0.21 0.08 0.004 0.27 0.002 Statin 0.21 0.14 0.19 0.45 −0.07 0.49 0.31 −0.12 0.25 Serum 0.36 −0.11 0.35 0.19 −0.18 0.12 0.07 0.23 0.05 VLDL 0.21 0.18 0.48 0.22 0.07 0.78 0.47 −0.25 0.32 Age 0.37 0.09 0.50 0.98 0.23 0.09 −0.23 −0.21 0.08 LDL 0.02 −0.19 0.56 0.05 0.80 0.01 0.11 −0.23 0.45 BMI 0.84 0.04 0.71 0.73 0.02 0.84 0.67 −0.06 0.58 Total cholesterol 0.02 −0.10 0.77 0.18 −0.86 0.01 0.06 0.60 0.08 Smoking history 0.51 −0.03 0.78 0.02 0.32 0.006 0.61 −0.14 0.21 Hypertension 0.49 −0.03 0.81 0.12 0.16 0.15 0.94 −0.06 0.60 Triglyceride 0.71 0.06 0.81 0.74 0.22 0.31 0.82 −0.19 0.39 Gender 0.96 0.01 0.99 0.12 0.14 0.28 0.42 −0.08 0.54 Total model R2 21% 0.040 24% 0.035 25% 0.031 Table 3 Correlation between HDL subgroups and lipid index. Small HDL Medium HDL Large HDL R P value R P value R P value HDL 0.060 0.56 0.68 <0.0001 0.80 <0.0001 Total cholesterol 0.39 <0.0005 0.60 <0.0001 0.24 0.023 LDL 0.30 <0.005 0.43 <0.0001 0.11 0.30 VLDL 0.42 <0.0001 0.22 0.042 −0.22 0.035 Triglycerides 0.36 <0.005 0.02 0.86 −0.37 <0.0005 Small HDL – 0.01 0.91 −0.26 0.014 Medium HDL 0.01 0.91 – 0.63 <0.0001 Large HDL −0.26 0.014 0.63 <0.0001 – HDL – high density lipoprotein; LDL – low-density lipoprotein; VLDL – very low density lipoprotein. The significant differences are indicated in bold. ==== Refs References 1 Barter P Gotto AM LaRosa JC Treating to New Targets I. 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==== Front Med Sci MonitMed. Sci. MonitMedical Science MonitorMedical Science Monitor : International Medical Journal of Experimental and Clinical Research1234-10101643-3750International Scientific Literature, Inc. 2658148810.12659/MSM.894476894476Animal StudyMarchantin M Induces Apoptosis of Prostate Cancer Cells Through Endoplasmic Reticulum Stress Zhang Tian-Wei 12ABDXing Li 2FGTang Jun-Long 34FGLu Jing-Xiao 34DELiu Chun-Xiao 5BCD1 Department of Urology, The Third Affiliated Hospital of Guangzhou Medical University, Guangzhou, Guangdong, P.R. China2 The First Clinical Medical College, Southern Medical University, Guangzhou, Guangdong, P.R. China3 Shenzhen Tumor Immuno-Gene Therapy Clinical Application Engineering Lab, Shenzhen Second People’s Hospital, The First Affiliated Hospital of Shenzhen University, Shenzhen, Guangdong, P.R. China4 Department of Urology, Shenzhen Second People’s Hospital, The First Affiliated Hospital of Shenzhen University, Shenzhen, Guangdong, P.R. China5 Department of Urology, Zhujiang Hospital, Second Affiliated Hospital of Southern Medical University, Guangzhou, Guangdong, P.R. ChinaCorresponding Author: Chun-Xiao Liu, e-mail: [email protected] Study Design B Data Collection C Statistical Analysis D Data Interpretation E Manuscript Preparation F Literature Search G Funds Collection 2015 19 11 2015 21 3570 3576 28 4 2015 22 7 2015 © Med Sci Monit, 20152015This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported LicenseBackground Apoptosis is mediated by the endoplasmic reticulum (ER) stress pathway, mitochondrial pathway, and death receptor. Data herein suggested an inhibitory effect of marchantin M on tumor formation in nude mice as well as the impact on CHOP and GRP78 expression. Material/Methods The role of marchantin M on proliferation and apoptosis of DU145 cells were measured by MTT and flow cytometry, respectively. Western blot was applied to detect the expression of GRP78 and CHOP. The mice received abdominal injection at 1 time/2 d and 2 ml/time. Tumor volume was measured every 6 days. The mice were euthanatized 30 days after marchantin injection and tumor weight was measured. Cell apoptosis was determined by TUNEL. The expressions of CHOP and GRP78 were detected by immunohistochemistry. Results Tumor size and weight in marchantin groups were significantly lower than in the control group (A, B) (P<0.05), and the inhibitory rate presented a dose-dependent increase. Compared with controls, the levels of CHOP and GRP78 expression elevated obviously following the treatment with marchantin (P<0.05). It showed statistically significant difference among groups C, D, E, with different levels of apoptosis indexes incremented in groups of marchantin H, M, L, compared with groups A and B (P<0.05). Conclusions Overall, this study shows that marchantin M circumvents the growth of prostate cancer PC-3 tumor and up-regulates expressions of CHOP and GRP78. Our data also indicate that marchantin M limits the proliferation and favors apoptosis of DU145 cells in a time- and dose-dependent manner. MeSH Keywords Aspergillosis, Allergic BronchopulmonaryCalcitonin Gene-Related PeptideProstatic Neoplasms ==== Body Background Prostate cancer is a common malignant tumor of the male reproductive system; it has a high mortality rate. Endocrine treatment is the main therapy for advanced prostate cancer. It usually develops to hormone-independent prostate cancer in most of the patients during the course of therapy, and hormone-independent transformation poses a serious obstacle to the efficacy of the treatment [1,2]. In malignant tumor cells, proteasome activity is increased, with disordered protein metabolism and dysregulated ubiquitin-proteasome. Evidence demonstrated that proteasome is a novel target for tumor treatment [3,4]. Tumor malignancy degree, including prostate cancer and colon cancer, correlates with proteasome activity up-regulation. Proteasome inhibitor decreases antiapoptotic proteins and up-regulates proapoptotic pathways, which leads to tumor cell apoptosis by blocking cell cycles. The proteasome exhibits potent induction of apoptosis in comparison to standard cytotoxic drugs. As a sensitization agent for chemoradiotherapy, it generates litter toxicity to normal tissue with selectivity and low resistance [5,6]. As a novel proteasome inhibitor, marchantin M is a type of double benzyl compound obtained from bryophytes. Priming studies illustrated a significant inhibitory role of marchantin in prostate cancer cell proliferation, which blocks PC-3 in G0/G1, induces tumor cell apoptosis, down-regulates antiapoptotic protein Bcl-2, and up-regulates proapoptotic protein Bax expression. However, its antitumor mechanism has not been fully elucidated [7,8]. Apoptosis is mediated by the endoplasmic reticulum (ER) stress pathway, mitochondrial pathway, and death receptor. ER stress appears in the early stage of apoptosis and CHOP and GRP78 are the markers of ER stress [9,10]. This study aimed to investigate the effect of marchantin M on tumor formation in nude mice and impact on CHOP and GRP78 expression. Material and Methods Human prostate cancer cell line PC-3 and DU145 were gifts from the Chinese Academy of Sciences, Shanghai Institute of Cell Biology, and were maintained in RPMI1640 medium containing 10% fetal bovine serum, 100 U/ml penicillin-streptomycin at 37°C and 5% CO2. Marchantin M was provided by Sigma (purity over 95%). Dimethyl sulfoxide was bought from Xi’an Tianzheng Co., LTD. TUNEL apoptosis detection kit, CHOPm and GRP78 monoclonal antibody, and CHOP and GRP78 immunohistochemistry kits were supplied by the Wuhan Boster Biotechnology Co., LTD. Animals Fifty male SPF BALB/c nude mice weighing 18–20 g at 4–6 weeks were provided by the Institute of Chinese Academy of Medical Sciences Tumor Animal Center and fed according to experimental animal standards. The experimentation with animals was governed by the Regulations of Experimental Animals of Hunan Authority and approved by the Animal Ethics Committee of Southern Medical University. Experimental methods Subcutaneous prostate cancer PC-3 nude mice model We subcutaneously injected 0.1 ml PC-3 cells in logarithmic phase at 1×107/ml to the left anterior axillary inoculation of nude mice. Mice phenotype and survival were observed daily, and tumor diameter was measured to draw the tumor growth curve. Animal grouping and administration When the tumor volume increased to 300 mm3, the mice were randomly divided into group A (saline), group B (DMSO), and group marchantin C, D, E (marchantin M 200, 100, 50 μmol/kg) with 10 animals in each group. The mice received abdominal injection at 1 time/2 d and 2 ml/time. Marchantin M was given to the mice at 200, 100, 50 μmol/kg in group C, D, E, respectively, according to previous results [11,12]. Tumor volume was measured every 6 days, V=π/6(a×b2). The mice were euthanatized at the 30th day after injection and tumor weight was measured. Tumor inhibitory rate (%)=(mean weight in control – mean weight in test)/mean weight in control ×100%. Cell apoptosis was determined by TUNEL according to manufacturer’s instructions. We observed 100 nucleuses in each field with 5 fields chosen in each slide. Apoptosis index AI=apoptotic cell number/cell number × 100%. CHOP and GRP78 expression detection in tumor tissue Immunohistochemistry SP (streptavidin-peroxidase) method was applied to detect CHOP and GRP78 expression. The paraffin section was prepared after PBS washing, primary antibody incubation, repairmen, DAB colorization (1 drop of DAB was added into 1ml water, before adding 1 drop of H2O2, and then mixed with 1 drop of phosphate buffer. The mixture was dripped to the section, and was shaken well for staining 5 to 10 min controlled by microscope), and dehydration. Result determination: positive staining of GRP78 and CHOP was found in cytoplasm and nucleus, respectively. Yellow or tan particles were observed in positive cell nuclei or cytoplasm. Dyeing strength was quantitatively analyzed and the average integral absorbance of positive area was determined. Dyeing strength and the percentage of positive cells were scored together for the evaluation. The score of the percentage of positive cells: 0 point, negative, 1 point, 0–25%, 2 points, 26–50%, 3 points, 51–75%, 4 points, ≥76%. The score of dyeing strength is determined by color depth: 0 point, colorless, 1 point, canary yellow, 2 points, brown yellow, 3 points, brown. Evaluation: negative, 0 point, weakly positive (+), 1–3 points, moderately positive (++), 4–5 points, strongly positive (+++), 6–7 points. The measurement of optical density was performed by using Image-Pro Plus 6.0 for immunohistochemical staining analysis. DU 145 cells proliferation measurement by MTT DU 145 cells were seeded into 96-well plate (5×103/well) followed by treatment with Marchantin M (5, 10, 20 μmol/L) after 24h culture for 24 h, 48 h and 74 h. At each time point, cell proliferation was measured. DU 145 cells apoptosis measurement by flow cytometry DU 145 cells were seeded into 96-well plate (1×106/well) followed by treatment with marchantin M (5, 10μmol/L) after 24 h culture. After that, cells were washed twice with PBS followed by fixation with 70% cold ethanol and subsequent flow cytometric analysis. Western blot Cell supernatants were isolated from lysed cells with lysis buffer, and protein concentration was measured by Bradford method. We loaded mg proteins into 8% SDS-PAGE followed by transferring into cellulose nitrate film and block for 1 h at room temperature. After that, primary antibody anti-β-actin, CHOP, or GRP78 was added and incubated overnight at 4°C, followed by addition of HRP-conjugated secondary antibody and incubated for 1 h. The protein band was visualized by enhanced chemiluminescence. Statistical analysis All statistical analyses were performed using SPSS19.0 software. Numerical data were presented as means and standard deviation (χ̄±S). Differences between multiple groups were analyzed using one-way ANOVA. P<0.05 was considered as significant difference. Results Tumor growth and nude mice general information No animals died during the test. The mass of tumor could be felt by touch after 1 week, and all the mice had a tumor of about 0.5–0.6 cm after 2 weeks. Tumor formation rate was 100%. Nude mouse activities, hair, and eating showed no significant abnormality among each group. Seven days after abdominal injection, tumor size increased gradually in group A and B, but it grew more slowly in group C, D, E. After administration for 14 days, tumor size in group C, D, and E began to decrease, while it kept increasing in group A and B. The tumor size showed significant difference among different groups after 30 days (P<0.05). The nude mice in group A and B had less hair, no activity and less eating. Their health status in group C, D, E was dose-dependent (Figure 1). Marchantin M’s impact on tumor weight and tumor inhibitory rate The nude mice were euthanized after intraperitoneal injection at 30 days. The tumor weight and size were significantly lower in groups of marchantin treatment with higher tumor inhibitory rates than those in group A and B (P<0.05). In addition, the tumor inhibitory rates presented statistical differences among different marchantin groups (P<0.05). Group H showed most significant inhibitory effect, in a time- and dose- dependent manner (Figure 2). Marchantin M’s effect on CHOP and GRP78 protein expression GRP78 is mainly expressed in the cytoplasm, while CHOP is mostly expressed in the nucleus. CHOP and GRP78 conferred no noticeable increased expression in group A and B, but the expression increased significantly in group C, D, and E (P<0.05). They presented statistical differences among different marchantin groups (P<0.05). Their expression showed time- and dose-dependence (Figure 3). Marchantin M’s effect on PC-3 cell apoptosis Apoptosis indexes in group marchantin H, M, and L increased to varying extents compared with group A and B (P<0.05) (Figure 4). Effect of Marchantin M on DU145 cell proliferation After treatment with Marchantin M, DU145 cell proliferation wa dose- and time-dependent. Marchantin M inhibited the growth of DU 145 cells, which was obvious 24 h after treatment of Marchantin M at a dose of 10 μmol/L. Furthermore, 20 μmol/L Marchantin M could significantly block the proliferation of DU145 cells (Figure 5). Effect of Marchantin M on DU145 cell apoptosis After treatment with Marchantin M, there were an increased number of apoptotic DU145 cells (Figure 6), suggesting Marchantin M is a promoter of cancer cells apoptosis. Discussion Currently, effective drugs are insufficient for clinical treatment of prostate cancer. As a double-benzyl drug, marchantin M is a proteasome inhibitor that has a variety of pharmacological activities. In vivo and in vitro studies showed that marchantin M presents a potential antitumor effect on numerous tumor cells, especially for prostate cancer PC-3 cell line. Marchantin M functions to induce p21 protein expression, block the cell cycle, and inhibit cell proliferation. By regulating the expression of apoptosis-related genes, it imposes cell apoptosis [13,14]. Studies reported that mRNA of XBP-1, as well as the expression of CHOP and GRP78, in marchantin M-treated DU145 cells increased in a time- and dose- dependent manner. The ER stress-initiated IRE1 pathway and XBP-1 was changed to XBP-1s, which stimulated the transcription of GRP78, indicating that marchantin M is as a direct inducer of ER and in turn promotes the apoptosis of prostate cancer cells [19,20]. Apoptosis is mediated by the endoplasmic reticulum (ER) stress pathway, mitochondrial pathway, and death receptor pathway. ER stress appears in the early stage of apoptosis [15,16]. GRP78 is a major molecular chaperone of ER stress, located on the ER, and is known to be an anti-apoptotic factor. It is overexpressed in prostate cancer and plays an essential role in the blockade of tumor cell invasion and tumor proliferation [17,18], possibly due to conferring nutrient deprivation or hypoxia in the microenvironment of tumors ([23]). CHOP is an ER stress-specific transcription molecule with normally low expression. In the context of the expressions of ER associated chaperon, GRP78 and CHOP, we found that the levels were elevated in marchantin M-treated prostate cancer cells. Sustained ER stress was reported to contribute to activation of CHOP and induce tumor cell apoptosis [19,20]. Our data are consistent with previous studies and illuminates an inhibitory effect of marchantin M on tumor formation in nude mice and CHOP and GRP78 expression. The results from recent work now indicate that ubiquitin – proteasome activity is up-regulated in the tumor cells [16,17]. Tumor cells are unraveled to be more sensitive to proteasome inhibitors. Marchantin M, but not trypsin, reverses tumor drug resistance, and inhibits peptidyl glutamine peptide hydrolyase and protease activities. ER stress initiates the expression of GRP78, and then activates IRE1, PERK, and AFT6 signaling pathways. As an upstream regulatory protein of PI3K, GRP78 affected the expression of AKT/PKB via PI3K/PKB signaling pathway. Abnormal regulation in the upstream signaling pathway leads to higher levels of KT/PKB activity in tumors. At the end of ER stress, CHOP, a small-molecule protein in the nucleus, is produced to induce cell apoptosis [17,18]. CHOP-induced apoptosis may be related to Akt phosphorylation reduction and TRB3 protein up-regulation. CHOP is likely to mediate the death receptor pathway through up-regulating DR5 expression. Ischemia and hypoxia caused by tumor cell excessive proliferation activate ER stress and induce cell apoptosis [21,22]. Once the degradation pathway of ER-associated proteins is impaired, misfolded proteins and unfolded proteins are prone to be accumulated in the endoplasmic reticulum lumen, leading to ER stress. Higher expression of CHOP and GRP78 was observed after marchantin M treatment, suggesting marchantin M could induce apoptosis of prostate cancer cells through ER stress. Through binding to cAMP response element protein, up-regulated CHOP is able to be involved in the regulation of Bcl-2, including up-regulation of Bax/Bak, down-regulation of Bcl-2 (causing imbalance of Bcl-2 and Bax), and subsequent increase of cytosolic calcium concentration (leading to apoptosis). However, whether marchantin M-induced cell apoptosis is involved in other pathways still needs to be determined. Conclusions We propose that marchantin M facilitates CHOP and GRP78 expression to suppress prostate cancer PC-3 tumor growth, and it also induces apoptosis of DU145 cell through ER stress. Source of support: Departmental sources Figure 1 PC-3 tumor growth curve in each group. 0.1 ml PC-3 cells in logarithmic phase at the concentration of 1×107/ml were subcutaneous injected to the left anterior axillary inoculation of nude mice. Tumor diameter was measured to evaluate tumor volume. * P<0.05, compared with group A and B; # P<0.05, compared with group E; & P<0.05, compared with group D; $ P<0.05, compared with group C. A: Control (Saline); B: DMSO; C: Higher dose of marchantin (200 μmol/kg); D: Medium dose of marchantin (100 μmol/kg); E: Lower dose of marchantin (50 μmol/kg). Figure 2 Marchantin impact on tumor weight and tumor inhibitory rate. Thirty days after the treatment of Marchantin, mice were sacrificed and tumor was isolated for measurement of tumor weight. * P<0.05, ** P<0.01, compared with group A and B; # P<0.05, ## P<0.01, compared with group E; & P<0.05, && P<0.01, compared with group D; $ P<0.05, $$ P<0.01, compared with group C. A: Control (Saline); B: DMSO; C: Higher dose of marchantin (200 μmol/kg); D: Medium dose of marchantin (100 μmol/kg); E: Lower dose of marchantin (50 μmol/kg). Figure 3 Marchantin effect on CHOP and GRP78 protein expression. Thirty days after the treatment of Marchantin, mice were sacrificed and tumor tissue was isolated for immunohistochemical analysis of the expression of CHOP and GRP78. * P<0.05, ** P<0.01, compared with group A and B; # P<0.05, ## P<0.01, compared with group E; & P<0.05, && P<0.01, compared with group D; $ P<0.05, $$ P<0.01, compared with group C. GRP78 protein mainly express in cytoplasm. Compared with group A and B, its expression increased in marchantin groups in a dose dependent manner. CHOP protein mainly express in nucleus. Compared with group A and B, its expression increased in marchantin groups with dose dependence. A: Control (Saline); B: DMSO; C: Higher dose of marchantin (200 μmol/kg); D: Medium dose of marchantin (100 μmol/kg); E: Lower dose of marchantin (50 μmol/kg). Figure 4 Marchantin effect on PC-3 cell apoptosis. Thirty days after the treatment of Marchantin, PC-3 cell apoptosis was measured by flow cytometry. * P<0.05, ** P<0.01, compared with group A and B; # P<0.05, ## P<0.01, compared with group E; & P<0.05, && P<0.01, compared with group D; $ P<0.05, $$ P<0.01, compared with group C. A: Control (Saline); B: DMSO; C: Higher dose of marchantin (200 μmol/kg); D: Medium dose of marchantin (100 μmol/kg); E: Lower dose of marchantin (50 μmol/kg). Figure 5 Effect of marchantin on DU145 cell proliferation. Thirty days after the treatment of Marchantin, DU145 cell proliferation was measured by MTT. Figure 6 Effect of marchantin on DU145 cell apoptosis. Thirty days after the treatment of Marchantin, DU145 cell apoptosis was measured by flow cytometry. * P<0.05, compared with group A; # P<0.05, compared with 5μmol/L group. 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==== Front Med Sci MonitMed. Sci. MonitMedical Science MonitorMedical Science Monitor : International Medical Journal of Experimental and Clinical Research1234-10101643-3750International Scientific Literature, Inc. 2660305010.12659/MSM.894890894890Clinical ResearchMinimally Invasive Unilateral vs. Bilateral Pedicle Screw Fixation and Lumbar Interbody Fusion in Treatment of Multi-Segment Lumbar Degenerative Disorders Liu Xiaoyang ABEFLi Guangrun BDWang Jiefeng BCZhang Heqing CEDepartment of Spine Surgery, Yuhuangding Hospital, Yantai, Shandong, P.R. ChinaCorresponding Author: Xiaoyang Liu, e-mail: [email protected] Study Design B Data Collection C Statistical Analysis D Data Interpretation E Manuscript Preparation F Literature Search G Funds Collection 2015 25 11 2015 21 3652 3657 03 6 2015 08 7 2015 © Med Sci Monit, 20152015This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported LicenseBackground The choice for instrumentation with minimally invasive transforaminal lumbar interbody fusion (MIS-TLIF) in treatment of degenerative lumbar disorders (DLD) remains controversial. The goal of this study was to investigate clinical outcomes in consecutive patients with multi-segment DLD treated with unilateral pedicle screw (UPS) vs. bilateral pedicle screw (BPS) instrumented TLIF. Material/Methods Eighty-four consecutive patients who had multi-level MIS-TLIF were retrospectively reviewed. All data were collected to compare the clinical outcomes between the 2 groups. Results Both groups showed similar clinical function scores in VAS and ODI. The two groups differed significantly in operative time (P<0.001), blood loss (P<0.001), and fusion rate (P=0.043), respectively. Conclusions This study demonstrated similar clinical outcomes between UPS fixation and BPS procedure after MIS-TLIF for multi-level DLD. Moreover, UPS technique was superior in operative time and blood loss, but represented lower fusion rate than the BPS construct did. MeSH Keywords Spinal FusionSpineSpondylosis ==== Body Background Minimally invasive transforaminal lumbar interbody fusion (MIS-TLIF) is a popular and effective surgical technique for the treatment of degenerative lumbar disorders (DLD), including spondylolisthesis, lumbar spinal canal stenosis associated with deformities, and discogenic pain identified by provocative discography [1–4]. Compared with the traditional open surgery, MIS-TLIF has multiple advantages, such as the decreased approach-related muscle damage, lesser blood loss, lower postoperative pain, shorter length of hospital stay, and minor postoperative narcotic usage allowing for early activity [1,5]. Many previous studies have demonstrated that MIS-TLIF could achieve excellent clinical outcomes [6–9]. Although MIS-TLIF is widely performed with the treatment of DLD, the choice for instrumentation with spinal fusion procedures remains controversial. In general, bilateral pedicle screw (BPS) fixation for MIS-TLIF is preferred as a standard procedure due to its rigid fixation, great biomechanical stability and good clinical results [9,10]. However, some studies have indicated that increased number of implants and excessive rigidity can lead to more adverse clinical effects, such as reducing fusion rate and adjacent segment degeneration [11,12]. Recently, unilateral pedicle screw (UPS) fixation has been recommended by an increasing number of surgeons [13–16]. UPS fixation for the MIS-TLIF has multiple advantages in reduced soft tissue disruption of the contralateral side, shorter surgical time, and lower implant costs [17–19], but relatively provides less rotational stability and stiffness based on many biomechanical studies[20]. As far as we know, few previous clinical trials comparing UPS versus BPS fixation for open or mini-open TLIF in multi-segment DLD have been reported. Based on the previous studies which have shown similar clinical and fusion results of UPS as those of bilateral fixation, we conducted this retrospective study to compare clinical outcomes in consecutive patients with multi-segment DLD treated with UPS or BPS instrumented TLIF. Material and Methods This study was approved by the Committee of Medical Ethics and the institutional review boards of Yuhuangding Hospital. The study period was from January 2010 to April 2013. Informed consent was obtained from patients or their family members if the patient was unable to provide consent. A total of 84 consecutive patients who had undergone a multi-level MIS TLIF by the senior surgeon were enrolled. Patients treated with BPS fixation for MIS-TLIF were compared with those with UPS construct, based on age, sex, and body mass index (BMI). Indications for surgery were: 1) spinal stenosis, 2) lumbar disk herniation, and 3) spondylolisthesis. Only those subjects aged 18 to 70 years could be included. Patients enrolled in our study were excluded if they had the following: 1) active infection, 2) metabolic disease, 3) severe osteoporosis, 4) severe chronic disease, 5) symptomatic vascular disease, or 6) previous lumbar surgery. The merits and drawbacks of each procedure were thoroughly discussed with the patients and their family. All data, including patient demographics, examination results, and operative data, were obtained from hospital records. All patients received 2-year follow-up postoperatively. The radiographic data were assessed individually by 2 senior specialists. Surgical techniques The patients were placed in the prone position under general anesthesia. A C-arm image intensifier was used to determine the location of the interbody level. We used the local autograft and Capstone cages (Medtronic Sofamor Danek, Memphis, Tennessee) and pedicle screws (Legacy; Medtronic Sofamor Danek) in the surgery. UPS fixation placed at the time of MIS-TLIF applied in this study was previously described by Lee et al. [21], and BPS was introduced as by Choi et al. [22]. All operations were performed by the same surgeon (Figure 1). A standard postoperative protocol was used for all patients. Drainage was placed for 48 hours postoperatively and intravenously prophylactic antibiotics were given for 24 hours postoperatively. Waist muscle function exercises with the legs straight were required. Patients in the unilateral group were mobilized early out of bed 24 hours postoperatively if no contraindications existed. Outcomes assessment All parameters, including blood loss, operative time, duration of hospital stay, complication rate, visual analog scale (VAS), and Oswestry Disability Index (ODI) scores, were obtained and compared to evaluate efficacy between the 2 groups. All patients were asked to return for follow-up at 1 week and 3, 6, 12, and 24 months postoperatively. Preoperative and postoperative radiographs, including anteroposterior and lateral flexion-extension, were used to evaluate fusion status, screw failure, and other complications. Fusion rate was measured according to the method of Schulte et al. [23]. All statistical analyses were performed with SPSS version 17.0 software. Categorical variables were compared with the chi-square test. The outcomes between 2 groups were tested with a paired t test. The comparisons of continuous data presented as mean±standard deviation (SD) were analyzed with an independent-samples t test. A P value less than 0.05 was considered significant. Results In this study, a total of 84 patients were followed up for an average of 26.2 months (range, 23–36 months). Mean length of follow-up was 26.7 months for the UPS group and 23.6 months for the BPS group. In the UPS group, 7 patients (16.7%) were diagnosed with spinal stenosis, 25 (59.5%) were diagnosed with lumbar disk herniation, and 10 patients (23.8%) were diagnosed with spondylolisthesis. In the BPS group, 9 (21.4%) were diagnosed with spinal stenosis, 22 (52.4%) with lumbar disk herniation, and 11 (26.2%) with spondylolisthesis. No significant differences were found between the 2 groups in patient demographics (Table 1). Table 2 shows the clinical and functional outcomes for the 2 groups. Mean length of hospital stay was 12.6 days in the UPS group and 13.4 days in the BPS group. No significant difference was found in hospital stay between the 2 groups. However, the UPS group required a shorter operative time and had less blood loss than the BPS group (P<0.01). Mean VAS score was 1.8±1.2 in the UPS group postoperatively and 2.2±1.4 in the BPS group. Patients in the UPS group postoperatively had an average ODI score of 17.4±4.7 and patients in the BPS group with16.6±7.5. Both mean postoperative VAS and ODI improved significantly in each group, compared with preoperative VAS and ODI; however, no statistically significant differences were obtained between the 2 groups (Table 2). With respect to fusion rate, 81.0% of patients in the UPS group and 95.2% of patients in the BPS group achieved successful fusion, which showed a significant difference (P=0.043). Neither group showed device-related complications, such as screw loosening or breakage, or fusion cage migration. With regard to general complications, there was no difference between the BPS and the UPS group (P>0.05). One patient in the UPS group and 3 patients in the BPS group developed superficial wound infections. All infections were completely controlled by intravenous antibiotics and daily dressing. Discussion Spinal fusion surgery is an effective method in the treatment of painful DLD. With the development of advanced systems that can provide adequate access for decompression and instrumentation placement and reduce tissue disruption, the MIS-TLIF has become more popular in the treatment of DLD. Recent biomechanical studies have suggested the equivalence between UPS fixation and standard BPS constructs [24–26], and some clinical data [14,15,17] have demonstrated acceptably reliable fusion rates in patients with UPS fixation, which requires fewer pedicle screws. However, Slucky et al. [24] reported that UPS after MIS-TLIF led to less rotational stability and stiffness than BPS fixation. Thus, the choice between BPS or UPS fixation after lumbar fusion for the treatment of DLD remains controversial. The main aim of the pedicle screw is to stabilize the spine to promote fusion; therefore, the fusion rate is considered as the most important outcome. Several studies have revealed that the unilateral fixation for TLIF achieves good outcomes [27,28]. Deutsch et al. [28], in their studies in 2006, reported good outcomes of UPS fixation after TLIF with a greater than 20-point reduction in the Oswestry Disability Index (ODI) score, but they did not report the exact fusion rate and only evaluated the outcomes of short-term follow-up of less than 1 year. Both Suk et al. [14] and Xue et al. [17] compared the efficacy of UPS fixation vs. BPS fixation after MIS-TLIF and reported lower fusion rates for the former; however, all patients in their studies received single-level fusion procedure. Some studies have shown that unilateral instrumentation might be not be suitable for multilevel fusion due to its inadequate fixation strength. Zhang et al. [29], in their prospective randomized study, presented similar failed fusion rates of 2-level fusion in the UPS group (3/16) and the BPS group (2/11). They concluded that UPS fixation in multi-level DLD is similarly effective and safe. However, our study showed a different result – that patients with UPS had a lower fusion rate (81.0% vs. 95.2%) and that BPS fixation was significantly safer. As many biomechanical studies have reported, the negative impact of the fusion in unilateral instrumentation might be due to less biomechanical stability. Moreover, we found less blood loss and a shorter operative time in patients with UPS fixation after MIS TLIF, compared with BPS fixation. These findings are consistent with the results of previous studies [17,28,30]. With respect to functional scores, there were no differences between UPS and BPS fixation procedure in VAS and ODI. This finding is consistent with the results from many previous studies [31–33], although the outcomes of patients in some of these studies were assessed using other assessment systems, including the Japanese Orthopaedic Association (JOA), mProlo, and 36-Item Short Form Healthy Survey version 2 (SF-36v2) scores. Patients with UPS fixation procedure had significantly had less blood loss and experienced shorter operation time as compared with those the BPS fixation in our study. It is mainly due to dissection of soft tissue and insertion of pedicle screws only on 1 side for UPS fixation, which takes less time and decreases blood loss. Hardware-related complications often cause serious adverse effects in fusion surgery. In our study, some patients had infections. One patient in the UPS group and 3 patients in the BPS group developed superficial wound infections. Similar to results in previous studies [14–16,34,35], we also found there was no difference in terms of complication rate between the 2 procedures (UPS vs. BPS, 2.4% vs. 7.1%). Several meta-analyses also demonstrated that patients with UPS procedure experienced similar complication rates as those with BPS procedure. Several important limitations in this study should be considered. First of all, the relatively small sample size might limit the comparability and outcomes. Secondly, the follow-up durations in our study were not long enough to determine the results. Finally, the design of this study was not random, which could not adequately assess the outcomes of the 2 surgical methods. Further studies are required to compare the efficiency and safety of UPS fixation in multi-level DLD. Conclusions In summary, the present study demonstrated that MIS-TLIF with UPS fixation leads to similar clinical outcomes, compared with BPS procedure for multi-level DLD. Despite an association with decreased operative time and less blood loss, the UPS technique had a lower fusion rate than the BPS construct did. Due to the limitations of this study, multi-center studies with more patients and longer follow-up period are required to further evaluate the outcomes of the 2 systems. Source of support: Departmental sources Competing interests The authors declare that they have no competing interests. Figure 1 X-ray films showed MIS-TLIF with pedicle screw fixation. (A) MIS TLIF with BPS fixation. (B) MIS TLIF with UPS fixation. Table 1 Patient demographics and preoperative data. Parameter UPS group BPS group P value Sample size (n) 42 42 – Age (mean ±SD, years) 61.4±11.8 62.1±10.2 0.613 Gender (M/F) 26/16 25/17 0.823 BMI (kg/m2) 24.3±3.2 24.5±3.1 0.437 Diagnosis 0.659 Spinal stenosis 7 (16.7%) 9 (21.4%) – Lumbar disk herniation 25 (59.5%) 22 (52.4%) – Spondylolisthesis 10 (23.8%) 11 (26.2%) – Symptom duration (mo) 11.2±4.3 12.4±4.1 0.776 Follow-up (mo) 25.8±16.4 26.4±17.2 0.898 Preoperative VAS (mean ±SD) 7.7±1.2 7.8±1.5 0.813 Preoperative ODI (mean ±SD) 44.2±12.1 43.8±11.9 0.689 UPS – unilateral pedicle screw; BPS – bilateral pedicle screw; SD – standard deviation; BMI – body mass index; VAS – visual analog scale; ODI – Oswestry Disability Index. Table 2 Clinical and functional outcomes for the two groups. UPS group (n=42) BPS group (n=42) P value Hospital stay (d) 12.6±2.6 13.4±2.1 0.122 Operative time (min) 92.1±21.6 112.3±25.6 <0.001* Blood loss (mL) 254±48.2 467±43.3 <0.001* Complication rate (%) 1 (2.4%) 3 (7.1%) 0.306 Fusion rate (%) 34 (81.0%) 40 (95.2%) 0.043* VAS (mean ±SD) 1.8±1.2 2.2±1.4 0.162 ODI (mean ±SD) 17.4±4.7 16.6±7.5 0.558 * P value was significant. ==== Refs References 1 Liu Z Fei Q Wang B Lv P A meta-analysis of unilateral versus bilateral pedicle screw fixation in minimally invasive lumbar interbody fusion PLoS One 2014 9 11 e111979 25375315 2 Deyo RA Nachemson A Mirza SK Spinal-fusion surgery – the case for restraint N Engl J Med 2004 350 7 722 26 14960750 3 Yuan HA Garfin SR Dickman CA Mardjetko SM A Historical Cohort Study of Pedicle Screw Fixation in Thoracic, Lumbar, and Sacral Spinal Fusions Spine (Phila Pa 1976) 1994 19 20 Suppl 2279S 96S 7817243 4 Bridwell KH Sedgewick TA O’Brien MF The role of fusion and instrumentation in the treatment of degenerative 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==== Front J Reprod InfertilJ Reprod InfertilJRIJRIJournal of Reproduction & Infertility2228-54822251-676XAvicenna Research Institute jri-13-241Original ArticleCorrelation of Sperm Associated Antigen 11 (SPAG11) and its Isoforms with Varicocele in Rats Tian Hong 12Huo Yong-Wei 12Zhou Jin-Song 12Wang Li-Rong 12Zhang Qiu-Yang 2Qiu Shu-Dong 121. Department of Anatomy, Histology and Embryology, School of Medicine, Xi’an Jiaotong University, Xi’an, China2. Research Center of Reproductive Medicine, School of Medicine, Xi’an Jiaotong University, Xi’an, China Corresponding Author: Shu-Dong Qiu, Department of Anatomy, Histology and Embryology, Medical School of Xi’an Jiaotong University, Xi’an 710061, China, E-mail:[email protected] 2012 13 4 241 247 03 3 2012 30 7 2012 Copyright© 2012, Avicenna Research Institute.2012This work is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported License which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly.Background: We undertook this study to investigate the variation relationship of sperm associated antigen 11 (Spag11) mRNA expression and SPAG11E protein in the epididymis and spermatozoa of experimental left varicocele (ELV) rats. These findings could contribute to the understanding of the role of epididymal proteins in sperm functions and the mechanism of male infertility induced by varicocele. Methods: The ELV model was established in adolescent male Sprague-Dawley rats. Four weeks after the operation, tissue distribution and changes in the expressions of Spag11 mRNA and SPAG11E protein caused by ELV in the whole of left epididymis and spermatozoa were studied using quantitative reverse transcription-polymerase chain reaction (RT-QPCR), immunohistochemistry and immunofluorescence. Significant differences were identified using one-way ANOVA followed by Student-Newman-Keuls test. Significance level (p) was fixed at 0.05. Results: The expected product of Spag11 was 96 bp that amplified by RT-QPCR was detected in the epididymal tissue and spermatozoa. SPAG11E protein was confined mainly to the supranuclear region of the principal cells and the stereocilium of the epididymal epithelium, it was concentrated on the acrosome and the tail of spermatozoa except the terminal piece. Statistical analyses of the images and the data indicated that Spag11 mRNA and SPAG11E protein expressions in the left epididymis and spermatozoa of ELV rats presented a considerable decrease (p<0.001) compared with that of the corresponding control group. Conclusion: The expressions of Spag11 mRNA and SPAG11E protein declined markedly in ELV rats, which suggest that SPAG11E may not only play an important role in sperm maturation, but it may also be influenced by varicocele. EpididymisRatSperm associated antigen 11 proteinSpermatozoaVaricocele ==== Body Introduction Spermatozoa leaving the testis undergo post-testicular maturation and acquire fertilizing ability and forward motility during their passage through the epididymis that provides the microenvironment for their maturation. Numerous proteins synthesized and secreted by the epididymal epithelia are thought to be considerable members of epididymal microenvironment and be involved directly or indirectly in male reproductive activities including the initiation of sperm maturation, sperm-oocyte recognition and the acrosome reaction (1). A family of these proteins includes SPAG11E coded by Spag11 gene in various species (2). The Spag11 gene, also known as epididymal protein 2 (EP2), formed by the fusion of two ancestrally independent β-defensin genes (3), generates at least 19 alternative mRNA spliced transcripts initiated at A and B promoters (1, 4–6). These transcripts are transcribed and translated in different portions of the male reproductive tract and in different species, to produce a complex group of small androgen-dependent secretary proteins in which different exon-encoded modules are variously combined (3). Most primate SPAG11 protein isoforms consist of N-terminal common region and C-terminal peptides, which are encoded by different combinations of exons (7). SPAG11 is detected in the epithelial cells of male reproductive organs, predominantly in epididymis. It is believed to play an important role in epididymal immunity in addition to their role in sperm maturation on the basis of their structural features and specific expression pattern. The Spag11 gene in rodents is similar to that in primates but it possesses fewer splicing variants, whose intimate expression pattern and function are less well documented. Spag11 (known in rat and mouse as Bin1b) is an androgen-dependent gene cloned from the rat epididymis by Peng Li et al. using differential display analysis of mRNA. However, only one rodent isoform, SPAG11E (EP2E), has been well-characterized and its full-length cDNA is composed of 385 base pairs (bp), with an open reading frame of 204 bp nucleotides, encoding a 68-amino acid protein (8). SPAG11E is exclusively expressed in rat epididymis, strongly in the distal caput and proximal corpus (4, 8), and it can bind the sperm head in all epididymal regions except the initial segment (9). SPAG11E exhibits a structural and antimicrobial similarity to β-defensins, and has become one member of the innate defensive system in epididymal epithelia. Moreover, as an essential component in the epididymal environment, SPAG11E is believed to be relevant to the acquisition and maintenance of progressive motility in sperm (9). Varicocele (VC), a tortuosity and dilation of the pampiniform plexus in the spermatic cord, has been identified to be one of the main causes of male infertility. However, the detailed mechanisms of varicocele-induced male subfertility and infertility are not well-elucidated. It was reported that varicocele might cause a series of pathophysiological changes in the testis and epididymis, with an adverse effect on the environment for spermatogenesis and sperm maturation. Therefore, we designed our study to observe the variation in the expression of Spag11 mRNA and SPAG11E protein in the epididymis and spermatozoa of the experimental left varicocele (ELV) in a rat model created via partial ligation of the left renal vein, revealing the correlation of Spag11 and its transcripts with VC. This study was also intended to provide the basis for understanding the mechanism of VC-induced infertility. Methods Animals: In total, 70 male 6–7 week-old Sprague-Dawley (SD) rats, weighing 130–150 g, were obtained from the Laboratory Animal Center of the School China. This study was approved by ethical commitee of Science and Technology Department of Shaanxi Province of China in 2008 and Medical School of Xi’an Jiaotong University in 2010. Groups and Treatments: The animals were randomly divided into the experimental left varicocele (ELV) group (n=35) and the sham-operated control group (n=35). The rats were sacrificed four weeks after the operation for various experiments. Procedure for partial left renal vein ligation: The rats were anesthetized with 20% ethylurethanm (5 ml/kg) i.p. and a midline incision was made to disconnect and expose the left renal vein. A 4–0 silk ligature was made around the vein with a metal probe interposed lateral to inferior vena cava and medial to the entrance of the adrenal and spermatic vein. The diameter of the metal probe was selected in a way to reduce the diameter of the renal vein by approximately 50%. Then the probe was removed and the renal vein could be recanalized via bypass circuit. The incision was sutured with 3–0 ligature. Every operated rat received an intraperitoneal injection of penicillin, 20 million units daily, for three consecutive days to prevent infection. Sham-operated animals, which served as the controls, were exposed to the medication only but no ligation of the left renal vein was made. Thirty-five male rats were operated successfully and subdivided into three groups, for immune-histochemistry (n=5), for immunofluorescence (n=10) and the remaining (n=20) for real-time quantitative reverse transcriptase polymerase chain reaction (RT-qPCR). Total RNA extraction: The whole epididymis and sperm mass gained from the cauda epididymis were immediately snap-frozen in liquid nitrogen, respectively. Total RNA of each sample was extracted by Trizol reagent. All the procedures were carried out according to the manufacture’s protocol (Invitrogen, Carlsbad, CA, USA). RNA concentration and quality were determined by measuring the absorbance at 260 nm and 280 nm. RT-qPCR: The RT-qPCR protocol was as follows: DNase-treated total RNA (3 μg) was reverse transcribed in a volume of 20 μl with Moloney Murine Leukemia Virus reverse transcriptase (Fermentas, Lithuania) and random hexamer primer (Fermentas) at 42°C for 60 min. Subsequently, the amplifications of Spag11 (GenBank accession NM 145087) and Gapdh (GenBank accession 017008) used as an internal control, were carried out by qPCR (TaqMan). Primers and probes were designed according to their mRNA sequences: Spag 11 forward primer 5′-CTGCTTGGTCCAAA GA AA CTCA-3′, reverse primer 5′-CGGCACA TGA AGA GCCTACA-3′, probe FCACCGTGTG CTT CATG CAGCGGP. Gapdh forward primer 5′-CA AGTTC AACGGCACAGTCAA-3′, reverse primer 5′-TG GTGAAGACGCCAGTAGACTC-3′, probe FTCT TCCAGGAGCGAGATCCCGC TA ACP. QPCR was conducted using PRISM 7000 Sequence Detector (ABI, Foster, USA). Each reaction mixture contained 12.5 μl 2×AmpliQ Real Time PCR Master Mix with 10 mM MgCl2 (Lifeson, Danmark), 0.9 μM forward primer, 0.9 μM reverse primer, 0.5 μM probe, 1 μl cDNA, and deionized water was added to reach a total volume of 25 μl. Unless indicated, all reagents used in qPCR were purchased from Genecore Biotechnologies Company (Genecore, Shanghai, China). Cycling parameters were kept at 50°C for 2 min, at 95°C for 5 min to initially denature and hold, then at 95°C for 30 s and at 61°C for 1 min for 45 cycles. All reactions including No Template Controls were repeated twice. Immunohistochemistry: Immunohistochemistry was performed as previously described (10). All the epididymides were fixed in freshly prepared Bouin’s solution, then embedded in paraffin wax and cut in 5 μm sections. Slices were stained with SPAG11E antibody raised against the peptide ERKGDISSDPWNRC. For control staining, normal rabbit serum was incubated with the sections (data not shown). To demonstrate immunoreactive SPAG11E a Streptavidin/ Peroxidase (SP) HistostainTM-Plus Kit (ZSJB, Beijing, China) was employed with diaminobenzidine as chromogen, resulting in brown products. Sections were counterstained with Harris hematoxylin. Photographs were taken by digital microscopy system (Olympus BX-1, Tokyo, Japan). The corrected gray value of immunopositive areas in 5 fields per slide at ×400 magnification was detected by an image analysis software (Image Pro Plus 5.1, Bethesda, MD, USA). The corrected gray value is proportional to the intensity of positive staining. Immunofluorescence: Sperm smears were produced on the basis of the protocol of Hamil KG et al. (11). The caudae were cut up, and were then placed in 0.01 M phosphate-buffered saline (PBS, pH=7.4) for 15 min. The supernate was centrifuged at 2,200 ×g for 15 min. Depositions (including sperm) were washed twice with 0.01 M PBS (pH=7.4) and were fixed in 4% paraformaldehyde for 30 min. Spermatozoa were washed with 50 mM glycine in 0.01 M PBS (pH=7.4), smeared on slides after adjusting sperm density, and air-dried. We used 1% Triton X-100 to increase the penetration of primary antibody. After washing twice in 0.01 M PBS (pH=7.4), nonspecific binding sites were blocked with normal goat serum for 25 min at 37°C. The primary antibody, rabbit anti-mouse SPAG11E antibody (1:600) was incubated with spermatozoa overnight at 4°C in 1% BSA, and 0.4% Triton X-100 in PBS. Unbound antibody was removed by three washes with 0.01 M PBS (pH=7.4), and FITC-conjugated AffiniPure goat anti-rabbit secondary antibody was added at 37°C for 1 hr. The unbound secondary antibody was removed by three washes with 0.01 M PBS (pH=7.4). The sections were mounted by glycerin-bicarbonate buffer, and observed using a digital microscopy system (Olympus BX-1, Tokyo, Japan) with a green filter before taking photos. For control staining, normal rabbit serum was incubated with the sections (data not shown). The corrected average fluorescence intensity of each picture at ×1000 magnification was examined by image analysis software as mentioned above. The optical density indirectly reflects the amount of antigens that can combine with homologous antibodies. The higher the corrected optical density is, the higher binding of the proteins and antibodies will be. Statistical analysis: The target gene expression difference was shown as a multiple of Spag11 mRNA quantifiability in ELV group relative to the normal control after homogenization by the reference gene. The relative expression of Spag11 in ELV groups was described using the equation: R= 2−ΔΔCT, ΔΔCT=(CTSPAG11−CTGAPDH)ELV– (CT SPAG11−CTGAPDH)CONTROL. Where R is the differences in gene expression level, CT is the threshold cycle. The data related to immunohistochemical staining was shown as mean±SD. The changes in expression between the cases and controls were evaluated using One-way ANOVA with SPSS, version 13.0, statistical software (SPSS Inc., Chicago, IL, USA). The p<0.05 was considered statistically significant. Results Animal Model: There was no death among the rats following the surgery. Four weeks after the operation, the left spermatic vein of model rats showed expansion, even beyond twice in diameter than before but the weights of the two kidneys were similar (Figure 1). Figure 1. The photograph of cirsoid left vena spermatica vein in ELV model rat. A: control group, normal vena spermatica interna; B: ELV group, varicose vena Effects of ELV on the Spag11 gene expression: We performed RT-qPCR to measure Spag11 gene expression levels and compared the corrected relative quantification between ELV and control groups. The results are shown in figure 2, table 1 and table 2. Figure 2. Amplification plot of RT-QPCR. Table 1. CT value of target amplification Group CT value of target amplification Gapdh Spag11 Control epididymis 24.32±3.15 25.25±2.37 spermatozoa 28.14±1.89 31.62±0.33 ELV epididymis 24.91±3.48 27.22±1.39 30.00±2.09 34.71±0.49 Table 2. The differences in the expression levels of Spag11 mRNA in the left epididymis and spermatozoa of ELV group compared with that of the corresponding control group Group Fold-change of ELV group Fold-change of control group Left epididymis 0.3842 1 Left spermatozoa 0.4273 1 96 bp PCR products of Spag11 in the epididymis and spermatozoa were observed on 2% agarose gel electrophoresis in 1×TAE buffer (data not shown). According to the definition of the equation: R=2−ΔΔCT, the multiple variance of control group is 1. As expected, ELV caused the down-regulation of Spag11 mRNA expressions of left epididymis and spermatozoa. Effect of ELV on the SPAG11E protein expression: In the ductal epithelium of epididymis, SPAG11E protein was expressed mainly in the supranuclear region of principal cells and it was associated with the apical stereocilia. There was no immunostaining in the clear, halo or basal cells. Maximum levels of SPAG11E expression were detected in the proximal, middle corpus, and the cauda, and a weak immunopositive staining was observed in the distal caput, while negative staining occurred in the initial segment (Figure 3). The protein was concentrated in the acrosome of the sperm. SPAG11E was less abundant on the tail (Figure 3). Figure 3. The expressions of SPAG11E protein in the epididymis and spermatozoa of ELV adolescent rats and corresponding control group. Original magnification ×40 (C, D, E, F, G, H and I) and ×100 (A, B, J, K and L). A, B: the cell-specific pattern of localization of SPAG11E in the epididymal epithelium (hematoxylin couterstaining). The supranuclear cytoplasm of principle cells exhibited immunopositive staining, the clear cells, halo cells and basal cells showed immunonegative. •: clear cell; ☆: halo cell; Δ: basal cell. C, D, E, F: the region-specific expression pattern of SPAG11E in the epididymis. Except for the initial segment (C), the remaining parts were immumopositive, weak in the distal caput (D), strong in the proximal, middle corpus (E) and the cauda (F). G, H, I: The expressions of SP AG11E in the distal caput, proximal corpus and the cauda of ELV rats. J: the localization of SPAG11E in spermatozoa of control rats. Anti-SPAG11E immunofluorescent staining was most intense in the acrosome of head and neck segment, middle segment and principal segment of spermatozoa tail. K: the expression of SPAG11E in spermatozoa from the right epididymis of ELV rats. L: the expression of SPAG11E in spermatozoa from the left epididymis of ELV rats, the intensity was obviously decreased when compared with J Consistent with changes in gene expression, immunohistochemistry showed a considerable decrease in left epididymal SPAG11E expressions in ELV groups (p<0.001) compared with that of the control group (Table 3). Moreover, lower binding of SPAG11E proteins and corresponding antibodies occurred in the spermatozoa from left epididymis of ELV groups (p<0.001) by optical density analysis (Table 4). Table 3. The analytical results of corrected gray value of SPAG11E by immunohistochemical staining in the epididymis of the ELV rats (Mean±SD) Group Corpus Cauda Control 0.2901±0.0231 0.2801±0.0087 ELV left 0.2388±0.0156 a,d 0.2314±0.0017 a ELV right 0.2617±0.0105 b 0.2791±0.0038 c Vs. corresponding control group in ELV group, a: p<0.001; vs. corresponding control group in ELV group; b: p<0.05; vs. right side in ELV group, c: p>0.05, vs. right side in ELV group, d: p>0.05 Table 4. The analytical results of corrected optical density of SPAG11E by immunofluorescence on the spermatozoa from the epididymal cauda in the ELV rats (Mean±SD) Group Spermatozoa Control 1.2517±0.2679 ELV from left epididymis 0.9241±0.2274 a,c from right epididymis 1.2683±0.1302 b Vs. the corresponding control group in ELV group, a: p<0.05; vs. the corresponding control group in ELV group; b: p>0.5; vs. right side in ELV group, c: p<0.05 Discussion The epididymis plays a crucial role in regulating spermiotelcosis (12). Region-specific, as well as cell-specific patterns of gene expression within its epithelium contribute to the spermiotelcosis microenvironment of the epididymis, in which there are rich specialized proteins interacting with spermatozoa. SPAG11, an androgen-dependent and epididymis-specific expressed gene, gives rise to multiple mRNAs that encode a group of small secretary peptides (2). The SPAG11 gene codes for at least five different message variants in Chimpanzees and for at least six different message variants in humans (13). However, Spag11 codes for fewer message variants in rodents, which the predominant one of is SPAG11E homologous to Bin-1b. Localization of Spag11 mRNA within the defensin gene cluster and identification of the specifically expressed SPAG11E protein in the male reproductive tract suggest that this androgen-dependent secreted protein is likely involved in sperm maturation as a component of the innate antimicrobial peptides in epididymal microenvironment (14). Varicocele, a common disorder in the field of andriatria, is present in approximately 15% of the general male population. Varicocele accounts for up to 40% of male factor infertility; therefore World Health Organization (WHO) has listed it as the first item for investigation in male infertility. Varicocele frequently occurs in the left side, often involving the contralateral side, and damages the function of reproductive system in a time-progressive pattern. It should be noted that adolescent varicocele is especially serious. Many studies have demonstrated that experimental left varicocele could result in atrophy of the epididymal ducts, degeneration of the epididymal epithelium, apoptosis within principal cells and edema of the interstitial tissue of rats (15, 16). To confirm the effects of varicocele on SPAG11 protein and its transcript expressions and to study their correlation with VC and infertility caused by VC, we used ELV rats. We investigated the effects of varicocele on the expression of Spag11 and its isomers in the epididymis and spermatozoa at both nucleic acid and protein levels. In 35 rats subjected to varicocele surgery in this study left spermatic vein showed the expected dilation, which testified that incomplete ligation of the left renal vein was a classic and effective method for inducing ELV and performing the follow-up experiments. We had reported that Spag11 mRNA expression was epididymis-specific. Among six rat tissues tested by RT-PCR, only the epididymis provided a detectable 376 bp PCR product (10). In this study, 96 bp specific strip of Spag11 gene was obtained from the frozen spermatozoa mass by RT-qPCR in addition to the same band in the epididymis. This was confirmed by the results of RT-qPCR in which the Spag11 mRNA expressions in the left epididymis and spermatozoa of ELV groups presented remarkable decreases compared with that of the corresponding control groups. The above-mentioned data illustrate that ELV may have an early impact on the Spag11 gene expression in the left side of the epididymis of the experimental groups. The direct damage to the epididymal epithelium caused by ELV may lead to the decline in Spag11 expression. In parts these changes are likely, to be the result of secondary decline in androgen secretion by varices. Although the expression of Spag11 variants in rodents is less well-understood, only a single rodent isoform, SPAG11E has been characterized. In rats, SPAG11E was reported to be regionally- and cell-specifically expressed in the epididymis and to be an androgen dependent secretary peptide containing the defensin-like six-cysteine motif. Recent research suggests that SPAG11E could bind sperm within different regions of epididymis (16). Our immunohistochemistry and immunofluorescence findings prove that SPAG11E possesses distinct cell- and region-specific expression patterns in the epididymis and sperm binding, which reveals that SPAG11E may play an essential role in post-testicular sperm maturation as a specific molecule of epididymal origin. After inducing varicocele in the hibateral epididymis, the expression of SPAG11E protein exhibited a sharp reduction. These changes in the expression rate were more obvious in the left than the right epididymis following ELV. No statistical differences in SPAG11E expression were found in right cauda epididymis of ELV rats, which indicates that the damage caused by varicocele on the left side of epididymis is more serious than that on the right side. These changes are in accordance with the results of its mRNA study. Conclusion According to our findings, varicocele may affect the synthesis of some specific proteins with anti-inflammatory properties within the epididymal epithelium. This effect evokes an early but a continuous change in the environment for post-testicular sperm maturation, which may later result in male subfertility or infertility. As defensin-like peptides, Spag11 mRNA and SPAG11E protein expressions showed a considerable decline in left epididymis at an early stage of ELV, which might be one of the reasons why varicoceles are prone to cause genital infection and then male infertility. However, we still need to do further research in order to fully understand how Spag11 and its isomers are involved in the mechanism of VC-induced infertility. Acknowledgement We especially thank Dr. Susan H. Hall in the Laboratories for Reproductive Biology, University of North Carolina for offering anti-SPAG11E primary antibody without payment, and for her practical guidance. This study was supported by Sci-Technical Development Project of Shaanxi Province, China (2006K15-G4) and the innovative fund of Xi’an Jiaotong University. Conflict of Interest Authors declare no conflict of interest. ==== Refs Reference Yenugu S Hamil KG Grossman G Petrusz P French FS Hall SH Identification, cloning and functional characterization of novel sperm associated antigen 11 (SPAG11) isoforms in the rat . Reprod Biol Endocrinol . 2006 ; 4 : 23 . 16643671 von Horsten HH Derr P Kirchhoff C Novel antimicrobial peptide of human epididymal duct origin . Biol Reprod . 2002 ; 67 (3 ): 804 – 13 . 12193388 Fröhlich O Po C Young LG Organization of the human gene encoding the epididymis-specific EP2 protein variants and its relationship to defensin genes . Biol Reprod . 2001 ; 64 (4 ): 1072 – 9 . 11259252 Fröhlich O Ibrahim NM Young LG EP2 splicing variants in rhesus monkey (Macaca mulatta) epididymis . Biol Reprod . 2003 ; 69 (1 ): 294 – 300 . 12606416 Avellar MC Honda L Hamil KG Yenugu S Grossman G Petrusz P Differential expression and antibacterial activity of epididymis protein 2 isoforms in the male reproductive tract of human and rhesus monkey (Macaca mulatta) . Biol Reprod . 2004 ; 71 (5 ): 1453 – 60 . 15229135 Fröhlich O Po C Murphy T Young LG Multiple promoter and splicing mRNA variants of the epididymis-specific gene EP2 . J Androl . 2000 ; 21 (3 ): 421 – 30 . 10819450 Hall SH Yenugu S Radhakrishnan Y Avellar MC Petrusz P French FS Characterization and functions of beta defensins in the epididymis . Asian J Androl . 2007 ; 9 (4 ): 453 – 62 . 17589782 Li P Chan HC He B So SC Chung YW Shang Q An antimicrobial peptide gene found in the male reproductive system of rats . Science . 2001 ; 291 (5509 ): 1783 – 5 . 11230693 Zhou CX Zhang YL Xiao L Zheng M Leung KM Chan MY An epididymis-specific beta-defensin is important for the initiation of sperm maturation . Nat Cell Biol . 2004 ; 6 (5 ): 458 – 64 . 15122269 Tian H Qiu SD Zhang QY Xue X Ge L Wang LR [Effects of experimental left varicocele on SPAG11 mRNA and SPAG11E in the testis and epididymis of adolescent rats] . Zhonghua Nan Ke Xue . 2008 ; 14 (3 ): 200 – 5 . Chinese. 18488329 Hamil KG Sivashanmugam P Richardson RT Grossman G Ruben SM Mohler JL HE2 beta and HE2gamma, new members of an epididymis-specific family of androgen-regulated proteins in the human . Endocrinology . 2000 ; 141 (3 ): 1245 – 53 . 10698202 Rodríguez CM Kirby JL Hinton BT Regulation of gene transcription in the epididymis . Reproduction . 2001 ; 122 (1 ): 41 – 8 . 11425328 Ibrahim NM Young LG Fröhlich O Epididymal specificity and androgen regulation of rat EP2 . Biol Reprod . 2001 ; 65 (2 ): 575 – 80 . 11466228 Yenugu S Hamil KG French FS Hall SH Antimicrobial actions of human and macaque sperm associated antigen (SPAG) 11 isoforms: influence of the N-terminal peptide . Mol Cell Biochem . 2006 ; 284 (1–2 ): 25 – 37 . 16411022 Zhang QY Qiu SD Ma XN Yu HM Wu YW Effect of experimental varicocele on structure and function of epididymis in adolescent rats . Asian J Androl . 2003 ; 5 (2 ): 108 – 12 . 12778320 Ozturk U Kefeli M Asci R Akpolat I Buyukalpelli R Sarikaya S The effects of experimental left varicocele on the epididymis . Syst Biol Reprod Med . 2008 ; 54 (4–5 ): 177 – 84 . 18942025	26807387	PMC4706104	NO-CC CODE	2021-01-05 05:06:45	yes	J Reprod Infertil. 2012 Oct-Dec; 13(4):241-247
==== Front BioinformationBioinformationBioinformationBioinformation0973-2063Biomedical Informatics 9732063001151610.6026/97320630011516RetractionRetraction for Singh et al. Performance evaluation of DNA Motif discovery programs 2015 30 11 2015 11 11 516 516 © 2015 Biomedical Informatics2015This is an Open Access article which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. This is distributed under the terms of the Creative Commons Attribution License. ==== Body This retracts the article “ Performance evaluation of DNA Motif discovery programs” in volume 3 and Issue 5 on page 205 Reader Feedback The article “Performance evaluation of DNA motif discovery programs”, by Singh et al. Bioinformation 2008 3(5): 205-212 [1], has text that was taken directly from the article “Limitations and potentials of current motif discovery algorithms” by Hu et al. Nucleic Acids Res. 2005 33(15): 4899-4913. [2] Editorial Decision Investigation suggests that significant portions of text are copied as stated above. This is clearly in violation of “ethics in publishing”. Therefore, it is decided to retract this article from Bioinformation as on November 30, 2015. Thus, this information is brought to the notice of the following authors: Chandra Prakash Singh Feroz Khan Bhartendu Nath Mishra Durg Singh Chauhan Citation:Bioinformation 11(11): 516-516 (2015) ==== Refs References 1 Singh Bioinformation 2008 3 5 205 PMC2646190 19255635 2 Hu Nucleic Acids Res 2005 33 15 4899 PMC1199555 16284194	26912955	PMC4748024	NO-CC CODE	2021-01-05 05:27:29	yes	Bioinformation. 2015 Nov 30; 11(11):516
==== Front 04104626011NatureNatureNature0028-08361476-46872667572110.1038/nature16193nihpa735485ArticleDDX5 and its associated lncRNA Rmrp modulate Th17 cell effector functions Huang Wendy 1Thomas Benjamin 2Flynn Ryan A. 3Gavzy Samuel J. 1Wu Lin 1Kim Sangwon V. 1Hall Jason A. 1Miraldi Emily R. 1456Ng Charles P. 1Rigo Frank 7Meadows Sarah 8Montoya Nina R. 1Herrera Natalia G. 1Domingos Ana I. 9Rastinejad Fraydoon 10Myers Richard M. 8Fuller-Pace Frances V. 11Bonneau Richard 456Chang Howard Y. 3Acuto Oreste 2Littman Dan R. 1121 The Kimmel Center for Biology and Medicine of the Skirball Institute, NYU School of Medicine2 Sir William Dunn School of Pathology, University of Oxford3 Center for Personal Dynamic Regulomes, Stanford University, Stanford, CA4 Center for Genomics and Systems Biology, Department of Biology, New York University, New York, NY5 Courant Institute of Mathematical Sciences, Computer Science Department, New York University, New York, NY6 Simons Center for Data Analysis, Simons Foundation, New York, NY7 Isis Pharmaceuticals, Carlsbad, CA8 HudsonAlpha Institute for Biotechnology, Huntsville, AL9 Instituto Gulbenkian de Ciencia, Oeiras, Portugal10 Integrative Metabolism Program, Sanford Burnham Prebys Medical Discovery Institute, Orlando, FL11 Division of Cancer Research, University of Dundee; Dundee, UK12 Howard Hughes Medical InstituteCorrespondence and requests for material should be addressed to [email protected] 2 2016 16 12 2015 24 12 2015 16 6 2016 528 7583 517 522 Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:http://www.nature.com/authors/editorial_policies/license.html#termsTh17 lymphocytes protect mucosal barriers from infections, but also contribute to multiple chronic inflammatory diseases. Their differentiation is controlled by RORγt, a ligand-regulated nuclear receptor. We identified the DEAD-box RNA helicase DDX5 as a RORγt partner that coordinates transcription of selective Th17 genes and is required for Th17-mediated inflammatory pathologies. Surprisingly, the ability of DDX5 to interact with RORγt and co-activate its targets depends on its intrinsic RNA helicase activity and binding of a conserved nuclear long noncoding RNA (lncRNA), Rmrp, which is mutated in Cartilage-Hair Hypoplasia (CHH) patients. A targeted Rmrp mutation in mice, corresponding to one in CHH patients, abrogated the lncRNA’s chromatin recruitment, ability to potentiate DDX5-RORγt interaction and RORγt target gene transcription. Elucidation of the link between Rmrp and the DDX5-RORγt complex reveals a role for RNA helicases and lncRNAs in tissue-specific transcriptional regulation and promises new opportunities for therapeutic intervention in Th17-dependent diseases. ==== Body T-helper 17 (Th17) cells are CD4+ lymphocytes that help protect mucosal epithelial barriers against bacterial and fungal infections 1, and that are also critically important in multiple autoimmune diseases 2–7. In murine models, attenuation of RORγt activity results in protection from experimental autoimmune encephalomyelitis (EAE), T cell transfer-mediated colitis, and collagen-induced arthritis 2–5. The Th17 cell differentiation program is defined by the induced expression of RORγt 2, a sterol ligand-regulated nuclear receptor that focuses the activity of a cytokine-regulated transcriptional network upon a subset of key genomic target sites, including genes encoding the signature Th17 cytokines (IL-17A, IL-17F, IL-22) as well as IL-23R, IL-1R1, and CCR6 8. Like other nuclear receptors, RORγt interaction with its ligands results in recruitment of co-activators at regulated genomic loci 9. We identified two new RORγt partners in Th17 cells, an RNA helicase and a long noncoding (lnc) RNA, which together associate with RORγt to confer target locus-specific activity in enabling the T cell effector program. The RNA helicase DEAD-box protein 5 (DDX5) functions in multiple cellular processes 10, including transcription and ribosome biogenesis 11–17 in both a helicase activity-dependent and -independent manner. The lncRNA Rmrp, RNA component of Mitochondria RNA-processing endoribonuclease (RNase MRP), is highly conserved between mouse and human and is essential for early murine development 18. Rmrp was first identified as a component of the RNase MRP complex that cleaves mitochondrial RNAs 19. In yeast, Rmrp contributes to ribosomal RNA processing and regulates mRNA degradation 20. In humans, mutations located in evolutionarily conserved nucleotides at the promoter or within the transcribed region of RMRP result in cartilage-hair hypoplasia (CHH), a rare autosomal recessive disorder characterized by early childhood onset of skeletal dysplasia, hypoplastic hair, defective immunity, predisposition to lymphoma, and neuronal dysplasia of the intestine 21,22. Immune deficiency in CHH patients is associated with recurrent infections, hematological abnormalities, and autoimmune pathologies in the joints and kidneys 23. The precise mechanisms by which Rmrp functions in the immune system have yet to be elucidated. Here we show that DDX5, through its helicase activity, mediates Rmrp-dependent binding to RORγt and recruitment to a subset of its chromatin target sites, thus controlling the differentiation of Th17 cells at steady state and in animal models of autoimmunity. DDX5 regulation of RORγt target genes To identify novel interacting partners of RORγt in Th17 cells, we enriched for endogenous RORγt-containing protein complexes and subsequently determined protein composition using LC-MS/MS (workflow diagramed in Extended Data Fig. 1a). Among the top hits of RORγt-interacting proteins was the RNA helicase DDX5. We validated this interaction through conventional co-immunoprecipitation (coIP) experiments followed by immunoblot analysis (Extended Data Fig. 1b). We investigated the function of DDX5 in T cells by breeding ddx5 conditional mutant mice with CD4Cre mice to generate T cell-specific DDX5-deficient animals (Ddx5fl/fl CD4Cre mice, designated DDX5-Tko). DDX5-Tko mice were born at the expected Mendelian ratio, were fertile, and did not display any gross phenotypic abnormalities. Activation status of T cells in the periphery was similar between Ddx5+/+CD4Cre+ (designated wildtype, WT) and mutant mice (Extended Data Fig. 1c) that had no DDX5 protein in spleen and lymph node CD4+ T cells (Extended Data Fig. 1d). Sorted naïve CD4+ T cells from WT and DDX5-Tko mice did not display significant differences in polarization towards Th1, Th2, and iTreg phenotypes in vitro (Fig. 1a). In contrast, DDX5-Tko naïve T cells cultured under Th17 polarizing conditions produced substantially less IL-17A than WT cells (Fig. 1a). RORγt protein expression and nuclear localization were similar between WT and DDX5-Tko Th17-polarized cells (Extended Data Fig. 1d–e) and, like RORγt, DDX5 protein localized mainly to the nucleus (Extended Data Fig. 1f). These results suggest that DDX5 is not required for Th17 lineage commitment, but contributes to Th17 cell effector functions. DDX5 can function as a transcriptional coactivator 12,24,25, augmenting the activities of other nuclear receptor family members, including the estrogen and androgen receptors 12,26. To determine if DDX5 partners with RORγt to facilitate the Th17 cell transcriptional program, we performed RNA-seq on in vitro polarized Th17 cells from WT or DDX5-Tko mice. Among the 325 genes that were significantly dysregulated in DDX5-deficient T cells 96hrs post polarization, approximately 40% had been previously identified as RORγt targets in Th17 cells (Extended Data Fig. 2a) 8. Ingenuity Pathway Analysis of DDX5-RORγt-coregulated genes revealed enrichment in “T helper cell differentiation program” as well as “interleukin production” (Extended Data Fig. 2b). Co-regulated genes (Fig. 1b) included those for the Th17 cytokines (Il17a, Il17f, and Il22) (Extended Data Fig. 2c). Independent biological samples were used to validate a subset of RORγt target genes with and without altered expression in DDX5-deficient Th17 cells (Extended Data Fig. 2d). We used anti-DDX5 antibodies in ChIP-seq studies to identify DDX5-occupied loci genomewide. A specific subset of previously published RORγt-occupied loci, including Il17a and Il17f, were enriched for DDX5 co-localization, as determined by SeqMiner clustering analysis (Extended Data Fig. 3a–b). Conventional ChIP-qPCR was used to validate DDX5 enrichment at the Il17a and Il17f loci and its dependency on RORγt in polarized Th17 cells (Extended Data Fig. 3c). These results suggest that DDX5 overlaps with RORγt in modulating a specific subset of the Th17 cell transcriptional program. DDX5 function in vivo in Th17 cells At steady state, cytokine-producing Th17 cells populate the small intestinal lamina propria of animals colonized with Segmented Filamentous Bacteria (SFB), a commensal microbe 27. When colonized with SFB, DDX5-Tko and their WT littermates had similar numbers of ileal-residing Foxp3−RORγt+CD4+ Th17 cells (Fig. 1c). However, the number and proportion of IL-17A–producing cells among RORγt+CD4+ cells from DDX5-Tko animals were markedly reduced compared to WT littermate controls (Fig. 1d–e). To evaluate the role of DDX5 in Th17-driven inflammation, we employed a T cell transfer model of colitis, in which disease severity is dependent on RORγt expression in donor T cells 3,28. Following transfer of CD4+CD45RBhi T cells into Rag-deficient recipients, mice that received WT T cells experienced weight loss (Fig. 2a) and developed colitis (Fig 2b), whereas recipients of DDX5-Tko cells did not. Total RNA harvested from large intestine lamina propria mononuclear cells revealed a significant reduction of both IL-17A and IFNγ transcripts from recipients of DDX5-Tko cells compared to WT controls (Extended Data Fig. 4a). Interestingly, there were comparable proportions of IFNγ-producing CD4+RORγt−Tbet+ (conventional Th1) cells in recipients of either WT or DDX5-Tko cells (Extended Data Fig. 4b). However, recipients of DDX5-Tko cells displayed a significant reduction in CD4+Foxp3−RORγt+ T cells co-expressing IL-17A and IFNγ, a cardinal feature of pathogenic T cells in several inflammatory disease settings (Fig. 2c and Extended Data Fig. 4b) 2,29,30. Consistent with a loss of pathogenic capacity, DDX5-Tko mice also exhibited attenuated disease compared to WT controls during experimental autoimmune encephalomyelitis (EAE) (Fig. 2d). Analysis of spinal cord infiltrates post immunization revealed a reduced proportion of IL-17A–producing CD4+ T cells (Fig. 2e and Extended Data Fig. 4c). In concert with our in vitro findings, these results in animals indicate that DDX5 selectively regulates the Th17 effector program, both in steady state and under inflammatory conditions. Function of DDX5-associated LncRNA RNA helicases are highly conserved enzymes that utilize the energy derived from ATP hydrolysis to unwind RNA duplexes, facilitate RNA annealing, and displace proteins from RNA. It was previously shown that DDX5 transcriptional coactivator activity for estrogen receptor, androgen receptor, and RUNX2 is independent of RNA helicase activity 12,24,26. We tested this requirement in the context of RORγt by retrovirally transducing DDX5-deficient T cells cultured under Th17 polarizing conditions with expression constructs for WT or mutant DDX5 with an inactivated helicase domain (helicase-dead). Surprisingly, only WT DDX5 rescued IL-17A and IL-17F production in these polarized Th17 cells (Fig. 3a–b and Extended Data Fig. 5a). This result suggested that perhaps RNA substrate(s) for the helicase activity of DDX5 contribute to its transcriptional coactivator role in Th17 cells. We next searched for RNA molecules that might participate in DDX5-RORγt-mediated transcription in Th17 cells. To this end, we first depleted ribosome-bound mRNAs undergoing active protein synthesis. Lysates pre-cleared of ribosomes were then subjected to immunoprecipitation with antibodies specific for DDX5 or RORγt, followed by deep sequencing of the associated RNAs (RIP-seq). Among 49,893 annotated lncRNAs in the mouse RefSeq and NONCODE database, 2,533 ncRNAs were expressed in Th17 cells (FPKM>1, Extended Data Fig. 5b). Interestingly, ncSRA, previously found to be associated with DDX5 in muscle cells 15, was not enriched in DDX5-containing protein complexes in Th17 cells. Instead, we found Rmrp to be the most enriched RNA associated with DDX5 and, to a lesser degree, RORγt, in Th17 cells (Fig. 3c and Extended Data Fig. 5c). Conventional RIP-qPCR with independent biological samples confirmed enrichment of Rmrp RNA in DDX5 pull-downs from Th17 cells, but not from thymocyte lysates (Extended Data Fig. 5d). RNA FISH revealed that Rmrp is localized in the nucleus of Th17 cells (Extended Data Fig. 6a). To evaluate the functional role of Rmrp, we transiently depleted Rmrp RNA from primary murine Th17 cells using an RNAseH-dependent antisense oligonucleotide (ASO). Similar to the DDX5-deficient Th17 cells, cells depleted for Rmrp expressed reduced IL-17A and IL-17F mRNA (Fig. 3d and Extended Data Fig. 6b). Human Th17 cells also displayed reduced cytokine production upon depletion of Rmrp or DDX5 (Fig. 3E and Extended Data Fig. 6c), suggesting that this regulatory mechanism is evolutionarily conserved. Importantly, Rmrp knockdown in DDX5-deficient murine Th17 cells did not further reduce IL-17A and IL-17F expression (Fig. 4a). Expression of RORγt-dependent, but DDX5-independent, CCR6 was unaffected by the reduction in Rmrp. Transduction of Rmrp into T cells cultured in Th1 polarization conditions had little effect on IFNγ production, but there was marked enhancement of IL-17A and IL-17F production in WT, but not DDX5-Tko, cells cultured in Th17 polarization conditions (Fig. 4b and Extended Data Fig. 7a–b). Thus, Rmrp-dependent cytokine gene expression requires the presence of DDX5. Th17 program in Rmrp mutant mice In contrast to wildtype Rmrp, a mutant Rmrp carrying a single nucleotide change (270 G>T), corresponding to an allele identified in CHH patients (262 G>T), failed to potentiate IL-17A production following transduction into Th17-polarized cells (Extended Data Fig. 7c–d). To ask whether G270 of Rmrp contributes to RORγt transcriptional output in vivo, we generated mice homozygous for the Rmrp G270T point mutation, using CRISPR-Cas9 technology (Fig. 4c). These animals were born at the expected Mendelian ratios and had no gross defects. ROR element-regulated luciferase activity was reduced in transiently transfected Th17 cells from DDX5-deficient and RmrpG270T mice and upon ASO-mediated knockdown of Rmrp (Fig. 4d). Comparison of transcription profiles of in vitro polarized Th17 cells from WT, RORγt-deficient, DDX5-deficient, and RmrpG270T/G270T mice indicated that 96 RORγt-dependent Th17 cell genes were co-regulated by Rmrp together with DDX5 (Extended Data Fig. 7e and Fig. 4e). RT-qPCR analysis of independent biological samples from in vitro polarized T cells from WT and RmrpG270T/G270T mice confirmed reduced IL-17F mRNA expression in the latter (Extended Data Fig. 7f), despite similar amount of RORγt binding to known cis-regulatory loci (Extended Data Fig. 7g). The proportion of RORγt+Foxp3− Th17 cells among total ileal LP CD4-lineage cells was unaffected in RmrpG270T/G270T animals, but these cells expressed relatively little IL-17A compared to those in wildtype littermates (Fig. 4f). Transfer of RmrpG270T/G270T T cells into Rag−/− mice resulted in reduced colitis, as determined by weight loss and colon histology, compared to transfer of wildtype cells (Extended Fig. 8a). These phenotypes are similar to what was observed in animals with a T cell-specific Ddx5 deletion (Fig. 2a–c), which is consistent with an important role of G270 of Rmrp in executing the Th17 effector program in vivo. RORγ/γt perform distinct functions in diverse tissues. RORγt is critical for thymocyte development, regulating survival of double positive cells, and for lymphoid tissue inducer cell-mediated development of secondary and tertiary lymphoid organs 31. While DDX5 and Rmrp are ubiquitously expressed, Rmrp was less enriched in thymocyte-derived than in Th17 cell-derived DDX5 immunoprecipitates (Extended Data Fig. 5d). When Ddx5 was inactivated at the common lymphoid progenitor stage, by breeding the conditional mutant mice with IL7R–Cre mice, there was no apparent defect in thymocyte subset development (Extended Data Fig. 8b). Similarly, RmrpG270T knockin animals displayed normal thymocyte subsets and, additionally, had intact secondary lymphoid organ development (Extended Data Figure 8c). Together, these results suggest that the DDX5-Rmrp complex performs Th17-specific functions. Rmrp in RORγt-DDX5 complex formation We next asked how Rmrp contributes to the DDX5-RORγt-regulated transcriptional circuit in Th17 cells. RORγt-DDX5 complex assembly was severely compromised in Th17 cells harboring RmrpG270T (Fig. 5a). Moreover, Rmrp recruitment to the RORγt protein complex was significantly reduced in Th17 cells from Rmrp mutant animals (Fig. 5b). In vitro, Rmrp binds directly to recombinant DDX5 (Extended Data Fig. 9a). Intriguingly, Rmrp was recruited to RORγt in the presence of wildtype, but not helicase-dead, DDX5. Furthermore, in vitro transcribed Rmrp RNA promoted RORγt interaction with wildtype, but not helicase-dead, DDX5 in the presence of ATP (Fig. 5c and Extended Data Fig. 9b). Mutant Rmrp was also defective in mediating DDX5-RORγt complex assembly in vitro (Extended Data Fig. 9c–d). To determine if Rmrp is associated with specific genomic loci, we performed chromatin isolation by RNA purification (ChIRP) followed by either locus-specific quantitative PCR or by deep sequencing (ChIRP-seq) 32. We employed two orthogonal antisense probe sets that specifically and robustly recovered Rmrp from Th17 cells (Extended Data Fig. 10a). When combined for Rmrp ChIRP-qPCR, the probes recovered RORγt-bound regions in the Il17a and Il17f loci from Th17 polarized cells of wild type but not DDX5Tko or RmrpG270T/G270T mice, in an RNase-sensitive manner (Fig. 5d and Extended Data Fig. 10b). For ChIRP-seq, we focused our analysis on signals that overlapped with use of the two probe sets. HOMER motif analyses of Rmrp peak regions identified the ETS, DR2/RORE, and AP1 transcription factor (TF) motifs to be the most highly enriched (Extended Data Fig. 10c). Consistent with this, Rmrp ChIRP-seq significantly overlapped with RORγt-bound loci, but not with sites occupied by CTCF or by other Th17 TFs, such as BATF, IRF4, STAT3, and c-Maf (Extended Data Fig. 10d). There was also significant overlap with RNAPol-II- and H3K4me3-associated chromatin, which mark actively transcribed regions. Concordantly, ChIRP-seq of Rmrp in DDX5-Tko Th17 cells revealed a loss of called Rmrp peaks despite similar amount of RNA recovery (Extended Data Fig. 10e), consistent with a DDX5 contribution to Rmrp assocation with chromatin. Rmrp association with RORγt bound sites was also reduced in polarized Th17 cells from RmrpG270T/G270T mice (Extended Data Fig.10f). Together, these results indicate that G270 of Rmrp is critical for DDX5-RORγt complex assembly and Rmrp recruitment to RORγt-occupied loci to coordinate the Th17 effector program in trans. Discussion Nuclear lncRNAs have key roles in numerous biological processes 33, including adaptive and innate immunity 34,35, but how individual lncRNAs perform their activities and whether they contribute to immunological diseases remain unknown. We identified nuclear Rmrp as a key DDX5-associated RNA required to promote assembly and regulate the function of RORγt transcriptional complexes at a subset of critical genes implicated specifically in the Th17 effector program (model in Fig. 5e). Rmrp thus acts in trans on multiple RORγt-dependent genes, and does so only upon interaction with enzymatically active DDX5 helicase. RNA helicase-dependent functions of lncRNAs have been described, e.g. the Drosophila male cell-specific lncRNAs roX1 and roX2 that are modified by the MLE helicase to enable expression of X-chromosome genes 36,37. In addition, DDX21 helicase activity in HEK293 cells is required for 7SK RNA regulation of polymerase pausing at ribosomal genes 38. Our results extend the concept of RNA helicase/lncRNA function to lineage-specific regulation of transcriptional programs. It is noteworthy that, unlike most lncRNAs, Rmrp is highly conserved among mammals. In humans, mutations of evolutionarily conserved nucleotides at the promoter or within the transcribed region of RMRP result in Cartilage-Hair Hypoplasia (CHH) 21,22. T cells from mice carrying a single nucleotide change (270 G>T) in Rmrp, corresponding to one found in CHH patients (262 G>T), had a compromised Th17 cell effector program. CHH patients have been noted to have defective T cell-dependent immunity, which may reflect, at least in part, reduced Rmrp-dependent activity at RORγt target genes. Since forced expression of either DDX5 or Rmrp enhanced Th17 cytokine production, it is also possible that gain-of-function mutations in either of these molecules may contribute to Th17-dependent inflammatory diseases. RORγt is an attractive therapeutic target for multiple autoimmune diseases 5,39. However, RORγt and its closely-related isoform RORγ have multiple other functions that would likely be affected by targeting of their shared ligand-binding pocket. RORγt is required for the development of early thymocytes, lymphoid tissue inducer (LTi) cells that initiate lymphoid organogenesis 31, type 3 innate lymphoid cells (ILC3) that produce IL-22 and protect epithelial barriers, and for IL-17 production by “innate-like” T cells, including TCRγδ and NKT cells 40–43. In the liver, RORγ contributes to regulation of metabolic functions 44. Mechanisms by which RORγ/γt differentially regulates transcription in these diverse cell types remain poorly understood. DDX5 and Rmrp are abundantly expressed in developing T cells in the thymus and in peripheral naïve and differentiated T helper subsets (Huang, unpublished). Intriguingly, the contribution of DDX5-Rmrp to RORγt-dependent functions appears to be confined to Th17 cells, as their loss of function did not affect thymocyte or lymphoid organ development. Our results raise the prospect that tissue- or cell type-specific mechanisms exist to regulate how RNA helicases and their associated lncRNAs are assembled with distinct transcriptional complexes to promote diverse gene expression programs. We speculate that the function of DDX5-Rmrp may be induced in response to specific tissue microenvironments in vivo. Th17 cells differentiate at mucosal barriers in response to signals from the microbiota, and upregulate their expression of IL-17A locally 45,46. Regional signals may induce DDX5/Rmrp association with RORγt, resulting in the transcriptional activation of multiple loci that endow Th17 cells with effector functions15. Our finding that DDX5 was required for the differentiation of “pathogenic” Th17 cells 2,29,30 suggests that strategies to interfere with this function may be of therapeutic benefit. A better understanding of this transcriptional regulatory system may provide new approaches for therapeutic intervention in autoimmune diseases and immune deficiencies in CHH patients. Methods Mice EF1a: Lox-stop-lox-GFP-L10, RORγ/γt-deficient animals, and Ddx5fl/fl mice were previously described 47–49. Conditional mutant mice were bred to CD4-Cre transgenic animals (Taconic) and maintained on the C57BL/6 background. We bred heterozygous 6–8wk old mice to yield Ddx5+/+CD4Cre+ (subsequently referred to as WT) and Ddx5fl/flCD4Cre+ (referred to as DDX5-Tko) littermates for experiments examining DDX5 in peripheral T cell function. DDX5 conditional mutant mice were also bred to IL7R–Cre transgenic animals (Jackson Laboratory) for experiments examining DDX5 during T cell development in the thymus. RmrpG270T knockin mice were generated using CRISPR-Cas9 technology by the Rodent Genetic Engineering Core (RGEC) at NYULMC. Guide RNA and HDR donor template sequences are provided in Supplementary Table 1. Heterozygous crosses provided Rmrp+/+ (WT) and RmrpG270T/G270T littermates for in vivo studies. All animal procedures were in accordance with protocols approved by the Institutional Animal Care and Use Committee of the NYU School of Medicine (Animal Welfare Assurance Number: A3435-01). In vivo studies Steady state small intestines were harvested for isolation of lamina propria mononuclear cells as described 50. For detecting SFB colonization, SFB-specific 16S primers were used 51. Universal 16S and/or host genomic DNA were quantified simultaneously to normalize SFB colonization of each sample. All primer sequences are listed in Supplementary Information Table. For the adoptive transfer model of colitis, 5×105 CD4+CD45RBhiCD62LhiCD44lowCD25− T cells were isolated from murine splenocytes by FACS sorting and administered intraperitoneally (i.p.) into Rag2−/− mice as previously described 52. Animal weights were measured approximately weekly. At the time of sacrifice (between weeks seven and eight), large intestines were harvested for H&E staining and isolation of lamina propria mononuclear cells as described 50. The H&E slides from each sample were examined in a double-blind fashion. The histology scoring (scale 0–24) was based on the evaluation of criteria described previously 53. For induction of active experimental autoimmune encephalomyelitis (EAE), mice were immunized subcutaneously on day 0 with 70 µg of MOG 35–55 peptide, emulsified in CFA (Complete Freund’s Adjuvant supplemented with 200 mg/mL Mycobacterium tuberculosis), and injected i.p. on days 0 and 2 with 100 ng/mouse of pertussis toxin (Calbiochem). The EAE scoring system was as follows: 0-no disease, 1-limp tail; 2-weak/partially paralyzed hind legs; 3-completely paralyzed hind legs; 4-complete hind and partial front leg paralysis; 5-complete paralysis/death. In transfer colitis and EAE experiments, animals of different genotypes were cohoused and were weighed and scored blindly. For statistical power level of 0.8, probability level of 0.05, anticipated effect size of 2, minimum sample size per group for two-tailed hypothesis is 6. Two-tailed unpaired Student’s t-test was performed using Prism (GraphPad Software). We treated a P-value of less than 0.05 as a significant difference. ; p<0.05, ; p<0.001, and ; p<0.005. All experiments were performed at least twice. In vitro T cell culture and phenotypic analysis Mouse T cells were purified from lymph nodes and spleens of six to eight week old mice, by sorting live (DAPI−), CD8−CD19−CD4+CD25−CD62L+CD44low/Int naïve T cells using a FACSAria (BD). Detailed antibody information is provided in Supplementary Table 1. Cells were cultured in IMDM (Sigma) supplemented with 10% heat-inactivated FBS (Hyclone), 50 U penicillin-streptomycin (Invitrogen), 4 mM glutamine, and 50 µM β-mercaptoethanol. For T cell polarization, 200 µl of cells was seeded at 0.3 × 10^5 cells per mL in 96-well plates pre-coated with goat anti-hamster IgG at a 1:20 dilution of stock (1mg/ml, MP Biomedicals Catalog # 55398). Naïve T cells were activated with anti-CD3ε (2.5 µg/mL) and anti-CD28 (10 µg/mL). Cells were cultured for 4−5 days under Th17 polarizing conditions (0.1–0.3 ng/mL TGF-β, 20 ng/mL IL-6), Th1 (10 ng/mL IL-12, 10 U/mL IL-2, and 2 ng/mL anti-IL-4), Th2 (10 ng/mL IL-4), or Treg conditions (5 ng/mL TGF-β, 10 U/mL IL-2). Human T cells were isolated from peripheral blood of healthy donors using anti-human CD4 MACS beads (Miltenyi). Human CD4 T cells were cultured in 96-well U bottom plates in 10 U/ml of IL-2, 10 ng/mL of IL-1β, 10 ng/ml of IL-23, 1 µg/ml of anti-IL-4, 1 µg/mL of anti-IFNγ and anti-CD3/CD28 activation beads (LifeTechnologies) at a ratio of 1 bead per cell, as previously described 54. For cytokine analysis, cells were incubated for 5 h with phorbol PMA (50 ng/mL; Sigma), ionomycin (500 ng/mL; Sigma) and GolgiStop (BD). Intracellular cytokine staining was performed according to the manufacturer’s protocol (Cytofix/Cytoperm buffer set from BD Biosciences and FoxP3 staining buffer set from eBioscience). A LSR II flow cytometer (BD Biosciences) and FlowJo (Tree Star) software were used for flow cytometry and analysis. Dead cells were excluded using the Live/Dead fixable aqua dead cell stain kit (Invitrogen). Nucleic acid reagents and T cell transduction Custom Rmrp and predesigned Malat1 Stellaris RNA FISH probes were purchased from BiosearchTech and used to label mRmrp and mMalat1 RNA in cultured Th17 cells according to the manufacturer’s protocol. Control and human DDX5 specific siRNAs (#8626) were obtained from Cell Signaling. Synthesis of ASOs was performed as previously described 55. All ASOs were 20 nts in length and had a phosphorothioate backbone. The ASOs had five nucleotides at the 5’ and 3’ ends modified with 2’-O-methoxyethyl (2’-MOE) for increased stability. ASOs and siRNA sequences are provided in Supplementary Table 1. siRNA and ASOs were introduced into murine Th17 cells by Amaxa nucleofection as previously described 56. WT and helicase dead mutant DDX5 were described previously 57. DDX5 and Rmrp were subcloned into the MSCV Thy1.1 vectors for retroviral overexpression and rescue assays in T cells. Retrovirus production was carried out in PlatE cells as described 58. Spin transduction was performed 24h after in vitro T cell activation by centrifugation in a Sorvall Legend RT at 2000rpm for 90min at 32° C. Aqua−Thy1.1+ live and transduced cells were analyzed by flow cytometry after 5 days of culture in Th17 polarizing conditions. RORγt Transcriptional Activity in Polarized Th17 Cells A ROR luciferase reporter was constructed with 4 RORE sites replacing the Gal4 (UAS) sites from the pGL4.31 vector (luc2P/GAL4 UAS/Hygro) from Promega (C935A) as described in 59. Naïve CD4+ T cells were cultured in Th17 polarizing conditions for 72h. Nucleofection (Amaxa Nucleofector 4D, Lonza) was then used to introduce 1 µg RORE-firefly luciferase reporter construct and 1 µg control renilla luciferase construct according to the manufacturer’s instructions. Luciferase activity was measured using the dual luciferase reporter kit (Promega) at 24h after transfection. Relative luciferase units (RLU) were calculated as a function of firefly luciferase reads over those of renilla luciferase. Co-immunoprecipitation and mass spectrometry 100×106 cultured Th17 cells were lysed in 25mM Tris (pH 8.0), 100mM NaCl, 0.5% NP40, 10mM MgCl2, 10% glycerol, 1X protease inhibitor and PhosphoSTOP (Roche) on ice for 30min, followed by homogenization with a 25g needle. The RORγ/γt-specific antibody used for pull down assays was previously described 56. Co-immunoprecipitated complexes were harvested with Protein G Dynabeads (Dynal, Invitrogen). Detailed antibody information is provided in Supplementary Table 1. Mass spectrometry and the Mascot database search to identify protein complex composition were both performed by the Central Proteomics Facility at the Dunn School of Pathology, Oxford, UK. Ribosome TRAP-seq, RIP-seq, and RNA-seq 20 million cells cultured in Th17 polarizing conditions for 48 h were lysed in 10 mM HEPES (pH 7.4), 150 mM KCl, 0.5 mM dithiothreitol (DTT), 100 ug/ml cycloheximide, 1% NP40, 30 mM DHPC, 1X protease inhibitor, and PhosphoSTOP (Roche). Ribosome-TRAP IP was first performed using 2 µg of anti-GFP antibody (Invitrogen) and harvested in 20 µl of Protein G magnetic Dyna beads. The supernatant was removed for subsequent RIP pull down using anti-DDX5 (Abcam) or anti-RORγt antibodies and harvested with Protein G Dyna beads. TRAPseq samples were washed with high-salt wash buffer (10 mM HEPES pH 7.4, 350 mM KCl, 5 mM MgCl2, 1% NP-40, 0.5 mM dithiothreitol (DTT), and 100 ug/ml cycloheximide). RIPseq samples were washed three times with 25mM Tris (pH 8.0), 100mM NaCl, 0.5% NP40, 10mM MgCl2, 10% glycerol, 1X protease inhibitor, and PhosphoSTOP (Roche). Enrichment of target proteins was confirmed by immunoblot analysis. Complementary DNAs (cDNAs) were synthesized from TRIzol (Invitrogen) isolated RNA, using Superscript III kits (Invitrogen). RNA-seq libraries were prepared and sequenced at Genome Services Laboratory, HudsonAlpha. Sequencing reads were mapped by Tophat and transcripts called by Cufflinks. Pulldown enrichment was calculated for each transcript as a ratio of FPKM recovered from TRAP-seq and RIP-seq samples compared to those from 5% input. For RNA-seq analysis, volcano scores for WT, DDX5-Tko, and RORγt knockout Th17 cells were calculated for each transcript as a function of its p-value and fold-change between mutant and WT controls. BAM files were converted to tdf format for viewing with the IGV Browser Tool. Ingenuity Pathway Analysis was used to identify enriched Gene Ontology (GO) terms in the DDX5-RORγt co-regulated gene set. ChIRP-seq and ChIRP-qPCR The ChIRP-seq assay was performed largely as described previously 60. Mouse Th17 cells were cultured as above and in vivo RNA-chromatin interactions were fixed with 1% glutaraldehyde for 10 minutes at 25°C. Anti-sense DNA probes (designated odd or even) against Rmrp were designed by Biosearch Probe Designer (#1: taggaaacaggccttcagag, #2: aacatgtccctcgtatgtag, #3: cccctaggcgaaaggataag, #4: aacagtgacttgcgggggaa, #5: ctatgtgagctgacggatga). Probes modified with BiotinTEG at the 3-prime end were synthesized by Integrated DNA Technologies (IDT). Isolated RNA was used in qRT-PCR analysis (Stratagene) to quantify enrichment of RMRP and depletion of other cellular RNAs. Isolated DNA was used for qPCR analysis or to make deep sequencing libraries with the NEBNext DNA Library Prep Master Mix Set for Illumina (NEB). Library DNA was quantified on the High Sensitivity Bioanalyzer (Agilent) and sequenced from a single end for 75 cycles on an Illumina NetSeq 500. Sequencing reads were first trimmed of adaptors (FASTX Toolkit) and then mapped using Bowtie to a custom bowtie index containing single-copy loci of repetitive RNA elements (rRNA, snRNAs, and y-RNAs 61). Reads that did not map to the custom index were then mapped to mm9. Mapped reads were separately shifted towards the 3’ end using MACS and normalized to a total of 10 million reads. Even and Odd replicates were merged as described previously60 by taking the lower of the two read density values at each nucleotide across the entire genome. These processing steps take raw FASTQ files and yield processed files that contain genome-wide RMRP-chromatin association maps, where each nucleotide in the genome has a value that represents the relative binding level of the RMRP RNA. MACS parameters were as follows: band width = 300, model fold = 10,30, p-value cutoff = 1.00e-05. The full pipeline is available at https://github.com/bdo311/chirpseq-analysis. ChIRP-qPCR was performed on DNA purified after treatment with RNase (60 min, 37°C) and proteinase K (45min, 65°C). The primers used for qPCR can be found in Supplementary Information Table 1. For enrichment analysis, we tested for the enrichment of Rmrp ChIRP peaks among ChIP peak sets for key Th17 transcription factors, CTCF, RNA pol II, and several histone marks (Ciofani et al., 2012). ATACseq, according to published protocol62, was performed on cultured Th17-polarized cells in vitro for 48h (unpublished). Because of differences in ChIP antibody affinities and the bias in the selection of ChIP and ChIRP factors, we used peaks generated from ATACseq data as a background set for the enrichment analysis. In our analysis, we considered all ChIRP and ChIP peaks that fell within +/−500bp of ATACseq peaks, and then calculated the overlap among the ChIRP and ChIP sets, using the hypergeometric distribution to estimate significance. In vitro binding assay For in vitro binding assays, pcDNA3.1-Rmrp vectors were used for T7 polymerase-driven in vitro transcription reactions (Promega). HA-DDX5 and FLAG-RORγt were in vitro transcribed and translated using an in vitro transcription and translation (TNT) system according to the manufacturer’s protocol (Promega). Alternatively, pGEX4.1-DDX5 (wildtype and helicase-dead mutant) constructs were transformed into BL21 to synthesize recombinant full-length GST-hDDX5 proteins. Full-length His-tagged human RORγt was purified in three steps through Ni-Resin, S column, and gel-filtration on AKTA. 0.5µg of each recombinant protein was incubated in the presence or absence of 200 µM ATP, 300ng in vitro transcribed Rmrp in coIP buffer containing 25mM Tris (pH 8.0), 100mM NaCl, 0.5% NP40, 10mM MgCl2, 10% glycerol, 1X protease inhibitor, RNaseInhibitor (Invitrogen), and PhosphoSTOP (Roche). GST-DDX5 was enriched on glutathione beads (GE), HA-DDX5, FLAG-RORγt, and His-RORγt were enriched using anti-HA, (Covance), anti-FLAG (Sigma), and anti-His antibodies (Santa Cruz Bio) coupled to Anti-Mouse immunoglobulin Dynabeads (Dynal, Invitrogen). Microscopy Th17 cells were cultured on glass coverslips for 48 h and fixed in 4% paraformaldehyde in PBS for 5min in room temperature. Fixed cells were permeabilized with 0.1% BSA, 0.1% Triton, 10% normal serum in PBS for 1 h. Cells were then incubated with primary antibodies (DDX5, Abcam or RORγt, eBiosciences) in 0.1% BSA, 0.2% Triton PBS overnight at 4°C. Secondary antibodies (anti-goat-Alexa 488 or anti-rat-Alexa647, Molecular Probe) were incubated in 4°C for 1 h. Stained cells were washed three times with 0.5% tween, 0.1% BSA in PBS. DAPI was used to stain for DNA inside the nucleus. Immunofluorescence images were captured on a Zeiss 510 microscope at 40x. ChIP and RT-qPCR analysis Th17 polarized cells were crosslinked with 1% paraformaldehyde (EMS) and incubated with rotation at RT. Crosslinking was stopped after 10 min with glycine to a final concentration of 0.125M and incubated 5 min further with rotation. Cells were washed with ice cold PBS 3X and pellets were either flash frozen in liquid N2 or immediately resuspended in Farnham Lysis buffer (5mM PIPES, 85mM KCl, 0.5% NP-40). Hypotonic lysis continued for 10 min on ice before cells were spun down and resuspended in RIPA buffer (1X PBS, 1% NP-40, 0.5% SDS, 0.5% Na-deoxycholate), transferred into TPX microtubes, and lysed on ice for 30 minutes. Nuclear lysates were sonicated for 40 cycles of 30 sec ON and 30 sec OFF in 10 cycle increments using a Biorupter (Diadenode) at high setting. After pelleting debris, chromatin was precleared with protein G dynabeads (Dynabeads, TFS) for 2h with rotation at 4°C. For immunoprecipitation, precleared chromatin was incubated with anti-RORγt antibodies (1µg per 2 million cells) overnight with rotation at 4°C and protein G was added for the final 2h of incubation. Beads were washed and bound chromatin was eluted. ChIP-qPCR was performed on DNA purified after treatment with RNase (30 min, 37°C) and proteinase K (2h, 55°C) followed by reversal of crosslinks (8–12h, 65°C). The primers used for qPCR were described in 63. For analysis of mRNA transcripts, gene specific values were normalized to the GAPDH housekeeping gene for each sample. All primer sequences are listed in Supplementary Information Table. Extended Data Extended Data Figure 1 Identification of DDX5 as a RORγt-interacting partner a, Mass spectrometry experimental workflow. Sorted naïve CD4+ T cells from WT mice were cultured in vitro in Th17 polarizing conditions for 48h. Immunoprecipitation of endogenous RORγt was performed using RORγ/γt-specific antibodies on whole cell lysates. RORγt enrichment in pull-down was confirmed by immunoblot. Immunoprecipitated proteins were digested and analyzed by mass spectrometry. The listed DDX5 peptides were identified in the Th17 RORγt immunoprecipitate. b, Co-immunoprecipitaton of DDX5 with anti-RORγt in lysates of in vitro polarized Th17 cells. For gel source data, see Supplementary Figure 1. c, Cell surface phenotype of splenic and lymph node DAPI−CD19−CD8α−CD4+T cells from WT and DDX5-Tko animals, examined by flow cytometry. d, Immunoblot of RORγt protein expression whole cell lysate of cultured Th17 cells from WT or DDX5-Tko animals. For gel source data, see Supplementary Figure 1. e, Immunofluorescence staining of RORγt in cultured Th17 cells from WT or DDX5-Tko animals. f, Immunofluorescence staining of DDX5 revealed nuclear localization in Th17 cells. Extended Data Figure 2 DDX5 co-regulates a subset of RORγt transcriptional targets in polarized Th17 cells a, Venn diagram of distinct and overlapping genes regulated by RORγt and/or DDX5, as determined from RNA-seq studies. b, Ingenuity Pathway Analysis of DDX5- and RORγt-coregulated genes. c, IGV browser view showing biological replicate RNA-seq coverage tracks of control, DDX5-Tko, or RORγt-deficient in vitro polarized Th17 cell samples at the Il17a, Il22, Ddx5, and Rorc loci. d, Independent qRT-PCR validation of RNA-seq results confirming effects of DDX5 deletion on RORγt target gene expression. Graph shows mean ± s.d. Extended Data Figure 3 DDX5 chromatin localization in Th17 cells a, ChIP-seq-generated heatmap of DDX5 occupancy in regions centered on 16,003 RORγt-occupied sites (+/− 2kb). KMeans linear normalization was used for clustering analysis by SeqMiner. Metagene analysis on cluster 1 depicts RORγt-occupied regions with DDX5 enrichment in wildtype but not DDX5-Tko cells; cluster 2 represents RORγt-occupied regions without DDX5 enrichment. b, IGV browser view of Il17a, Il17f, and Rorc loci with ChIP-seq enrichment for RNAPol-II, RORγt, and DDX5. c, Independent ChIP-qPCR of DDX5 in polarized Th17 cells. DDX5 occupancy at the Il17a and Il17f loci (as identified by RORγt ChIP-seq MACS peak called #32 and #39 respectively from b.) in control, DDX5-Tko, or RORγt-deficient cells. Results are representative of two independent experiments. Each experiment was performed with two technical replicates. Graph shows mean ± s.d. p<0.01 (Prism, t-test). Extended Data Figure 4 Influence of DDX5 on T cell phenotypes in autoimmune disease models a, At 8 weeks after T cell transfer, LILP mononuclear cells were evaluated for amounts of IL-17A and IFNγ mRNA by qRT-PCR. Results are representative of two independent experiments. Each experiment was performed using large intestines from 3 animals in each condition. qRT-PCR was performed with two technical replicates. Graph shows mean ± s.d. * p<0.03 (Prism, t-test). b, Gating strategy for analysis of Th17 and Th1 cells from large intestine of Rag2-deficient recipients of WT or DDX5-Tko naïve T cells analyzed at 8 weeks after T cell transfer. c, Representative IL-17A and IFNγ intracellular staining of Aqua−CD4+RORγt+T helper 17 cells in spinal cord of MOG immunized animals on Day 21. Extended Data Figure 5 ncRNAs enriched in DDX5 and RORγt RIP-seq studies a, DDX5-Tko cells were transduced with WT or helicase-mutant DDX5 and evaluated for DDX5 expression by immunofluorescence (left) and immunoblot (right) with anti-DDX5 antibody. For gel source data, see Supplementary Figure 1. b, Venn diagram of ncRNAs detected by deep sequencing following co-immunoprecipitation (RIP-seq) of ribosome-depleted Th17 cell lysates with anti-DDX5 and anti-RORγt antibodies. c, Abundance of top ncRNAs enriched in DDX5 and RORγt immunoprecipitates from polarized Th17 cell lysates depleted of ribosomes. Top panel indicates abundance of the ncRNAs in total lysate. d, Conventional RIP-qRT-PCR experiments to compare Rmrp association with DDX5 in Th17 and developing thymocytes. Results are representative of three independent experiments. Each experiment was performed with two technical replicates. Graph shows mean ± s.d. p<0.001 (Prism, t-test). Extended Data Figure 6 Rmrp and DDX5 knockdown in mouse and human Th17 cells a, RNA FISH analysis, using probes specific for Rmrp (green) and Malat1 (red) lncRNAs, in Th17 cells at 72h following nucleofection with control (CTL) or Rmrp ASOs. b, Effect of Rmrp ASOs targeting different regions of Rmrp transcript on levels of Rmrp, IL-17F, and CCR6 RNAs in polarized Th17 cells. c, Knockdown of DDX5 reduced IL-17A production in in vitro polarized human RORγt+ Th17 cells. p<0.01 (Prism, t-test). Representative result in left panel. Each dot represents a different healthy donor (n=4). Graphs show mean ± s.d. Extended Data Figure 7 Effects of WT and mutant Rmrp in T cell differentiation a, IL-17A mRNA in cell lysates of in vitro polarized murine Th17 cells at 96 h following transduction of control vector or WT Rmrp. Results are representative of two independent experiments. b, IFNγ production in polarized murine Th1 cells at 96 h after transduction of control or Rmrp-encoding vector. Representative of two independent experiments. Each experiment was performed with two technical replicates. c, Comparison of human and mouse Rmrp sequences. Several mutations identified in CHH patients are highlighted. d, IL-17A production in polarized murine Th17 cells at 96 h after transduction of WT or mutant Rmrp vectors. Representative of two independent experiments. e, The Venn diagram depicts number of distinct and overlapping genes regulated by RORγt, DDX5, and Rmrp in in vitro-polarized Th17 cells. f, Expression of cytokine and Foxp3 mRNAs in T cells from WT or RmrpG270T/G270T mice cultured ex vivo in Th17, iTreg, Th1 and Th2 polarizing conditions. Results are representative of two independent experiments. Each experiment was performed with two technical replicates. * p<0.001 (Prism, t-test). g, ChIP-qPCR experiment using anti-RORγ/γt antibodies on chromatin of Th17 cells from WT or mutant animals cultured for 48 h in vitro. Each dot represents a different biological sample. WT, n=2; RmrpG270T, n=2. Result is representative of three separate independent experiments. Graphs show mean ± s.d. N.S. not significant (Prism, t-test). Extended Data Figure 8 Effect of Ddx5 and Rmrp mutations in inflammation and thymocyte development a, Left panel: Percent weight change in Rag2−/− recipients of WT (black circles) or RmrpG270T/G270T (gray squares) naïve CD4+ T cells in the transfer model of colitis. Animal weight was measured on day 56. (WT: n=8; RmrpG270T/G270T: n=8, combined from three independent experiments). Graphs show mean ± s.d. * p<0.001 (Prism, t-test). Middle panel: histology score (scale of 0–24) (WT: n=8; and RmrpG270T/G270T: n=5), combined from two independent experiments. p<0.01 (Prism, t-test). Right panel: representative H&E staining of large intestine of Rag2−/− animals on day 56 after naïve T cell transfer. b, Mice with deletion of Ddx5 in early common lymphoid progenitors have normal thymic development. Left: immunoblot of thymocyte lysates with anti-DDX5 antibody confirmed depletion of DDX5; right: percent CD4 single positive (SP), CD8α SP, double positive (DP), and double negative (DN) cells among total thymocyte. Each bar is result from one animal (wt/het, n=9; DDX5-clpKO, n=6). For gel source data, see Supplementary Figure 1. c, Thymocyte and peripheral T cell surface phenoptypes of WT and RmrpG270T/G270T knock-in mice at steady state. Peripheral T cell gate: DAPI−CD19−CD8α−CD4+. Extended Data Figure 9 Association of Rmrp lncRNA with DDX5 and RORγt in vitro a, In vitro translated HA-tagged wildtype or helicase-dead DDX5 and FLAG-tagged RORγt were incubated with in vitro transcribed Rmrp. After capture on anti-HA or anti-FLAG beads, the amount of lncRNA was determined by qRT-PCR. Data are representative of two independent experiments. Each experiment was performed with two technical replicates. Graphs show mean ± s.d. * p<0.001 (Prism, t-test). b, Helicase requirement for in vitro interaction of DDX5 and RORγt. Recombinant GST-DDX5 (wildtype or helicase-dead mutant) and His-RORγt full-length protein were synthesized in E. coli, purified, and assayed for binding with or without in vitro transcribed Rmrp RNA in the presence exogenous ATP. For gel source data, see Supplementary Figure 1. c, Association of in vitro transcribed WT and mutant Rmrp with recombinant GST-DDX5 captured on glutathione beads (left) or with recombinant GST-DDX5 and His-RORγt captured with anti-His antibody. Amounts of associated Rmrp were quantified using qRT-PCR. Data are representative of two independent experiments. Each experiment was performed with two technical replicates. Graphs show mean ± s.d. * p<0.001 (Prism, t-test). d, Comparison of ability of in vitro transcribed WT and RmrpG270T lncRNA to promote interaction between recombinant RORγt and DDX5 in vitro. For gel source data, see Supplementary Figure 1. Extended Data Figure 10 Rmrp chromatin localization in Th17 cells a, ChIRP-seq sample validation of Rmrp RNA pull-down over other nuclear ncRNAs using pools of Even or Odd capture probes. Graphs show mean ± s.d. b, ChIRP-qPCR of Rmrp RNA pull-down from WT Th17 cell lysate treated with or without RNase (n=2). qPCR for each sample was performed with two technical replicates. Graph shows mean ± s.d. p<0.001 (Prism, t-test). c, HOMER motif analysis reveals top three DNA motifs within Rmrp-enriched peaks. d. Significance of peak overlaps between Rmrp ChIRP-seq and ChIP-seq for BATF (n=2), IRF4 (n=7), STAT3 (n=2), c-Maf (n=2), RORγt (n=2), CTCF (n=2), RNA Pol II (n=2), H3K27me3 (n=4), and H3K4me3 (n=3) in Th17 cells (hypergeometric distribution). Each dot represents a separate biological replicate of ChIP-seq experiments. e, Venn Diagram depicting changes in peaks called from Rmrp (ChIRP-seq) experiments in WT and DDX5-Tko Th17 cells. f, Comparison of Rmrp chromatin occupancy (ChIRP-seq) at known RORγt occupied loci in in vitro-polarized Th17 cells from WT and RmrpG270T/G270T mice. Supplementary Material 1 Extended Data Figure 7 2 Extended Data Figure 1 Extended Data Figure 10 Extended Data Figure 2 Extended Data Figure 3 Extended Data Figure 4 Extended Data Figure 5 Extended Data Figure 6 We thank Maria Pokrovskii for unpublished ATAC-seq data and Lana X. Garmire for suggestions on our manuscript. This work was supported by a Cancer Research Institute Irvington Postdoctoral Fellowship (W.H.), Institutional NRSA T32 CA009161_Levy (W.H.), National Multiple Sclerosis Society postdoctoral fellowship FG 2089-A-1 (L.W.), Career Development Award (#329388) from the Crohn’s and Colitis Foundation of America (S.V.K.), Dale and Betty Frey Fellowship of the Damon Runyon Cancer Research Foundation 2105-12 (J.A.H.), HHMI Exceptional Research Opportunities Program (N.R.M and N.H.), NIH F30 1F30CA189514-01 (R.A.F.), NIH P50-HG007735 and R01-HG004361 (H.Y.C.), NIH RO1 AI080885 (D.R.L), NIH R01DK103358 (R.B. and D.R.L.), and the Howard Hughes Medical Institute (H.Y.C. and D.R.L.). Author Contributions W.H. and D.R.L. designed experiments, analyzed data and wrote the manuscript with input from the co-authors, B.T. and A.O. performed mass spectrometry studies, F.W.R. designed and synthesized control and Rmrp ASOs, S.J.G. and L.W. performed MOG-EAE immunization and scoring, S.V.K. performed blinded histology scoring on colitis sections, S.M. and R.M.M. performed library preparation for RNA sequencing studies, N.R.M. and N.G.H. performed microscopy studies, F.R. provided recombinant full length His-tagged hRORγt, and F.F.P. generated DDX5 conditional mutant animals. J.A.H. performed RORγt ChIP studies. C.P.N performed DDX5 studies in the thymus. R.A.F., W.H. and H.Y.C. performed ChIRP-seq experiments. E.R.M and R.B. performed statistical analyses on ChIRP-seq experiments. RNA-seq, TRAP-seq, RIP-seq, and ChIRP-seq data have been uploaded on GEO (GSE70110) and will be made available upon manuscript publication. Reprints and permission information are available at www.nature.com/reprints. Supplementary Information is linked to the online version of the paper at www.nature.com/nature. Figure 1 Requirement for DDX5 in Th17 cytokine production in vitro and at steady state in vivo a, Selective Th17 cell differentiation defect in DDX5-deficient T cells after polarization for 96 h. Representative of three independent experiments. b, Volcano plot of RNA-seq of cultured Th17 cells from DDX5-Tko mice and littermate controls. Black dots: differentially expressed genes (minimum fold change of two with p-value < 0.05). Blue dots: known RORγt-dependent genes. Red dots: top RORγt-DDX5 co-regulated genes. c, SFB colonization and percentage and number of RORγt+ CD4+ T cells and d, number of IL-17A-producing CD4+ T cells in ileal lamina propria (LP) of co-housed WT (n=5) and DDX5-Tko (n=5) animals. Graphs show mean ± standard deviation (s.d.) from two independent experiments, combined. N.S., not significant. ** p<0.01 (Prism, Paired t-test). e, Representative IL-17A expression in CD4+Foxp3−RORγt+ Th17 cells from ileal LP of WT and DDX5-Tko animals after restimulation. Figure 2 Role of DDX5 in mouse models of Th17 cell-mediated autoimmune disease a, Weight change in Rag2−/− recipients of WT or DDX5-Tko CD4+ naïve T cells in the transfer model of colitis measured on days 0, 10, 25, 37, and 45. (PBS: n=4; WT: n=9; DDX5-Tko: n=13, combined from three independent experiments). b, H&E staining and analysis of large intestine (LI) at day 45. Representative sections (black bar = 100µm) and histology scores (scale of 0–24) are shown. Scores for PBS (n=3), WT (red, n=8) and DDX5-Tko (blue, n=7) mice are from two independent experiments. c, Cytokine production defect in DDX5-Tko Th17 (RORγt+) but not Th1 (RORγt T-bet+) cells in LILP at day 45 (n=4 per group). d, EAE disease scores (scale of 0–5) in co-housed MOG-immunized littermates. WT (n=13) and DDX5-Tko (n=11) mice, combined from three independent experiments. e, Defective IL-17A production in DDX5-Tko CD4+RORγt+ cells in spinal cord of MOG-immunized mice. n=7 per group. Graphs show mean ± s.d. N.S. not significant, p < 0.05, * p < 0.01, * p < 0.001 (Prism, t-test). Figure 3 Requirement for helicase-competent DDX5 and its associated lncRNA Rmrp in induction of Th17 cell cytokines a, Cytokine production in DDX5-Tko cells transduced with WT or helicase-mutant DDX5 and subjected to sub-optimal Th17 cell polarization. b, Results from four independent experiments shown (a). c, IGV browser view of Rmrp showing coverage of mapped RNA reads from total Th17 lysate, Ribosome TRAP-seq (described in Methods), DDX5 RIP-seq, and RORγt RIP-seq. d, Effect of mouse Rmrp-specific ASO. Results are representative of three independent experiments with two technical replicates. e, IL-17A production following Rmrp knockdown in in vitro polarized human Th17 cells. Each symbol (right panel) represents cells from a healthy donor (n=5). Graphs show mean ± s.d. CTL, control; p<0.01, ** p<0.0001 (Prism, t-test). Figure 4 Analysis of DDX5-dependent Rmrp function in Th17 cell differentiation a,b, Cytokines in WT and DDX5-Tko in vitro polarized Th17 cells upon Rmrp knockdown (a) or overexpression (b). Representative of three independent experiments. c, Sequence of Rmrp (nucleotides 258–275) from WT and RmrpG270T/G270T littermates. d, Rmrp-dependent expression of a RORE-directed firefly luciferase reporter nucleofected into polarized Th17 cells at 72 h. Firefly and control Renilla luciferase activities were measured 24 h later. Each dot represents the result from one nucleofection. Results from two independent experiments. e, Top RORγt targets co-regulated by DDX5 and Rmrp. f, Proportion of CD4+Foxp3− T cells expressing RORγt (left) and numbers of Th1 (IFNγ+RORγt−Tbet+), Th17 (IL-17A+RORγt+Foxp3−), and Tγδ17 (Tγδ+RORγt+) cells (right) in ileum. Symbols represent cells from one animal. Graphs show mean ± s.d. p<0.01, *p<0.001, P < 0.0001 (Prism, t-test). Figure 5 Rmrp localization at RORγt-occupied genes and role in RORγt-DDX5 assembly a, RORγt association with immunoprecipitated (IP) DDX5 in polarized Th17 cells. IB, immunoblot. Representative of three independent experiments. b, Rmrp quantification by qRT-PCR in RORγt immunoprecipitates from polarized Th17 cells. Representative of two independent experiments with two technical replicates. c, Rmrp requirement for ATP-dependent in vitro interaction of recombinant GST-DDX5 and His-RORγt. Representative of three independent experiments. For gel source data (a,c), see Supplementary Figure 1. d, Rmrp occupancy at RORγt genomic target loci in polarized Th17 cells. Rmrp ChIRP-qPCR amplicons (bottom) are indicated in IGV browser view of RORγt ChIP at the Il17 locus (top). Data from 2–4 experiments with two technical replicates. e, Model for DDX5-Rmrp complex recruitment to RORγt-occupied chromatin in Th17 cells. 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==== Front Am J Case RepAm J Case RepamjcaserepThe American Journal of Case Reports1941-5923International Scientific Literature, Inc. 2689736010.12659/AJCR.896798896798ArticlesA Boy with Relentless Pruritus: Job’s Syndrome Khan Kamran DEF1Wozniak Susan E. E1Giannone Anna Lucia BE2Abdulmassih Maria Elena E31 Department of General Surgery, Sinai Hospital of Baltimore, Baltimore, MD, U.S.A.2 Aureus University School of Medicine, Oranjestad, Aruba3 Psychoanalysis, Private Practice, Lechería, Anzoategui, VenezuelaAuthors’ Contribution: A Study Design B Data Collection C Statistical Analysis D Data Interpretation E Manuscript Preparation F Literature Search G Funds Collection Conflict of interest: None declared Corresponding Author: Kamran Khan, e-mail: [email protected] 21 2 2016 17 104 110 22 11 2015 02 12 2015 © Am J Case Rep, 20162016This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported LicensePatient: Male, 6 Final Diagnosis: Job’s Syndrome (hyper IgE syndrome) Symptoms: Pruritus Medication: — Clinical Procedure: None Specialty: Allergology Objective: Rare disease Background: Job’s syndrome (hyper IgE syndrome) is a very rare primary immunodeficiency disease that has an annual approximate incidence of less than 1/1,000,000. This manuscript aims to provide education regarding diagnosis and management strategies of this syndrome worldwide. Case Report: A 6-year-old boy was seen at the clinic secondary to persistent pruritus interfering with sleep. At the age of 2 months, the patient developed diffuse eczematous and desquamating skin lesions. He was subsequently diagnosed with atopic dermatitis and managed conservatively. From 2 months to 7 years of age, intermittent exacerbations of dermatitis persisted despite an aggressive treatment regimen. The serum IgE level increased exponentially over a period of 7 years, with a peak value of 57,400 IU/ml. Molecular genetic testing revealed a dominant negative mutation within the SH2 domain of the Signal Transducer and Activator of Transcription (STAT3) gene. The patient was subsequently diagnosed with Job’s syndrome. Management included proper skin care, prophylactic antibiotics, immunomodulating agents, and psychotherapy. Conclusions: Job’s syndrome can often go unrecognized and masquerade as atopic dermatitis. Therefore, genetic testing for this condition should be obtained in all patients with treatment-refractory AD. Additionally, psychotherapy can be a successful management strategy for the grating psychological impact that can be imposed on children with excessive pruritus. MeSH Keywords Job SyndromePruritusPsychotherapy ==== Body Background Job’s syndrome is a very rare primary immunodeficiency disease that has an approximate annual incidence of less than 1/1,000,000 [1]. Fewer than 300 cases have been reported in the literature [1]. This report demonstrates how Job’s syndrome can often go unrecognized and masquerade as atopic dermatitis. We also demonstrate the refractoriness to treatment and subsequent psychosocial impact. The aim of this article is to provide education regarding management strategies of this syndrome worldwide. Case Report A 6-year-old Hispanic boy was seen at the clinic secondary to persistent pruritus interfering with sleep. The patient was born at another hospital via an uncomplicated caesarean section, to a 32-year-old primigravid female. The mother received prenatal care and standard screening tests were reportedly normal throughout pregnancy. The birth weight was 3,600 grams and the length 54 cm. The patient progressed well and continued to receive appropriate perinatal care. At age the age of 2 months, the patient developed diffuse, eczematous, and desquamating skin lesions. He was diagnosed with atopic dermatitis and managed conservatively. When skin lesions continued to worsen, topical fluticasone was attempted, yielding minimal relief. Ultimately, a combination of topical fluticasone, cyclosporine, acitretin, oral prednisone, and intramuscular methotrexate yielded mild to moderate improvement for periods of time. Despite this aggressive treatment regimen, the patient continued to experience intermittent exacerbations of pruritus and dermatitis. Of note, during this time he also developed 5 upper respiratory infections. At 1 year of age, the patient was admitted to another hospital for pneumonia. On examination, the temperature was 38°C, the blood pressure 99/56 mm Hg, and the pulse was 140 beats per minute. Erythematous, eczematous lesions were present on the trunk and upper/lower extremities. There was mild leukocytosis (10.4 mm3) and eosinophilia (7%); other laboratory test results were reportedly negative (Table 1). A chest radiograph revealed patchy consolidation and a diagnosis of pneumonia was made. The patient was placed on appropriate antibiotics and shortly thereafter was discharged. Intermittent exacerbations of dermatitis persisted, and at the age of 3 years he was referred to an immunologist for further workup. Allergen testing revealed severe allergies to eggs, peanuts, and soy. There was a serum IgE level of 386 IU/mL. The administration of topical fluticasone, cyclosporine, and oral methotrexate were continued, under the presumed diagnosis of atopic dermatitis. However, continued workup and varying treatment regimens remained unfruitful. At subsequent follow-up visits to the Immunology clinic, a continued upsurge in the serum IgE level was noted; laboratory findings are shown in Table 1. The patient explained that the pruritus did not remit with sleep and interfered with daily activities. He would also frequently avoid bathing due to the pain associated with the lesions, and stopped going to school for the same reason. Further, given the exasperating nature of the lesions, the patient’s continued scratching only worsened the pruritic lesions into large, bloody, excoriations to oftentimes infected lesions. This unremitting exasperation caused insomnia, ultimately even giving way to suicidal ideation. With countless hospital admissions for pneumonia and infected skin lesions, the psychological impact was very severe. The patient was born in Venezuela, and had resided there for the entirety of his life. The mother reported that there were no known sick contacts, pets in the household, or insect bites. He did not have a history of travelling outside of Venezuela. The patient did not smoke, drink alcohol, or use illicit drugs. His father had a history of asthma, allergic conjunctivitis, and allergic rhinitis. During yet another exacerbating episode, he was admitted to another facility. On examination, the temperature was 39°C, the blood pressure 105/70 mm Hg, and the pulse 143 beats per minute. The patient appeared agitated and irritable. There were vesicular lesions forming pustules, with multiple white plaques on an erythematous base, covering approximately 80% of the skin surface (Figure 1). Dennie Morgan folds, allergic shiners, hyper-linear palms (Figure 2), and edema were noted. There was diffuse alopecia, madarosis, and clubbed fingers present. The patient had palpable, non-painful axillary and inguinal lymphadenopathy bilaterally; the remainder of the examination was normal. There was leukocytosis (19,900 mm3), with a prominent left shift, and 44% eosinophilia; other laboratory results are shown in Table 1. Immunological workup included a serum IgE level that was 817 IU/mL; serum complement, natural killer cells, lymphocyte count, and other immunoglobulins were normal. Th17 cells were quantified via flow cytometry and revealed a markedly decreased level (0–0.20% of CD4 T cells). Skin biopsy revealed superficial perivascular dermatitis with psoriasiform changes, hypogranulosis/agranulosis with parakeratosis, and fragmented neutrophils, suggestive of atopic dermatitis versus psoriasis. A pediatric hematologist was consulted and ruled out hematological malignancy. Upon review of these results and discussion with an infectious disease specialist, antimicrobial therapy was initiated. Over a period of 2 weeks, there was mild improvement of symptoms, with complete relapse at 3 weeks. The patient continued to experience persistent, fractious pruritus, despite the use of 1st and 2nd generation anti-histamines, triple doses of H2 receptor antagonists, and amitriptyline. Methotrexate (10 mg/m2, weekly) was administered for 4 months, with modest improvement of the skin lesions, but without alleviation of the pruritus. He was discharged on oral cefadroxil, chlorpheniramine, hydroxyzine, omalizumab, ranitidine, topical fluticasone/unibase, and moisturizers. During the next year, there was modest improvement of the dermatitis, but the pruritus remained. A psychotherapist also became involved in the patients care at this time, perhaps providing some of the greatest relief. At the age of 7 years, a second skin biopsy was obtained. Histopathological examination revealed superficial perivascular dermatitis with psoriasiform epidermal changes, compatible with atopic dermatitis versus lichen simplex chronicus. The serum IgE level was 57,400 IU/ml; laboratory values are shown in Table 1. Upon reviewing the patient’s previous serum IgE levels, and recent spike, molecular genetic testing was obtained. This revealed a dominant negative mutation within the SH2 domain of the STAT3 gene, subsequently diagnosing the patient with Job’s syndrome. The patient is presently doing well with improvement of the skin lesions over 80% of the skin surface. This was largely contributed to his refraining from scratching and daily showers after multiple sessions of psychotherapy; however, the pruritus has persisted. His current treatment includes monthly IVIG, deflazacort (9 mg orally), and hyperbaric oxygen therapy. The patient is also being considered for anti-interleukin 4 therapy (Dupilumab). Discussion Job’s faithfulness was tested when God permitted Satan to “smote Job with boils from the sole of his feet unto his crown.” In 1966, 2 females with recurrent skin abscesses, dermatitis, and pneumonia were described by Davis et al. [2]. These became the first 2 cases coined as Job’s syndrome. Six years later, 2 cases with similar symptoms, and the addition of eosinophilia and elevated serum IgE levels, were described by Buckley et al. [3]. Job’s syndrome (hyper IgE syndrome) is characterized by the triad of elevated serum IgE level (>2000 IU/ml), pneumonia with formation of pneumatoceles, and recurrent staphylococcal skin abscesses [4]. Characteristic facial features present in Job’s syndrome have a reported incidence of 83%, and 100% for patients older than 16 years [4]. These include a prominent forehead, deep-set eyes, mild prognathism, a broad nasal bridge, and increased inter-alar distance [4]. Delayed tooth eruption, bone fractures, hyperextensible joints, and scoliosis are also frequently present [4]. This patient’s facial features resembled those frequently present in Job’s syndrome (Figure 3). In the majority of patients, heterozygous mutations of the STAT3 gene are found. Autosomal dominant forms of the disease are associated with mutations in the STAT3 gene, whereas autosomal recessive forms are associated with mutations and deletions in dedicator of cytokinesis 8 (DOCK8) and tyrosine kinase 2 (TYK2) genes [5]. There can be similarities to atopic dermatitis, Wiskott-Aldrich syndrome, Netherton syndrome, and severe combined immunodeficiency; therefore, these should be considered as a differential diagnosis. Atopic dermatitis (AD) is a common condition of early childhood, affecting 15% to 30% of children; 85% develop the disease within 5 years of life [6]. The hallmark of the diagnosis is a relapsing-remitting pruritic dermatitis. Patients typically have elevated serum IgE levels, eosinophilia, and profound pruritus that can cause insomnia, analogous to this patient. The diagnosis of AD seems likely. However, due to the presence of recurrent pneumonia, an exponentially rising serum IgE level, and early onset of symptoms, other differential diagnoses can be considered. The second diagnostic consideration was if the patient had a mutation in the Wiskott-Aldrich syndrome protein (WASp) gene. Wiskott-Aldrich syndrome (WAS) is an X-linked immunological disease, characterized by the triad of eczema, recurrent infections, and microthrombocytopenia [7]. Patients generally have elevated levels of serum IgE, IgA, IgG, and variable levels of serum IgM [7]. In this case, the patient’s platelets were of normal quantity and quality, making WAS an improbable diagnosis. Thus, the patient did not undergo genetic testing for WAS. In this case, recurrent pneumonia and cutaneous infections prompted the consideration of a severe combined immuno-deficiency (SCID). Etiologies of SCID include cytokine receptor defects, adenosine deaminase (ADA) deficiency, and multiple histocompatibility complex (MHC) II deficiency, which lead to a defect in cell-mediated and humoral immunity [8]. Classic features include recurrent bacterial, fungal, viral, and protozoal infections [8]. In the preponderance of cases, patient’s T cells are absent [8]. Findings in this case resembling SCID included elevated serum IgE level, skin rash, and recurrent infections. However, flow cytometry revealed the patient’s T cells to be of normal quantity, ruling out the disease. In summary, we favored the diagnosis of Job’s syndrome. This was confirmed with the findings of elevated serum IgE (>2000 IU/mL), characteristic facial features, and a dominant negative mutation within the SH2 domain of the STAT3 gene. In addition to conservative pharmacological therapy (steroids, vitamin A derivatives), it is imperative that these patients receive proper skin care to prevent the development of recurrent Staphylococcal skin infections. Effective treatments include immersion in bleach baths (1/2 cup of bleach in a tub of water for 15 minutes, 3 times a week), or chlorinated pools [9]. Prevention also involves the prompt administration of anti-Staphylococcal antibiotics at the first sign of infection. Prophylactic therapy with trimethoprim-sulfamethoxazole has been shown to be highly effective [4]. Intravenous immunoglobulin (IVIG) and Interferon (IFN)-gamma have been used anecdotally, with favorable results. There have been no randomized controlled trials described in the literature supporting the use of IVIG for Job’s syndrome. One study reported improvement of eczema in patients with Hyper IgE syndrome, after receiving IVIG (400 mg/day for 5 days) [10]. This study also exhibited diminished enhanced IgE production in vivo and in vitro [10]. The resolution of pneumonia after administration of IVIG in Job’s syndrome has been described [11]. However, 1 study concluded that IVIG was of no clear benefit, and did not significantly decrease serum IgE levels or IgE synthesis [12]. IFN-gamma has demonstrated effectiveness in decreasing serum IgE levels [13]. However, serum IgE levels returned to pre-treatment levels within 1–3 months after completion of treatment [13]. In addition to these treatments, psychotherapy was an integral part of the patient’s treatment regimen. Dr. Maria Elena Abdulmassih: I initially met this patient after he experienced an episode of intense agitation that caused him to scratch himself until he bled. During the course of family therapy sessions, I observed that this patient would use his constant state of pain and scratching to manipulate the parents into obeying him. The patient would use scratching and pain as a tool for social isolation. He did not want anyone to see him in his current state and also wished that he was dead. This led the parents to isolate the boy from normal social life. During family therapy, I was able to demonstrate to the parents that this defense mechanism of isolation was detrimental to the patient’s mental health. I was able to explain to the patient that blaming others for his condition was not helpful, and that he had to cope and hopefully overcome it in life. Reinforcing the “ego” of the patient was an integral part of the treatment in an effort to control his anger. The same mechanism of manipulating his parents was seen while bathing. Due to complaints of agonizing pain, he refused to bathe. This contributed enormously to the vicious cycle of his recurrent infections and secondary pruritus. After multiple interventions during family therapy, this patient began to demonstrate an improved attitude towards his condition. We were then able to start an aggressive plan consisting of daily showers, wound care, and hyperbaric oxygen therapy. Conclusions We conclude that further research is necessary regarding treatment regimens for Hyper-IgE syndrome (Job’s syndrome). This condition can be identical in presentation to AD; therefore, genetic testing should be obtained in all children with treatment refractory AD. Psychotherapy can be a successful management strategy for the severe psychological impact that can be imposed on children with excessive pruritus, in addition to pharmacological therapy. Statement We have no sources of financial support and no conflicts of interest to disclose. Figure 1. Clinical photographs of the patient: There are vesicular lesions forming pustules, with multiple white plaques on an erythematous base covering approximately 80% of the skin surface (A–F). Diffuse excoriations secondary to scratching are seen (A–C). Figure 2. Hyper-linear palms: A feature commonly present in atopic conditions. Figure 3. Characteristic facial features: Note the presence of a prominent forehead, broad nasal bridge, increased inter-alar distance, and mild prognathism. Table 1. Laboratory data. Variable Reference Range Hospital admission for pneumonia at 1 year of age Presentation to an Immunology clinic at 3 years of age Presentation to the emergency department at 6 years of age Presentation to an Immunology clinic at 7 years of age Hematocrit (%) 36.0–46.0 34 39.1 40.6 44.5 Hemoglobin (g/dl) 12.0–16.0 10.6 12 13 12.9 White-cell count (per mm3) 4,500–11,000 10.4 13.5 19.9 15.1 Differential count (%) Neutrophils 40–70 36 45 42 37 Band forms 0.0–7.0 2 1 Lymphocytes 22–44 57 32 30 37 Monocytes 4.0–11 4 3 Eosinophils 0–8 7 17 33 44 Basophils 0–3 Platelet count (per mm3) 150,000–400,000 300,000 580,000 582,000 381,000 Erythrocyte sedimentation rate (mm/hr) 1.0–17 33 40 C-reactive protein (mg/l) <5 17.87 3.9 Protein (mg/dl) Total 6.4–8.2 7.5 Albumin 3.4–5.0 3.8 Lactate dehydrogenase (U/L) 81–234 407 Vitamin D (ng/ml) 13.0–47.8 2.1 Parathyroid hormone (pg/ml) 11.0–67 286 Calcium (mg/dl) 8.5–10.1 7.4 Phosphate (mg/dl) 2.4–4.9 3.9 Immunoglobulins IgE (IU/mL) Variable* 26 386 817 57,400 IgG (mg/dl) Variable* 1,130.00 1240 IgA (mg/dl) Variable* 104 119 IgM (mg/dl) Variable* 154 IgD (mg/dl) Variable* 1.9 Total T3 (ng/dl) 94–269 254 Total T4 (ug/dl) 4.5–12.5 9.13 TSH (µIU/mL) 0.5–4.7 4.93 2.49 Anti-nuclear antibody Negative Anti-smooth muscle antibody Negative Anti-transglutaminase antibody Negative Serum protein electrophoresis Normal pattern HIV-1/HIV-2 Negative Negative HTLV-1/HTLV-2 Negative VDRL Negative EBV Negative Mycoplasma IgM Negative Chlamydia pneumoniae IgM Negative ==== Refs References: 1. Job syndrome Genetics Home Reference [serial online] 2008 2 [cited 2015 Sep 10]. Available from: http://ghr.nlm.nih.gov/condition/job-syndrome 2. Davis SD Schaller J Wedgewood RJ Job’s Syndrome. Recurrent, “cold”, staphylococcal abscesses Lancet 1966 1 7445 1013 15 4161105 3. Buckley RH Wray BB Belmaker EZ Extreme hyperimmunoglobulinemia E and undue susceptibility to infection Pediatrics 1972 49 1 59 70 5059313 4. Grimbacher B Holland SM Gallin JI Hyper-IgE syndrome with recurrent infections – an autosomal dominant multisystem disorder N Engl J Med 1999 340 9 692 702 10053178 5. Farmand S Sundin M Hyper-IgE syndromes: recent advances in pathogenesis, diagnostics and clinical care Curr Opin Hematol 2015 22 1 12 22 25469836 6. Bieber T Atopic dermatitis N Engl J Med 2008 358 14 1483 94 18385500 7. Massaad MJ Ramesh N Geha RS Wiskott-Aldrich syndrome: A comprehensive review Ann NY Acad Sci 2013 1285 26 43 23527602 8. Rivers L Gaspar HB Severe combined immunodeficiency: recent developments and guidance on clinical management Arch Dis Child 2015 100 7 667 72 25564533 9. Cruz-Portelles A Estopinan-Zuniga D A new case of Job’s syndrome at the clinic: a diagnostic challenge Rev Port Pneumol 2014 20 2 107 10 24560409 10. Kimata H High-dose intravenous gamma-globulin treatment for hyperimmunoglobulinemia E syndrome J Allergy Clin Immunol 1995 95 3 771 74 7897163 11. Bilora F Petrobelli F Boccioletti V Pomerri F Moderate-dose intravenous immunoglobulin treatment of Job’s syndrome. Case report Minerva Med 2000 91 5–6 113 16 11084845 12. Wakim M Alazard M Yajima A High dose intravenous immunoglobulin in atopic dermatitis and hyper-IgE syndrome Ann Allergy Asthma Immunol 1998 81 2 153 58 9723561 13. King CL Gallin JI Malech HL Regulation of immunoglobulin production in hyperimmunoglobulin E recurrent-infection syndrome by interferon gamma Proc Natl Acad Sci USA 1989 86 24 10085 89 2513574	26897360	PMC4763797	NO-CC CODE	2021-01-05 05:34:52	yes	Am J Case Rep. 2016 Feb 21; 17:104-110
==== Front TheranosticsTheranosticsthnoTheranostics1838-7640Ivyspring International Publisher Sydney 10.7150/thno.14306thnov06p0594Research PaperNovel Bioluminescent Activatable Reporter for Src Tyrosine Kinase Activity in Living Mice Leng Weibing 12Li Dezhi 12Chen Liang 1Xia Hongwei 2Tang Qiulin 2Chen Baoqin 1Gong Qiyong 3Gao Fabao 3Bi Feng 12✉1. Department of Medical Oncology, West China Hospital, Sichuan University, Chengdu 610041, Sichuan, China;2. Laboratory of Signal Transduction & Molecular Targeted Therapy, State Key Laboratory of Biotherapy, Sichuan University, Chengdu 610041, Sichuan, China;3. Department of Radiology, West China Hospital, Sichuan University, Chengdu 610041, Sichuan, China.✉ Corresponding author: Feng Bi, E-mail: [email protected] Interests: The authors have declared that no competing interest exists. 2016 24 2 2016 6 4 594 609 3 11 2015 27 1 2016 © Ivyspring International Publisher. Reproduction is permitted for personal, noncommercial use, provided that the article is in whole, unmodified, and properly cited. See http://ivyspring.com/terms for terms and conditions.2016Aberrant activation of the Src kinase is implicated in the development of a variety of human malignancies. However, it is almost impossible to monitor Src activity in an in vivo setting with current biochemical techniques. To facilitate the noninvasive investigation of the activity of Src kinase both in vitro and in vivo, we developed a genetically engineered, activatable bioluminescent reporter using split-luciferase complementation. The bioluminescence of this reporter can be used as a surrogate for Src activity in real time. This hybrid luciferase reporter was constructed by sandwiching a Src-dependent conformationally responsive unit (SH2 domain-Srcpep) between the split luciferase fragments. The complementation bioluminescence of this reporter was dependent on the Src activity status. In our study, Src kinase activity in cultured cells and tumor xenografts was monitored quantitatively and dynamically in response to clinical small-molecular kinase inhibitors, dasatinib and saracatinib. This system was also applied for high-throughput screening of Src inhibitors against a kinase inhibitor library in living cells. These results provide unique insights into drug development and pharmacokinetics/phoarmocodynamics of therapeutic drugs targeting Src signaling pathway enabling the optimization of drug administration schedules for maximum benefit. Using both Firefly and Renilla luciferase imaging, we have successfully monitored Src tyrosine kinase activity and Akt serine/threonine kinase activity concurrently in one tumor xenograft. This dual luciferase reporter imaging system will be helpful in exploring the complex signaling networks in vivo. The strategies reported here can also be extended to study and image other important kinases and the cross-talks among them. Src tyrosine kinasenoninvasive molecular imagingbioluminescence imaging (BLI)kinase activitydrug development. ==== Body Introduction Src plays a crucial role in the crosstalk and mediation of many signaling pathways, including cell cycle progression, apoptosis, angiogenesis, adhesion and migration 1-3. It is not surprising that aberrant activation of Src kinase contributes to diverse aspects of carcinogenesis. Deregulation and increased activity of Src has been observed in a variety of human cancers and usually correlates with poor clinical prognosis 4-7. Growing evidence shows that inhibition of Src kinase activity has preclinical anti-tumor effects resulting in the development of many Src activity inhibitors 8,9. However, Src inhibitors have shown disappointing therapeutic efficacy in clinical trials 9-11. It remains challenging to realize the potential of basic research into preclinical or clinical application 12. To overcome these challenges in the case of Src tyrosine kinase, we must improve early drug discovery strategies by combining our knowledge of the basic biology of the enzyme with the use of appropriate preclinical modeling. This approach would better mimic and thereby predict clinical response in the target tissue environment, rather than in reductionist systems. Molecular imaging, especially optical imaging, provides a new platform for noninvasive visualization of biological processes at the molecular level in vivo. Optical imaging, including fluorescence imaging and bioluminescence imaging (BLI) 13, is helping to bridge the gap between our understanding of critical biologic events and their clinical applications. Fluorescence resonance energy transfer (FRET) analysis has been widely used in characterizing the spatiotemporal dynamics regulation of Src activity in a single living cell 14-17. Using fluorescence lifetime imaging microscopy (FLIM), the FRET-Src biosensor has also been used as a preclinical tool to assess drug delivery and efficacy in tumors 18. However, the FRET assay suffers from several weaknesses, including the need for an external excitation source, insufficient detection sensitivity and dynamic range, the challenge for stable expression, and autofluorescence 19. Furthermore, FRET assay requires sophisticated and expensive instrumentation and comprehensive post-imaging data analysis 20,21. These demerits potentially limit its usefulness for in vivo application and for high-throughput screening (HTS) in drug development. As an alternative to fluorescence assay, bioluminescence assay is capable of ironing out these flaws and provides complementary advantages for preclinical applications in vivo. Bioluminescence imaging has emerged as a sensitive technology to advance our understanding of disease mechanisms at the molecular level and accelerate drug discovery and development 22,23. In particular, bioluminescence can circumvent cell and tissue autoluminescence resulting in a better signal to noise ratio 24. This provides a complementary and alternative approach to traditional biochemical assays and the FRET assay, for preclinical evaluation of anticancer therapeutics in experimental cancer models. We reasoned that developing a method to noninvasively monitor and image Src activity in animals would be a valuable tool to further understand the biology of the Src kinase signaling pathway. To this end, we developed and characterized a novel bioluminescent activatable reporter based on the split-luciferase fragment complementation assay. This reporter system also allows monitoring of Src-targeted drug efficacy in intact cells and mice. The new imaging reporter would provide a better understanding of the role of the functional Src kinase in cancer biology and help accelerate the discovery and development of new anti-Src drugs. Materials and Methods Ethics statement All experimental procedures with animals used in this study had been given prior approval by the Experimental Animal Manage Committee of Sichuan University under Contract 2015012A. Animal handling and all procedures on animals were carried out strictly according to the guidelines of the Animal Care and Use Committee of Sichuan University and the Animal Ethics Committee Guidelines of the Animal Facility of the West China Hospital. The nude mice were maintained under specific pathogen-free (SPF) conditions. Mice were gas anesthetized with isofluorane (2% isoflurane in 100% oxygen, 1 L/min) using the XGI-8 Gas Anesthesia Unit (Caliper Life Sciences) during all injection and imaging procedures. Construction of plasmids The N and C fragments of luciferase (Firefly or Renilla) were amplified by PCR from pGL3-Basic or pRL-tk (Promega), and the Myc-tag sequence was introduced by primer design. The fragment 370bp fragment, including nucleotide sequences of SH2 domain derived from Shc (aa 374-465), linker, and Src consensus substrate peptide, was synthesized by Genewiz Company (Su-zhou, China). This cassette and the luciferase fragments were seamlessly cloned into a HIV-1-based lentiviral expression vector, which was derived from pLVX-puro (Clontech). Gaussia luciferase, which was amplified from pGLuc-Basic (NEB), was cloned into the 5′ end of the puromycin resistance gene with a “self-cleaving” T2A sequence. For the Src reporter based on Renilla luciferase (rBSR), Firefly luciferase fragments were replaced by Renilla luciferase fragments, and the neomycin resistance gene was used for selection of stable transfectants. For the Akt reporter, the hybrid Firefly luciferase was cloned into pLVX-puro. Site-directed mutagenesis was performed using the QuickChangeTM site-directed mutagenesis kit (Stratagene). The GenBank accession numbers for Src reporters, Akt reporters, and Gluc-T2A-puro sequence are KT986061-KT986067. Cell culture HEK293T, Hela, HT29, MDA-MB-435S, Lovo, Colo320, SW480, SW48, SW1116, HCT116 and Caco-2 cells were purchased from ATCC. All cell lines were cultured in Dulbecco modified Eagle medium (DMEM, Gibco Laboratories, Grand Island, NY) supplemented with 10% fetal bovine serum (Gibco). Cell cultures were maintained in a 37°C incubator with 5% CO2. Lentivirus production The lentiviral plasmids of the reporters psPAX2 and pMD2.G were co-transfected into HEK293T cells in a 10cm dish using Lipofectamine 2000 (Invitrogen). Twelve hours after transfection, the medium was changed to 2% FBS-DMEM. Two days after transfection, the conditioned medium was collected, filtered through 0.4 μ filter, and used for infection. Western blotting and coimmunoprecipitation For Western blots, cells expressing the reporter were cultured in 6-well plates and were treated with stimulant, inhibitor or vehicle for the indicated times. Total protein lysate was prepared using lysis buffer containing protease inhibitors and phosphatase inhibitors. Protein was quantified using the BCA protein assay (Pierce Chemical Co.). Western blotting was performed as described previously 25. Proteins were visualized using florescent-labeled secondary Abs and quantified by Odyssey infrared imaging system. The antibodies used in our study were as follows: EGFR, p-EGFR(Tyr1173), Src, p-Src(Tyr416), Akt, p-Akt(Ser473), Erk, p-Erk(Thr202/Tyr204), p-FAK(Tyr397) and p130Cas (Tyr410) antibodies were obtained from Cell Signaling Technology. β-actin and FAK antibodies were purchased from Santa Cruz Biotechnology Inc. For coimmunoprecipitation, cells stably transfected with reporter were cultured on 60-mm culture dishes. After treatment, the cells were harvested in cell lysis buffer. One part of the whole-cell lysates was used for input. The proteins in the remaining lysates were coimmunoprecipitated with mouse anti-Myc antibody (clone 4A6; Millipore). The immune complexes were captured using protein G-coupled magnetic beads (Millipore) and then fractionated by SDS-PAGE. Phosphorylation of the Src reporter was detected with the anti-phospho-tyrosine antibody (Upstate). Cells-based in vitro assay Cells expressing the reporter were cultured in 24-, 48-, or 96-well plates and treated with stimulants, inhibitors or the vehicle. The stimulants used in our study were EGF (peprotech), PP1 (Cayman), dasatinib, and saracatinib (Selleck). All the bioluminescence was obtained in living cells. For the internal control bioluminescence, Gaussia luciferase activity was measured by adding coelenterazine (Regis, 1.5uM in D-PBS, 100ul/well) with the parameters: 1-min exposure; emission filter, 500nm; f-stop, 1; binning, 8; field of view, 15 cm. For the complemented Firefly activity, after administration of D-luciferin (Xenogen, 50ug/ml in Cell Culture Medium, 100ul/well), luminescence intensity (photons/second/square centimeter/steridian or p/s/cm2/sr) was measured by the charge-coupled device (CCD) camera of IVIS spectrum (Caliper Life Sciences, Hopkinton, MA) using the following parameters: 1-min exposure; emission filter, 600nm; f-stop, 1; binning, 8; field of view, 15 cm. The measure of Gluc activity was preferential to avoid mutual interference, because Gluc emission signal intensity is almost negligible at 600nm after minutes due to the rapid kinetics of coelenterazine. The value of each well is expressed in the normalized activity, which is calculated as the ratio of Firefly luciferase (Fluc) activity at 600nm to Gaussia luciferase (Gluc) activity at 500nm (Fluc/Gluc). In vivo mouse imaging experiments To establish xenograft tumors, cells (1x106 cells/sample) stably transfected with the wild type or mutant reporter(s) were implanted subcutaneously in the bottom left or right flanks of 4-week old female nude mouse. BLI was performed pretreatment and after treatment with vehicle or inhibitors for indicated times when the xenografts reached a volume of 40 mm3. Mice were gas anesthetized with isofluorane (2% isoflurane in 100% oxygen, 1 L/min) using the XGI-8 Gas Anesthesia Unit (Caliper Life Sciences) during all injection and imaging procedures. The Renilla and Gaussia luciferases react with the same substrate, coelenterazine, to produce blue light with peak emission at approximately 480nm. The bioluminescence of luciferin-dependent and coelenterazine-dependent luciferases was obtained from the same mouse with different emission filters. For Firefly luciferase luminescence, the mice were imaged after i.p. injection of D-luciferin (150 mg/kg BW) using the following parameters: 2-min exposure; emission filter, 600nm; f-stop, 1; binning, 8; field of view, 15 cm. For Gaussia or Renilla luciferase activity, luminescence was measured after i.p. injection of coelenterazine (1mg/kg BW) with the parameters: 3-min exposure; emission filter, 500nm; f-stop, 1; binning, 8; field of view, 15 cm. The measure of Gluc or Rluc activity was preferential to avoid mutual interference, because Gluc or Rluc emission signal intensity is almost negligible at 600nm after minutes due to the rapid kinetics of coelenterazine. Although Firefly luciferase has glow kinetics, the bioluminescence intensity maintains only about 1 hour due to luciferin consumption. Little luminescence signal from last substrate injection was detected at emission filters 500nm and 600nm after 2 hours. New substrates needed for the bioluminescence at each time point. High-throughput screening against a kinase inhibitor library with BSR A compound library containing 84 small molecular weight kinase inhibitors from National Compound Resource Center in China was used to validate the efficacy of BSR for HTS. HT29-BSRwt treated with vehicle (DMSO) served as a normalized control. HT29-BSRwt cells cultured in 96-well plates were treated with 30uM of each compound for 60min in triplicates, according to the official recommendation for the preliminary screening concentration. BLI was performed as mentioned in vitro assay, and HT29-BSRmut as a positive control. The result of each compound sample was the average of normalized bioluminescence activity (Fluc/Gluc) in the triplicates. Data analysis For in vitro analyses, the value of each well is presented as normalized bioluminescence activity, which is calculated as the ratio of Firefly luciferase (Fluc) activity at 600nm to Gaussia luciferase (Gluc) activity at 500nm (Fluc/Gluc). The Gaussia luciferase activity was used as an internal control to normalize the cell number and the expression efficacy. For in vivo analyses, the value of the mutant on the right flank was used as a reference point for each bioluminescence imaging. The result was presented as normalized bioluminescence activity ratio of the wild-type value and the mutant value. Data were collected from at least 3 independent experiments with 3 or more replicates per experiment. Values are reported as mean±SD. All statistical analyses were performed with SPSS 14.0 for Windows software (SPSS Inc). In case only two groups were compared, a Student's t test was used. For multiple comparisons at different time points, analysis of variance (ANOVA) was performed. The EC50 values were fitted with sigmoidal dose-response curves using GraphPad Prism 5.0 software. Results Schematic of the bioluminescence-based activatable reporter for noninvasive observation of Src activity Our novel bioluminescent activatable reporter takes advantage of two facts. First, the crystal structure analysis of Firefly luciferase (PDB ID: 1LCI) has shown that the enzyme has a globular structure with a large N-terminal domain and a small C-terminal domain joined by a flexible hinge region 26. Second, the active Src kinase can phosphorylate the potential Src substrate on tyrosine residues and then the phosphotyrosine (p-Tyr) -containing peptide can be recognized and bound by the SH2 domain 27. To develop this intra-molecular activatable reporter, we constructed a hybrid luciferase by inserting the SH2 domain and the Src consensus substrate peptide (Src-pep) between the amino-(Nluc) and carboxyl-(Cluc) terminal domains of the Firefly luciferase molecule (Fig. 1A). Analogous reporters have been successfully developed to noninvasively image phosphorylation events of many kinases, such as AKT 28,29, FADD 30, EGFR 31, C-MET 32 and TGFβ 33. The functional basis of this approach is that the proximity of the N and C terminals of the SH2 domain 31,32,34 and the flexible linkers enable the inactive luciferase fragments to reassemble the active luciferase molecule. In the presence of Src kinase activity, phosphorylation of the Src consensus substrate at tyrosine residues (Tyr 662 and 664) would result in its intra-molecular interaction with the docking pocket of the SH2 domain, thus sterically preventing reconstitution of a functional luciferase. Thus, the activatable reporter is designed to increase bioluminescent activity following inhibition of the Src substrate phosphorylation (Fig. 1B). Apart from the hybrid luciferase, the bioluminescent Src reporter (BSR) also constitutes some other essential elements (Fig. 1C): (a) a lentiviral vector-based HIV for stable transformation, (b) a coelenterazine-dependent luciferase for internal control (considering the capacity of the lentiviral vector, Gaussia luciferase (Gluc), the smallest known coelenterazine-using luciferase, was the ideal candidate; Gaussia luciferase was co-expressed with the puromycin resistance gene via the “self-cleaving” T2A sequence), (c) flexible linker sequences for minimizing steric hindrance, and (d) a Myc-tag for Western blot analysis and immunoprecipitation. To validate that the reporter was indeed correctly expressed in living cells, we transfected BSR into HT29 cells containing high Src expression and activity 35. The expressions of the recombinant protein and many signaling molecules were examined through Western blotting. The activities of several important molecules of Src signaling cascades, such as Erk, Akt, FAK, and p130Cas, were not affected, suggesting that the reporter did not perturb endogenous cellular signaling (Fig. 1D). We immunoprecipitated BSR using anti-Myc antibody to validate that the reporter is a substrate for the Src kinase. An increase in phosphorylation of the reporter was observed when the immunoprecipitated BSR was probed with phosphotyrosine antibody, indicating that the hybrid luciferase didn't disturb the phosphorylation of the Src consensus substrate peptide (Fig. 1E). Characterization of the Src-dependent, phosphorylation-sensitive bioluminescent reporter in living cells According to the schemes presented above, construction of a specific Src reporter required a specific substrate peptide. The Src consensus substrate peptide WMEDYDYVHLQG, derived from p130cas, was considered to be the best candidate for two reasons: first, it is not a substrate for other kinases 14 and second, this peptide contains the YDYV motif, which serves as a Shc-SH2 binding site with moderate binding affinity when phosphorylated 36. Therefore, after careful experimentation, this substrate peptide and Shc-SH2 domain (amino acids 374-465) 31,32 were selected as the best partners to construct the bioluminescent Src reporter (BSRwt). Two phenylalanine mutations of the corresponding phosphorylation sites were also generated by site directed mutagenesis as a control vector for imaging experiments (BSRmut) (Fig. 1A). Since Src is overexpressed and highly activated in the HT29 colorectal cell line, we generated stable HT29 cell lines expressing the BSRwt or BSRmut to validate the phosphorylation dependence of the BSR reporter. First, we generated stable HT29 cell line expressing the full-length Firefly luciferase for the control of direct drug effect on FL activity. The results showed no significant changes among the vehicle, dasatinib, saracatinib and EGF treatments (Fig. 2A). The Firefly luciferase requires ATP to catalyze the oxidation of luciferin, and the kinase phosphorylation also consumes ATP. These data demonstrated that the change of ATP levels following the kinase activity did not induce increase or decrease of the firefly luciferase activity. For the BSR reporter, the bioluminescence intensity showed a nearly 5-fold difference (p < 0.01) between the wild-type and mutant reporters; treatment with the Src selective inhibitor PP1 could reverse the Src-induced bioluminescence decrease. In contrast, HT29-BSRmut cells showed no significant change in bioluminescence activity in response to the PP1 treatment (Fig. 2B). A corresponding difference in phosphorylation of BSR between the wild-type and the mutant was also observed when the immunoprecipitated BSR was probed with phosphotyrosine antibody. Immunoprecipitation also revealed a decrease in phosphorylation of BSRwt in response to the PP1 treatment (Fig. 2C). These results further confirmed that Src-directed tyrosine phosphorylation occurred at the putative Src phosphorylation sites (Tyr 662 and 664) of consensus substrate peptide in the reporter. However, because the steady-state Src activity is low in HEK293T cells, the Src reporter showed little or no change in the complementation activity of the luciferase (data not shown). To further validate the specificity of the reporter for Src kinase, we generated stable HEK293T cell lines expressing the BSRwt or BSRmut. The activated mutant of Src family kinase (Src Y527F, Yes Y535F or Fyn Y531F) was transfected in HEK293T-BSR cells. The results demonstrated that only HEK293T-BSRwt cells transfected with Src Y527F showed a robust nearly 5-fold Src-mediated decrease of bioluminescence compared with HEK293T-BSRmut cells, indicating BSR has the specificity for the Src reporter in mammalian cells (Fig. 2D). Next, to image the steady-state level of Src phosphorylation in cultured cells, we transiently transfected panels of colorectal cancer cells with BSRwt. The results revealed low Src activity in SW480 and Caco-2 cell lines and high Src activity in HT29, SW48, and SW1116 cell lines. The normalized complemented luciferase activity (Fluc/Gluc) in SW480-BSRwt was the highest -- about 5.3-fold higher than that in HT29-BSRwt (Fig. 2E). The bioluminescence was also correlated with the differential steady-state levels of Src phosphorylation in all cell lines, as revealed by Western blot (Fig. 2F). Dynamic imaging of the kinetics of EGF-induced Src activation in living cells Treatment of cells with EGF can trigger Src activity through the corresponding receptor EGFR 14. To validate the activation of Src kinase activity by the BSR (rather than inhibition), we examined the effects of EGF stimulation on BSR bioluminescence activity. For the dose-dependent assay, HEK293T-BSRwt cells were serum-starved overnight and stimulated with increasing concentrations of EGF for 1 hour. The results indicated a dose-dependent decrease of bioluminescence, which correlated with the increase of Src phosphorylation (Fig. 3A-B). Then, for the time-dependent assay, HEK293T-BSRwt cells were serum-starved overnight and stimulated with 100ng/ml of EGF for the indicated times. The bioluminescence activities were detected at different times. The results demonstrated that HEK293T-BSRwt cells underwent a significant EGF-mediated reduction of luciferase activity (about 62.1%) after 30 min of treatment. The reduction in luciferase activity correlated with the observed increase in Src phosphorylation over this time period, but not for total Src, as confirmed by Western blotting (Fig. 3C-D). In contrast, HEK293T-BSRmut cells showed little change in the bioluminescence activity (data not shown). After confirming the decrease of bioluminescence activity induced by Src activation, we further explored whether this reduction could be blocked by PP1, a selective inhibitor of Src kinase. As expected, the results demonstrated that the decrease in luciferase activity induced by EGF stimulation was largely eliminated in cells pretreated with PP1. Western blot analysis of these samples indicated that the increase in bioluminescence activity correlated with a decrease in Src activity (Fig. 3E-F). These results further confirmed that the reporter was specific for Src kinase and that the observed decrease of BSR luciferase activity was caused by the Src-dependent conformation change resulting in the separation of the luciferase fragments. Dose- and time-dependent imaging of Src activity in response to specific inhibitors in cultured cells The utility of the reporter system was further evaluated in testing the efficacy of Src inhibitors in cultured cells. We used HT29-BSR cells for more detailed analyses of the Src reporter in cell-based assays under various experimental conditions. We also generated stable HT29 cell line expressing the full-length Firefly luciferase for the control of direct drug effect on the enzyme activity. The results showed no significant changes (data were shown in Fig. 2A). In dose response studies, HT29-BSRwt cells were treated with increasing concentrations of two inhibitors under clinical investigation (dasatinib or saracatinib) for 2h following which bioluminescence activity in cells was monitored with IVIS spectrum system. Luciferase activity increased in a dose-dependent manner for both inhibitors, but saracatinib induced a greater increase in complemented Firefly luciferase activity than dasatinib. Bioluminescence was maximally induced 3.3 ± 0.18 fold over untreated controls by 150nM dasatinib and 4.5 ± 0.24 fold by 2uM saracatinib. These luciferase signal increases were correlated with the decrease of endogenous phospho-Src, but not with total Src, as detected by Western blotting (Fig. 4A-B). In time course studies, Src reporter cells were pretreated with 150nM dasatinib and 2uM saracatinib at various times. Luciferase activity increased in a time-dependent manner for both inhibitors; however, an early increase was observed in bioluminescent activity following the inhibition with saracatinib. Western blot analysis indicated that the activation of reporter correlated with phosphorylation of endogenous Src (Fig. 4C-D). Thus, the activatable design renders the reporter as a novel molecular tool for the measurement of Src inhibition. Our results demonstrated that saracatinib showed earlier induction and reached a higher plateau. This may be caused by the higher affinity of saracatinib for Src kinase 37. When the normalized luciferase activity of the vehicle control was set as 0% and the maximum increased activity of drug treatments as 100%, the EC50 of dasatinib for Src activity inhibition in our intracellular context was about 42.36nM. The inhibitory EC50 of phospho-Src, calculated by Western blotting using phospho-specific antibodies, was reported to be about 20nM 38. This difference may be due to different cell lines and experimental conditions, because the EC50 value estimated by the relative band intensities (% of vehicle control) in our Western blotting analysis was about 40.45nM. For saracatinib, the EC50 values were 0.89uM as measured by the luciferase activity and 1.04uM by Western blotting (Fig. S1). These results demonstrated that the BSR reporter exhibits similar or even higher sensitivity compared with Western blot. Noninvasive imaging of Src activity in mice The ability of an agent to modulate its target's activity in vivo depends on multiple factors. The pharmacokinetics and bioavailability of the drug in the whole body and in the tumor itself have a substantial impact on the optimal dosing and administration schedule required for maximal target modulation 28. So far, it has not been possible to measure the Src activity in vivo due to the lack of appropriate technologies. Bioluminescence imaging is one of the most widely used imaging technologies for interrogating cellular and molecular events in rodent models of human biology 39,40. We have demonstrated that the BSR reporter efficiently detects Src tyrosine kinase inhibition in vitro. We therefore hypothesized that this reporter could be further developed into a model system to measure the inhibition of Src activity in vivo. To test this concept, we implanted HT29-BSRwt or HT29-BSRmut cells into the bilateral flanks of nude mice to establish human tumor xenograft models. The HT29-BSRmut on the right flank was used as a control. When the tumors reached a volume of 40 mm3, the mice were intraperitoneally treated with vehicle alone, dasatinib (20mg/kg) or saracatinib (25mg/kg). Serial imaging was performed at 0, 2, 6, 12, and 24 hours following drug administration and compared with the corresponding mutant control bioluminescence. These results demonstrated that the bioluminescence activity of BSRwt remained essentially flat over a 24-h period in vehicle-treated mice. On the other hand, in mice treated with saracatinib, the bioluminescence activity of BSRwt increased within the first 2-h and reached a peak at approximately 12 hours after treatment with about 4.5-fold increase compared to pretreatment. As for dasatinib, the bioluminescence slowly increased and reached about 4.2-fold at approximately 24 hours after treatment (Fig. 5). These results demonstrate that the BSR reporter is a novel molecular tool to measure Src inhibition in vivo. This technology also facilitated the assessment of pharmacokinetics and pharmacodynamics of drugs in mice that may be of relevance in humans. This function provides an immediate and sequential opportunity to optimize therapeutic routes of drug administration for maximal tumor control and minimal normal tissue toxicity. High-throughput screening against a kinase inhibitor library using BSR To examine the performance of the reporter for high-throughput drug screening, bioluminescence activities of HT29-BSRwt cells were screened against a kinase inhibitor library. This compound library was obtained from National Compound Resource Center in China, containing 84 small molecular weight kinase inhibitors. Src kinase inhibitors as well as upstream modulators of Src kinase activity would lead to increase in complemented Firefly luciferase activities in HT29-BSRwt cells. HT29-BSRmut cells were used as a negative control for compounds that led to changes in bioluminescence signals independent of inhibition of Src kinase activity (data not shown). The application of the bioluminescent activatable reporter for HTS has demonstrated that variability in seeding densities minimally affects the signal-to-noise ratio, thereby enabling flexibility in cell plating 30. In our experiment, each screening plate also included intra-plate controls (vehicle treatment and HT29-BSRmut) to assess the quality of the assay protocol in a 96-well plate format. Among all the 84 kinase inhibitors screened, a compound (Tyrphostin AG1478) led to significant bioluminescence activity increase compared with the vehicle control treatment (about 3.4-fold) comparable with the known Src-specific Kinase inhibitors (PP1 and PP2, about 4.5-fold and 3.7-fold, respectively) (Fig. 6A). To further validate the effect of Tyrphostin AG1478 on Src kinase, HT29-BSRwt and HT29-BSRmut cells cultured in 24-well plates were treated with Tyrphostin AG 1478 (0.1mM) for 60min prior to the BLI analysis. The results showed that Tyrphostin AG 1478 led to significant increase (about 2.78 fold) in complemented Firefly luciferase activities in HT29-BSRwt cells (Fig. 6B). Since Tyrphostin AG1478 is a highly specific inhibitor of the EGFR tyrosine kinase, it is possible that the effect of AG1478 on Src activity is caused by the inhibition of EGFR (Fig. 6C). Monitor two kinase activities in one subject by monitoring of Firefly and Renilla luciferase complementation assays Complex signaling networks, characterized by cross-talk between various components, underlie the malignant phenotype of cancer. Therefore, it is important to develop a multi-reporting system to simultaneously monitor the activities of various kinases both in vitro and in vivo. Since Firefly and Renilla luciferases have different substrate specificities and emission spectra 41, we hypothesized that it is possible to apply imaging of both enzymes in the same model based on split luciferase complementation strategy. To test this concept, a new Src reporter based on split Renilla luciferase complementation assay (indicated as rBSR) was constructed by replacing the Firefly luciferase fragments with Renilla luciferase fragments. In our preliminary experiment, we noticed that higher luciferase activity was reconstructed when C-terminal fragment of Renilla luciferase was place on the N-terminal of the reporter. Therefore, the structure of the Renilla Src reporter was Crluc-Sh2-peptide-Nrluc (Fig. 7A). The bioluminescent AKT reporter (indicated as fBAR), which was developed as reported by Zhang et al. 28, was chosen as the other kinase reporter based on split Firefly luciferase complementation assay (Fig. 7B). Since MDA-MB-435S cell line has high levels of Src kinase and Akt kinase 42,43, we generated stable MDA-MB-435S cell lines expressing both rBSR and fBAR after G418 and puromycin selections. Cells expressing the wild-type or the mutant reporters were implanted into the bilateral flanks of nude mice to establish xenograft models. The mutant on the right flank was used as a control. Firefly and Renilla luciferase bioluminescence imagings were concurrently performed after treatment with vehicle control, perifosine (30mg/kg), or dasatinib (20mg/kg) for 6 hours. The results demonstrated that the bioluminescence activities for both reporters increased when administrated with the corresponding inhibitors. Interestingly, treatment of Src inhibitor dasatinib induced significant increases of both Renilla luciferase and Firefly luciferase activities by about 2.14-fold and 3.92-fold, respectively. In contrast, the Akt inhibitor only significantly activated the fBAR reporter about 3.29-fold compared to pretreatment (Fig. 7C-D). This effect may be caused by the cross talks between the signaling pathways and also the fact that Akt operates downstream of the Src signaling pathway. Discussion Extensive preclinical evidence warrants targeting Src as a therapeutic approach for cancer. The common methods for the elucidation of Src kinase activity, such as radioimmunoassay (RIA) and Western blot, require destroying large amounts of cells or tissue, and are not accessible within the relevant cellular microenvironment. The advent of optical-based molecular imaging paradigm makes it possible to visualize, characterize, and quantitatively measure biological processes at the cellular and molecular levels in living subjects. Previous studies have utilized FRET-based reporter molecules for non-invasive imaging of Src kinase 14,15,44. However, it is difficult to employ these reporters in living animals due to their high degree of autofluorescence and poor signal penetration depth through biologically heterogeneous tissues 45. Therefore, in this manuscript, we described a novel activatable reporter based on split luciferase complementation. Luciferase based complementation assay has been widely applied to visualize the phosphorylation-dependent interactions. This has been achieved through either an inter-molecular or an intra-molecular complementation strategy. For inter-molecular reporters, also called bimolecular reporters, the luciferase is split into two non-functional fragments (Nluc and Cluc), each of which is fused to one of the two binding partners. When the binding partners interact in a phosphorylation-dependent manner, the two luciferase fragments are brought into proximity, leading to luciferase reconstitution 46. For the intra-molecular complementation strategy, the single-chain reporter is constructed by sandwiching a conformationally responsive unit between the split luciferase fragments. This conformationally responsive unit usually consists of a kinase-specific substrate and a phosphoaminoacid-binding domain. The reporter design predicts that in the absence of kinase phosphorylation the N- and C- terminal luciferase domains interact and reconstitute enzyme activity. In contrast, the conformational changes induced by the phosphorylation event can sterically prevent the complementation 28. We first attempted the inter-molecular complementation strategy by fusing the Firefly luciferase fragments to the Src consensus substrate peptide and the SH2 phosphopeptide binding domain, respectively, expecting to yield a large dynamic range from low to high bioluminescence. By using a mechanistically similar strategy, other investigators have successfully monitored the phosphorylation events of IRS-1 47, Cdc25C 46, c-Myc 48 and PKA 49. However, in our experience, the inter-molecular Src reporters showed marginal bioluminescence increase (data not shown). We also attempted targeting the reporter to the cytoplasm with a nuclear export signal (NES) 49 or using different configuration of the fused protein at different dissection sites 50; none of these strategies resulted in a satisfactory bioluminescence signal. This can be explained by the too short length of the Src substrate peptide to interact with the SH2 domain. Alternatively, upon interaction of the phosphorylated Src substrate peptide and SH2 domain, the configuration may not be suitable for the complementation and reconstitution of luciferase. It is also known that the geometry of Nluc and Cluc in the interacting protein complex can largely influence how the active site is reconstituted 50. For the kinase-activatable reporter, another important aspect is to ensure the specificity. In our study, we chose the peptide derived from p130cas as the Src consensus substrate. This peptide has previously been demonstrated to be specifically phosphorylated by activated Src 14. It is of note that SH2 domain also plays an important role in the design of the intra-molecular activatable reporter. We have shown that Shc-SH2 domain (aa 374-465) has a better dynamic range than Src-SH2 domain (aa 167-267), which was used for FRET-Src reporter 14. This phenomenon may be due to the different binding affinity for p-Tyr-containing peptides. A plausible explanation is that the SH2 phosphotyrosine binding domain competes with phosphatase and prevents tyrosine dephosphorylation of the substrate peptide 51. The SH2 domain of Shc can also interact with the YDYV motif 36 but displays a relatively low binding affinity for p-Tyr-containing peptides compared with the SH2 domain of c-Src 52. A similar phenomenon was reported in the process of developing FRET RhoA sensor 53. The Src reporter we describe here represents a novel tool for the dynamic measurement of Src activity without labor-intensive and time-consuming tasks for tissue analysis and longitudinal studies of biological processes. Our study has illustrated the general utility of the bioluminescent Src reporter for the analysis of kinase inhibitors undergoing clinical studies in cancer cells cultures and xenografts of mice. We demonstrated that inhibition of Src kinase activity resulted in activation of reporter's bioluminescence activity in a dose- and time-dependent manner. Also, the simplicity, cost-effectiveness, high sensitivity, and wide dynamic range of bioluminescent cell-based assays make them highly attractive for the development of high throughput screening to accelerate drug discovery and development of Src inhibitors. Contrary to current early stage drug discovery research depending on various in vitro biochemical and mass spectrometry protocols, mammalian cell-based functional screening assays have shown promise in predicting the in vivo dynamics. Furthermore, compared to other molecular imaging modalities, such as FRET, bioluminescent imaging is more suitable for simultaneously monitoring two molecules in one subject 19. In our study, we employed two molecular bioluminescent reporters based on Firefly and Renilla luciferase complementation with different substrate specificities and emission spectra 41 to investigate two signaling pathways (Src kinase and Akt kinase) in one subject. This work opens up new opportunities to evaluate diverse aspects of complicated biological processes with molecular imaging in living subjects. We have furnished an important example of the power of the luciferase-based optical imaging reporter in studying sophisticated signal transduction pathways and drug efficacy. Unlike the fluorescence complementation, luciferase complementation does not assemble irreversibly. Thus, the BSR reporter can detect not only the phosphorylation but also the subsequent dephosphorylation. Phosphorylation induces the decrease of bioluminescence, whereas, dephosphorylation increases the bioluminescence. In the continuous biological processes of living cells, the phosphorylation and dephosphorylation are in dynamic balance. And the activity change of BSR was due to a shift in the phosphorylation/dephosphorylation balance. However, it is noteworthy that monitoring molecular events in a single cell by bioluminescent reporters, requires a complicated and expensive bioluminescence microscope 54,55. Conclusion We have developed a novel, inter-molecular, Src-activatable reporter based on split-luciferase complementation. The reporter allows non-invasive, real time, and dynamic imaging as well as quantification of Src kinase activity in living cells and in vivo. It will greatly facilitate the investigation of Src phosphorylation events and the evaluation of anti-Src drug efficacies in preclinical models. It will also provide an approach to identify lead compounds targeted to Src kinase from libraries using cell-based, high-throughput screening. Supplementary Material Supplementary Figure S1. Click here for additional data file. This work was supported by the National Basic Research Program of China (973 Program, 2011CB935800), and the Program for Changjiang Scholars and Innovative Research Team in University (PCSIRT, Grant No. IRT1272) of China. Abbreviations BLIbioluminescence imaging FRETfluorescence resonance energy transfer FLIMfluorescence lifetime imaging microscopy HTShigh-throughput screening NESnuclear export signal FlucFirefly luciferase GlucGaussia luciferase RlucRenilla luciferase BSRbioluminescent Src reporter Src-pepSrc consensus substrate peptide EGFepidermal growth factor rBSRbioluminescent Src reporter based on Renilla luciferase fBARbioluminescent Akt reporter based on Firefly luciferase. Figure 1 Schematic of the activatable reporter based on split-luciferase complementation assay for detecting Src activity. (A) The model shows a diagrammatic representation of the domain structure of the activatable reporter and the sequences of the linker and Src kinase substrate. Two versions of the reporter were developed: the BSRwt molecule, which contains the wild-type Src-pep sequences and the BSRmut molecule, which contains two tyrosine-to-phenylalanine substitutions at the putative phosphorylation sites. (B) BSR functions in Src-dependent phosphorylation of the Src-pep substrate. In the presence of Src kinase (Src on), the phosphorylation of Src-pep results in its intra-molecular interaction with the SH2 domain, sterically preventing the complementation of split luciferase fragments and generating minimal bioluminescence activity. In the absence of Src activation (Src off), the reconstitution of N- and C-terminal luciferase domains restores the bioluminescent activity. (C) The lentiviral vector map of BSR reporter. Via the “self-cleaving” T2A sequence, Gaussia luciferase and the puromycin resistance gene were co-expressed under the control of hPGK promoter to serve as an internal control and selection of stable transductants, respectively. (D) Cell lysates from HT29 and HT29-BSRwt cells were analyzed by Western blotting using antibodies specific for total EGFR, p-EGFR(Tyr1173), total Src, p-Src(Tyr416), total Akt, p-Akt(Ser473), total Erk, p-Erk(Thr202/Tyr204), total FAK, p-FAK(Tyr397) and p130Cas (Tyr410). β-actin was used as a loading control and myc as the BSR expression control. (E) Myc antibody was used to immunoprecipitate the BSR reporter molecule and Western blot analysis was performed by using phospho-tyrosine antibody. Figure 2 Characterization of the BSR reporter in vitro (A) The changes of the full-length Firefly luciferase activity in HT29 cells with the vehicle, dasatinib, saracatinib and EGF treatments. (B) HT29-BSRwt or HT29-BSRmut cells cultured in 24-well plates were treated with the vehicle or 10uM PP1 for 60min. A pseudocolor data of Firefly luciferase imaging represents four separate experiments performed with quadruple culture wells. The changes in normalized bioluminescence activity (Fluc/Gluc) were plotted as fold induction over the value of BSRmut/pp1 (-) (line2). Data are presented as means ± S. E. Statistical analysis was done by one group t test for significance at the p< 0.01 level. (C) Western blotting analysis was performed using the antibody specific for phospho-tyrosine, phospho-Src, and total Src. For coimmunoprecipitation, Myc antibody was used to immunoprecipitate the BSR reporter molecule and Western blot analysis was performed by using phospho-tyrosine antibody. (D) Bioluminescence imaging of HEK293T-BSRwt co-expressed with the activated mutant of Src family kinase (Src Y527F, Yes Y535F or Fyn Y531F). (E and F) Bioluminescence imaging of the steady-state level of Src phosphorylation in a panel of colorectal cancer cells (SW480, Caco-2, HCT116, HT29, SW48, Colo320, SW1116 and Lovo) with the BSR reporter. Western blot analysis of the indicated cells was performed using antibodies specific for phospho-Src and total Src, β-actin and myc as the controls. Figure 3 In vitro characterization of EGF-induced activation of BSR (A) HEK293T-BSRwt cells were serum-starved overnight, stimulated with increased concentration of EGF for 1h, and then subjected to bioluminescence imaging. A pseudocolor data of Firefly luciferase imaging represents of four separate experiments performed with quadruple culture wells. The changes in normalized bioluminescence activity (Fluc/Gluc) compared with vehicle-treated value were expressed as the percentage change. (B) Cells from (A) were analyzed by Western blotting using the antibody specific for phospho-tyrosine, phospho-Src and total Src, β-actin and myc as controls. (C) HEK293T-BSRwt cells were serum-starved overnight, stimulated with 100ng/ml of EGF for the indicated times, and then subjected to bioluminescence imaging. (D) Cells from (C) were analyzed by Western blotting using the antibody specific for phospho-tyrosine, phospho-Src and total Src, β-actin and myc as controls. (E) Bioluminescence imaging of the effect of PP1 on EGF-induced activation of Src phosphorylation. HEK293T-BSRwt cultured on 48-well plates were serum-starved overnight, pretreated with vehicle or PP1 (10uM) for 2 hours, and then stimulated with vehicle or EGF (100ng/ml) for 30min. The changes in normalized bioluminescence activity (Fluc/Gluc) over vehicle treatment levels (line 1) were determined and reported as fold induction. (F) Cells from (E) were analyzed by Western blotting using antibodies specific for phospho-tyrosine, phospho-Src and total Src, β-actin and myc as controls. Figure 4 BSR reporter in response to Src inhibitors in vitro (A and B) Dose response studies. HT29-BSRwt cells cultured on 48-well plates were treated with increasing concentrations of dasatinib or saracatinib for 2h. The changes in normalized bioluminescence activity (Fluc/Gluc) were plotted as fold induction over vehicle-treated values. Western blotting analysis with phospho-tyrosine, phospho-Src, and total Src-specific antibodies was performed to confirm the inhibition of Src activity. (C and D) Time course studies, HT29-BSRwt cells cultured on 48-well plates were treated with dasatinib (150nM) or saracatinib (2uM) for the indicated times. The changes in normalized activity (Fluc/Gluc) were plotted as fold induction over vehicle-treated values. Cells were analyzed by Western blotting using antibodies specific for phospho-tyrosine, phospho-Src, and total Src, β-actin and myc were used as controls. Figure 5 Noninvasive imaging of inhibition of Src kinase activity in mice (A) Xenograft models were established by subcutaneous injections in the bilateral flanks of nude mice with HT29-BSRwt (left) and HT29-BSRmut (right) cells. The mutant was used as a control for each of the mice. BLI activity of pretreated and intraperitoneally treated mice with vehicle control (20% DMSO in PBS), dasatinib, (20mg/kg) or saracatinib (25mg/kg) was monitored at various times. Representative images of serial Firefly luciferase bioluminescence imaging at indicated times of treatment are shown. (B) Graph represents induction of bioluminescence by vehicle, dasatinib, or saracatinib at each time point. The value was calculated using the mutant reporter as a control at each time point for each of the mice. For each xenograft, we first calculated the normalized bioluminescence activity by the ratio of Firefly luciferase activity and Gaussia luciferase activity. We then calculated the normalized bioluminescence activity ratio of the wild-type and mutant values at the same time point in the same mouse. Figure 6 High-throughput screening against a kinase inhibitor library using BSR (A) A representative pseudocolor image in the HTS assays. HT29-BSRwt cells cultured in 96-well plates were treated with 30uM of each compound for 60min in triplicates (for example: 1A~1C, 1D~1F, 2A~2C…). In this assay, PP1, PP2, and Tyrphostin AG 1478 induced significant increases in the bioluminescence activity compared to the vehicle control. (B) Further validation of the effect of Tyrphostin AG 1478 on Src activity with BSR in 24-well plates. (C) Western blotting analysis of the phosphorylation of EGFR and Src kinases after Tyrphostin AG 1478 treatment in HT29 cells. Figure 7 Monitoring two kinase activities in a single mouse using both Firefly and Renilla luciferase complementation assays. (A) Schematic representation of the bioluminescent Src reporter based on Renilla luciferase complementation strategy (rBSR). N- and C-terminal Firefly luciferase domains in the BSR reporter were replaced by C- and N-terminal Renilla luciferase domains, respectively. (B) The lentiviral vector structures of Src reporter based on split Renilla luciferase complementation assay (rBSR) and Akt reporter based on split Firefly luciferase complementation assay (fBAR). (C) MDA-MB-435S cells expressing both rBSR and fBAR were used to establish xenograft tumors by subcutaneous injections in the bilateral flanks of nude mice with 435S-rBSRwt/fBARwt cells on the left and 435S-rBSRmut/fBARmut1cells on the right cells (1x106 cells/sample). Firefly and Renilla luciferase bioluminescence imagings were performed after treatment with vehicle control, perifosine (30mg/kg) or dasatinib (20mg/kg) for 6 hours. (D) The histogram of Firefly and Renilla luciferase bioluminescence imaging described in C. 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==== Front Vaccine Vaccine Vaccine 0264-410X 1873-2518 Elsevier Ltd. Published by Elsevier Ltd. S0264-410X(14)00862-7 10.1016/j.vaccine.2014.06.061 Article Modified Newcastle disease virus vectors expressing the H5 hemagglutinin induce enhanced protection against highly pathogenic H5N1 avian influenza virus in chickens Kim Shin-Hee a Paldurai Anandan a Xiao Sa a Collins Peter L. b Samal Siba K. [email protected]⁎ a Virginia-Maryland Regional College of Veterinary Medicine, University of Maryland, 8075 Greenmead Drive, College Park, MD, United States b Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, MD, United States ⁎ Corresponding author. Tel.: +1 301 314 6813; fax: +1 301 314 6855. [email protected] 23 6 2014 31 7 2014 23 6 2014 32 35 4428 4435 9 4 2014 18 5 2014 11 6 2014 Copyright © 2014 Elsevier Ltd. Published by Elsevier Ltd. All rights reserved.2014Elsevier LtdSince January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active.Highlights • Mesogenic Newcastle disease virus (NDV) strain Beaudette C (BC) was modified to enhance the protective efficacy of the foreign antigen. • The modified NDV vectors were compared for their ability to express the HA protein of H5N1 HPAIV. • The modified NDV vectors expressed enhanced levels of the HPAIV HA protein. • Two of the modified NDV vectors induced higher levels of immunogenicity and protective efficacy against HPAIV. • Two of the modified vectors were found to be superior to conventional rLaSota vector. Naturally-occurring attenuated strains of Newcastle disease virus (NDV) are being developed as vaccine vectors for use in poultry and humans. However, some NDV strains, such as Beaudette C (BC), may retain too much virulence in poultry for safe use, and more highly attenuated strains may be suboptimally immunogenic. We therefore modified the BC strain by changing the multibasic cleavage site sequence of the F protein to the dibasic sequence of avirulent strain LaSota. Additionally, the BC, F, and HN proteins were modified in several ways to enhance virus replication. These modified BC-derived vectors and the LaSota strain were engineered to express the hemagglutin (HA) protein of H5N1 highly pathogenic influenza virus (HPAIV). In general, the modified BC-based vectors expressing HA replicated better than LaSota/HA, and expressed higher levels of HA protein. Pathogenicity tests indicated that all the modified viruses were highly attenuated in chickens. Based on in vitro characterization, two of the modified BC vectors were chosen for evaluation in chickens as vaccine vectors against H5N1 HPAIV A/Vietnam/1203/04. Immunization of chickens with rNDV vector vaccines followed by challenge with HPAIV demonstrated high levels of protection against clinical disease and mortality. However, only those chickens immunized with modified BC/HA in which residues 271–330 from the F protein had been replaced with the corresponding sequence from the NDV AKO strain conferred complete protection against challenge virus shedding. Our findings suggest that this modified rNDV can be used safely as a vaccine vector with enhanced replication, expression, and protective efficacy in avian species, and potentially in humans. Keywords Newcastle disease virusVectored vaccineHemagglutininH5N1 highly virulent avian influenza virus ==== Body 1 Introduction Newcastle disease virus (NDV) is a member of the family Paramyxoviridae and has a nonsegmented, negative-sense RNA genome that contains six genes (3′-N-P-M-F-HN-L-5′) [1]. NDV isolates are categorized into three pathotypes based on their virulence in chickens: lentogenic (avirulent), mesogenic (moderately virulent), and velogenic (virulent) [2]. Lentogenic and mesogenic strains of NDV are widely used as live attenuated vaccines to control Newcastle disease in poultry. In addition, NDV can be modified by reverse genetics to express one or more protective antigens representing heterologous pathogens. One of the applications has been to modify NDV to express a protective antigen from another poultry pathogen to create a bivalent vaccine. For example, NDV has been engineered to express the hemagglutinin HA gene of H5N1 highly pathogenic avian influenza (HPAIV) virus, or the VP2 protein of infectious bursal disease virus (IBDV), to create a bivalent vaccine against NDV and HPAIV or IBDV, respectively [3], [4]. Another application has been used to develop NDV strains as a potential vaccine vector in humans against emerging and re-emerging pathogens, such as H5N1 HPAIV and severe acute respiratory syndrome coronavirus (SARS-CoV) [5], [6]. Although both lentogenic and mesogenic strains have potential as veterinary or human vaccine vectors, in nonhuman primates the mesogenic strain Beaudette C (BC) replicated to a higher titer and induced a substantially higher antibody response compared to the lentogenic strain LaSota, and thus appeared to be more effective [7]. However, NDV strains that have a polybasic cleavage site in the F protein and an intracerebral pathogenicity index (ICPI)>0.7 have been classified as Select Agents. Most mesogenic strains, including strain BC, fall into this category even though their pathogenic potential in chickens is limited. The Select Agent classification renders them impractical for vaccine development and use. Therefore, we have endeavored to develop NDV vaccine vectors that would be more effective than lentogenic strains and yet would have the avirulent phenotype. We further evaluated these new NDV vectors by using them to express the HA protein of H5N1 HPAIV. HPAIV is an economically important disease of poultry worldwide. In particular, HPAIV subtype H5N1 infections have resulted in the culling or death of more than 500 million poultry in more than 62 countries. Furthermore, H5N1 strains have been found to cause disease in humans. Therefore, vaccination of poultry or humans against HPAIV could play an important role in reducing virus shedding and raising the threshold for infection and transmission [9]. However, development of vaccines against HPAIV has been hampered due to poor immunogenicity of the virus [10]. The development and manufacture of HPAIV vaccines based on inactivated virus are costly due to the requirement of enhanced biosafety level 3 containment. Therefore, vaccine platforms that can avoid these shortcomings are needed. Live NDV vectors represent an alternative approach. Remarkably, our modified versions of the BC strain expressing the HA protein of H5N1 HPAIV were found to be avirulent by the ICPI test. We evaluated selected constructs for replication, immunogenicity, and protective efficacy in chickens. Our results showed that two of the modified vectors are superior to the commonly used rLaSota vector. 2 Materials and Methods 2.1 Construction and in vitro characterization of modified versions of NDV strain BC We modified a previously described full length cDNA of the antigenome of strain BC [11] to change its naturally-occurring F protein cleavage site motif (RRQKR↓F) to that of strain LaSota (GRQGR↓L) to create rBC/LascFc. This backbone was further modified by replacing various regions in the F gene with those of velogenic NDV strain AKO [8] or the complete HN protein gene of strain LaSota (Fig. 1A). Particularly, we replaced the region of the F gene encoding amino acids 271–330, or 275–330, or 331–390 of rBC/Las Fc with the corresponding segment of the AKO strain. Strain AKO was chosen because this strain replicates to high titer in vitro and in vivo [15]. We chose these segments, because our preliminary work showed that these modifications enhanced virus replication and syncytium formation in vitro (data not shown). Consequently, this can also enhance the in vivo replication and immunogenicity of vaccine vectors. Infectious viruses were generated using a reverse genetics established in our laboratory [12].Figure 1 Construction of modified versions of NDV strain rBC and virus replication and induction of serum antibodies in response to infection of 2-week-old chickens. (A) rBC and rLaSota are recombinant versions of the respective biological strains. The other four viruses are versions of rBC that each have the polybasic F protein cleavage site of the BC strain replaced with the cleavage site of LaSota. In addition, in modified rNDV/AKO F 271-330, AKO F 275-330, and AKO F 331-390, the regions of the F gene encoding the indicated F protein amino acids have been swapped with that of virulent strain AKO to improve replication. (B) Chickens (eight birds per group) were inoculated with each virus (264 HA units) by the intranasal route. Three birds in each group were sacrificed on 3 dpi, and virus titers in the collected tissue samples (brain, trachea, lung, and spleen) were determined by limiting dilution in DF1 cells. (C) Serum samples were collected at 1 and 2 wpi and evaluated for virus-specific antibodies by a hemagglutination inhibition assay using chicken erythrocytes. The in vivo replication and immunogenicity of the modified rNDVs were evaluated in 2-week-old chickens (eight birds per group). Birds were inoculated with 200 μl of each virus (256 HA units/bird) by the intranasal route. Three birds from each group were sacrificed at 3 days post-infection (dpi) and tissues samples (lung, trachea, spleen, and brain) were collected for virus titration. Serum samples collected on days 7 and 14 were evaluated for seroconversion by hemagglutination inhibition (HI) assay [2]. 2.2 Construction and characterization of modified versions of NDV vectors expressing the HA protein of HPAIV The HA gene ORF of HPAIV strain A/Vietnam/1203/04 (H5N1) was modified by PCR and inserted between the P and M genes in the antigenomic cDNAs of rLaSota and the modified rNDVs. In addition, the original polybasic cleavage site of the HA gene (PQRERRRKKG) was replaced by that of the low-pathogenicity influenza virus strain A/chicken/Mexico/31381/94 (PQRETG) [13]. Infectious viruses were generated by reverse genetics [12]. The expression of the HA protein by rNDVs and its incorporation into the vector particles were evaluated by Western blotting [4]. Surface expression of the HA protein was evaluated on virus-infected DF1 cells (MOI of 0.1) by immunofluorescence microscopy and flow cytometry. The multicycle growth kinetics of rNDVs was evaluated in DF1 cells in the presence of 10% chicken egg allantoic fluid [12], [14]. Pathogenicity of rNDV/HA vectors was evaluated by mean embryo death time (MDT) in embryonated chicken eggs and ICPI assay in 1-day-old chicks [2]. 2.3 Immunogenicity and protective efficacy of the NDV/HA vectors in 2-week-old chickens Groups (n = 16 per group) of 2-week-old SPF chickens were infected by the oculonasal route with 106 EID50 per bird of rLaSota/HA, rNDV-AKO F 271–330/HA, or rNDV-Las HN/HA, and an additional group of birds (n = 6) was left uninfected. Following immunization, pre-challenge serum samples were collected weekly from all of the birds. Eight birds from each group were challenged with 104 ELD50 of HPAIV strain A/Vietnam/1203/2004 at 1 week post-immunization (wpi), and the remaining eight birds were challenged in the same way at 3 wpi. For the immunized control group, 3 birds were challenged 1 and 3 wpi. To monitor shedding of the challenge virus, oral and cloacal swabs were collected on days 4 and 7 post-challenge, inoculated into 9-day-old SPF embryonated chicken eggs, and confirmed by HA assay using chicken erythrocytes. Three chickens from each group were sacrificed at 4 days post-challenge to evaluate challenge virus replication in different organs (brain, trachea, lungs, and spleen). All experiments involving virulent NDV and HPAIV were performed in our USDA approved enhanced Biosafety Level-3 facility. 3 Results 3.1 Generation of new NDV vectors A series of modified versions of rBC (Fig. 1A) were recovered and passaged seven times in 1-day-old chicks. The sequence of the F gene of these viruses was confirmed by RT-PCR and sequence analysis to be correct. The in vivo replication and immunogenicity of these rNDVs were evaluated in 2-week-old chickens (Fig. 1B and C). When birds were sacrificed at 3 dpi, rBC replication was detected in the trachea, lung, and spleen, but not in the brain, whereas replication of rLaSota was detected only in the trachea (Fig. 1B). Two of the modified viruses, rBC/Las Fc and rNDV-AKO F 275–330, remained restricted to the trachea while the other two modified viruses, rNDV-AKO F 271–390 and rNDV-AKO F 331–390, replicated in both the trachea and the lung (Fig. 1A). Each of the modified vectors replicated more efficiently than rLaSota (p < 0.05). rNDV-AKO F 271–390 and rNDV-AKO F 331–390, the two viruses that replicated in both the trachea and the lungs, induced higher levels of NDV-specific serum antibodies than rLaSota and the other modified viruses at 1 wpi. All of the modified viruses induced higher levels of serum antibodies than rLaSota at 2 wpi (p < 0.05) (Fig. 1C). 3.2 Generation of and characterization of NDV vectors expressing the HA protein of H5N1 HPAIV We then engineered rLaSota and three of the modified rBC vectors described above as well as rBC in which the HN gene was replaced by that of LaSota (Fig. 2A) to express the HA protein of H5N1 HPAIV strain A/Vietnam/1203/2004 (Fig. 2A). The recovered viruses were passaged seven times in 9-day-old embryonated chicken eggs. All of the viruses replicated efficiently in the eggs (>28 HAU/ml), and the sequence of F and HA genes were found to be correct. The multi-cycle growth kinetics of the rNDVs bearing the H5N1 HPAIV HA gene showed that each of the modified rBC viruses replicated more efficiently than rLaSota or rLaSota/HA for the first 24 hpi (Fig. 2B). Specifically, the rBC-derived viruses reached 8 log10 TCID50/ml at 24 hpi, whereas rLaSota and rLaSota/HA had titers of 6.5 and 5.4 log10 TCID50/ml, respectively, at 24 hpi. The expression of HA by the rNDVs was evaluated in DF1 cells in the presence of 10% allantoic fluid. Despite the presence of the added protease, under these conditions HA was detected only as the uncleaved precursor (HA0, molecular weight of 66 kDa). The expression of the HA0 protein could be detected at 12 h post-infection (hpi) in the case of three modified rNDVs (AKO F 271–330, AKO F 275–330, and Las HN) (Fig. 3A). Subsequently, the expression of HA0 was detected for all the rNDVs at 24 hpi. At 24 hpi, modified rNDV containing AKO F 331–390 or Las HN expressed higher levels of HA0 than the other viruses.Figure 2 Construction of modified NDVs expressing the H5N1 HPAIV HA gene and multi-cycle replication in vitro. (A) The HA ORF of HPAIV H5N1 strain A/Vietnam/1203/04 was engineered to be flanked by the NDV gene-start and gene-end signals and was inserted into the intergenic region between the P and M genes in the rNDV vectors shown. Genes derived from rBC or rLaSota are shown as black or gray rectangles, respectively. F gene segments derived from the NDV AKO strain are shown as white bars with designated location. All viruses contained the F protein cleavage sequence of LaSota, GRQGRL, as shown. (B) Monolayer cultures of the chicken fibroblast DF-1 cell line were infected with the indicated viruses at an MOI of 0.01 PFU/cell. The medium contained 10% uninfected egg allantoic fluid as a source of exogenous protease. Aliquots of the media were taken at 8-h intervals and viral titers were determined by limiting dilution on DF1 cells. Figure 3 Expression of H5N1 HPAIV HA protein in vitro by rNDV/HA vectors and incorporation of HA into rNDV virions. (A) Expression of the HA protein in DF1 cells. DF1 cells were infected with each virus at MOI 1, and cell lysates were collected at 12 or 24 h post-infection for Western blot analysis using convalescent serum from chickens that had been infected with H5N1 HPAIV to visualize the HA protein and monoclonal antibody to visualize the NDV HN protein. (B) Incorporation of the HA protein into the NDV vector particle. The viruses were harvested from allantoic fluids of infected eggs at 72 hpi, purified through a 30% sucrose cushion, and analyzed by Western blot as in part A. (A, B) Lanes: rLaSota (lane 1), rLaSota/HA (lane 2), modified rNDV-AKO 271-330/HA (lane 3), modified rNDV-AKO 275-330/HA (lane 4), modified rNDV-AKO 331-390/HA (lane 5), and modified rNDV-Las HN/HA (lane 6). (C, D) Expression of the HA protein on the surface of DF-1 cells infected with the indicated NDV/HA vector. DF1 cells were infected with each virus (MOI of 0.1) and, at 24 h post-infection, were stained with anti-peptide antiserum against the HA protein followed by anti-Alexa Fluor 488 antibody, fixed with 4% paraformaldehyde and were analyzed by immunofluorescence microscopy (C) and flow cytometry (D). The incorporation of HA into the vector particle was further analyzed by Western blotting (Fig. 3B). The HA protein was detected in the form of HA0 as well as the larger subunit of the cleaved form, HA1 (molecular weight 40 kDa). Incorporation of HA into the vector particle was observed to various extents for the rLaSota parent vector and the modified rNDV vectors, in particular those containing F 271–330 and Las HN (Fig. 3B). There was a two-fold increase in the incorporation of HA1 protein into the vector particle for rNDV-Las HN/HA compared to rLaSota/HA. We next evaluated the surface expression of the HA protein in DF1 cells by immunofluorescence microscopy (Fig. 3C). Similar to the results with the Western blots, modified rNDV containing AKO F 271–330 or Las HN expressed higher levels of the HA protein on the surface of DF1 cells. Furthermore, this was quantitatively confirmed by flow cytometry (Fig. 3D). These results identified rNDV vector containing AKO F 271–330 or Las HN to have enhanced HA expression and incorporation of the HA protein into virus particles. The pathogenicity of rNDVs expressing the HA protein was evaluated by the MDT and ICPI assays (Table 1 ). The avirulent nature of rLaSota was not affected in either assay by expression of the HPAIV HA protein. The MDT values of the modified rNDVs expressing the HA protein were more than 90 h, and the ICPI values were 0.00, indicating that these modified rNDV vectors are avirulent in chickens. Chicks infected with rNDVs had no apparent clinical signs during the 8-day period of the ICPI test, confirming the attenuation of all modified rNDVs.Table 1 Pathogenicity of parental and modified rNDVs in embryonated eggs and 1-day-old chicks. Virus MDTa (h) ICPIb rBC 58 1.57 rLaSota 117 0.00 rLaSota/HA 121 0.00 Modified rNDV-AKO F 271-330/HA 112 0.00 Modified rNDV-AKO F 275-330/HA 114 0.00 Modified rNDV-AKO F 331-390/HA 98 0.00 Modified rNDV-Las HN/HA 102 0.00 a Mean embryo death time (MDT): the mean time (h) for the minimum lethal dose of virus to kill all of the inoculated embryos [2]. Pathotype definition: virulent strains, <60 h; intermediate virulent strains, 60 to 90 h; avirulent strains, >90 h. b Intracerebral pathogenicity index (ICPI): evaluation of disease and death following intracerebral inoculation in 1-day-old SPF chicks [2]. Pathotype definition: virulent strains, 1.5–2.0; intermediate virulent strains, 0.7–1.5; and avirulent strains, 0.0–0.7. 3.3 Immunogenicity and protective efficacy of rNDV/HA vectors in chickens against HPAIV challenge Two-week-old chickens in groups of 16 were infected intranasally with rLaSota/HA, rNDV-AKO F 271–330/HA, or rNDV-Las HN/HA, or were left uninfected (n = 6). To determine the ability of the modified viruses to induce rNDV-specific or HPAIV-specific immune responses, serum samples were collected at 1, 2, and 3 wpi and tested against HPAIV A/Vietnam/1203/2004 (Fig. 4A) and highly virulent NDV strain GB Texas (Fig. 4B) using HPAIV-specific and NDV-specific HI assays. In this study, immune response against GB Texas was evaluated for potential use of NDV vectors as a dual vaccine. In the HPAIV-specific assay, HI titers increased with time, reaching the highest titer at 3 wpi. The two modified rNDV vectors induced higher HA-specific antibody titers than the rLaSota vector (p < 0.05). In the NDV-specific assay, the increase in antibody titer with time was somewhat less and the differences between the viruses were somewhat less. The highest NDV-specific antibody titers were observed with the modified rNDV vectors, although this was less evident at 3 wpi.Figure 4 Induction of serum antibodies in 2-week-old chickens in response to infection with rNDV vector vaccines. Chickens were inoculated with each virus (106 EID50) by the intranasal route. Serum samples were collected at 1, 2, and 3 wpi. Virus-specific antibodies were determined by a hemagglutination inhibition assay using HPAIV A/Vietnam/1203/2004 (A) or NDV strain GB Texas (B). To evaluate the protective efficacy of rNDVs, the immunized chickens were divided into two groups and challenged at two different time points, 1 wpi and 3 wpi. Each group (eight birds each; three for the unimmunized control) was challenged with 104 ELD50 of homologous HPAIV A/Vietnam/1203/2004 via the oculo-nasal route. For the unimmunized chickens, challenge with the HPAIV virus resulted in clinical signs at 1 day post-challenge (dpc), and 100% mortality at 2 dpc. In contrast, all of the immunized chickens were completely protected from clinical disease and mortality following HPAIV challenge. We determined shedding and replication of HPAIV challenge virus by collecting oral and cloacal swabs (4 and 7 dpc) and tissue samples (brain, lung, trachea, spleen; 4 dpc) from the immunized chickens. For the groups challenged at 1 wpi, challenge HPAIV shedding was detected 4 dpc in oral and cloacal swabs in some chickens immunized with rLaSota/HA or modified rNDV-Las HN/HA, whereas no shedding was detected 4 dpc from chickens immunized with modified rNDV-AKO F 271–330/HA virus (Table 2 ). No shedding was observed in any of the swabs taken at 7 dpc, and no virus was recovered from the tissue specimens collected 4 dpc (data not shown). For the groups that were challenged at 3 wpi, only a single bird (in the rLaSota/HA group) had detectable shedding (in an oral swab) at 4 dpc (Table 2). No shedding was detected from any bird on 7 dpi, and no virus was detected in any of the tissue samples collected 4 dpc (data not shown). These data indicated that all of the immunized groups of the chickens were efficiently protected against a dose of HPAIV that was rapidly fatal in unimmunized birds. The modified rNDV vectors appeared to be more protective than rLaSota.Table 2 Oral and cloacal shedding of A/Vietnam/1203/2004 HPAIV challenge virus in 2-week-old chickens immunized 1 or 3 weeks earlier with rNDV expressing the HPAIV HA protein. Chicken groupa 1 week post-immunization 3 weeks post-immunization Oral Cloacal Oral Cloacal rLaSota/HA 2/5 1/5 1/5 0/5 Modified rNDV-AKO F 271-330/HA 0/5 0/5 0/5 0/5 Modified rNDV-Las HN/HA 1/5 1/5 0/5 0/5 a Two-week-old chickens (16 birds per group) were immunized intranasally with the indicated NDV strain. One week post-immunization, eight birds from each group were challenged with HPAIV A/Vietnam/1203/2004 by the intranasal route, and at 3 weeks post-immunization the remaining eight birds were challenged in the same way. Oral (A) and cloacal (B) swabs were collected from five birds in each group on day 4 and 7 post challenge. To identify the shedding of challenge virus, aliquots (100 μl each) of the collected samples were inoculated into three eggs per sample, and allantoic fluids were collected 3 dpi. Virus replication was determined by hemagglutination assay. Detection of virus in any of the three eggs was considered positive. Only the day 4 swabs are shown because no shedding was detected in any of the day 7 swabs. Note that this study included a mock-immunized group for each challenge (n = 3), but these birds died on day 2 post-challenge and were not sampled. 4 Discussion NDV is being developed as a bivalent vaccine vector for protection of chickens against NDV and other poultry pathogens [3], [4], [9], [16], [17]. Additionally, NDV has shown promising results as a potential vaccine vector for human use [18], [19], [20]. NDV is attractive as a vaccine vector in humans because it is highly attenuated in non-human primates, it is not a common human pathogen and is antigenically distinct from common human pathogens and thus should be unaffected by pre-existing immunity, and it can be given topically to the respiratory tract to induce both mucosal and systemic immunity. Although both lentogenic and mesogenic NDV strains can be used as vaccine vectors, the mesogenic strains are more easily grown in vitro and are more immunogenic in vivo. However, mesogenic strains might cause disease in poultry, and most strains are classified as Select Agents, which precludes their development as a vaccine vector. Therefore, we changed the polybasic F cleavage site of mesogenic strain BC to the dibasic F cleavage site of strain LaSota. It would eliminate one of the criteria in classifying BC as a Select Agent. We also confirmed that the recovered virus stably maintained modified F protein cleavage sites during seven passages in chickens. Since reversion of the F protein cleavage site sequence will require mutation of 11 nucleotides, there exists a distance possibility of reversion to virulence. Further, the genetic stability of the cleavage site sequence and virus attenuation need to be carefully evaluated with insertion of different foreign genes into this vaccine vector prior to its use in the field. In addition, we swapped regions of the F gene with strain AKO, or swapped the HN gene with rLaSota. This was done because the fusogenic activity of NDV can affect virus replication in vitro and in vivo, and its modulation can enhance the potential of rNDVs for use as vaccine vectors [21]. The resulting modified rBC viruses were found to be more restricted for replication than their rBC parent and were more immunogenic than rLaSota strain in 2-week-old chickens. We then used these vectors to express the HA glycoprotein of H5N1 HPAIV. H5N1 HPAIV viruses have been a major concern both in poultry industry and public health. There is a need to develop effective vaccines against HPAIV. Our results showed that all the modified rNDV/HA vectors were avirulent in 1-day-old chicks, with an ICPI value of 0.00. Thus, these vectors are not Select Agents and would not pose a threat to the poultry industry. In this study, two modified rNDV vectors, one containing Las HN and the other containing AKO F 271–330, showed enhanced expression of the HPAIV HA protein and incorporation of the HA protein into the NDV particles. Thus, these two modified rNDV/HA vectors were chosen for evaluation of immunogenicity and protective efficacy against H5N1 HPAIV in parallel with the LaSota/HA vector. Our immunization study showed that most serum samples from immunized chickens with the three rNDVs at 1 wpi had high levels of HI titers against NDV and HPAIV, suggesting that these vectors can be efficient as early as 1 wpi. At 3 wpi, titers of serum samples from all immunized chickens remained high. Subsequent challenge with HPAIV H5N1 showed that each of the vectors induced substantial protection against HPAIV challenge. Specifically, whereas all of the unimmunized chickens died by 2 dpc, all of the immunized chickens remained free of disease without replication of detectable infectious challenge virus in tissue samples. However, evaluation of tracheal and cloacal swabs taken at 4 and 7 dpc did reveal differences in the level of protection. Specifically, only the rNDV-AKO F 271–330/HA vector completely prevented detectable HPAIV shedding in all of the birds. The rNDV-Las HN/HA vector was the next most protective, with shedding detected in 2 swabs. The rLaSota/HA was the least protective, with shedding detected in 4 swabs. These results suggest that the two modified rNDV vectors can be more efficient in restricting replication of HPAIV challenge virus than the current rLaSota vector. Since virus shedding can transmit HPAIV to unvaccinated flocks and to humans, an ideal HPAIV vaccine should completely prevent any challenge virus replication and shedding. This suggests that the rNDV-AKO F 271–330/HA vector represents an improved H5N1 HPAIV vaccine for poultry. In summary, our finding showed that the mesogenic NDV strain BC can be modified for possible use as an avirulent, safe, and effective vaccine vectors in chickens. These rNDV vectors can replicate to higher levels in vitro and in chickens than the rLaSota virus, without causing any clinical signs. Our study has identified two improved rNDV vectors that induce higher levels of immunogenicity and protective efficacy against HPAIV. Whether these two rNDV vectors would also provide increased immunogenicity and protection against HPAIV in mammalian species remains to be identified. Acknowledgements We thank Daniel Rockemann, Girmay Gebreluul, Yonas Araya, Andrea Ferrero-Perez, and our laboratory members for excellent technical assistance; and Dr. Bernard Moss (NIAID, NIH) for providing the vaccinia T7 recombinant virus and the pTM1 plasmid. This research was supported by National Institute of Allergy and Infectious Diseases (NIAID) contract no N01A060009 (85% support) and NIAID, National Institutes of Health (NIH) Intramural Research Program (15% support). The views expressed herein do not necessarily reflect the official policies of the Department of Health and Human Services, nor does mention of trade names, commercial practices, or organizations imply endorsement by the U.S. Government. ==== Refs References 1 Samal S.K. Newcastle disease and related avian paramyxoviruses Samal S.K. The biology of paramyxoviruses 2011 Caister Academic Press 69 114 2 Alexander D.J. Newcastle disease Purchase H.G. Arp L.H. Domermuth C.H. Pearson J.E. 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==== Front Int J Biol SciInt. J. Biol. SciijbsInternational Journal of Biological Sciences1449-2288Ivyspring International Publisher Sydney 10.7150/ijbs.14151ijbsv12p0594Research PaperRevealing the Effects of the Herbal Pair of Euphorbia kansui and Glycyrrhiza on Hepatocellular Carcinoma Ascites with Integrating Network Target Analysis and Experimental Validation Zhang Yanqiong 1✉Lin Ya 12Zhao Haiyu 1Guo Qiuyan 1Yan Chen 1Lin Na 1✉1. Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, China.2. College of Pharmacy, Fujian University of Traditional Chinese Medicine, Fuzhou 350122, China.✉ Corresponding authors: Prof. Na Lin & Yanqiong Zhang. Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, No. 16, Nanxiaojie, Dongzhimennei, Beijing 100700, China. E-mail: [email protected] & [email protected]; Phone:+861064014411-2869; Fax: +861064013996.These authors contribute equally to this study. Competing Interests: The authors have declared that no competing interest exists. 2016 25 3 2016 12 5 594 606 18 10 2015 22 2 2016 © Ivyspring International Publisher. Reproduction is permitted for personal, noncommercial use, provided that the article is in whole, unmodified, and properly cited. See http://ivyspring.com/terms for terms and conditions.2016Although the herbal pair of Euphorbia kansui (GS) and Glycyrrhiza (GC) is one of the so-called "eighteen antagonistic medicaments" in Chinese medicinal literature, it is prescribed in a classic Traditional Chinese Medicine (TCM) formula Gansui-Banxia-Tang for cancerous ascites, suggesting that GS and GC may exhibit synergistic or antagonistic effects in different combination designs. Here, we modeled the effects of GS/GC combination with a target interaction network and clarified the associations between the network topologies involving the drug targets and the drug combination effects. Moreover, the "edge-betweenness" values, which is defined as the frequency with which edges are placed on the shortest paths between all pairs of modules in network, were calculated, and the ADRB1-PIK3CG interaction exhibited the greatest edge-betweenness value, suggesting its crucial role in connecting the other edges in the network. Because ADRB1 and PIK3CG were putative targets of GS and GC, respectively, and both had functional interactions with AVPR2 approved as known therapeutic target for ascites, we proposed that the ADRB1-PIK3CG-AVPR2 signal axis might be involved in the effects of the GS-GC combination on ascites. This proposal was further experimentally validated in a H22 hepatocellular carcinoma (HCC) ascites model. Collectively, this systems-level investigation integrated drug target prediction and network analysis to reveal the combination principles of the herbal pair of GS and GC. Experimental validation in an in vivo system provided convincing evidence that different combination designs of GS and GC might result in synergistic or antagonistic effects on HCC ascites that might be partially related to their regulation of the ADRB1-PIK3CG-AVPR2 signal axis. herb-herb combinationEuphorbia kansuiGlycyrrhizahepatocellular carcinoma ascitesnetwork pharmacology. ==== Body Introduction Hepatocellular carcinoma (HCC) represents one of the most frequent malignancies, and the incidence of HCC is increasing worldwide 1. Although great progress has been made in medical and surgical treatments for this disease, its prognosis has not conspicuously improved in recent years 2. As one of the severe complications of HCC, malignant ascites, which is an abnormal accumulation of fluid in the peritoneal cavity caused by tumor infiltration or secretion 3, is a recognized indication of end-stage events in this malignancy and often deteriorates the patient's quality of life 4. Currently, several treatments, such as salt restriction, peritoneal catheter drainage, intraperitoneal chemotherapy and paracentesis, have been performed for the remission of ascites 5. However, the therapeutic efficacies of these treatments are unsatisfactory, and the treatment of malignant ascites remains a difficult problem to solve. Therefore, it is extremely necessary to develop novel and effective therapeutic strategies for the management of HCC-induced malignant ascites to improve patient quality of life. Traditional Chinese medicine (TCM) is one of the most popular complementary and alternative medicine modalities worldwide and has been practiced in China for more than 2,000 years. TCM is increasingly used for the treatment of malignant ascites. For example, the roots of Euphorbia kansui T. N. Liou ex T. P. Wang (Gansui, GS), which is recorded in the Shennong-Bencao, has been used for centuries in China for the treatment of ascites, edema and asthma 6. Recent studies have demonstrated that GS also has a variety of pharmacological actions including anti-fertility, anti-viral, and immune system-regulating effects. Moreover, GS is effective in the treatment of cancer, pancreatitis, and intestinal obstruction 7. However, GS is strongly toxic to the liver and kidneys and can induce symptoms such as diarrhea, stomachache, dehydration and respiratory failure 8, which have seriously restricted the clinical application of GS. Glycyrrhiza (Licorice, Gancao, GC) is another Chinese herb. GC is the root of Glycyrrhiza uralensis Fisch. or Glycyrrhiza glabra L., Leguminosae and has been prescribed in many TCM formulas for its medical activities, which include anti-inflammatory, immunoregulatory, and anti-allergic effects 9. In TCM, GC is commonly combined with other herbs into a single prescription and functions as a unique “guide drug” to enhance the effectiveness of other ingredients or to reduce toxicity and improve flavor in almost half of all Chinese herbal formulas 10, 11. In the Chinese medicinal literature, GS and GC compose one herbal pair of the so-called "eighteen antagonistic medicaments", in which two herbs are mutually incompatible and thus theoretically should not be applied simultaneously. However, the GS/GC combination is prescribed in a classic TCM formula, Gansui-Banxia-Tang, which has been used for the treatment of cancerous ascites 12, 13, which implies that the two herbs may exhibit synergistic or antagonistic effects in different combination designs. TCM emphasizes the integrity of the human body and the interaction between human individuals and their environment. TCM herbs are multi-component and multi-target agents that essentially achieve therapeutic effectiveness by collectively modulating the molecular network of the body system using its active components 14. System biology is a useful tool for explaining and predicting various events related to the efficacy of drugs 15 and is valuable for evaluating the rationality and compatibility of herbs or prescriptions. Because herbs manifest their actions via their targets, the effects of herb-herb combinations should depend on the interactions of their targets in a network manner. To the best of our knowledge, the rationality of the herbaceous compatibility of GS and GC has not been fully elucidated. In the current study, we modeled the effects of the GS/GC combination with a target interaction network and clarified the associations between the network topologies of the drug targets and the drug combination effects. Materials and Methods The technical strategy of this study is presented in Figure 1. Data preparation Structural information about the chemical components of GS and GC The TCM Database@Taiwan 16 (http://tcm.cmu.edu.tw/, Updated in Jun 28, 2012), which is currently the largest non-commercial TCM database in the world, was used to collect the structural information (.mol or .sdf files) of 4 GS compounds and 82 GC compounds. Protein-protein interaction (PPI) data PPI data were collected from the following eight existing PPI databases 17-24. The interactions used in this study include direct (physical) and indirect (functional) associations derived from high-throughput experiments, genomic context, co-expression correlation and manually extraction from the published data. Detailed information about these PPI databases is provided in Supplementary Table S1. Prediction of putative targets for GS and GC Putative targets of GS and GC were predicted based on structural similarity comparisons as described in our previous study 25. Detailed information about the prediction method of putative targets for GS and GC is provided in Supplementary Material. Network construction and analysis The PPI data about the putative targets of GS and GC, known therapeutic targets of ascites and other human proteins were used to construct the drug target PPI network which was visualized by Navigator software (version 2.2.1). According to the previous study of Li et al. 26, we defined a node as a hub if its degree is greater than twice the median degree of all nodes in the network. Subsequently, the PPIs among the hubs were used to construct the hub network. Moreover, four topological features, 'Degree', 'Node betweenness', 'Closeness' and 'K value' (definitions are provided in Supplementary Material), were calculated to identify the major putative targets of GS and GC. These targets were defined as those with values of the four features that were higher than the corresponding median values. Molecular docking simulation Molecular docking simulation was performed to evaluate the direct binding efficiencies of the main chemical components of GS and GC (Supplementary Figure S1 and Table S2) with ADRB1 and PIK3CG, respectively, using the electronic high-throughput screening (eHiTS) docking module, which is a flexible ligand docking system 27. The structures of ADRB1 and PIK3CG were obtained from the previous studies of Cushing et al. 28 and Warne et al 29. The docking score represents the direct binding energy of the ligand to the receptor in units of log Kd. The higher the absolute value of a docking score is, the stronger the direct binding efficiency of the ligand to the receptor. According to the scoring system of the eHiTS algorithm, the direct binding efficiencies of compound-target pairs were classified into three levels: strong, i.e., the absolute value of the docking score is higher than 5.66 for GS or 5.70 for GC; middle, i.e., the absolute value of the docking score ranges from 4.66 to 5.66 for GS or 4.70 to 5.70 for GC; and weak, i.e., the absolute value of the docking score is lower than 4.66 for GS or 4.70 for GC. Experimental validation H22 mouse HCC ascites models were used to examine the synergistic or antagonistic effects of the actions of GS and GC on HCC ascites in different combination designs and to validate their possible combination principles (ADRB1-PIK3CG-AVPR2 signal axis). Drug preparation GC (Lot: 120713) and GS (Lot: 121106) were both purchased from Anhui Fengyuan Tongling Herbal Pieces Co. Ltd. High performance liquid chromatography-mass spectrometry (HPLC-MS) was used to determine the main chemical components of GS and GC. As shown in Supplementary Figure S1 and Table S2, 11 and 36 chemical components were identified in GS and GC, respectively. GC was decocted to a mother liquor with a dosage of 1.34 g/kg. In brief, 20.1 g GC was soaked in a ten-fold volume of water for 1 h, heated with an electric heater and decocted for 1.5 h. After standing and filtrating, the residue was decocted again with the same amount of water for 1 h. After standing and filtrating, the solution was combined, concentrated and distillated at 60 ℃ to a designated volume (300 mL) and kept at 4 ℃. GS was crushed with a grinder and filtered with an 80-mesh sifter. The powder was retained for further use. The GS powder and GC decoctions (mother liquors) were mixed at different ratios and heated before intragastric administration to the different groups. Cell culture and animal model The experimental protocol was approved by Medical Experimental Animal Care Committee of Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences. The murine H22 HCC ascitic cell line was purchased from the Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences (Shanghai, China). H22 cells were cultured in RPMI-1640 medium supplemented with 2 mM L-glutamine, 100 IU/mL penicillin, 100 μg/mL streptomycin, and 10% FCS at 37 ℃ under a humidified atmosphere of 5% CO2, and the cultures were passaged every 2 or 3 days. Male BALB/C mice (6 weeks old) weighing 18-22 g were obtained from the Laboratory Animal Center of China Academy of Chinese Medical Sciences (Beijing, China) and were acclimatized for 1 week prior to use in the experiment. All mice were bred in laminar flow cabinets under specific pathogen-free conditions. The H22 HCC ascites model was prepared according to previous studies 35-38. In brief, the needle was inserted into the left lower abdomen, and H22 cells were inoculated intraperitoneally. Each mouse was inoculated with 1×10 7 H22 cells. The procedure was not associated with mortality or morbidity. Grouping and treatment In our previous study, we investigated the synergistic and antagonistic effects of GS/GC combinations with different ratios in the treatment of HCC ascites based on uniform design (the data have not been published; please see details in Supplementary Tables S3 and S4). In this study, all H22 HCC ascites models were divided randomly into 4 groups (10 mice in each group): (1) the model control group; (2) the GS alone group (0.93 g/kg; the diuretic effect of GS on HCC ascites was strongest); (3) the GS/GC_synergy group (GS/GC ratio was 1/0.39, i.e., 0.69 g/kg to 0.27 g/kg this combination had the strongest effects in treating HCC ascites) (see Group 5 in Supplementary Tables S3 and S4); and (4) the GS/GC_antagonism group (GS/GC was 1/1.11, i.e., 0.93 g/kg to 1.03 g/kg, the strongest antagonistic effects in treating HCC ascites) (see Group 6 in Supplementary Tables S3 and S4). The H22 HCC ascites mice in the GS, GS/GC_synergy and GS/GC_antagonism groups were intragastrically administered the appropriate doses of GS and GC mentioned above. The mice in the model control group received an equal volume of normal saline. The treated mice were cared for under standard laboratory conditions and used in different experiments. On the 10th day, the surviving mice were sacrificed by cervical dislocation. The mice were weighed, and abdominal circumferences were measured. Their ascites and blood were collected for physical examinations and enzyme-linked immunosorbent assay (ELISA). Next, the liver and kidney tissues were immediately dissected and fixed in paraformaldehyde overnight for hematoxylin-eosin (H&E) staining to detect the toxic effects of GS and GC combinations. Specifically, the kidney tissues were used for western blot and quantificational real-time polymerase chain reaction (qRT-PCR) analyses. Physical examinations To measure the ascites volume, the ascites fluid was collected from the opened abdominal wall following cervical dislocation and measured via syringe. Serum biochemical analysis The serum biochemical analyses included alanine aminotransferase (ALT, related to the liver), aspartate amino transferase (AST, related to the liver), creatinine (CREA, related to the kidney), and blood urea nitrogen (BUN, related to the kidney) and were determined by routine kinetic and fixed-rate colorimetric methods using common hospital laboratory equipment. All assays were conducted in triplicate using fresh serum. ELISA The ascites and sera were diluted to different concentrations and analyzed using mouse ADRB1, PIK3CG, AVPR2 ELISA kits obtained from Beijing Xinfangcheng Biotechnology (Beijing, China) following the manufacturer's instructions. Immunohistochemical staining Expression patterns and subcellular localizations of ADRB1, PIK3CG and AVPR2 proteins in kidney tissues obtained from different groups were examined by immunohistochemical staining according to our previous studies 34. The following antibodies were used in this study: anti-ADRB1 (lot: GR174027-3, Abcam; Cambridge, MA), anti-PIK3CG (lot: GR104307-9, Abcam; Cambridge, MA) and anti-AVPR2 (lot: 9A08V2, Bioss; Beijing, China). Western blot analysis The western blot procedures are described in our previous studies 29, 39. The primary antibodies included anti-ADRB1 (lot: GR174027-3, Abcam; Cambridge, MA), anti-PIK3CG (lot: GR104307-9, Abcam; Cambridge, MA), anti-AVPR2 (lot: GR146393-1, Abcam; Cambridge, MA), anti-pPIK3CG (Phospho-Ser1101, lot: AD 071823, Biorbyt; Cambridge, MA), anti-pAKT (Phospho-S473, lot: GR-139335-14, Abcam; Cambridge, MA), and anti-β-actin (lot: I1o813, Beijing TransGen Biotech Co. Ltd., Beijing, China). All experiments were performed in triplicate. The mean normalized protein expressions ± SEs were calculated from independent experiments. QRT-PCR The qRT-PCR procedures are described in our previous studies 40-41. GAPDH was used as the internal control for the normalization and quantification of the target gene expression. The primer sequences for the ADRB1, PIK3CG and AVPR2 mRNAs are listed in Supplementary Table S5. The relative quantification of the target gene expression was evaluated using the comparative cycle threshold (CT) method as in a previous study 42. All experiments were performed in triplicate. The mean normalized gene expressions ± the SEs were calculated from independent experiments. Statistical analysis The SPSS software version 13.0 (SPSS Inc., Chicago, IL, USA) for Windows and SAS 9.1 (SAS Institute, Cary, NC) were used for the statistical analysis. The continuous variables are expressed as the means ± the S.E. For comparisons of the means of multiple groups, one-way ANOVAs followed by LSD tests were performed. Differences were considered statistically significant at P <0.05. Results & Discussion Putative targets for GS and GC Based on our previously developed target prediction system 25, the numbers of putative targets for GS and GC were 13 and 102, respectively. Eleven of the putative targets of the two herbs overlapped, which was suggestive of potential interactions between GS and GC. Detailed information about putative targets is provided in Supplementary Table S6. Combination principles of the herbal pair GS and GC in its action on HCC ascites To shed light on the combination principles of the actions of herbal pair GS and GC on the HCC ascites, we first constructed a drug target network using the PPI information of the putative targets of GS and GC, known therapeutic targets for ascites and other human proteins. The network consisted of 3955 nodes and 7735 edges. For detailed information about this network, see Supplementary Table S7. The hubs in a drug target network have extremely high levels of degree and tend to encode essential genes. According to the previous studies of Li et al 26 and our research group 38-40, we identified a node as a hub if its degree was more than twice the median degree of all nodes in the network. Consequently, 936 nodes were identified as hubs. Next, we constructed the interaction network of these hubs, which consisted of 934 nodes and 3976 edges as shown in Figure 2A. Please see the detailed information about this network provided in Supplementary Table S8. Based on the hub interaction network, four topological features, i.e., 'Degree,' 'Node betweenness', 'Closeness' and 'K value' were calculated to screen the major putative targets of GS and GC. In total, we identified 3 and 39 major putative targets for GS and GC, respectively (Supplementary Table S9). Modularity has been reported to be another important aspect of a PPI network. Nodes that are highly interconnected within the network are usually involved in the same biological modules or pathways. Using a Markov clustering algorithm, we divided the interaction network of major putative targets into 3 functional modules containing 36, 8 and 8 nodes (Figure 2B). According to an enrichment analysis based on the GO annotation system and the KEGG pathway, the biggest functional module was significantly associated with pathways in cancer. The other modules were involved in cyclins and cell cycle regulation and second-messenger-mediated signaling. Because an interaction with a high 'edge-betweenness' has been defined as a bottleneck with many 'shortest paths' going through it that controls the rate of information flow, we further calculated the 'edge-betweenness' of each interaction in the network of major putative targets. As shown in Supplementary Table S10, the interaction between ADRB1 and PIK3CG exhibited the greatest edge-betweenness value (52.08), which suggested its importance in connecting the different modules in the network. As illustrated in Figure 2B, ADRB1 and PIK3CG were major putative targets of GS and GC, respectively, and they both had functional interactions with AVPR2 according to the information of existing interaction databases (ADRB1-AVPR2: reaction from String/HAPPI; ADRB1-PIK3CG: direct interaction from HPRD; PIK3CG-AVPR2: reaction from I2D). AVPR2 has been recognized as a known therapeutic target for ascites; therefore, we propose that the effects of the GS/GC combination on ascites might be associated with the ADRB1-PIK3CG-AVPR2 signal axis. Prior to the experimental validation, a molecular docking simulation was performed using the eHiTS software to investigate whether the main chemical components of GS and GC could bind directly to ADRB1 and PIK3CG, respectively. This structure-based method has been reported to play important roles in the discovery of ligand-protein interactions and the elucidation of binding mechanisms. Consequently, seven compound-target pairs were deleted either because their structural information was unavailable or negative results were output from eHiTS. The positive docking results obtained from the eHiTS software are summarized in Supplementary Table S11. Among the 12 chemical components of GS, 8 (66.67%) exhibited middle-strong binding efficiencies with ADRB1. Regarding to 30 chemical components of GC, 20 (66.67%) exhibited middle-strong binding efficiencies with PIK3CG. These findings imply that two-thirds of the chemical components of GS and GC might have strong potential to directly bind with ADRB1 and PIK3CG, respectively; this supposition should be validated experimentally in further studies because molecular docking remains a computer simulation procedure that is used to predict the conformation of a receptor-ligand complex. Experimental Validation The synergistic and antagonistic effects of GS and GC acting on HCC ascites under different combination designs Compared to the model control group, the ascites volumes (Figure 3A), body weights (Figure 3B) and abdominal circumferences (Figure 3C) in the GS, GS/GC_synergy and GS/GC_antagonism groups were all reduced. Notably, the differences in these three physical features between the GS/GC_synergy and model control groups were greater than those between the GS and model control groups (Figure 3). However, there were no significant differences between the GS/GC_antagonism and model control groups in terms of ascites volume, body weight or abdominal circumference (all P>0.05, Figure 3). Because GS is strong toxic to the liver and kidney (which has seriously restricted its clinical application), and GC is extensively used to enhance the effectiveness and reduce the toxicity of other drugs in TCM, we sought to observe the changes in toxicity of the GS/GC combinations of different ratios on the HCC ascites mice. Figure 4A illustrates the serum levels of two enzymes related to liver metabolism. No increases in AST or ALT were observed in response to any of the combinations of GS/GC compared to the ascites model group, although these enzymes did decrease slightly in the GS/GC_synergy group (Figure 4A). Additionally, evaluation of the liver sections stained with H&E revealed that GS and GS/GC combination treatment did not aggravate the damage of ascites to the liver tissues, which was shown in the model control group, such as inflammation or necrosis (Figure 4B). Moreover, the kidney toxicities of the different drug treatments were evaluated in H22 HCC ascites mice with serum biochemical and histological analyses. We found that the serum levels of BUN and CREA were not altered significantly in any of the treatment groups compared to the model control group (Figure 5A). Furthermore, no specific pathological symptoms were observed in any of the groups. Nearly all the kidney cells maintained normal structures, the degeneration and necrosis of the glomerulus and renal tubes induced by toxicants and immunological factors were not observed in any region of the kidney, no edema or swelling were observed in the renal tubes of the kidney tissue (Figure 5B). These results suggest that the GS/GC combinations at different ratios did not aggravate the pathological changes in either the livers or kidneys of the H22 HCC ascites mice. The GS/ GC combination attenuates HCC ascites partially by regulating the ADRB1-PIK3CG-AVPR2 signal axis We further detected the regulatory effects of the GS/GC combinations of different ratios on the expressions of ADRB1, PIK3CG and AVPR2 at both the protein and mRNA levels in the ascites, sera and kidney tissues of the different groups by ELISA, immunohistochemistry, western blot and qRT-PCR analyses. ADRB1 (official full name: adrenoceptor beta 1, ADRβ1) is an adrenergic receptor that belongs to the prototypic family of guanine nucleotide-binding regulatory protein-coupled receptors that mediate the physiological effects of the hormone epinephrine and the neurotransmitter norepinephrine 41. Previous animal studies have revealed direct relationships of the lymph flow rate and liquid clearance with adrenergic receptors activity in the lymphatic system 42. It also has been reported that beta-adrenergic receptors may play a role in ascites susceptibility 43. In this study, ADRB1 was predicted to be a major putative target of GS. As shown in Figure 6A, the positive staining of ADRB1 protein was localized in cellular cytoplasm of kidney tissues. Statistically, immunohistochemistry, western blot and qRT-PCR all showed that the ADRB1 protein and mRNA levels in the ascites, sera and kidney tissues of the GS group were significantly decreased compared to the model control group (all P<0.01, Figure 6B and Figure 7A), which implies a regulatory effect of GS on ADRB1. Notably, the ADRB1 protein and mRNA levels in the GS/GC_antagonism group were higher than those in the GS group (all P<0.01, Figure 6B and Figure 7A), suggesting that GC reverse the downregulation of ADRB1 caused by GS treatment when the ratio of GS and GC was 1:1.11. Moreover, we found that the GS/GC combination ratio of 1:0.39 (GS/GC_synergy group) also dramatically reduced the expression of ADRB1 in the ascites, sera and kidney tissues (all P<0.01, Figure 6B and Figure 7A), but no significant differences compared to the GS/GC_antagonism group, were noted, which suggests that GC might not have affected the regulation of ADRB1 expression by of GS in this combination design. Phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit gamma (PIK3CG) is an enzyme that phosphorylates phosphoinositides on the 3-hydroxyl group of the inositol ring. PIK3CG is an important modulator of extracellular signals, including those elicited by E-cadherin-mediated cell-cell adhesion, which plays an important role in the maintenance of the structural and functional integrity of the epithelia. Functionally, PIK3CG is responsible for coordinating a diverse range of cellular processes that includes proliferation, cell survival, degranulation, vesicular trafficking and cell migration 44. As a main component of PI3K/AKT pathway that has been reported to be involved in carcinogenesis and the formation of malignant ascites 45, 46, PIK3CG may also contribute to these pathogeneses. Here, we identified PIK3CG as a major putative target of GC. The immunohistochemistry showed the cytoplasmic staining of PIK3CG protein in kidney tissues (Figure 6C). Our data also indicated that the expression levels of PIK3CG protein and mRNA in the ascites, sera and kidney tissues of the GS/GC_synergy group were significantly lower than those in the model control and GS groups, which suggests a regulatory effect of GC on PIK3CG. However, the levels of PIK3CG in the ascites, sera and kidney tissues of the GS/GC_antagonism group exhibited no significant changes compared to the model control or GS groups possibly because the dosage of GC in this group was too low to downregulate its putative target (Figure 6D and Figure 7B). More interestingly, similar results regarding the changes in phosphorylated PIK3CG (p-PIK3CG) and p-AKT in the different groups are presented in Supplementary Figure S2 and imply a role of GC in the activation of the PI3K/AKT pathway. Arginine vasopressin receptor 2 (AVPR2) belongs to the seven-transmembrane domain G protein-coupled receptor (GPCR) superfamily and couples to Gs to stimulate adenylate cyclase. AVPR2 expressed in the kidney tubule, predominantly in the distal convoluted tubule and collecting ducts, where its primary function is to respond to the pituitary hormone arginine vasopressin (AVP) by stimulating mechanisms that concentrate the urine and maintain water homeostasis in the organism 47. Notably, AVPR2 has been recognized as a therapeutic target for the treatment of malignant ascites. Increasing evidence indicates that several AVPR2 antagonists, such as satavaptan, tolvaptan and lixivaptan, can improve the control of ascites in cirrhosis 48-50. More interestingly, our network analysis identified interactions between ADRB1 and AVPR2 and between PIK3CG and AVPR2, which suggest a role of the ADRB1-PIK3CG-AVPR2 signal axis in attenuating ascites. Similar to ADRB1 and PIK3CG protein, the positive staining of AVPR2 protein was localized in cellular cytoplasm of kidney tissues. As shown in Figure 6F and Figure 7C, the expression levels of AVPR2 in the ascites, sera, and kidney tissues of the GS and GS/GC_synergy groups were significantly lower than those of the model control group (all P<0.05), which implies a diuretic function of the two-drug combination. However, the levels of AVPR2 in the ascites, sera, and kidney tissues of the GS/GC_antagonism group were not significantly different from those of the model control group, which suggests that the GS/GC combination at the ratio 1:0.39 might exhibit antagonistic effects on malignant ascites. Conclusion This systems-level investigation that integrated drug target prediction, network analysis and experimental validation provided the convincing evidence that GS and GC under different combination designs might exert synergistic or antagonistic effects on HCC ascites, which might be partially related to their regulations of the ADRB1-PIK3CG-AVPR2 signal axis. These findings also indicated that the herbal pair of the 'eighteen antagonistic medicaments' could exhibit synergistic effects with rational drug combination design. Therefore, additional studies should explore promising drug combinations of other herbal pairs. Supplementary Material Additional File 1 Supplementary information, Figures S1-S2. Click here for additional data file. Additional File 2 Table S1-Table S5. Click here for additional data file. Additional File 3 Table S6-Table S7. Click here for additional data file. Additional File 4 Table S8-Table S9. Click here for additional data file. Additional File 5 Table S10-Table S11. Click here for additional data file. This study was supported by the National Basic Research Program of China (973 Program) (2011CB505300, 2011CB505305), the National Natural Science Foundation of China (81303153), Beijing Nova program (Z1511000003150126), Beijing Joint Project Specific Funds and the Fundamental Research Funds for the Central public welfare research institutes (ZZ2014008) and the Prospective Study Platform Project of Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences (QZPT002). All authors have read the journal's authorship agreement and policy on disclosure of potential conflicts of interest. Figure 1 A schematic diagram of the systems biology-based strategies for revealing the combination principles of the herbal pair of Euphorbia kansui (GS) and Glycyrrhiza (GC) used in the therapy of hepatocellular carcinoma ascites based on target network analysis and experimental validation. Figure 2 Putative targets of the Euphorbia kansui (GS) and Glycyrrhiza (GC) interaction networks. (A) Interaction network of hub nodes in the interaction network of the putative targets of GS and GC. (B) Three functional modules of the interaction network of the major putative targets of GS and GC. The yellow and blue nodes denote the putative targets of GS and GC, respectively. The red node denotes the known therapeutic target for the treatment of ascites. The purple nodes denote other human proteins that directly interact with the putative targets of GS and GC. The blue edges denote the ADRB1-PIK3CG-AVPR2 signal axis. Figure 3 Changes in ascites volumes. (A) body weights (B) and abdominal circumferences (C) in the H22 HCC ascites model group (n=9), Euphorbia kansui (GS) alone treatment group (n=8), GS/Glycyrrhiza (GC) combination_synergistic effect group (GS/GC_synergy, n=8) and GS/GC combination_antagonistic effect group (GS/GC_antagonism, n=9). The data are represented as the means ± the S.E. '' P<0.05 compared with the model group, '*' P<0.01 compared with the model group. Figure 4 Liver toxicity in the H22 HCC ascites mice. (A) No increases in AST or ALT levels were observed in response to any of combinations of GS/GC, although these levels did decrease slightly in the GS/GC_synergy group. (B) Evaluation of the liver sections stained with Hematoxylin-eosin (H&E) revealed that GS and GS/GC combination treatment did not aggravate the damage of ascites to the liver tissues, which was shown in the model control group, such as inflammation or necrosis. H&E staining, 200×magnification. Figure 5 Kidney toxicity in the H22 HCC ascites mice. (A) The serum levels of BUN and CREA were not significantly altered in any of the treatment groups. (B) No specific pathological symptoms were detected in any of the groups. Almost all the kidney cells maintained their normal structures, degeneration and necrosis of the glomeruli and renal tubes induced by toxicants and immunological factors were not observed in any region of the kidney, and no edema or swelling were observed in the renal tubes of the kidney. Hematoxylin-eosin (H&E) staining, 200×magnification. Figure 6 Expression patterns and subcellular localizations of ADRβ1. (A), PI3Kγ (B) and AVPR2 (C) protein in kidney tissues of the different groups as detected by immunohistochemistry. The data are represented as the means ± the S.E. '' and '*' P<0.05 and P<0.01, respectively, compared with the model group; '#' and '##' P<0.05 and P<0.01, respectively, compared with the GS group. Figure 7 Expressions of ADRβ1. (A) PI3Kγ (B) and AVPR2 (C) at the protein and mRNA levels in the ascites, sera and kidney tissues of the different groups as detected by ELISA, western blot and qRT-PCR analyses. The data are represented as the means ± the S.E. '' and '**' P<0.05 and P<0.01, respectively, compared with the model group; '#' and '##' P<0.05 and P<0.01, respectively, compared with the GS group. 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==== Front Med Sci MonitMed. Sci. MonitMedical Science MonitorMedical Science Monitor : International Medical Journal of Experimental and Clinical Research1234-10101643-3750International Scientific Literature, Inc. 2723654310.12659/MSM.896428896428Lab/In Vitro ResearchMicroRNA-208b Alleviates Post-Infarction Myocardial Fibrosis in a Rat Model by Inhibiting GATA4 Zhou Chaoyuan BCEFCui Qintao BCFSu Guobao BDFGuo Xiaoliang BFLiu Xiaochen DFZhang Jie ADepartment of Cardiovascular Surgery, The First Affiliated Hospital of Xinxiang Medical University, Weihui, Henan, P.R. ChinaCorresponding Author: Jie Zhang, e-mail: [email protected] Study Design B Data Collection C Statistical Analysis D Data Interpretation E Manuscript Preparation F Literature Search G Funds Collection 2016 29 5 2016 22 1808 1816 26 10 2015 17 11 2015 © Med Sci Monit, 20162016This work is licensed under Creative Common Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0)Background Myocardial infarction affects the health of many people. Post-infarction myocardial fibrosis has attracted much attention, but details of the mechanism remain elusive. In this study, the role of microRNA-208b (miR-208b) in modulating post-infarction myocardial fibrosis and the related mechanism were investigated. Material/Methods A rat model of myocardial infarction induced by ligating the left anterior descending artery was used to analyze the expression and roles of miR-208b by overexpression with the lentivirus vector of pre-miR-208b. Myocardial function was assessed and the expression of fibrosis-related factors type I collagen (COL1) and ACTA2 (alias αSMA) was detected. Myocardial fibroblasts isolated from newborn rats were transfected with luciferase reporter vectors containing wild-type or mutant Gata4 3′ UTR to verify the relationship between Gata4 and miR-208b. We then transfected the specific small interference RNA of Gata4 to detect changes in COL1 and ACTA2. Results miR-208b was down-regulated in hearts of model rats (P<0.01). Overexpressing miR-208b improved myocardial functions, such as reducing the infarction area (P<0.05) and promoting LVEF and LVFS (P<0.01), and inhibited COL1 and ACTA2 (P<0.01). Luciferase reporter assay proved Gata4 to be the direct target of miR-208b, with the target sequence in the 3′UTR. Inhibiting GATA4 resulted in the down-regulation of COL1 and ACTA2, suggesting that the role of miR-208b was achieved via regulating GATA4. Conclusions This study demonstrates the protective function of miR-208b via GATA4 in post-infarction myocardial fibrosis, providing a potential therapeutic target for treating myocardial fibrosis. MeSH Keywords Collagen Type IFibrosisGATA4 Transcription FactorMicroRNAsMyocardial Infarction ==== Body Background Myocardial infarction damages heart muscle by causing blood flow stoppage, which may lead to heart failure or cardiac arrest, and causing discomforts similar to heartburn. As a manifestation of coronary artery disease, myocardial infarction impacts the health of many people, with about 90% of the cases attributable to modifiable risk factors such as smoking and hypertension [1]. Environmental pollution, including noise and air pollution, also contribute to myocardial infarction [2]. In terms of the pathogenic mechanism, the most evident feature of myocardial infarction is the accumulation of collagen, which in the heart is primarily synthesized by cardiac fibroblasts, leading to post-infarction myocardial fibrosis. Collagen is expressed at low levels in healthy hearts, but is increased at infarction sites and the peripheral zone with the progression of myocardial infarction [3]. Myocardial fibrosis can be clearly observed at the infarction sites at day 7 after myocardial infarction in rats, contributing to heart dysfunction [4]. Promising detection and evaluation techniques, such as ultrasonic backscatter, have been reported [5]. Improved therapy for cardiac diseases, especially the stem cell transplantation strategy [6], has attracted attention, and related molecular mechanisms, like the role of C3G protein, have been revealed [7]. However, there is still a need for more detailed information on myocardial infarction and fibrosis. microRNAs are short RNAs encoded by the genome, inhibiting translation or promoting mRNA degradation via binding to the 3′ untranslated region (UTR) of mRNAs [8]. A single microRNA can recognize various target mRNAs, and a single mRNA can be bound by several microRNAs simultaneously, constituting an elaborate regulatory system. Recent studies have suggested the association and use of microRNAs in myocardial infarction and fibrosis. Overexpression of microRNA-21 (miR-21) decreases myocardial infarction area [9], and miR-1 and mir-206 are up-regulated in myocardial infarction [10], with miR-1 also being a potential biomarker for acute myocardial infarction [11]. miR-29 is a repressor of myocardial fibrosis after infarction [12], as well as miR-24, which modulates myocardial fibroblast functions after infarction via the furin-TGF-β pathway [13]. It thus appears that exploitation and utilization of these powerful microRNAs are of great significance in treatment of myocardial infarction and myocardial fibrosis. miR-208 is primarily expressed in the heart and its elevated concentration in plasma is correlated with myocardial injury [14], which can be a biomarker of acute myocardial infarction [15,16]. However, its relationship with post-infarction myocardial fibrosis and the mechanism remain unclear. This study aimed to reveal the role of miR-208b, a member of the miR-208 family, in modulating post-infarction myocardial fibrosis and the possible regulatory mechanism. The expression of miR-208b was examined in rat hearts in an induced myocardial infarction rat model. miR-208b was overexpressed by the lentivirus vector of pre-miR-208b, and the myocardial function after the model construction was monitored. Fibrosis-related factors, including type 1 collagen (COL1) and actin alpha 2, smooth muscle, and aorta (ACTA2, alias αSMA), were detected to analyze the influence of miR-208b on myocardial fibrosis. Myocardial fibroblasts isolated from newborn rats were used to verify the direct regulatory relationship of miR-208b and GATA binding protein 4 (GATA4). All these experiments were performed to provide valid information regarding the regulatory role of miR-208b in post-infarction myocardial fibrosis. Material and Methods Animals The specific pathogen-free grade Sprague-Dawley (SD) rats used in this study were purchased from Vital River Laboratories (Beijing, China), and raised at 24°C and 50% humidity. Sixty adult rats (weight 250±20 g) were used for the construction of a myocardial infarction model – 15 for the sham-operated group and 45 for the operated group. Ten newborn rats (age 2 days) were prepared for the culture of myocardial fibroblasts. The animal experiments were approved by a local ethics committee and performed according to the rules of our institute. Myocardial infarction rat model The rats for model induction were anesthetized by diethyl ether supplemented with intraperitoneal injection of ketamine (75 mg/kg). Atropine (20 μg/kg) was injected to reduce the airway secretion. After being affixed to the operating table, the rats were connected to an electrocardiograph and a ventilator with respiratory rate of 80 times per min, inspiration and expiration ratio of 1: 2, and tidal volume of 18 mL/kg. The pleura were opened to expose the heart, and the left anterior descending (LAD) was ligated with Prolene and a needle (0.5 mm in diameter). Then the chest and skin were closed and sterilized with iodophor. The ventilator was removed after the rats revived. Electrocardiograph indicating an elevation of J point at 10 min after the operation indicated successful model induction. Rats in the sham-operated groups underwent all of the same procedures detailed above except for the ligation step, making a ringer instead of the ligation. To prevent infections, all rats were injected with penicillin (Thermo Scientific, Carlsbad, CA) during the 3 days after the operation. Myocardial fibroblast culture The newborn rats were anesthetized and sacrificed for heart sampling. The heart samples were cut into pieces of about 1 mm3 and digested by trypsin (Gibco, Carlsbad, CA) at 37°C for 15 min. Then cells were collected and cultured in Dulbecco’s modified Eagles medium (DMEM)/F12 (Gibco) supplemented with fetal bovine serum (Gibco) at 37°C with 5% CO2 for adherence. After 90 min, the cell suspension was removed and the adherent cells were collected and cultured. The purity of myocardial fibroblasts was examined by anti-vimentin, anti-Von Willebrand factor, and anti-desmin (ABcam, Cambridge, UK), and the fibroblasts were used for further analysis when the purity reached 95%. Lentivirus transfection Lentivirus to overexpress pre-miR-208b and blank lentivirus were designed and constructed by GenePharma (Shanghai, China). For animal transfection, the lentivirus was injected through the coronary artery of the operated group after the induction of the rat model. The rats were raised for 4 weeks for further sampling and detection. For cell transfection, the cells were pre-cultured in 24-well plates (1×105/well) for 24 h and then treated with 6 μg/mL Polybrene (Sigma-Aldrich, Shanghai, China) and lentivirus suspension, and cultured for 24 h. Then the medium was replaced by fresh medium, and BD FACSCanto (BD Biosciences, San Jose, CA) was used to detect the transfection efficiency after 48 h. Myocardial function assessment At 14 d after the operation, the rats were anesthetized, and left ventricular fractional shortening (LVFS) and left ventricular ejection fraction (LVEF) were examined by ultrasonic cardiogram (10S probe, image depth of 2–4 cm, frequency of 11.4 MHz). Then 3 rats from each group were sacrificed for sampling. The remaining heart samples were cut and fixed in 4% paraformaldehyde for 8 h, embedded in paraffin, cut into 6-μm slices, and stained with the Masson staining method. The slides were then deparaffinized, stained in iron hematoxylin for 7 min, immersed in 1% hydrochloric acid for 30 s, stained in ponceau for 5 min, immersed in phosphomolybdic acid buffer and aniline blue for 5 min each, and immersed in phosphomolybdic acid buffer for 1 min. After treatment in 1% acetic acid for 1 min and dehydration, the slides were integrated with neutral balsam and observed with an optical microscope (Leica Microsystems, Wetzlar, Germany). Three visual fields were randomly chosen for each sample, and the infarction area was calculated with Qwin software (Leica Microsystems). Luciferase reporter assay The regulatory relationship between Gata4 and miR-208 was predicted by use of the online database TargetScanHuman 7.0. The binding site in the Gata4 3′ untranslated region (UTR) was mutated by use of the QuikChange Multi Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA). Then the mutant-type or wild-type of Gata4 3′UTR was ligated into pGL3-basic vector (Promega, Madison, WI) and transfected to cultured myocardial fibroblasts (overexpressed miR-208b or not) in 12-well plates (3×104/well). phRL-TK vector (Promega) was co-transfected as the internal control. Luciferase activity was measured by GloMax (Promega). siRNA transfection The specific knockdown of Gata4 was achieved by transfecting its siRNA or siRNA control designed by RiboBio (Guangzhou, China) to the cultured myocardial fibroblasts using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. The cells were pre-cultured in 24-well plates (1×105/well) for 24 h before transfection, and then in the serum-free medium during transfection. Transfected cells were collected for further analysis at 36 h after transfection. Real-time quantitative PCR (qPCR) At 1, 2, and 4 weeks after model construction, myocardial infarction (I) zone and infarction border (B) zone of rat heart samples were collected. The siRNA-transfected cells were also collected. TRIzol (Invitrogen) was used for total RNA extraction, and RNAiso for Small RNA (TaKaRa, Dalian, China) was used for miRNA extraction. PrimeScript 1st Strand cDNA Synthesis (TaKaRa) was used in reverse transcription, and the specific reverse primer for miR-208b-3p was 5′-CTC AAC TGG TGT CGT GGA GTC GGC AAT TCA GTT GAG ACC TTT TG-3′. qPCR was conducted on a LightCycler 480 (Roche, Basel, Switzerland) with specific primers for miR-208b-3p (Fw: 5′-ACA CTC CAG CTG GGA TAA GAC GAA CA-3′ and Rv: 5′-TGG TGT CGT GGA GTC G-3′), Col1a1 (Fw: 5′-CAA GAT GGT GGC CGT TAC TAC-3′ and Rv: 5′-GCT GCG GAT GTT CTC AAT CT-3′), Acta2 (Fw: 5′-AGG GAG TGA TGG TTG GAA TG-3′ and Rv: 5′-GGT GAT GAT GCC GTG TTC TA-3′) and Gata4 (Fw: 5′-GGA GCT GGC CAG GAC TGT CG-3′ and 5′-TGC GCA GGC CTT CGG ATC AC-3′). U6 and Gapdh were used as the endogenous controls. Data were analyzed with the 2−ΔΔCt method. Western blot Tissue samples of I zone at 2 weeks after model induction and transfected cells were collected for protein extraction. The protein samples were lysed with RIPA Lysis Buffer (Beyotime, Shanghai, China) and quantified using the BCA Protein Assay kit (Beyotime). Protein samples of 20 μg were loaded in each lane and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Protein bands on the gel were then transferred to a polyvinylidene fluoride membrane, which was then blocked in 5% skim milk for 2 h at room temperature. The blot was incubated in specific primary antibodies (anti-COL1, anti-αSMA, and anti-GATA4, ABcam) overnight at 4°C and then in horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. Positive signals were developed by ECL Plus Western Blotting Substrate (Thermo Scientific) and analyzed with ImageJ software version 1.49 (National Institutes of Health, Bethesda, MD) using GAPDH as the endogenous control. Statistical analysis All the experiments were performed at least 3 times and data are shown as the mean ± standard deviation. The t test was performed using SPSS 20 (IBM, New York, USA) and differences were considered significant at P<0.05. Results miR-208b is down-regulated in hearts of myocardial infarction rats The changes in miR-208b-3p were detected at 1, 2, and 4 weeks postoperatively (Figure 1). Compared to the sham-operated group, miR-208b-3p expression in the operated group was significantly down-regulated (P<0.001 or P<0.01) in both I zone and B zone. The reduced expression was the most significant at 2 weeks postoperatively and had slightly increased when detected at 4 weeks after the operation. The reduction was more severe in I zone than in B zone, indicating that miR-208b was reduced in myocardial infarction tissues, which suggests its potential regulatory role in myocardial infarction. miR-208b overexpression mitigates myocardial infarction damages To analyze the role of miR-208b in myocardial infarction, miR-208b was overexpressed by transfecting its precursor, pre-miR-208b, and was verified at 2 weeks after the operation and transfection (Figure 2A). qPCR results indicated miR-208-3p levels were significantly increased compared to the transfection control group (P<0.001), suggesting the successful overexpression of miR-208b and the suitability of the transfected samples for further tests. The status of heart samples was measured in 3 aspects: infarction area, LVEF, and LVFS. Infarction area was assessed using the Masson staining method and results showed that the infarction area of the transfection control group was approximately 50%, but after the overexpression of miR-208b-3p the infarction area was significantly reduced to approximately 30% (P<0.05, Figure 2B). LVEF and LVFE are used to reflect the conditions of left ventricular and the prognosis of myocardial infarction. LVEF in the sham-operated groups was approximately 60%, which was significantly reduced to approximately 20% after the operation (P<0.001, Figure 2C), and increased to over 30% with the overexpression of miR-208b (P<0.01). Similarly, LVFS was significantly reduced from approximately 30% in the sham-operated group to approximately 15% percent in the operated group (P<0.01, Figure 2D), and was obviously recovered by miR-208b (P<0.01). These results indicate that overexpression of miR-208b can alleviate the effects of myocardial infarction, suggesting its vital role in modulating myocardial infarction progression. miR-208b inhibits Col1a1 and Acta2 Because miR-208b improved the heart functions and reduced infarction area after the induction of myocardial infarction, it was assumed to affect the synthesis of fibrosis-related factors, such as COL1 and ACTA2, which were detected in this study. Expression of Col1a1, a gene encoding the pro-alpha1 chains of COL1, and Acta2 in I zone, were detected by qPCR (Figure 3A), and COL1 and ACTA2 were detected by Western blot (Figure 3B). Their mRNA levels were significantly up-regulated after myocardial infarction induction (P<0.001), and both were inhibited when miR-208b was overexpressed (P<0.01). The protein expression level of these 2 factors showed consistently changing patterns, indicating that miR-208b might inhibit the expression of Col1a1 and Acta2, and further suppress the synthesis of protein COL1 and ACTA2, thus mitigating post-infarction myocardial fibrosis. miR-208b functions via targeting Gata4 The functional mechanism of miR-208b in myocardial fibroblasts was further analyzed from the molecular level. Gata4 was predicted to be a target of miR-208b-3p by use of the online database TargetScan, which was verified in cultured myocardial fibroblasts by dual-luciferase reporter assay with the wild-type (wt) and the mutant-type (mut) of Gata4 3′UTR (Figure 4A). miR-208-3p was supposed to bind to position 1363 to 1369 of wt-Gata4 3′UTR, but not mut-Gata4 3′UTR. As predicted, the luciferase activity of wt-Gata4 3′UTR vector could be inhibited by miR-208b overexpression (P<0.01, Figure 4B). However, when the target sequence was mutated (mut-Gata4 3′UTR), overexpression of miR-208b had little effect on the luciferase activity (no significant difference was found). These results show that Gata4 is a direct target of miR-208b, with the sequence GUCUUA in its 3′UTR being the interaction sites. Overexpression of miR-208b led to the down-regulation of GATA4 protein expression (P<0.01, Figure 4C), suggesting that miR-208 is capable of directly binding to Gata4 to suppress its expression. Based on these demonstrated roles of miR-208b in inhibiting factors related to myocardial fibrosis, the association between GATA4 and these factors was analyzed to verify whether miR-208b acted via GATA4, before which Gata4 was inhibited by the small-interference RNA (si-GATA4), and the effects were examined (Figure 4D). Data showed that si-GATA4 could effectively down-regulate Gata4 mRNA levels compared to the si-control group (P<0.001). Then the protein levels of COL1 and ACTA2 were detected within these cell samples (Figure 4E), both of them being inhibited when GATA4 was suppressed. Taken together, these findings show that GATA4 was directly inhibited by miR-208b, and was able to suppress the expression of the fibrosis-related factors COL1 and ACTA2, which might be the regulatory mechanism of miR-208b in post-infarction myocardial fibrosis. Discussion In this study, miR-208b was found to be down-regulated in hearts of the myocardial infarction rat model and its roles in modulating myocardial fibrosis post infarction were revealed; besides the alleviating effects in myocardial infarction, miR-208b can also inhibit the expression of fibrosis-related factors, including COL1 and ACTA2. Further mechanism analysis revealed that miR-208b directly targets Gata4, through which it suppresses the progression of post-infarction myocardial fibrosis. Two fibrosis-related factors, COL1 and ACTA2, were used as the indicators of myocardial fibrosis in this study. COL1 consists of a triple helix composed of two alpha 1 chains (encoded by Col1a1) and one alpha 2 chain (encoded by Col1a2). It is generally used as a marker of fibrosis in various diseases, including myocardial fibrosis [17,18]. Low levels of COL1 are found in normal rat hearts, but after induction of myocardial infarction, COL1 is increased [19]. In the present study, Col1a1 was detected from the mRNA level and COL1 from the protein level using an antibody recognizing the helical COL1, and results showed that both the gene Col1a1 and protein COL1 were up-regulated in I zone after induction of myocardial infarction, indicating aggravated myocardial fibrosis. Overexpression of miR-208b inhibited Col1a1 transcription and COL1 synthesis. Similarly, ACTA2, which is highly correlated with fibrosis, as proven in different diseases [20], was also promoted at both mRNA and protein levels after myocardial infarction, and was repressed by miR-208b overexpression. Taken together, these results show that the fibrosis-related factors COL1 and ACTA2 were both up-regulated by the induction of myocardial infarction, suggesting anabatic myocardial fibrosis, which was nevertheless mitigated by miR-208b overexpression. The regulation of COL1 and ACTA2 by miR-208b was shown to be achieved via GATA4. GATA4 is an important transcription regulator, playing extensive roles in regulating genes involved in heart developmental processes and diseases [21,22], and serving as an indicator of myocyte hypertrophy and fibrosis [23,24]. Human GATA4 binds to the DNase I hypersensitive sites of COL1A2 gene and down-regulates COL1A2 expression [25]. Consistently, results of this study showed GATA4 was inhibited by miR-208b overexpression, which was also correlated with down-regulated COL1 and ACTA2 in si-GATA4 myocardial fibroblasts. Together with the protective roles of miR-208b overexpression in myocardial fibrosis, these results suggest the promoting effects of GATA4 in post-infarction myocardial fibrosis. However, GATA4 may play distinct roles as a fibrosis suppressor in other cell types or tissues, such as the hepatic mesenchymal cells of fetal mice, in which Gata4 knockout leads to advanced liver fibrosis [26]. A possible explanation for the distinct functions of GATA4 in different cells might be the disparity in regulatory pathways involved. Influences of GATA4 on myocardial fibrosis in the present study were exerted via COL1 and ACTA2, but its roles in other cells might be achieved via some other factors. miR-208b was detected to be down-regulated in I zone and B zone of the heart after myocardial infarction in this study, while its up-regulation in plasm after myocardial infarction has been demonstrated in previous studies. For example, the increasing level of miR-208b can serve as a biomarker of myocardial injury [14] and left ventricular remodeling after acute myocardial infarction [16,27]. The decreased level in myocardial fibroblasts and the increased level in plasm might be caused by the stimulated exportation and release of miR-208b from cells. The existence of microRNAs in plasm has been found in various animal species besides humans, and is derived from circulating blood cells and other cells affected by diseases [28,29]. Cells selectively release some premature and mature microRNAs to the blood [30] via exosomes, microvesicles, or protein complexes [31]. From this point of view, the down-regulated miR-208b level found in this study did not contradict its up-regulated level in the plasm; therefore, it could be conjectured that the release of miR-208b to the plasm might be promoted during myocardial infarction. However, it was uncertain whether the altered intracellular miR-208b level during myocardial infarction resulted from its transcription changes or the cellular release activities, and this needs to be investigated by further comprehensive studies. The protective roles of miR-208b are undeniable. In addition to its functions in cardiovascular diseases, as shown in previous studies [32], it was further demonstrated by the present study to have anti-fibrosis functions during myocardial infarction that reduce damage to the heart. Now that numerous microRNAs have been identified to be the possible therapeutic targets of diseases [33,34], the potential use of miR-208b is an alternative in alleviating post-infarction myocardial fibrosis, provided that a comprehensive understanding of the regulatory network is achieved. Conclusions In summary, miR-208b was demonstrated to alleviate heart damage in a myocardial infarction rat model and to inhibit post-infarction myocardial fibrosis, which is possibly achieved via regulating an important transcription factor, GATA4. These findings reveal the potential use of miR-208b as a therapeutic target for treating post-infarction myocardial fibrosis. Source of support: Departmental sources Conflicts of interest There are no conflicts of interest. Figure 1 miR-208b-3p is down-regulated in myocardial infarction tissues of the rat model. Detection of miR-208b-3p expression by qPCR was performed in heart samples of the sham-operated group and I zone and B zone of the operated group at 1, 2, and 4 weeks after the operation. Significant expression reduction was observed in I zone and B zone compared to the sham-operated group. P<0.01. * P<0.001. U6 was used as the endogenous control. Sham, the sham-operated group. I zone, myocardial infarction zone. B zone, infarction border zone. Figure 2 Overexpression of miR-208b in rat hearts mitigates myocardial infarction damage. Detection was conducted at 2 weeks after the operation. miR-208b was overexpressed by transfecting the lentiviral vector overexpressing pre-miR-208b, and blank lentiviral vector was used in the control group. * P<0.05; P<0.01; * P<0.001. Sham, the sham-operated group. Lv-control, the transfection control group with blank lentivirus. Lv-pre-miR-208b, the transfection group with pre-miR-208b lentivirus. (A) Increase in miR-208b-3p levels indicates the successful overexpression of miR-208b. (B) Percentage of infarction area was reduced by miR-208b overexpression. (C) Myocardial infarction operation reduced LVEF, which was alleviated by overexpression of miR-208b. LVEF, left ventricular ejection fraction. (D) Myocardial infarction operation reduced LVFS, which was alleviated by overexpression of miR-208b. LVFS, left ventricular fractional shortening. Figure 3 Overexpression of miR-208b inhibits the synthesis of myocardial fibrosis-related factors COL1 and ACTA2. Sham, the sham-operated group. Lv-control, the transfection control group with blank lentivirus. Lv-pre-miR-208b, the transfection group with pre-miR-208b lentivirus. Col1a1, gene encoding the pro-alpha1 chains of COL1. COL1, type 1 collagen. ACTA2, actin, alpha 2, smooth muscle, aorta, alias αSMA. (A) qPCR showing that Col1a1 and Acta2 are up-regulated in myocardial infarction and inhibited by miR-208b. P<0.01; * P<0.001. (B) Western blot showing that expression of COL1 and ACTA2 were up-regulated in myocardial infarction and inhibited by miR-208b. GAPDH was the endogenous control. Figure 4 Gata4 was a direct target of miR-208b and inhibited COL1 and ACTA2. Lv-control, the transfection control group with blank lentivirus. Lv-pre-miR-208b, the transfection group with pre-miR-208b lentivirus. si-GATA4, small interference RNA to inhibit Gata4. si-control, the control group of GATA4 inhibition. UTR, untranslated region. wt-Gata4 3′UTR, wild-type Gata4 3′UTR. mut-Gata4 3′UTR, mutant Gata4 3′UTR. (A) miR-208b-3p is predicted to bind to the GUCUUA sequence in wt-Gata4 3′UTR, and mutations in this sequence are supposed to cause failure in miR-208b and Gata4 interaction. The 4 mutated sites are underlined. (B) Luciferase activity detection indicated only wild-type-Gata4 3′UTR can be bound by miR-208b. No significant difference was found between the 2 groups using mut-Gata4 3′ UTR. GAPDH was the endogenous control. (C) si-GATA4 led to the down-regulation of Gata4 mRNA levels. (D) GATA4 and these factors was analyzed to verify whether miR-208b acted via GATA4, before which Gata4 was inhibited by the small-interference RNA (si-GATA4), and the effects were examined. Data showed that si-GATA4 could effectively down-regulate Gata4 mRNA levels compared to the si-control group (P<0.001). (E) Western blot showing COL1 and ACTA2 were inhibited when GATA4 was suppressed. GAPDH was the endogenous control. COL1, type 1 collagen. 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==== Front Med Sci MonitMed. Sci. MonitMedical Science MonitorMedical Science Monitor : International Medical Journal of Experimental and Clinical Research1234-10101643-3750International Scientific Literature, Inc. 2735649110.12659/MSM.898702898702Lab/In Vitro ResearchTargeting Transforming Growth Factor-Beta1 (TGF-β1) Inhibits Tumorigenesis of Anaplastic Thyroid Carcinoma Cells Through ERK1/2-NF-κB-PUMA Signaling Yin Qiang 1DELiu Shan 1AFDong Anbing 2AFMi Xiufang 3BHao Fengyun 4ACFZhang Kejun 2ACD1 Department of Oncology, People’s Hospital of Rizhao, Rizhao, Shandong, P.R. China2 Department of Thyroid Surgery, The Affiliated Hospital of Qingdao University, Qingdao, Shandong, P.R. China3 Department of Internal Medicine, People’s Hospital of Zhangqiu, Jinan, Shandong, P.R. China4 Department of Pathology, The Affiliated Hospital of Qingdao University, Qingdao, Shandong, P.R. ChinaCorresponding Author: Kejun Zhang, e-mail: [email protected] Study Design B Data Collection C Statistical Analysis D Data Interpretation E Manuscript Preparation F Literature Search G Funds Collection 2016 30 6 2016 22 2267 2277 25 3 2016 12 6 2016 © Med Sci Monit, 20162016This work is licensed under Creative Common Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0)Background The transforming growth factor-beta (TGF-β) signaling pathway plays a critical role in promoting tumor growth. TGF-β1was found to be overexpressed in anaplastic thyroid cancer (ATC). We therefore tested our hypothesis that targeting TGF-β1 inhibits tumorigenesis of ATC cells. Material/Methods Effects of TGF-β1 stimulation or TGF-β1 inhibition by small interfering RNA (TGF-β1siRNA) on proliferation, colony formation, and apoptosis in 8505C cells in vitro was detected using siRNAs and inhibitors to examine the TGF-β1 signaling pathway. A subcutaneously implanted tumor model of 8505C cells in nude mice was used to assess the effects of TGF-β1 inhibition on tumorigenesis development. Results TGF-β1siRNAs decreased proliferation and colony formation, and increased apoptosis in 8505C cells in vitro and inhibited tumor growth in vivo. TGF-β1siRNA inhibited phosphorylation ERK1/2 (pERK1/2) and increased p65-dependant PUMA mRNA and protein expression. Knockdown of p65 or PUMA by siRNA reduced TGF-β1siRNA-induced apoptosis, as well as caspase-3 and PARP activation. Upregulation of p65 or PUMA expression by TGF-β1siRNA requires pERK1/2 inhibition. TGF-β1 shRNA inhibited tumor growth in vivo. Conclusions Therapies targeting the TGF-β1 pathway may be more effective to prevent primary tumor formation. The ability of this therapy to decrease tumorigenesis may be related to ERK1/2/NF-κB/PUMA signaling. MeSH Keywords Apoptosis Inducing FactorGenes, NeoplasmMAP Kinase Signaling SystemNF-kappa BTransforming Growth Factor beta1 ==== Body Background Anaplastic thyroid cancer (ATC) is responsible for the majority of deaths from all thyroid malignancies and has a median survival of 6 months. The resistance of ATC to conventional thyroid cancer therapies, including surgery, radioiodine, and thyroid-stimulating hormone [TSH] suppression, contributes to the very poor prognosis of this malignancy [1,2]. Currently, there is no standard or effective therapy for ATC, and patient survival has not improved in over 6 decades [3]. Therefore, investigation of novel antiproliferative and gene therapies has been an ongoing interest. TGF-β is part of a large family of structurally related cytokines that include bone morphogenetic proteins, growth and differentiation factors, activins, and inhibins. There are 3 isoforms of TGF-β ligand (TGF-β1–3), and as ubiquitous cytokines, they play an important role in numerous cellular processes, including proliferation, adhesion, motility, apoptosis, differentiation, and immune regulation [4]. Many tumor suppressors functions by TGF-β1 signals [5–7], and TGF-β1 functions by various mechanisms, such as ERK1/2 [8,9], NF-κB [10,11], PUMA [12], and p21WAF1[13]. TGF-β1 is highly expressed in various cancers such as prostate cancer [14], ovarian carcinoma [15], hepatocellular carcinoma [16,17], bladder carcinoma [18], breast cancer [19], and cholangiocellular carcinoma [20], and aberrant TGF-β1 expression is associated with more aggressive tumors and poor prognosis. In thyroid cancer, high expression of TGFβ1 was found to be closely related with the occurrence of thyroid cancers [21]. In this study, we used TGFβ1 gene-silenced ATC cells to determine if TGFβ1 silencing can inhibit the proliferation and induce apoptosis of ATC cells in vitro and in vivo. The results of these studies indicate that small interfering RNA (siRNA) -mediated silencing of TGFβ1 in the ATC cells decreased the proliferation and induces apoptosis in vitro, and decreased the growth in an orthotopic model. TGF-β/ERK1/2/NF-κB/PUMA are involved in the mechanisms by which TGFβ1 regulates the growth and apoptosis of ATC cells, providing a novel therapeutic target for the pathogenesis and gene therapy of ATC. Material and Methods Cell line and culture The human anaplastic thyroid cancer cell line 8505C cells were purchased from DSMZ (Beijing, China) and were cultured according to the supplier’s directions. Briefly, the cells were grown in RPMI1640 medium supplemented with 10% fetal bovine serum (FBS), penicillin, sodium pyruvate, and non-essential amino acids. Adherent monolayer cultures were maintained on plastic and incubated at 37°C in 5% carbon dioxide and 95% air. The cultures were free of Mycoplasma species. In all of the assays, a monolayer of cells that was 50–70% confluent was used. All the methods used were according to the manufacturer’s instructions. Agents The following primary antibodies were used from Santa Cruz Biotechnology: pERK (T202/Y204), ERK1/2, TGF-β1–3, TGF-β1 siRNA, SMAD2 siRNA, siRNA, control siRNA, and β-actin. The following primary antibodies were used from Cell Signaling: NF-κBp65 (p65), PUMA, bcl-2, Mcl-1, Bcl-XL, PARP, Bax, Bak, Bid, Bim, and Caspase-3. NF-κBp65 siRNAs and hrTGF-β1 were from Applied Biosystems. MEK/ERK inhibitor U0126 was purchased from Cell Signaling Technology. Anti-Ki67 Ab was from Thermo Scientific. Inhibitors were tested for monotherapy and combination therapy: U0126:10 mM. Cells incubated with culture medium or culture medium with DMSO served as controls. Plasmids Short-chain oligonucleotide was designed according to the TGF-β1 mRNA sequence provided by Genebank. The 2 oligonucleotides were selected as: forward, 5′-GATCCCCTGCCGCTGCTGCTACCttcaagagaGGTAGCA GCGGCAGCATTTTTGGAAA-3′; reverse, 3′-AGCTTTTCCAAAAATGCTGCTGCCGCTGCTGCTACCtctctt gaaGGTAGCAGCGGCAGCAGGG-5′. It was chemosynthesized by Shgong.com. It was ligated and then we inserted the 2 oligonucleotides above into the pcDNA3.1 plasmid (which encodes GFP as a reporter protein). The recombinant TGF-β1 shRNA expression vector was evaluated by using enzyme cutting. The negative control plasmid had a sequence inserted at the same place using the following 2 oligonucleotides: 5′-GCTACGCCTTCATAAGGCACGTGCTTCAAACGGGCATGCGCC ATAT GCAGTCTTTTTTGTCGACA-3′; reverse, 3′-GGCTAAGATTTCCGCGGACGAAGCCTTG CCGTACCCC GAGCACTTCACGAAAAACAGCTGCGAGA-5′. The recombinant TGF-β1-shRNA plasmid was confirmed by digestion and gene sequencing. Plasmid pcDNA3.1 was as the control plasmid. Transient/stable siRNA transfection Cells were seeded in 6-well plates, grown to 50–80% confluence. The cell density is not too high and that the cells are in optimal physiological condition on the day of transfection. 8505C cells were transfected with TGF-β1siRNA (2 μg) in OptiMEM (Gibco, BRL) for 24–72 h using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. To determine the signaling pathways involved in the production of NF-κBp65 (p65) and PUMA, 8505C cells were transfected with p65 siRNA or PUMA siRNA or their control siRNA for 24 h, then transfected with TGF-β1siRNA or its control siRNA for 48 h using Lipofectamine 2000 according to the manufacturer’s instructions above. For stable TGF-β1siRNA transfection, 24 h after TGF-β1siRNA or control siRNA transfection, the cells were split into 96-well plates and subjected to the G418 (1 mg/ml) selection for 2 weeks. The transcriptional silencing TGF-β1 protein and mRNA were screened using reverse transcription-PCR (RT-PCR) and Western blot, as described below. Different agents used in the transfection assay had no effect on target protein expression. All transfection experiments were done at least 3 times. Western blot assay For total protein extraction, cells were washed once with phosphate-buffered saline (PBS) and lysed with RIPA buffer (150 mM NaCl, 50 mM Tris-HCl, 1% Nonidet P-40, supplemented with complete protease inhibitor tablets (Roche Diagnostics) for 30 min on ice. For in vivo study, the tumor tissues were homogenized for tissue lysate extraction. Both cell lysate and tissue lysate were centrifuged and the supernatants were collected. Nuclear and cytoplasmic extracts were prepared using the Nuclear Extract Kit (Active Motif), according to manufacturer’s instructions. Protein concentration was quantified with Coomassie Plus (Bradford) Protein Assay Reagent according to the manufacturer’s instructions. Extracts (40 μg) were resolved on 10% SDS-PAGE and transferred to Hybond-C Extra nitrocellulose membranes (GE Healthcare; Germany). Membranes were probed with primary antibodies against TGF-β1, TGF-β2, TGF-β3, ERK1/2, Perk1/2, p65, PUMA, caspase-3, cleaved caspase-3, PARP, cleaved PARP, Noxa, Bak, Bid, Bim, bcl-2, Mcl-1, and Bcl-XL followed by incubation for 1 h at room temperature with HRP-conjugated anti-rabbit IgG or anti-goat IgG, respectively. Immunoblotting for β-actin served as protein loading control. All experiments were performed at least 3 independent times. Reverse transcriptase polymerase chain reaction (RT-PCR) RNA from transfection or inhibitors treated cells was extracted using Purification Kit (Roboklon, Germany). We used 2 μg of total RNA for cDNA synthesis with the Reverse Transcriptase Kit (Promega, Shanghai, China). The resulting cDNA was used for RT-PCR analysis with the G-Taq Kit according to the manufacturer’s instructions. RT-PCR was performed using appropriate gene-specific forward and reverse primers, which were selected using the Blast Primer tool. The results were analyzed using the ImageJ software. The relative change in the ratio of the target protein to the DMSO control was determined. Cell viability assay 8505C cells transfected with TGFβ1 siRNA or control siRNA were seeded in 96-well plates at a density of 5000 cells/well in 100 mL of medium and incubated for 24 h. To determine the effect of p65 and PUMA, 8505C cells were transfected with p65 siRNA or PUMA siRNA or control siRNA for 24 h before the TGFβ1 siRNA or control siRNA transfection, then the cells were also seeded in 96-well plates at a density of 5000 cells/well in 100 mL of medium and incubated for 24 h. Cells incubated with culture medium with or without DMSO served as controls. Determination of viable cells was performed by adding 3-(4,5-dimethyl-2-thiazol)-2,5-diphenyl-2H-tetrazolium bromide (MTT; Sigma-Aldrich) to a final concentration of 0.5 mg/ml. After 3-h incubation at 37°C and 5% CO2 in a humidified incubator, the formazan crystals were resolved by 10% SDS in 10 mM HCl and the absorption was measured at 560 nm. MTT measurements were performed daily for 5 consecutive days, sometimes at 72 h, as triplicates in 3 independent experiments. The absorption data were used to calculate the doubling time of each cell line. Apoptosis assay 8505C cells were transfected with TGFβ1 siRNA or control siRNA for 72 h. 8505C cells were transfected with p65 siRNA or PUMA siRNA or control siRNA for 24 h, then transfected with TGFβ1 siRNA or control siRNA for 72 h. After 72 h, transduced cells were collected and fixed overnight at 4°C with 75% ethanol for propidium iodide staining and flow cytometry analysis on a FACSCalibur (Becton Dickinson Immunocytometry Systems) to evaluate sub-G1 cell populations (apoptotic rate). Soft-agar colony formation assay 8505C, stable 8505C/control siRNA, and stable 8505C/TGFβ1 siRNA cells were resuspended in RPMI-1640, supplemented with 10% FCS and 0.33% (w/v) agarose (Life Sciences Corporation). One ×103 cells/ml were seeded into soft agar and colony growth was analyzed after 7 days by light microscopy. Only colonies with more than 3 cells were counted. Independent colony formation experiments were repeated twice, each in triplicate. In vivo studies Animal studies were approved by Animal Care and Use Committee and conducted in accordance with NIH guidelines. 8505C or TGFβ1 shRNA/8505C or control shRNA/8505C cells suspended in Matrigel (5×106 cells/200 mL) were inoculated subcutaneously into the right flank of 4- to 6-week-old female athymic nude (nu/nu) mice. The mice were sacrificed, and tumors were dissected after 7 weeks. The primary tumors were divided into 3 portions for cell lysate production, and for making paraffin blocks for Ki-67 immunohistochemistry and TUNEL staining. Proliferative index Paraffin sections (4-μm thick) were routinely stained with hematoxylin and eosin. To assess tumor cell proliferation, immunohistochemical staining for Ki67 antigen (DakoCytomation, Hangzhou, China) was performed. Briefly, after deparaffinization and rehydration, the sections were immersed in 0.3% hydrogen peroxide to quench intrinsic peroxidase activity. The diluted antibodies were then added to the sections and incubated at 37°C for 1 h. The labeled antigen was visualized with the Histofine kit (Bosde, Wuhan, China), followed by reaction with 3,3′-diaminobenzidine. Finally, the sections were counterstained with hematoxylin. Ki67-positive cells were determined by counting about 500 nuclei in randomly selected microscopic fields, and the Ki67 labeling index was expressed as the ratio of Ki67-positive cells to total cells. TUNEL assay The TUNEL staining was performed with the In Situ Cell Death Detection kit according to the manufacturer’s instructions. In brief, DNA strand breaks were labeled with fluorescein dUTP and TdT in the dark at 37°C for 1 h. Subsequently, the sections were counterstained with Hoechst 33258 dye. TUNEL-positive (apoptotic) cells were detected as localized bright green cells by using scanning laser confocal microscopy (Leica, Beijing, China). Data were expressed as the ratio of apoptotic cells to total cells. Statistical analysis Data are expressed as the mean ±SD. SPSS 11.0 software was used for analysis. The statistical significance of a difference between 2 groups was assessed using the t test (2-tailed). ANOVA was used when more than 2 groups were involved, and then the t test was further applied to analyze difference between groups. P<0.05 was considered to indicate a statistically significant result. All experiments were repeated at least 3 times. Results Effect of siRNA on TGF-β1 expression in 8505C cells 8505C cells were transfected with TGF-β1 siRNA and control siRNA for 24–72 h. Western blot and RT-PCR analysis was used to detect the TGF-β1 protein and mRNA level after transfection. Cells transfected with TGF-β1 siRNA displayed a time-dependent reduction in the expression levels of TGF-β1 protein (Figure 1A) and mRNA (Figure 1B). Control siRNA did not exhibit any effect on protein levels of TGF-β1 (Figure 1A) and mRNA levels of TGF-β1 (Figure 1B). These data confirmed the suppression effect of siRNA and established the efficiency of siRNA transfection. We also detected TGF-β2 and TGF-β3 protein and mRNA expression before and after siRNA transfection. Results from our in vitro experiments showed no change in expression for the 2 isoforms, TGF-β2 and TGF-β3, illustrating the specificity of the siRNA sequence designed for this study (Figure 1C, 1D). Effect of TGF-β1 gene silencing on colony formation, cell survival, and apoptosis 8505C cells were transfected with the siRNA plasmid and selected for stable clones demonstrating inhibition of TGF-β1 expression. Silencing of TGF-β1 transcripts in positive clones was confirmed by RT-PCR, and TGF-β1 protein expression in positive clones was confirmed by Western blot assay. As shown in Figure 2A–2B, TGF-β1 mRNA and protein levels were decreased in both the 8505C/TGF-β1siRNA1 clone and the 8505C/TGF-β1siRNA2 clone when compared with control siRNA transfected cells. To investigate the effect of TGF-β1 gene silencing on the growth of 8505C cells, we performed a soft-agar colony-formation assay. As shown in Figure 2C, TGF-β1siRNA1/2-transfected cells formed a significantly reduced (P<0.01) number of colonies compared with untreated or control siRNA transfected cells. Next, the effects of TGF-β1 silencing on both cell survival and apoptosis were determined in vitro. Analysis of cell survival using the MTT assay showed a significant decrease in cell survival for TGF-β1siRNA1 and TGF-β1siRNA2 transfected cells when compared with the control siRNA transfected cells, and at 72 h the survival rate reached the lowest value (Figure 2D). Cell apoptosis was determined using propidium iodine staining followed by flow cytometry analysis. Cells transfected with TGF-β1/siRNA1 and TGF-β1/siRNA2 for 72 h showed significant apoptosis, and the non-transfected cells showed less apoptosis, suggesting TGF-β1 targeting induced cell apoptosis (Figure 2E). Upregulation of PUMA by TGF-β1 silencing correlates with apoptosis induction in 8505C cells To study the mechanism of TGF-β1 silencing to induce apoptosis in ATC cells, protein expression of PUMA, caspase-3, PARP, other proapoptotic Bcl-2 family members, and antiapoptotic proteins were detected by Western blot assay. As shown in Figure 3A, TGF-β1siRNA transfection for 48 h in the 8505C cells markedly induced PUMA protein expression in a time-dependent manner. The peaks of PUMA protein induction were detected at 36 h following TGF-β1 siRNA1 transfection. Cleavage of PARP, a 116-kDa protein involved in DNA repair, is a characteristic marker in the detection of apoptotic cells. As shown in Figure 3B, in TGF-β1/siRNA1-transfected cells, full-length PARP was efficiently cleaved, and the characteristic apoptotic 89-kDa cleavage product was detectable 36 h after TGF-β1/siRNA transfection. In addition, proteolytic activation of pro-caspase-3 was detectable by Western blotting with an anti-caspase-3 antibody 36 h after TGF-β1/siRNA1 transfection (Figure 3C). In contrast, TGF-β1/siRNA transfection did not upregulate proapoptotic protein Noxa, Bak, Bid, or Bim, but reduced the expression of the antiapoptotic proteins bcl-2, Mcl-1, and Bcl-XL (Figure 3A). To determine if TGF-β silencing induced cell apoptosis and inhibited proliferation by inducing PUMA upregulation, 8505C-TGF-β1/siRNA1 cells were transfected with siRNAs (siRNA 1 and siRNA 2) targeting PUMA using Lipofectamine 2000. Figure 3D shows that the silencing efficiency of the PUMA protein induced by PUMA siRNA1 and PUMA siRNA2 reached approximately 90%, (p<0.01). 8505C-TGF-β1/siRNA2 cells has the same results as 8505C-TGF-β1/siRNA1 (data not shown). By flow cytometry analysis, cells transfected with PUMA/siRNA1 and PUMA/siRNA2 showed 5.2–4.6% apoptosis, which was significantly lower compared with TGF-β1/siRNA transfection groups (P<0.01) (Figure 3E). Analysis of cell survival using the MTT assay showed 80–75% cell survival in the PUMA/siRNA1- and PUMA/siRNA2-transfected cells when compared with the TGF-β1/siRNA-transfected cells (P<0.01) (Figure 3F). TGF-β1 silencing induced NF-κB-dependent PUMA upregulation We found that the TGF-β1 silencing inhibited proliferation and induced apoptosis, which is correlated to PUMA upregulation. In a previous study, the p65 subunit of NF-κB was recently identified as a transcriptional activator of PUMA in response to TNF-α [22], sorafenib [23], and regorafenib [24]. In the present study we transfected the p65siRNA into the 8505C-TGF-β1/siRNA cells. The results showed that in the presence of p65siRNA, PUMA protein was obviously inhibited in the 8505C-TGF-β1/siRNA cells (Figure 3G), suggesting that p65 by siRNA abrogated PUMA induction by TGF-β1 silencing. Cell apoptosis was also significantly decreased (Figure 3E) and cell survival rate was significantly increased (Figure 3F), which was similar to the PUMA siRNA transfection (Figure 3E, 3F). TGF-β1-induced NF-κB-PUMA regulation is MEK/ERK-dependent, but SMAD2-independent TGF-β1 silencing resulted in NF-κB-dependent PUMA upregulation, but the mechanisms remain largely unknown. Recent studies show that targeting Ras/Raf/MEK/ERK signaling induced PUMA upregulation, which is mediated by subsequent NF-κB p65 activation [23,24]. Therefore, we speculated that TGF-β1 silencing could induce inactivation of MEK/ERK signaling, which could promote the subsequent cell apoptosis of ATC cells. To test this, Western blotting analysis was performed, and the results showed that the phosphorylation of ERK1/2(pERK1/2) was inhibited in the 8505C-TGF-β1/siRNA1 cells compared to the control siRNA or non-transfected cells (Figure 4A). 8505C-TGF-b1/siRNA2 cells had the same results as 8505C-TGF-β1/siRNA1 cells (data not shown). TGF-β1 silencing did not have any effect on total ERK1/2 (data not shown). TGF-β1 silencing can inhibit pERK1/2 level and induce NF-κB-dependent PUMA upregulation, so we next tested whether TGF-β1 could elevate the pERK1/2 level and inhibit the NF-κB-dependent PUMA upregulation. 8505C cells were treated with human recombinant 1,5 and 10 ng/ml TGF-β1(rhTGF-β1) for 24 h, pERK1/2, p65, and PUMA protein was detected by Western blot assay. The results showed that TGF-β1 strongly promoted phosphorylation of ERK1/2. In addition, p65 and PUMA expression was decreased in the 8505C/TGF-β1 cells compared to the control cells (Figure 4B). However, when 8505C cells were treated with U0126 (10 uM) 6 h before TGF-β1 (rhTGF-β1) treatment, p65 and PUMA expression was restored (data not shown), suggesting p65 and PUMA was ERK1/2-dependent. TGF-β1 transduces signals from the cell membrane to the cell nucleus through serine/threonine kinase receptors and their downstream effectors, SMAD molecules. The TGF-β1-SMAD signaling pathway is involved in regulation of cell growth, differentiation, migration, apoptosis, and extracellular matrix remodeling. We next studied whether TGF-β1-SMAD signaling is involved in p65-PUMA regulation. To test this, Western blotting analysis was performed. 8505C cells were transfected with TGF-β1 siRNA and control siRNA for 48 h, the pSMAD2 was significantly decreased in the 8505C-TGF-β1/siRNA cells (Figure 4A). When 8505C cells were treated with TGF-β1 for 24 h, the pSMAD2 and ERK1/2 was increased, and p65 and PUMA expression was significantly decreased (Figure 4B). However, when 8505C cells were transfected with SMAD2 siRNA and control siRNA for 24 h before rhTGF-β1 (1,5 and 10 ng/ml) treatment, the pERK1/2, p65, and PUMA expression was not affected (Figure 4C), suggesting that ERK1/2, p65, and PUMA expression were not regulated by SMAD2. TGF-β1 silencing inhibits tumor growth TGF-β1 shRNA/8505C cells, control shRNA/8505C cells, and untreated 8505C cells were injected subcutaneously in nude mice and observed for a period of 7 weeks. Xenograft tumor growth and lung metastasis models were obtained as described in the Methods section. As shown in Figure 5A, TGF-β1 shRNA/8505C clones were much smaller compared with control shRNA/8505C cells and untreated 8505C cells groups (p<0.01). By Western blot assay, p65 and PUMA protein were exclusively detected in the TGF-β1 shRNA/8505C groups, and p-ERK1/2 was decreased to almost undetectable levels, compared to the control shRNA/8505C cells and untreated 8505C groups (Figure 5B). To determine the effects of TGF-β1silencing on the other 2 isoforms, Western blot analysis was done. As shown in Figure 5B, there was no significant difference in the expression levels of either TGF-β2 or TGF-β2 in the TGF-β1 shRNA/8505C groups when compared with controls. Xenotransplants of TGF-β1 shRNA/8505C clones revealed fewer cells positive for the proliferation marker Ki67 in the periphery of the tumors than the control shRNA/8505C cells and untreated 8505C cells, which has high positive Ki67 expression (Figure 5C). TUNEL analysis provided in vivo evidence that TGF-β1silencing significantly increased cell apoptosis compared to the non-treated or shRNA/8505C groups (p<0.05) (Figure 5D). Discussion In this study we first investigate the effect of reduction of TGFβ1 induced by TGFβ1 siRNA on the proliferation, colony formation, and apoptosis in vitro. We used 2 independent TGFβ1-knockdown clones (8505C/TGFβ1siRNA1 and 8505C/TGFβ1siRNA2) to perform MTT, colony formation, and apoptotic assay. Our results demonstrate that TGFβ1 silencing inhibits growth and colony formation and induces apoptosis of 8505C cells in vitro. In our study, the stable knockdown of TGFβ1 in the ATC cell lines 8505C, by plasmid based shRNA expression, resulted in reduced tumor growth in vivo. Although the 3 mammalian isoforms of TGFβ1, TGFβ2, and TGFβ3 share 60–80% identity at the amino acid level, the promoter regions of these isoforms are highly variable, suggesting that their expression is regulated by distinct mechanisms [25]. Results from our in vitro and in vivo experiments showed no change in expression for TGFβ2 and TGFβ3 isoforms by TGF-β1sliencing, illustrating the specificity of the siRNA/shRNA sequence designed for this study. In this study, knockdown of TGFβ1/TGF-βR signaling affected cellular growth and apoptosis, but the central signaling pathway, acting downstream of TGF-β and leading to cell death, is not clear. Nuclear factor-κB (NF-κB) is a nuclear transcription factor regulating the expression of various genes involved in cell proliferation, tumorigenesis, and inflammation [26]. PUMA (p53 upregulated modulator of apoptosis), a BH3-only Bcl-2 family member, functions as a critical initiator of apoptosis in cancer cells [27]. Our results demonstrate that TGF-β stimulation inhibited NF-κB p65 and PUMA expression, and knockdown of TGF-β activated NF-κB p65 and PUMA. TGF-β silencing-induced apoptosis and growth inhibition in ATC cells was inhibited by NF-κB p65 siRNA transfection. PUMA is necessary for TGF-β1 silencing and induces apoptosis as shown by PARP cleavage and pro-caspase-3 cleavage resulting in caspase-3 activation. To further characterize TGF-β silencing involvement in apoptosis, the expression of apoptosis-related proteins, such as bcl-2, Mcl-1, Bcl-XL, Noxa, Bak, Bid and Bim, was analyzed. Proapoptotic proteins Noxa, Bak, Bid, and Bim were not upregulated, but reduced the expression of the antiapoptotic proteins bcl-2, Mcl-1, and Bcl-XL. A recent study has shown that PUMA is a direct target of NF-βB and mediates TNF-a-induced apoptosis in vitro and in vivo [22]. Our study found that knockdown of NF-βBp65 reduced TGF-β1siRNA-induced apoptosis and PUMA upregulation. We therefore suggested that knockdown of TGFβ1/TGF-βR signaling induced apoptosis by NF-κBp65-dependant PUMA upregulation. However, the mechanism underlying the relation between TGFβ1/TGF-βR and NF-κBp65-/PUMA is unclear. The targeting of Ras/Raf/MEK/ERK signaling promoted PUMA-dependent apoptosis of tumor cells [23,24], suggesting PUMA was negatively regulated by ERK signaling. In our study, we found that TGF-β1 stimulating activated ERK1/2 and inhibited NF-κBp65/PUMA signaling, and vice versa. However, treatment with ERK inhibitor U0126 inhibited TGFβ1-induced ERK1/2 activity and NF-κBp65/PUMA downregulation. Although SMAD2 activation was shown after TGF-β1 stimulation, knockdown of SMAD2 did not affect the ERK1/2 activity level. We therefore concluded that growth inhibition of ATC cells by TGFβ1 silencing is dependent on the cell apoptosis induction, progressing from ERK inhibition and p65 nuclear translocation, leading to PUMA induction and onset of mitochondria-mediated apoptosis. Moore et al. [28] has reported that TGFβ1 silencing resulted in a 50% increase in proliferation of breast cancer MDA-MB-435 cells, and has no effect on cell apoptosis in vitro compared to the controls, which was contrary to our study. In Moore’s results, AKT and ERK signaling pathways were activated, suggesting a possible role of these signaling pathways in promoting growth signaling. In Moore’s study, there was no significant difference in the growth kinetics of the primary tumors between the TGFβ1siRNA and control siRNA groups in vivo. However, in our study, significant growth inhibition was found in TGFβ1siRNA groups compared to the control siRNA groups in vivo. In our study, ERK1/2 signal was decreased after the TGFβ1 silencing. ERK inhibition, leading to PUMA and apoptosis induction, may be the main causes of these contrary results. Conclusions Results of the present study suggests that therapies targeting TGF-β1 in tumor cells may be effective in decreasing tumorigenesis, which may be related to ERK1/2/NF-κB/PUMA signaling. Future studies on TGF-β signaling in various stages of tumor progression and metastasis may lead to the further development of more tumor-targeted therapies to decrease the incidence of tumorigenesis. Conflicts of interest No potential conflicts of interest were disclosed. Source of support: This study was supported by grants from the National Natural Scientific Research Fund, China (No. 81270648) and the Natural Science Research Foundation of Shandong Province (No. 2014ZRB01198) Figure 1 TGF-β1 gene knockdown by siRNA transfection in 8505C cells. 8505C cells were transfected with TGF-β1 siRNA and control siRNA for 24–72 h. (A) Representative images showing expression of TGF-β1 protein in control siRNA and TGF-β1siRNA transfected cells as analyzed by Western blot. (B) Representative images showing expression of TGF-β1 mRNA in control siRNA and TGF-β1siRNA-transfected cells as analyzed by RT-PCR. (C) Representative images showing expression of TGF-β2, TGF-β3 protein in control siRNA, and TGF-β1siRNA-transfected cells (72-h transfection) as analyzed by Western blot. (D) Representative images showing expression of TGF-β2 and TGF-β3 mRNA in control siRNA and TGF-β1siRNA-transfected cells (72-h transfection) as analyzed by RT-PCR. β-actin and GAPDH were controls. Figure 2 Effect of TGF-β1 gene targeting on colony formation, cell survival, and apoptosis. (A) Silencing of TGF-β1 transcripts in positive clones was confirmed by RT-PCR. (B) Silencing of TGF-β1 protein in positive clones was confirmed by Western blot assay. (C) Photomicrographs showing soft-agar colony formation and histogram showing number of colonies formed by 8505C cells transfected with siRNA. (D) 8505C cells were transfected with the siRNA plasmid for 120 h, cell survival rate was detected by MTT assay, 8505C cells were transfected with the siRNA plasmid for 120 h, and cell apoptotic rate was detected by flow cytometry assay. (E) 8505C cells were transfected with the siRNA plasmid for 120 h, and cell apoptotic rate was detected by flow cytometry assay. Vs. control, * p<0.05; ** p<0.01. Figure 3 Upregulation of NF-κB-dependent PUMA by TGF-β1 silencing correlates with apoptosis induction. (A) 8505C cells was transfected with TGF-β1/siRNA or control siRNA for 0–48 h. NF-κBp65, PUMA, or other bcl-2 family member proteins were detected by Western blot assay. (B) Cell lysates (40 ug of protein) were analyzed by Western blotting using a monoclonal anti-PARP antibody at 36 h. Concomitant with the induction of apoptosis, PARP was fragmented, resulting in the characteristic 89-kDa cleavage product. (C) Caspase-3 is activated in TGF-β1/siRNA 8505C cells at 36 h, as shown by the conversion of pro-caspase-3 to activated cleaved caspase-3. (D) 8505C cells were transfected with TGF-β1/siRNA or control siRNA for 24 h, then transfected with PUMA siRNA1 and PUMA siRNA2 for 48 h. PUMA mRNA induction by TGF-β1/siRNA was analyzed by RT-PCR. vs. TGF-β1/siRNA, * p<0.01. (E) 8505C cells were transfected with TGF-β1/siRNA or control siRNA for 24 h, then transfected with PUMA siRNA or P65 siRNA for 48 h. Cell apoptosis was detected flow cytometry assay. vs. TGF-β1/siRNA, * p<0.01. (F) 8505C cells were transfected with TGF-β1/siRNA or control siRNA for 24 h, then transfected with P65 siRNA and PUMA siRNA for 48 h. Cell viability was detected by MTT assay. vs. TGF-β1/siRNA, * p<0.01. (G) 8505C cells were transfected with TGF-β1/siRNA or control siRNA for 24 h, then transfected with P65 siRNA1 and P65 siRNA2 for 48 h. P65 and PUMA mRNA induction by TGF-β1/siRNA was analyzed by RT-PCR. Figure 4 Knockdown of TGF-β1 activates p65 to induce PUMA through ERK inhibition. (A) 8505C cells were transfected with TGF-β1/siRNA or control siRNA for 48 h. Expression of pERK1/2 and PSMAD2 was analyzed by Western blotting. (B) 8505C cells were treated with rhTGF-β1 (1,5 and 10 ng/ml) for 24 h, pSMAD2, pERK1/2, NF-κBp65, and PUMA protein was detected by Western blot assay. (C) 8505C cells were transfected with either a control siRNA or a SMAD siRNA for 24 h, and then treated with 10 ng/ml rhTGF-β1 for 24 h. pSMAD2, pERK1/2, NF-κBp65, and PUMA proteins were detected by Western blot assay. Figure 5 Effects of TGF-β1 silencing on 8505C cell tumor growth in vivo. (A) 8505C ATC cells (6×106) were inoculated subcutaneously into the right flank of 4-week-old female athymic nude (nu/nu) mice. The tumor growth curves represent 8505C cells, TGF-β1 shRNA-transfected 8505C cells, and shRNA-transfected 8505C cells, as labeled. Point, mean tumor volume (calculated from 6 mice); bars, upper 95% confidence intervals. (B) Protein expression of p-ERK1/2, NF-κBp65, PUMA, (PI3K)/Akt, and TGF-β1-3 were detected in the tissues in vivo. (C) Positive Ki67 staining of the tumor tissues. (D) TUNEL staining of the tumor tissues. 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==== Front Med Sci MonitMed. Sci. MonitMedical Science MonitorMedical Science Monitor : International Medical Journal of Experimental and Clinical Research1234-10101643-3750International Scientific Literature, Inc. 2756673110.12659/MSM.897699897699Lab/In Vitro ResearchEffect of Over-Expression of Zinc-Finger Protein (ZFX) on Self-Renewal and Drug-Resistance of Hepatocellular Carcinoma Zhang Shuhong ADShu Ronghua CEYue Meng BFZhang Shuhong AGDepartment of Gastroenterology, Jinan Central Hospital Affiliated to Shandong University, Jinan, Shandong, P.R. ChinaCorresponding Author: Shuhong Zhang, e-mail: [email protected] Study Design B Data Collection C Statistical Analysis D Data Interpretation E Manuscript Preparation F Literature Search G Funds Collection 2016 27 8 2016 22 3025 3034 22 1 2016 25 1 2016 © Med Sci Monit, 20162016This work is licensed under Creative Common Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0)Background X-chromosome-coupled zinc finger protein (ZFX) in the Zfy protein family is abundantly expressed in both embryonic and hematopoietic stem cells (HSCs). ZFX exist in various tumor cells and is correlated with proliferation and survival of tumor cells. As a malignant tumor with high invasiveness, hepatocellular carcinoma (HCC) may present resistance against chemotherapy and features of stem cells. This study aimed to explore the expression of ZFX in HCC cells, in an attempt to illustrate the role of ZFX in tumorigenesis. Material/Methods The expression of ZFX in tumor tissues was quantified by RT-PCR. The ZFX expression was then silenced to evaluate the stem cell-like features of HCC cells, including self-renewal, colony formation, and cell cycle, along with the sensitivity to cisplatin. Xenograft of ZFX-overexpressed HCC on nude mice was performed to evaluate the in vivo effect of ZFX on tumor growth. Results Quantitative RT-PCR showed over-expression of ZFX in 51.8% of HCC tumors. The silencing of ZFX gene inhibited the self-renewal, colony formation, and proliferation ability of HCC cells (p<0.05 in all cases) via the cell cycle arrest at G0/G1 phase, in addition to the elevated sensitivity of tumor cells to cisplatin (p<0.001). Further studies showed that binding between ZFX and promoter regions of Nanog or SOX-2 regulatory factor initiate their expression in HCC cells. The xenograft experiment indicated the potentiation of tumor growth by ZFX over-expression. Conclusions ZFX is over-expressed in HCC cells, and correlates with stem cell-like features and pleiotropic characteristics. MeSH Keywords Carcinoma, HepatocellularSelf AdministrationSelf-Injurious BehaviorTristetraprolin ==== Body Background Hepatocellular carcinoma (HCC) accounts for 80~90% of all liver cancers and is the third leading mortality factor among all cancers worldwide [1]. Due to its insidious onset and course, HCC is often at late stage at the time of primary diagnosis, causing its high invasiveness, unsatisfactory treatment efficacy, and high mortality [2,3]. Most HCC patients presented re-occurrence even after surgical resection and/or chemotherapy, and rapid progression into terminal stage cancer. It is now recognized that the stem cell-like feature of tumor cells during the progression is closely related with the chemo-resistance and reoccurrence [4–9]. Recently, there has been an increasing number of potential prognosis biomarkers associated with tumor progression of hepatocellular carcinoma [10–13]. Elevated ZFX expression level has been detected in both pleiotropic embryonic stem cells (ESCs) and hematopoietic stem cells (HSCs) to keep the self-renewal ability of cells. ZFX over-expression has also been found in various cancer cells, including esophagus, gastric and prostate cancer cells, whose stem cell-like feature, colony formation ability, drug resistance, cell survival, and metastasis have all been confirmed to be regulated by ZFX [14–17]. The function of ZFX in HCC, however, remains unknown. This study thus aimed to investigate the expressional and functional characteristics of ZFX in HCC. Material and Methods Tissue sample collection A total of 83 liver cancer patients were recruited in this study, including 48 cases of HCC, 22 cases of intrahepatic cholangiocarcinoma (ICC), and 13 cases of mixed hepatocarcinoma (MHC). All patients received confirmed diagnosis by pre-operative biopsy. Both tumor tissue and tumor-adjacent tissue (2 cm from the tumor) samples were collected during surgical resection. This study was pre-approved by the ethics committee of our hospital and we obtained written consent from all patients. Another 6 samples of normal liver tissues were recruited as the control group. Cell culture Human HCC cell lines HKCI-10, HKCI-C2, and HKCI-8 were cultured in RPMI 1640 medium (Invitrogen, US) containing 10% fetal bovine serum (FBS). Normal hepatocyte cell line L02 was cultured in DMEM medium (Invitrogen, USA) containing 10% FBS as previously reported [18]. RNA extraction and RT-PCR Total RNA was extracted from cultured cells by Trizol reagents (Invitrogen, USA). Residual DNA was degraded by RNase-free DNase I (Promega, USA). First-strand cDNA was synthesized using TaqMan Master Mix (ABI, US) followed by RT-PCR. The fluorescent intensity was detected by FAM490 system and replicated in triplicate. The gene expression level was normalized against 18s rRNA using 2−ΔCt method. When compared to the mean value of normal liver tissues, genes with more than 2-fold increase of expression were defined as having elevated expression. Western blot Total protein from lysis buffer was separated in 8% SDS-PAGE and transferred onto PVDF membrane. After blocking non-specific binding sites, the membrane was incubated in anti-ZFX, anti-SOX-2, anti-Nanog antibody (1:1 000, Cell Signaling, USA) or anti-glyceraldehyde-3-phosphate dehydrogenase antibody (1:25 000, Chemicon). Horseradish peroxidase (HRP) was again used to incubate the membrane. The signal was detected by ECL detector (GE Healthcare, USA). Packaging and transfection of lentivirus A FUGENE transfection kit (Roche, Switzerland) was used to transfect HEK293FT cells with pCMV-VSV-G, pRSC-Rev, pMDLg/pRRE, pLKO.1-NS Ctrl, or pLKO.1-hZFX shRNA (Sigma, USA) vectors. At 48 h after transfection, virus was precipitated from supernatants by buffer (1:4). At 24 h before transfection, HKCI-10 and HKCI-C2 cells were seeded into 6-well plates. At 48 h after transfection, cells were incubated in medium containing 2 mg/mL puromycin for 10-day selection as previously reported [19]. Expression of ZFX in HKCI-8 cell line We used pCMV6-ZFX (Origene, USA) vector containing neomycin-resistant gene or blank pCMV6 vector to transfect HKCI-8 cells with the help of Lipofectamine 2000 reagent (Invitrogen, USA). At 48 h after transfection, neomycin (500 mg/mL) was added into the culture medium for cell selection. Quantitative PCR was used to describe the over-expression of ZFX in cells. Cell cycle analysis Stable transfected cells (3×105) were digested by pepsin and rinsed in PBS. Cells were fixed using 3 mL 70% ethanol at –20°C overnight, followed by precipitation at 1000 g centrifugation for 5 min. PBS was used to gently wash cells twice. Cells were then stained by 50 mg/mL propidium iodide (PI) and 0.5 mg/mL nuclease A for 30 min, followed by flow cytometry analysis. The mean value of G0/G1, S, and G2/M stage was calculated from independent experiments performed 3 times each. Cell proliferation assay Stable transfected cells (1×103) were seeded into a 96-well plate, in which 8 replicates were performed for a certain time-point (D0~D7). Using MTT assay, the optical density value at 570 nm was measured on 7 consecutive days. The Click-iT EdU flow cytometry approach was used to detect the level of NDA replication, using the integration of uridine analogs 5-ethynyl-2-0-deoxyuridine (EdU) of newly synthesized DNA strands. In brief, stable transfected cells (3×105) were first incubated in complete medium containing 10 mM EdU for 2 h, followed by PBS washing and Click-iT fixation. After 15-min incubation at room temperature, cells were re-suspended in 500 mL 1× Click-iT buffer and 0.5-mL reaction cocktails were added. After rinsing and re-suspension, cells were analyzed in the FACS Calibur system (BD, US). The percentage of various fluorescent cells were compared by use of WinMDI 2.9 software. Colony formation assay Stable transfected cells were inoculated into 6-well plates (200 cells per well). After 7-day incubation, medium was removed and cells were washed twice with PBS. After staining by 1% crystal violet (in absolute methanol) for 15 min, the number of colonies (defined as more than 20 cells per colony) was counted and recorded. Single cell clonal analysis To evaluate the in vitro self-renewal ability of transfected cells, they were seeded into 96-well plates (1 cell per well) and cultured in DMEM medium containing 10% FBS. Twenty-four hours later, those wells with no cells or more than 1 single cell were excluded, leaving only wells with a single cell. Seven days later, the number of clones with more than 20 cells was counted. In vitro cytotoxicity assay Stable transfected cells (5×103) were inoculated into 96-well plates. Cisplatin (20 mM) was added for 48-h incubation, followed by MTT assay for cell viability at an absorbance value of 570 nm. ChIP-PCR assay Forty-eight hours after ZFX-flag transient transfection, L02 cells were fixed in 1% formaldehyde and lysed in RIPA lysis buffer containing proteinase inhibitor. Chromatin was fragmented into ~600 bp lengths by ultrasonic processing. After centrifugation, chromatin precipitation was diluted 10-fold by ChIP diluents. With 1-h pre-incubation in protein G-agarose, anti-flag antibody or IgG-controlled serum was used for 4°C overnight incubation. After immunoprecipitation, protein G-agarose beads were used to collect immunoprecipitation complex, which was rinsed by low- and high-saline buffer, LiCl rinsing buffer, and TE buffer, and was eluted by elution buffer. The eluted compounds were decoupled and processed in proteinase K, and purified using a PCR DNA purification kit (Qiagen, USA). Real-time ChIP-PCR was used to quantify ZFX level based on SYBR Green reaction mixtures, along with pre-designed primers flanking possible ZFX binding sites [20]. Xenograft of tumor cells Stable transfected HKCI-8 cells (1×107) were re-suspended in 0.1 mL PBS, which was subcutaneously injected into the backs of nude mice (7 weeks old). After 5 days, the tumor size (equals to width2 × length/2) was measured and calculated. Three weeks later, mice were sacrificed to extract the tumor tissue for measurement. Statistical analysis All collected data were analyzed by SPSS16.0 software. The paired t test or Mann-Whitney test, as appropriate, was used to compare the ZFX expression level between tumor and tumor-adjacent tissues. The non-paired t test was used to analyze the correlation between ZFX expression and clinical pathological parameters. The paired t test was used to compare the function of ZFX. Statistical significance was defined as p<0.05. Results ZFX is over-expressed in human HCC Quantitative RT-PCR revealed the significantly elevated ZFX expression in HCC tumor samples (T) when compared to normal liver tissues (NL). Among all 83 cases of HCC, 43 (51%) tumor samples had elevated ZFX expression compared to tumor adjacent (TN) tissues (Figure 1A). Both mRNA and protein levels of ZFX were elevated in tumor tissues (Figure 1B). No correlation existed between ZFX expression level and major clinical parameters, including age, sex, HBV level, histological type, number of lesions, and microvascular invasion. Stage III HCC tumors, however, had higher ZFX expression levels compared to stage I or II tumors (Figure 1C, p<0.05 by non-paired t test), suggesting the correlation between ZFX expression and tumor stage. Further analysis revealed elevated ZFX expression level in liver cirrhosis-induced HCC compared to those tumors not caused by liver cirrhosis (Figure 1D, p<0.05). Because tumor-adjacent liver cirrhosis lesions are often recognized as precancerous lesions, ZFX expression level may serve as an index for evaluating the risk of HCC. ZFX induced the G0/G1 phase arrest of HCC cells To explore the mechanism underlying up-regulation of ZFX in HCC tissues, we transfected 2 HCC cell lines, HKCI-10 and HKCI-C2, which have increased ZFX expression (Figure 2A, p<0.01), with lentivirus vector for stable ZFX-deficient clones. Both qRT-PCR and Western blotting revealed the efficiency of ZFX gene silencing (Figure 2B, p<0.001). PI staining showed significantly elevated numbers of cells at G0/G1 phase with fewer cells at S or G2/M phase (Figure 2C, p<0.01), suggesting the cell cycle arrest at G0/G1 phase of HCC cells by the silencing of ZFX gene. ZFX facilitated HCC cell growth and proliferation MTT assay showed decreased in vitro proliferation of HCC cells with ZFX gene silencing (Figure 3A, p<0.001), consistent with results from EdU staining (Figure 3B, p<0.001 in both cell lines). Colony formation assay revealed a decreased number and size of cell colonies after ZFX silencing (Figure 3C, p<0.001). The over-expression of ZFX facilitated HCC cell proliferation and colony formation ability (p<0.001 in both cases). These results collectively suggest the important role of ZFX on in vitro proliferation and growth of HCC cells. ZFX induced stem cell-like features of HCC Due to its critical role in preserving the pluripotency of cells, ZFX’s function in mediating stem cell-like characteristics of HCC cells was further studied. We mainly studied 2 major features, self-renewal and drug resistance, of stem cell-like features. The silencing of ZFX in HKCI-10 and HKCI-C2 cells lines inhibited self-renewal ability, with formation of fewer and smaller colonies (Figure 4A, p<0.001). Higher sensitivity to chemotherapy agents occurred in HCC cells after ZFX silencing, as IC50 value decreased from 16.86 μg/mL ±1.07 μg/mL (control) to 6.73 μg/mL ±1.10 μg/mL (shZFX-C1) or 7.03 μg/mL ±1.12 μg/mL (shZFX-C2) (Figure 4B, p<0.001). Therefore, ZFX modulates the stem cell-like features of HCC cells. ZFX induced stem cell-like features of HCC ESC pluripotent-related transcriptional factors include SOX-2, Nanog, OCT-4, and Notch1 [21,22]. In this study, we focused on the potential effect of ZFX on the expression of those transcriptional factors. Our study found significantly decreased mRNA and protein expressions of SOX-2 and Nanog after silencing ZFX (Figure 5A, 5B, p<0.001), leaving the expression of OCT-4 and Notch1 largely unchanged. The over-expression of ZFX in HCC cells significantly altered the expression level of SOX-2 and Nanog mRNA (Figure 5C, p<0.001). In total, there were 6 potential binding sites for ZFX within the 2k promoter region of SOX-2 gene, along with 3 binding sites in Nanog gene promoter (Figure 5D). To check if ZFX can actually bind onto the promoter region of those 2 genes, we used ChIP-PCR assay and showed the decreased DNA enriching in SOX-2 promoter binding sites by ZFX (Figure 5E, p<0.05). Although no DNA enriching phenomena occurred in the promoter region of Nanog gene, a positive relationship did exist between ZFX expression and Nanog (Figure 5F, p<0.05). These results collectively indicate the role of SOX-2 and Nanog as the downstream target genes of ZFX. ZFX induced stem cell-like features of HCC In a further in vivo xenograft study on nude mice, we found the over-expression of ZFX in HCC cells significantly facilitated the growth of xenograft tumors (Figure 6), suggesting the potentiation of ZFX on in vivo growth of HCC lesions. Discussion Zinc finger protein coupled with X chromosome (ZFX) is a highly reserved protein coded by a gene located in the X chromosome of mammals. As 1 family of zinc finger protein, ZFX contains acidic transcriptional activation domain, nuclear targeting sequence, and DNA structural domain [23,24]. Recent studies have supported the role of ZFX in tumorigenesis. In this study, we, for the first time, confirmed the up-regulation of ZFX in HCC tissues and the important role of ZFX on the stem cell-like properties of HCC. In liver cirrhosis-derived HCC cells, the elevation of ZFX expression was even more significant, which has not been reported previously. About 70~90% of HCC were derived from liver cirrhosis, which may be caused by viral hepatitis, alcohol abuse, and metabolic disorders. Our discovery that liver cirrhosis-derived HCC cells had elevated ZFX expression indicates the value of ZFX as a predictive index for evaluating cancer risk in patients with precancerous lesions. The silencing of ZFX significantly inhibited the self-renewal of HCC cells, in addition to the potentiation of its sensitivity to chemotherapy, thereby suggesting the novel role of ZFX as a target for gene therapy of HCC. The cancer stem cell theory has been postulated in recent years and has been strengthened by an increasing body of evidence. Some scholars recognize the primary tumor as a complex of heterogeneous cell populations, in which certain cancer stem cells keep various features, including low differentiation, unlimited cell division, resistance to chemotherapy, and self-renewal [13,25–27]. The existence of such tumor-initiated stem cells (TISCs), however, remains controversial as some researchers believe that the stem cell-like features existed not just in a certain sub-population of cancer cells, but in all solid tumor cells. Our findings generally agree with the latter opinion, as most HCC cells had potentiated stem cell features with the help of ZFX. The inhibition of ZFX expression led to broad suppression of cell growth and self-renewal ability. The over-expression of ZFX in HCC cells facilitated both in vitro proliferation of HCC cells and in vivo growth of xenograft. We also investigated the downstream effector of ZFX in HCC cells, focusing on the effects on expression of stem cell-related genes, including Nanog, Oct-4, Notch1, and SOX-2. No significant change of Oct-4 or Notch1 occurred. The expression of Nanog and SOX-2, however, was significantly altered by ZFX, which possibly binds onto the promoter region of SOX-2 gene as shown by ChIP-PCR analysis. Recent studies showed that the synergistic expression of Nanog and SOX-2 can re-program somatic cells for induction into pluripotent ESCs [28,29], suggesting the potency of co-expression of stem cell factors in keeping cells at the undifferentiated status. Moreover, both Nanog and SOX-2 have been suggested to be related with cancer progression because they may potentiate the expression of ZFX, further causing the oncogenesis of HCC [30–32]. Nanog has been shown to significantly potentiate to the chemo-resistance of both oral squamous cell carcinoma and prostate cancer [33,34]. In this study, we further demonstrated a positive relationship between ZFX expression and Nanog, indicating that the elevated sensitivity of HCC cells with ZFX silencing may be related with Nanog down-regulation. Conclusions The elevated expression of ZFX in HCC, plus its relationship with cancer stem cells and chemo-resistance, indicate the importance of further studies on the signal pathway of ZFX in tumor cells. Our results and further studies should provide a new potential target for novel therapy against HCC. Source of support: Departmental sources Figure 1 Overexpression of ZFX in HCC. (A) Up-regulation of ZFX in HCC tumor tissues by qRT-PCR. NL, normal liver tissue; TN, tumor adjacent tissue; T, HCC tumor. (B) Elevated ZFX protein levels in HCC tissue by Western blotting. (C) Stage III HCC had higher ZFX mRNA than stage I and II tumors. (D) Elevated ZFX levels in liver cirrhosis-related HCC tumors compared to non-cirrhosis-derived tumors. * p<0.05; p<0.01. Figure 2 Cell cycle arrest of HCC cell lines by ZFX silencing. (A) Up-regulation of ZFX in HCC cell line, HKCI-10, and HKCI-C2 compared to control cells. (B) Silencing of ZFX gene decreased both mRNA (top) and protein (bottom) levels. (C) G0/G1 phase arrest in cells with ZFX gene silencing. Right panels show decreased percentage of cells at S and G2/G1 phase. p<0.01; * p<0.001. Figure 3 Inhibition of in vitro HCC cell proliferation by ZFX gene silencing. (A) MTT analysis showed cell viability. (B) EdU staining for cell proliferation. Left panels, non-proliferated (P1) and proliferated (P2) cells; right panels, percentage change of proliferated cells. (C) Decreased colony formation ability after ZFX silencing. p<0.01; * p<0.001. Figure 4 ZFX regulated stem cell-like features of HCC cells. (A) Single-cell clone formation was compromised after ZFX silencing in HCC cell lines. A cell colony was defined as having more than 20 cells. (B) Chemotherapy agent sensitivity was increased after ZFX silencing. By MTT analysis, cell survival curve is shown in the left panel, while the right panel shows decreased IC50 values against cisplatin. p<0.01; *** p<0.001. Figure 5 ZFX regulated SOX-2 and Nanog expression. (A) qRT-PCR shows decreased SOX-2 and Nanog mRNA expressions after ZFX silencing. (B) SOX-2 and Nanog protein levels were down-regulated in Western blotting. (C) The over-expression of ZFX induced SOX-2 and Nanog expression. (D) Possible ZFX binding sites within promoter regions of SOX-2 and Nanog genes. (E) SOX-2 and Nanog promoter can bind with ZFX by ChIP-PCR assay. (F) A positive relationship existed between ZFX and Nanog mRNA levels from 83 cases of primary HCC tissues and adjacent tissues. * p<0.05; p<0.01; * p<0.001. Figure 6 ZFX over-expression facilitated in vivo tumor growth. (A) Nude mouse model showed over-growth of tumors derived from ZFX-overexpression HCC cells (far right panels). (B) Tumor volume of ZFX-overexpressed xenograft was significantly larger than control ones. * p<0.05. ==== Refs References 1 Li Y Tang ZY Hou JX Hepatocellular carcinoma: insight from animal models Nat Rev Gastroenterol Hepatol 2012 9 32 43 22025031 2 Motola-Kuba D Zamora-Valdes D Uribe M Mendez-Sanchez N Hepatocellular carcinoma. 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==== Front 10149973435773Nat ChemNat ChemNature chemistry1755-43301755-43492765786610.1038/nchem.2551hhmipa787794ArticleOligoarginine Peptides Slow Strand Annealing and Assist Nonenzymatic RNA Replication Jia Tony Z. 12Fahrenbach Albert C. 13Kamat Neha P. 1Adamala Katarzyna P. 1†Szostak Jack W. 12341 Howard Hughes Medical Institute, Department of Molecular Biology, and Center for Computational and Integrative Biology, Massachusetts General Hospital, 185 Cambridge Street, Boston, Massachusetts, 02114, USA2 Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford St., Cambridge, Massachusetts, 02138, USA3 Earth-Life Science Institute, Tokyo Institute of Technology, 2-12-1-IE-1 Ookayama, Meguro-ku, Tokyo 152-8550, Japan4 Department of Genetics, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, Massachusetts, 02115, USA Corresponding Author: Jack W. Szostak, Howard Hughes Medical Institute, Department of Molecular Biology, and Center for Computational and Integrative Biology, Massachusetts General Hospital, 185 Cambridge Street, Massachusetts, 02114, Tel: 617-726-5102, Fax: 617-726-6893, [email protected] Address: MIT Media Lab and McGovern Institute, Departments of Biological Engineering and Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, Massachusetts, 02139, USA 23 5 2016 27 6 2016 10 2016 27 12 2016 8 10 915 921 Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: http://www.nature.com/authors/editorial_policies/license.html#termsThe nonenzymatic replication of RNA is thought to have been a critical process required for the origin of life. One unsolved difficulty with nonenzymatic RNA replication is that template-directed copying of RNA results in a double-stranded product; following strand separation, rapid strand reannealing outcompetes slow nonenzymatic template copying, rendering multiple rounds of RNA replication impossible. Here we show that oligoarginine peptides slow the annealing of complementary oligoribonucleotides by up to several thousand-fold; however, short primers and activated monomers can still bind to template strands, and template-directed primer extension can still occur within a phase-separated condensed state, or coacervate. Furthermore, we show that within this phase, partial template copying occurs even in the presence of full-length complementary strands. This method for enabling further rounds of replication suggests one mechanism by which short, non-coded peptides could have enhanced early cellular fitness, potentially explaining how longer, coded peptides, i.e. proteins, came to prominence in modern biology. ==== Body RNA has been postulated to be the biopolymer from which early life on Earth evolved, due to the central role of RNA as a mediator of information transfer between DNA and proteins, and to the ability of RNA to act as both a propagator of genetic information and as a catalyst. Most notably, RNA is the catalyst responsible for the ribosomal synthesis of all coded proteins1–3, strongly suggesting that RNA-based catalysis preceded the evolution of coded peptide synthesis. Furthermore, recent findings point to a potential prebiotic pathway for the synthesis of ribonucleotides and thus RNA4–6. If RNA was indeed the original biopolymer of cellular life, then selective pressures for faster and more accurate RNA replication would likely have led to the evolution of an RNA polymerase ribozyme that could catalyze the replication of increasingly complex RNA genomes. However, prior to the evolution of the first RNA polymerase ribozyme, RNA must have replicated nonenzymatically3,7. This conclusion has motivated a long history of efforts to copy RNA templates nonenzymatically, and although the efficient copying of arbitrary template sequences has not yet been demonstrated, recent advances7–11 suggest that such template copying may well be possible. Given the potential for nonenzymatic template copying to generate seminal RNA strands, one must then ask what additional steps are required to enable repeated cycles of RNA replication. The nonenzymatic copying of a template strand results in the formation of an RNA duplex, which must then denature to provide templates for the next round of replication. We have previously shown that the thermal separation of the strands of an RNA a duplex is facilitated by the incorporation of a fraction of 2′–5′ linkages in the RNA backbone; these linkages form as a consequence of nonenzymatic template copying and significantly lower the melting temperature of the resulting duplex12. However, subsequent rounds of nonenzymatic RNA replication are inhibited by the rapid reannealing of the separated strands following heating and cooling13, preventing the weakly-binding RNA primers and activated monomers required for polymerization from associating with the template14 (Fig. 1a). In order for subsequent rounds of replication to be possible, reannealing of the separated single strands must occur on a time scale that is comparable to or slower than the rate of strand copying. In principle, this kinetic control could be accomplished by operating in a highly dilute regime (≤1 nM RNA)7,13; however, 1 nM RNA corresponds to only a few strands per protocell 3–4 μm in diameter. Protocells containing only a few strands of RNA would not have contained a sufficient concentration of an RNA with catalytic activity to confer a benefit to the protocell7. We therefore sought to identify conditions under which RNA strand reannealing at more relevant μM concentrations is significantly slowed, while minimally affecting template-copying chemistry. Considerable evidence supports the possibility that peptides and RNA could have been present together15–17 on the primitive earth. Atmospheric discharge experiments18,19, transport from meteors and cosmic dust20,21, and more recent scenarios for prebiotic amino acid synthesis16,17 all point towards the existence of amino acids on the earth’s surface shortly after its formation. Although early studies indicated that the basic amino acids arginine (Arg, R) and lysine (Lys, K) would have been among the least abundant22,23, recent studies illustrate a prebiotically plausible arginine synthesis from simple precursors in a cyanide-rich reducing environment in the presence of hydrogen sulfide24. Arginine is particularly interesting functionally, since arginine-rich oligopeptides are known to bind strongly to both DNA and RNA25,26; the DNA condensing protein protamine is mostly composed of arginines27,28 and Arg3 has been reported to condense DNA in vitro via electrostatic interactions29. If arginine-rich peptides could have assembled on the early Earth, it seems likely that they would have interacted strongly with RNA, potentially enhancing or inhibiting aspects of RNA replication and function. A variety of prebiotically plausible mechanisms for peptide bond formation have also been explored30–32 including the solution-phase elongation of the peptide chain by the stepwise addition of either N-carboxyanhydrides (NCA) or 2-thiono-5-oxazolidones, formed by the reaction of amino acids with the volcanic gases carbonyl sulfide33 or carbon disulfide34, respectively. Although most of these processes lack sequence specificity, efficient oligoarginine production via polymerization of the L-arginine-NCA has been reported through a unique mechanism that involves a six-membered ring intermediate35. The polymerization of arginine on acidic mineral surfaces provides an alternative experimentally demonstrated pathway to oligoarginine peptides that could operate in the presence of a complex mixture of amino acids36. Given the potential existence of arginine-rich peptides on the early Earth, coupled with the ability of these peptides to bind to RNA, we sought to explore the nature and consequences of such RNA-peptide interactions on nonenzymatic replication. In the course of these investigations, we discovered that short arginine-rich peptides can prevent the annealing of complementary RNA strands in a concentration- and length-dependent manner. In the present work, we simulate a post-replication round of nonenzymatic RNA polymerization by thermally denaturing an RNA duplex, and show that template-directed primer extension proceeds only in the presence of an oligoarginine peptide (Fig. 1b). We also show that the nonenzymatic primer extension reaction occurs within a phase-separated condensed state, i.e. a coacervate, formed by the electrostatic binding of oppositely charged RNA and oligoarginine polyelectrolytes. Taken together, these results show that cationic peptides could have enhanced the fitness of an emerging protocell by assisting in multiple rounds of replication. Results Binding of RNA to Oligoarginine Peptides As a prelude to exploring the functional consequences of the binding of arginine-rich peptides to RNA oligonucleotides, we used a series of independent assays to examine the binding of oligoarginine peptides (R10NH2 and R9NH2, NH2:C-terminal amide) to short RNA oligonucleotides. Initial gel electrophoresis assays showed that the oligoarginine peptide R10NH2 at concentrations ≥100 μM caused a visible shift in the mobility of an RNA 15-mer (Supplementary Fig. S1). Furthermore, the fluorescence emission intensity of a 2-aminopurine (2AP)-containing RNA 15-mer increased significantly upon addition of 100 μM R9NH2 (Supplementary Fig. S2). As 2AP fluorescence is quenched by the stacking of adjacent bases37, the increase in fluorescence suggests that peptide binding alters the conformation of ssRNA such that base-stacking is decreased. Finally, a solution containing a fluorescently labeled RNA 15-mer became turbid in the presence of 10 mM R9NH2 due to the formation of aggregated peptide-RNA complexes (Supplementary Fig. S3). The driving force for this complexation is most likely electrostatic as seen previously for the association of polylysine with DNA38. In order to examine the effect of peptide and RNA length on the concentration dependence of the interaction, we turned to fluorescence anisotropy titrations. We observed that the fluorescence anisotropy of a 2AP-containing RNA 15-mer increased with increasing peptide concentrations; an increase in fluorescence anisotropy indicates a decrease in the rate of rotational diffusion, consistent with peptide binding to RNA (Fig. 2a). The presence of R10NH2 resulted in a greater increase in the fluorescence anisotropy compared to R7 and R5 at the same peptide concentrations; addition of R5 did not result in any significant increase in the fluorescence anisotropy. Likewise, addition of R10NH2 to an RNA 15-mer resulted in a greater increase in the fluorescence anisotropy compared to addition of R10NH2 to a 10-mer, while R10NH2 addition to a 7-mer did not result in any significant increase in the fluorescence anisotropy (Fig. 2b). These data indicate that longer peptides and RNA bind more tightly to each other than do their shorter counterparts, a result which would be expected from association driven by electrostatic interactions39. To further explore the possibility of a conformational change in the RNA upon peptide binding, we monitored the circular dichroism (CD) spectrum of a single-stranded RNA 15-mer in the presence of increasing concentrations of R10NH2 (Fig. 2c). The spectrum in the absence of peptide is characteristic of an A-type helical conformation40, but the 270 nm peak decreases in intensity with increasing peptide concentration, indicating that the global helical structure of the single-stranded RNA is significantly disrupted upon peptide binding. Finally, we measured the effect of addition of Mg2+ to a pre-formed RNA-peptide complex. Both fluorescence anisotropy (Supplementary Fig. S4) and CD (Supplementary Fig. S5) measurements show that added Mg2+ displaces the peptide in the RNA-peptide complex, presumably by disrupting the electrostatic interactions between the peptide and RNA. Oligoarginine Peptides Interfere with RNA Annealing The formation of charge-neutralized RNA-peptide complexes suggested that peptide binding might increase the rate of annealing of complementary strands; on the other hand, the apparent change in RNA conformation upon peptide binding hinted that the rate of annealing might be decreased. To address these hypotheses we measured the effect of oligoarginine peptides on the second-order rate of annealing of complementary RNA 15-mers by stopped-flow fluorometry (Fig. 3a). Each RNA strand was separately incubated with peptide at room temperature for at least one hour to ensure maximum peptide-RNA binding before annealing. A single 2AP residue present in one RNA strand was employed as a reporter of the fraction of RNA in duplex form, as 2AP fluorescence is quenched upon duplex formation37. RNA melting analysis showed that replacing one A residue in one of the strands with 2AP does not compromise duplex stability at room temperature (Supplementary Fig. S6). We observed that the t1/2 (initial second-order annealing half-life) values for two RNA 15-mers increased from 0.9(1) s in the absence of peptides, up to a maximum of 5.2(5) × 103 s (SEM values in parentheses) in the limit of increasing concentrations and lengths of oligoarginines (Fig. 3b); for the most effective peptide, R10NH2, no further inhibition of annealing was observed above a concentration of ~15 μM (see Supplementary Table S1 for the annealing t1/2 for all conditions tested), perhaps due to the formation of a phase-separated coacervate at concentrations >15 μM R10NH2 (Supplementary Fig. S7). To confirm that this annealing inhibition was due to the cationic nature of the oligoarginine peptides, we tested the anionic peptide GDGEGDGEGD (G: glycine, D: aspartate, E: glutamate), which did not result in a significant increase in the RNA annealing t1/2 (Supplementary Fig. S8). As the fluorescence anisotropy results (Fig. 2b) indicate that R10NH2 binds to longer RNAs more strongly than shorter RNAs, we investigated whether R10NH2 could selectively inhibit the annealing of longer RNAs. Indeed, we observed that after R10NH2 addition the annealing rate of RNA decreased more for longer RNA oligonucleotides (Fig. 3c). Remarkably, a 15-mer anneals roughly two orders of magnitude more rapidly to a 7-mer (t1/2 = 71(7) s) than to its full-length complementary strand (t1/2 = 5.2(5) × 103 s) in the presence of 100 μM R10NH2. We then asked whether the ions and ionic compounds required for nonenzymatic replication9 would reduce the degree of peptide-conferred annealing inhibition. In the presence of 100 μM R10NH2, the addition of Mg2+ (up to 50 mM, Supplementary Fig. S9), the activated monomer cytidine 5′-phosphor-2-methylimidazolide (2-MeImpC, up to 50 mM, Supplementary Fig. S10), cytidine monophosphate (up to 50 mM, Supplementary Fig. S11), and sodium citrate (up to 25 mM, Supplementary Fig. S12) all significantly increased the annealing rate of two RNA 15-mers (by up to two orders of magnitude). High concentrations of Tris-Cl buffer (up to 500 mM) also increased the annealing rate of two RNA 15-mers in the presence of R10NH2 but to a lesser extent (Supplementary Fig. S13). These observations are consistent with the binding of oligoarginine peptides to RNA by electrostatic interactions, which are disrupted by monovalent and divalent cations. Finally, we also confirmed that the ability of R10NH2 to slow RNA annealing is not significantly affected by pH within the range of pH 6–9 (Supplementary Fig. S14). We suggest that the binding of oligoarginine to RNA changes the RNA structure significantly and contributes to annealing inhibition. At sufficiently high concentrations, added oligoarginine results in the formation of a condensed phase (Supplementary Fig. S7) driven by charge-neutralization, not unlike those observed for ATP in the presence of polyvalent cations41,42. Oligoarginine-Assisted Nonenzymatic RNA Replication The observation that R10NH2 greatly slows the annealing of two RNA 15-mers while having little effect on the rate of annealing of a 7-mer to a 15-mer led us to hypothesize that in the presence of R10NH2 a short primer would still be able to anneal to a longer template even in the presence of the complementary strand to the template, allowing primer extension to occur. Traditionally, high concentrations (≥50 mM) of Mg2+ and activated monomer are used in nonenzymatic replication experiments9. However, because both Mg2+ and activated monomers disrupt the electrostatic interactions between R10NH2 and RNA, we performed all nonenzymatic polymerization studies at lower concentrations of Mg2+ and monomer (10 mM each). The annealing of complementary 15-mers in buffer conditions simulating a nonenzymatic primer extension experiment (10 mM MgCl2, 10 mM 2-MeImpC, and 250 mM Na-HEPES pH 8) is still effectively slowed by 100 μM R10NH2 (t1/2 = 8.5(4) × 102 s). Because R10NH2 alters the conformation of ssRNA, we then asked whether R10NH2 would inhibit nonenzymatic primer extension by measuring the rate of primer extension on a 16-mer C4 template using guanosine 5′-phosphor-2-methylimidazolide (2-MeImpG). We found that the rate of primer extension decreased by only ~30% in the presence of 1 mM of the R10NH2 peptide (Figs. 4a–b and Supplementary Fig. S15)—in contrast to the greater than three order-of-magnitude increase in the annealing t1/2 of two RNA 15-mers in the presence of 100 μM R10NH2 (Fig. 3). To our satisfaction, a promising result was obtained when the complementary strand to a template, preincubated with R10NH2, was added to a preformed primer-template complex, also preincubated with peptide (Fig. 4c). Under these conditions, it appears that the peptide effectively inhibited the annealing of the complementary strand to the template, while still allowing the primer extension reaction to proceed, whereas in the absence of peptide, the addition of the complementary strand resulted in complete inhibition of the primer-extension reaction. We then turned our attention to simulating a post-replication round of nonenzymatic template-directed primer extension by starting with an RNA duplex, i.e. a template strand already bound to its complement. We added the other components of the nonenzymatic primer extension reaction (primer, Mg2+, and 2-MeImpG) and briefly heated the sample to 95 °C to melt the duplex followed immediately by cooling on ice. Rapid cooling is necessary (Supplementary Fig. S16), most likely to allow peptide-RNA binding to occur before the annealing of complementary RNA strands. The primer extension reaction was then allowed to proceed as the reaction mixture warmed to room temperature overnight (Fig. 4d). In the absence of the oligoarginine peptide, no primer extension was observed, as expected from rapid annealing of the complementary strand to the template. In the presence of peptide, however, the primer extension reaction did proceed (Fig. 4e); we propose that the thermally separated template and complement each tightly binds to the peptide, which prevents them from reannealing. However, the shorter primer binds more weakly to the peptide and is still able to hybridize to the template, thus allowing nonenzymatic primer extension to proceed under conditions approximating a post-replication round of RNA polymerization. Primer extension within the RNA-Peptide Condensed Phase As we observed the formation of phase-separated droplets when R10NH2 was added to both single-stranded (Fig. 5a) and duplex RNA (Fig. 5b), we suspected that the primer extension reaction was occurring in the highly concentrated RNA-peptide coacervate rather than in the very dilute aqueous phase. To begin to test this hypothesis, we used confocal fluorescence microscopy to monitor the localization of a 5′-cyanine 3 (Cy3)-labeled RNA 8-mer primer in the presence of R10NH2 and a complementary RNA 15-mer. As expected, the 8-mer and 15-mer phase-separate together into large (~5–10 μm) globular structures (Fig. 5c); this phase separation phenomenon is dependent on a variety of factors including the presence of peptide and a complementary RNA (Supplementary Fig. S17), the salt concentration (Supplementary Figs. S18–S19), and the length of the RNA itself (Supplementary Figs. S20–S21). A quantitative UV spectroscopic analysis also revealed that in the presence of R10NH2 and a complementary 15-mer, roughly 97% of a native RNA 7-mer resides in the condensed phase (Supplementary Figs. S22–S23, Supplementary Table S2). These observations suggest that the primer extension reaction occurs within the coacervate phase, since that is where most of the primer is localized. To show directly that the observed primer extension reaction was occurring in the coacervate droplets, we centrifuged a solution containing peptide and all components of a nonenzymatic primer extension experiment and separated the supernatant from the pellet containing the coacervate phase. We then allowed the primer extension reaction to proceed separately in both the supernatant and the coacervate phase. Subsequent analysis of the reaction products confirmed that in the presence of peptide, a majority of the primer extension reaction occurred within the isolated coacervate phase (Fig. 5d). We suggest that in the presence of peptide, shorter RNA strands, i.e. primers, that bind weakly to peptide are still able to diffuse freely, and hence are still able to hybridize to the template strands in the condensed phase and undergo polymerization. Discussion One of the seldom-addressed problems with the RNA world hypothesis is that in order for multiple generations of nonenzymatic RNA replication to occur, the new single-stranded templates generated by melting the duplex product of template copying must reanneal on a time scale comparable to or slower than the time scale of template copying. In practice, this is a formidable challenge, since the reannealing of complementary RNA strands at reasonable concentrations (~1 μM) is extremely fast (t1/2 ≈ 1 s), while RNA copying chemistry, at least in the current state of the art, is quite slow, occurring over hours to days10. We have demonstrated here that the binding of oligoarginine peptides to complementary strands of RNA selectively slows down strand reannealing by up to several thousand-fold. This kinetic property allows template-directed nonenzymatic RNA polymerization to occur in the presence of a full-length strand complementary to the template (Fig. 4e). Surprisingly this nonenzymatic primer extension reaction occurs in a condensed coacervate phase formed as a consequence of the electrostatic association of the oligoarginine peptides with RNA oligonucleotides (Fig. 5). Within the coacervate phase the annealing of longer RNA oligomers is selectively inhibited (Fig. 3), thus allowing time for primer extension to occur before strand annealing is complete. Further exploration and optimization of the selective inhibition of strand annealing has the potential to allow multiple rounds of RNA replication to occur. In light of previous research showing the potential plausibility of prebiotic arginine synthesis and peptide bond formation30, the possibility that arginine-rich peptides facilitated continued cycles of RNA replication in a prebiotic setting should be considered. We note that the prebiotic synthesis of arginine-rich peptides would be enhanced by the existence of local environments whose ambient chemistry favored arginine synthesis24; another potentially important factor is that the oligomerization of arginine specifically, even in the presence of other amino acids, could be templated by negatively charged surfaces such as minerals36, fatty acid membranes, or even RNA itself43. Assuming the simultaneous existence of arginine-rich peptides and RNA on the early earth, a primitive cell would gain a considerable evolutionary advantage if it were able to internally synthesize arginine-rich peptides, perhaps foreshadowing the evolution of coded translation and the later appearance of the arginine-rich DNA condensing proteins such as protamine27,28 and the histones44 found in modern cells. However, the concentrations of oligoarginine peptides used in this study for successful nonenzymatic template-directed primer extension in the presence of a competing complementary strand are quite high (1.5 mM) and it is unclear how such concentrations could be reached in a prebiotic system or whether lower concentrations of peptide could have had the same effect. As it is known that nucleic acids electrostatically bind to many cationic polymers45, the inhibition of RNA annealing could very well have been a result of the strong binding of RNA to any cationic polymer, some of which may be more effective than oligoarginine. Thus, we cannot discount the possibility that a presently unknown cationic prebiotic polymer, potentially with a simpler structure and/or synthesis than oligoarginine, e.g. a polyamine42,46, could also efficiently slow annealing. In addition, the recent discovery of complex macrostructures resulting from self-assembling cationic tripeptides47 suggests that the supramolecular assembly of simple prebiotic peptides to form a cationic RNA-binding matrix should also be considered as a future avenue of investigation. Although we have shown that oligoarginine binding to RNA slows complement-template annealing, the mechanism by which this occurs remains to be elucidated. In principle, the relevant RNA-peptide interaction could be either thermodynamically or kinetically controlled. In the thermodynamically controlled extreme, the addition of the peptide to double-stranded RNA would destabilize the duplex, forming peptide-single-stranded RNA complexes over time. In this scenario, binding of oligoarginine to a single strand of RNA must be much stronger than binding to the duplex, so that the peptide would cause the RNA duplex to dissociate. A short primer, being less strongly bound to peptide than the full-length RNA39, could then hybridize to the template-peptide complex and subsequently take part in template-directed primer extension. However, we do not observe primer extension products without a heating and immediate cooling step (Supplementary Fig. S16), which strongly argues for a kinetically controlled mechanism in which the reannealing of longer strands is selectively inhibited after strand separation. This retardation of hybridization could occur if, for example, peptide binding causes a conformational change in the RNA that prevents the nucleation of base-pairing required for efficient annealing48,49. As we have shown, the peptide binds only weakly to short primer strands, and this fact allows the primer to bind to the template, thus permitting the primer extension reaction to proceed. We also propose that a rapid cooling step immediately after strand separation is necessary as it promotes peptide-RNA binding on a timescale competitive with the reannealing of two longer strands. Thus the prebiotic accessibility of a steep temperature gradient is critical for successful replication in the system we studied. Thermal convection and thermophoresis within the porous rocks of hydrothermal vents50 and hydrothermal circulation generated by hydrothermal vents in ponds or lakes51 are two prebiotically plausible environments that would allow biomolecules to access both very hot and very cold aqueous environments on a timescale fast enough to promote successful nonenzymatic replication. In a search for further evidence that would distinguish between thermodynamically and kinetically controlled mechanisms, we consider the differences between the CD spectra of single- versus double-stranded RNA 15-mers upon addition of R10NH2. Single-stranded RNA in the absence of peptide exists predominantly in an A-form helical conformation, but this conformation is disrupted after binding to R10NH2 as evidenced by the disappearance of the 270 nm peak in the CD spectrum (Supplementary Fig. S5). In contrast the 270 nm peak in the CD spectrum of duplex RNA does not disappear after addition of R10NH2 (Supplementary Fig. S24); disappearance of the 270 nm peak would have indicated possible destabilization and/or denaturation of the duplex by peptide binding. Instead, the 270 nm peak shifts to a maximum at 310 nm, consistent with a conformational change. This spectral data provides evidence that the strands of the 15-mer RNA duplex do not separate upon addition of R10NH2, supporting a mechanism of peptide-mediated primer extension that is not thermodynamically controlled. In fact, the CD spectrum may suggest specific binding between an RNA duplex and R10NH2; further investigations into the nature of this complex are ongoing. It has recently been shown that a variety of phase-separated systems can be encapsulated inside model protocells41. The assembly of a fatty acid vesicle around an oligolysine-RNA coacervate has also been reported52, which is significant as lysine oligomers are also able to slow strand annealing, albeit less effectively than oligoarginine (Supplementary Fig. S25). These results, along with the observations that arginine-rich peptides can localize RNA to53 and are compatible with (Supplementary Fig. S26) fatty acid membranes, suggest that oligoarginine-RNA coacervates may also be encapsulated within fatty acid-based model protocell membranes. The encapsulation of coacervate compartments within a model protocell is particularly interesting as it could have been a mechanism by which primitive cells organized and concentrated compounds and functionality, perhaps resulting in the earliest organelles42,54. Remarkably, modern eukaryotic cells also utilize phase-separated RNA-protein granules for a variety of cellular functions, including segregation and concentration of RNA and proteins55–57. It is unknown whether these modern coacervates are a relatively recent innovation, or reflect an ancient evolutionary origin. In order to better understand the origin of life, we aim to design and construct a model protocell system9—for example a fatty acid vesicle containing templates, primers, activated mononucleotides, peptides, and catalytic metal ions—in which multiple rounds of nonenzymatic primer extension reactions can be induced by thermal cycling and can be iterated ad infinitum. Multiple rounds of replication would bring us one step closer to emulating the first primitive cells that were able to grow, divide, and evolve under early earth conditions. Methods Peptides, RNA, and Nucleotides Peptides (R5, R7, R9, R9NH2, and R10NH2; NH2: C-terminal amide) were purchased from either NeoBioLab (Woburn, MA) or GenScript (Piscataway, NJ) at >95% purity as trifluoroacetic acid salts. DNA and RNA were purchased from IDT (Integrated DNA Technologies, Coralville, IA) and were used without purification unless otherwise noted. See Supplementary Methods for a list of all other chemicals and their suppliers as well as for the synthesis of the GDGEGDGEGD peptide (Supplementary Fig. S8). The activated ribonucleotide monomers guanosine 5′-phosphor-2-methylimidazolide (2-MeImpG) and cytidine 5′-phosphor-2-methylimidazolide (2-MeImpC) were synthesized according to published procedues58 with minor modifications (Supplementary Methods). Experiments, Data Analysis, and Figures All experiments were performed in triplicate or greater. Curve fitting was performed using MATLAB (Natick, MA) and figures were prepared with Igor Pro (Wavemetrics, Lake Oswego, OR) and Adobe Illustrator (San Jose, CA). Fluorescence Anisotropy A 2-aminopurine37 (2AP)-containing RNA 15-mer (5′-CC(2AP)GUCAGUCUACGC-3′), 10-mer (5′-CC(2AP)GUCAGUC-3′), or 7-mer (5′-CC(2AP)GUCA-3′) was diluted to 10 μM in annealing buffer (10 mM Tris-Cl, 50 mM NaCl, and 1 mM EDTA in nuclease-free water at pH 8) to a total volume of 100 μL in a sample cuvette (Starna Cells, Inc., Atascadero, CA, 10 mm pathlength). We increased the concentration of peptide (R10NH2, R7, or R5) by adding small volumes (~1 μL) of concentrated solutions (100 μM–10 mM) to the sample cuvette and obtained data at each point (303 nm excitation, 370 nm emission) on a Cary Eclipse fluorescence spectrometer (Agilent Technologies, Santa Clara, CA). We modulated the magnesium and sodium concentration in the same way (Supplementary Fig. S4). See Supplementary Methods for further details. Circular Dichroism 5 μM of an RNA 15-mer (5′-GCGUAGACUGACUGG-3′) or a 15-mer duplex (5′-GCGUAGACUGACUGG-3′ and its complement) in annealing buffer (10 mM Tris-Cl, 50 mM NaCl, and 1 mM EDTA in nuclease-free water at pH 8) was prepared in a 300 μL quartz cuvette (Starna Cells, 10 mm pathlength). Data was obtained using an Aviv 202 circular dichroism spectrometer (Aviv Biomedical, Inc., Lakewood, NJ) from 200 nm to 350 nm with a 2 nm step size and an 8 nm bandwidth. See Supplementary Methods for further details. Stopped-Flow Annealing Experiments A typical stopped-flow annealing experiment consisted of mixing two RNAs, e.g. one 15-mer (5′-GCGUAGACUGACUGG-3′) and one fully complementary 2AP-containing 15-mer (5′-CC(2AP)GUCAGUCUACGC-3′). All 2AP bases were deoxyribonucleotides, as provided by IDT. 1 μM of each RNA strand was diluted in annealing buffer (10 mM Tris-Cl, 50 mM NaCl, and 1 mM EDTA in nuclease-free water at pH 8) and incubated separately at room temperature for at least one hour either in the presence of or absence of peptide (R5, R7, R9, or R10NH2, up to 100 μM). The peptide length and concentration, as well as the length of one of the strands of RNA, was varied as indicated in Fig. 3. The two solutions were injected into the mixing chamber of an SX20 stopped-flow spectrometer (Applied Photophysics, Leatherhead, Surrey, UK), and the spectral data was immediately recorded (303 nm excitation, WG 320 nm emission filter). See Supplementary Methods for further details. Nonenzymatic Primer Extension Nonenzymatic RNA primer extension reactions were performed with 2-MeImpG. See Supplementary Methods for further details. Confocal Fluorescence Microscopy All samples were imaged using a Nikon (Tokyo, Japan) A1R confocal microscope (100×, 1.49 N. A. Apochromat TIRF oil immersion objective) microscope at 561 nm (pinhole 0.5 AU). See Supplementary Methods for further details. Supplementary Material 1 We thank Aaron E. Engelhart, Christian Hentrich, Benjamin D. Heuberger, Aaron T. Larsen, Tivoli J. Olsen, Noam Prwyes, Ruth Saganty, Lijun Zhou, and all members of the Szostak Lab for helpful discussions and critical reading of the manuscript. We also thank Gary Ruvkun and Eric Rubin for their support and very helpful advice. J.W.S. is an Investigator of the Howard Hughes Medical Institute. A.C.F. is supported by a Research Fellowship from the Earth-Life Science Institute at the Tokyo Institute of Technology. N. P. K. is supported by an appointment to the NASA Postdoctoral Program, administered by Oak Ridge Associated Universities through a contract with NASA. This work was supported by grants from the Simons Foundation (290363) and NASA (NNX15AL18G) to J.W.S. Author Contributions T. Z. J., A. C. F., and J. W. S. conceived of experiments and wrote the manuscript. N. P. K. performed the vesicle leakage assays and T. Z. J. performed all other experiments. K. P. A. contributed intellectually. Competing Financial Interests No competing financial interests are declared. Figure 1 The reannealing problem and a proposed solution. a) Complete template-directed primer extension results in a full-length duplex (newly synthesized strand in maroon, original template in gray) (1). After strand separation by heating (2), subsequent cooling results in rapid reannealing of the newly synthesized complementary strand to the template strand (1); this prevents primer-template binding, outcompeting the slow process of nonenzymatic RNA polymerization (3) thereby preventing further rounds of RNA replication. b) RNA-binding oligoarginine peptides (green) inhibit strand annealing and promote further rounds of nonenzymatic replication. After an RNA duplex is formed (1), the strands are separated by heating (2). Subsequent cooling allows the peptide to bind to the separated complementary strands but not to the shorter RNA primers. This selectivity prevents reannealing of the full-length replicated strands, allowing each strand to act as a template, to which shorter primers can then bind (3). The nonenzymatic polymerization reaction is free to proceed, resulting in a complete replication cycle that would not be possible without the peptide. Figure 2 RNA-peptide binding measured by fluorescence anisotropy and circular dichroism. Experiments were performed in annealing buffer (Methods). Error bars indicate ± one SEM. a) Fluorescence anisotropy of a 2-aminopurine (2AP)-containing RNA 15-mer (5′-CC(2AP)GUCAGUCUACGC-3′, 10 μM) in the presence of three oligoarginine peptides of different lengths (R5, R7, and R10NH2; NH2: C-terminal amide) over increasing peptide concentrations. Higher peptide concentrations and greater peptide length lead to increased fluorescence anisotropy. b) Fluorescence anisotropy of 2AP-containing RNAs of increasing lengths (7-mer, 10-mer, and 15-mer, 10 μM; see Methods for sequences) with increasing R10NH2 concentrations. Longer RNAs show a greater increase in anisotropy. c) Circular dichroism (CD) traces of an RNA 15-mer (5′-CCAGUCAGUCUACGC-3′, 5 μM) with increasing concentrations of R10NH2. The initial spectrum is characteristic of an A-type helical conformation40. The molar circular dichroism (Δε) of the 270 nm peak decreases with increasing peptide concentration (up to 25 μM). Figure 3 RNA annealing rates in the presence of peptides. Initial second-order annealing half-lives (t1/2) were obtained for an RNA 15-mer (5′-GCGUAGACUGACUGG-3′) and its 2AP-containing complement (5′-CC(2AP)GUCAGUCUACGC-3′) in annealing buffer (Methods) at RNA concentrations of 1 μM (See Supplementary Methods for fitting parameters and Supplementary Table S1 for a list of all conditions tested). Error bars indicate ± one SEM. a) Kinetic traces showing the annealing of the two RNA 15-mers in the presence or absence of 100 μM R5, R7, R9, or R10NH2. b) t1/2 for RNA 15-mers with increasing concentrations of R5, R7, R9, and R10NH2. c) t1/2 for RNA of different lengths (7-mers to 15-mers, on x-axis; see Supplementary Methods for sequences) annealing to the 2AP-containing 15-mer with or without 100 μM R10NH2. Longer RNAs exhibit slower annealing kinetics in the presence of peptide. Figure 4 Nonenzymatic RNA polymerization. Reactions performed with 10 mM MgCl2, 250 mM Na-HEPES pH 8, and 10 mM 2-MeImpG. a) Polyacrylamide gel of nonenzymatic additions of 2-MeImpG to a 5′-cyanine 3 (Cy3)-labeled primer (5′-Cy3-CAGACUGG-3′, 2 μM) on a C4 template (5′-AACCCCCCAGUCAGUC-3′, 2.5 μM) with or without 1 mM R10NH2. Bolded cytosines represent 2-MeImpG binding sites on the template. b) log of the fraction of unreacted primer for gel lanes in a vs. time, with (red) or without (black) R10NH2. The slope of the lines (linear fit, R2 = 0.99) represents the pseudo-first-order rate constant, kobs, in h−1 for the respective reactions. With 1 mM R10NH2, kobs = 0.084(5) h−1. With no peptide, kobs = 0.110(5) h−1. SEM in parentheses. n = 5. c) Polyacrylamide gel of an overnight nonenzymatic primer extension experiment with 1.2 μM primer and 1.25 μM template (incubated with or without 1.5 mM R10NH2), after addition of 0 μM or 2 μM complementary strand to the template (5′-GACUGACUGGGGGGUU-3′) separately incubated with or without 1.5 mM R10NH2. d) Post-replication round of nonenzymatic primer extension. The template (gray) and its complement (red) were annealed, then peptide, monomer, and primer (blue) were added and the mixture was heated briefly to 95 °C. After cooling to 4 °C to allow primer-template binding, the system was allowed to warm to room temperature. e) Polyacrylamide gel of the overnight primer extension experiment described in d with 0.95 μM primer, 1 μM template, and with or without 1.2 μM complementary strand and 1.5 mM R10NH2, respectively. Figure 5 Condensed phase of RNA. Scale bars, 10 microns. a) Confocal fluorescence microscopy image (488 nm excitation, 525 nm emission) of a 5′-6-carboxyfluorescein (FAM)-labeled RNA 15-mer (5′-FAM-CCAGUCAGUCUACGC-3′, 5 μM) with 1 mM R10NH2 in annealing buffer (Methods). b) Image of the sample from a but with an added complementary RNA 15-mer (5′-GCGUAGACUGACUGG-3′, 5 μM). c) Confocal fluorescence microscopy image (561 nm excitation, 595 nm emission) of a 5′-Cy3-labeled RNA 8-mer (5′-Cy3-CAGACUGG-3′, 10 μM) with a complementary RNA 15-mer (5′-CCAGUCAGUCUACGC-3′, 10 μM) with 1 mM R10NH2, 100 mM Tris-Cl pH 8, and 10 mM MgCl2. d) Nonenzymatic primer extension experiment after 4 hours with the Cy3-labeled RNA primer (1.25 μM) and template (1.4 μM) from Fig. 4, 10 mM MeImpG, 10 mM MgCl2, 250 mM Na-HEPES pH 8, and with or without 1 mM R10NH2. 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==== Front Med Sci MonitMed. Sci. MonitMedical Science MonitorMedical Science Monitor : International Medical Journal of Experimental and Clinical Research1234-10101643-3750International Scientific Literature, Inc. 2793277810.12659/MSM.898574898574Molecular BiologyExpression of Long Non-Coding RNA (lncRNA) Small Nucleolar RNA Host Gene 1 (SNHG1) Exacerbates Hepatocellular Carcinoma Through Suppressing miR-195 Zhang Hui ABCDEFGZhou Dong BCEYing Mingang EFChen Minyong BDChen Peng DFChen Zhaoshuo DZhang Fan BFDepartment of Abdominal Surgery, Fujian Provincial Cancer Hospital, Fujian Medical University Teaching Hospital, Fuzhou, Fujian, P.R. ChinaCorresponding Author: Hui Zhang, e-mail: [email protected] Study Design B Data Collection C Statistical Analysis D Data Interpretation E Manuscript Preparation F Literature Search G Funds Collection 2016 09 12 2016 22 4820 4829 18 3 2016 26 4 2016 © Med Sci Monit, 20162016This work is licensed under Creative Common Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0)Background Aberrant expression of lncRNA has been suggested to have an association with tumorigenesis. Our study was designed to reveal the underlying connection between lncRNA SNHG1 and hepatocellular carcinoma (HCC) pathogenesis. Material/Methods A total of 122 pairs of HCC tissues (case group) and matched adjacent non-tumor liver tissues (control group) were collected for this study. RT-PCR and in situ hybridization were conducted to investigate differences in lncRNA SNHG1 expression between the case and control group. The expression levels of lncRNA SNHG1 and miR-195 in HepG2 cells transfected with SNHG1-mimic and SNHG1-inhibitor were measured by RT-PCR. The proliferation, invasion, and migration status of HepG2 cells after transfection were assessed through MTT assay, wound healing assay, and Transwell assay, respectively. Whether miR-195 is a direct downstream target of lncRNA SNHG1 was verified by both bioinformatics target gene prediction and dual-luciferase report assay. Results The expression level of lncRNA SNHG1 was remarkably upregulated in HCC tissues and cell lines compared with normal tissues and cell lines. High expression of lncRNA SNHG1 contributed to the downregulation of miR-195 in HepG2 cells. Also, lncRNA SNHG1 exacerbated HCC cell proliferation, invasion, and migration in vitro through the inhibition of miR-195. This suggests that miR-195 is a direct downstream target of lncRNA SNHG1. Conclusions lncRNA SNHG1 may contribute to the aggravation of HCC through the inhibition of miR-195. MeSH Keywords Carcinoma, HepatocellularHep G2 CellsIn Situ Hybridization, FluorescenceRNA, Long Noncoding ==== Body Background Hepatocellular carcinoma (HCC) is believed to be one of the most prevalent cancers, with high morbidity and mortality. HCC accounts for about 80%–90% of liver cancer cases and results in an annual death of 600,000 [1–3]. Previous studies have shown that contributors to HCC include liver cirrhosis, infection with hepatitis B/C virus (HBV/HCV), adiposity, aflatoxin contamination, excessive alcohol consumption, and environmental pollution [4]. Despite emerging therapies, including percutaneous ablation, liver transplantation, and the recently approved sorafenib (a systemic therapy for HCC), there is still no cure for HCC [1,5–8]. Therefore, the underlying molecular mechanisms involved in the development of HCC should be further explored to identify innovative therapeutic targets for HCC patients [9–11]. Long non-coding RNAs (lncRNAs) are transcriptional RNA molecules with more than 200 nucleotides; lncRNAs have limited or non-protein-coding capacity [12]. Functional lncRNA shares several specific sequence features, including fewer introns and low GC content, poor start codon, and open reading frame contexts. As suggested by previous studies, several important biological processes, including transcription, translation, and cellular differentiation, have been found to be regulated by lncRNAs at post-transcriptional levels. Also, lncRNAs are involved in chromatin modification and trafficking between nuclear and cytoplasmic structures [13]. Accumulating reports have suggested that lncRNAs are involved in the pathogenesis and progression of several cancers. For instance, a high expression level of metastasis triggered by lung adenocarcinoma transcript 1 (MALAT-1), a kind of lncRNA, has been suggested to be associated with the development of non-small-cell lung carcinomas (NSCLCs). In addition, MALAT-1 expression has been suggested to exhibit an upward trend in colorectal, prostate, pancreatic, and breast cancer tissues compared with normal tissues [14]. Therefore, it is hypothesized that lncRNA expression, which is aberrantly expressed in a variety of cancers, might have a significant impact on the development of HCC [13]. Previous reports have provided evidence that the small nucleolar RNA host gene (SNHG1) of lncRNA was significantly upregulated in NSCLC cell lines, and may act as a new potential therapeutic target for NSCLC interventions [15]. However, the association between lncRNAs and HCC has not yet been clarified. The lncRNAs are able to bind to specific structures of microRNAs (miRNAs), which also participate in the pathogenesis and progression of a variety of cancers, and miRNA motif regulates protein-coding genes at expression levels [12]. Previous studies showed that miRNAs play a crucial role in the angiogenesis and metastasis process of HCC [16]. For example, miR-135, miR-21, and miR-17 exhibited abnormally high expressions in HCC tissues compared to normal liver tissues, while expression levels of miR-101, miR-122, miR-125, miR-150, miR-375, and miR-195 in HCC tissues were markedly decreased [17,18]. As suggested by an online database (http://starbase.sysu.edu.cn/mirLncRNA.php), miR-195 may interact with SNHG1. However, the synergic effects of miR-195 and SNHG1 in suppressing carcinoma angiogenesis or metastasis remain unclear [16]. Since both SNHG1 and miR-195 expressions levels are altered in NSCLC, we suspected that SNHG1 may regulate miR-195 expressions, thereby affecting the carcinogenesis process of HCC. This research was conducted using both in situ hybridization and RT-PCR for the purpose of assessing whether lncRNA SNHG1 was overexpressed in HCC tissues and cell lines. More importantly, we aimed to investigate the association between SNHG1 and miR-195 with respect to HCC pathogenesis using bioinformatics target gene prediction, dual-luciferase report assay, RT-PCR, cell proliferation assay, wound healing assay, and Transwell assay. Material and Methods Ethical statement This research project was supported by the Medical Ethical Committee of Fujian Provincial Cancer Hospital, Fujian Medical University Teaching Hospital. All 122 patients were informed about the project and gave written consent to participate in the research. Human tissue samples and cell lines We obtained a total of 122 pairs of HCC and matched adjacent non-tumor tissues from patients admitted in the Fujian Provincial Cancer Hospital, Fujian Medical University Teaching Hospital (Fuzhou, China) between March 2014 and March 2015. Patients had not received chemotherapy or radiotherapy prior to surgery. The collected tissues were immediately frozen at −80°C after surgery. Human HCC cell lines HepG2 and normal human liver cell line L02 were obtained from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Shanghai) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Gibco, Shanghai) was prepared to cultivate these cell lines. The moist culture atmosphere was set at 37°C with 5% CO2. Hybridization in situ To detect lncRNA SNHG1 and miR-195 expression levels in liver tissues, hybridization in situ was conducted based on the manufacturer’s instructions. Digoxin (DIG)-labeled lncRNA SNHG1 and miR-195 probe (1:400 and 1:400, respectively) were added to tissue slices (embedded in paraffin) at 55°C for one hour. After washing, the tissue was sealed by reagents for one hour, the sealing reagent was removed, and Tris Buffered Saline Tween (TBST) containing anti-DIG antibody (1:200) was incubated with tissue slices at 37°C for one hour. Results were observed by microscope after staining with hematoxylin and eosin (H & E). We purchased the probes and reagent kit from Boster (Wuhan). Quantitative real-time PCR In order to quantify the expression levels of lncRNA SNHG1 and miR-195 in both tissues and cells, quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) was conducted. Total RNA was extracted by using TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA). Then a reverse transcription kit (Bio-Rad, Hercules, California, USA) was used to reverse transcribe lncRNA SNHG1, miR-195, and an internal control U6a into cDNA. Subsequently, RT-PCR reactions were performed using a preheated ABI 7500 RT-PCR System (Applied Biosystems, Carlsbad, USA) along with SYBR Premix Ex Taq (Takara, Japan). Analysis of relative gene expressions was independently performed three times and each sample was verified in triplicate. The relative expression levels of RNA were calculated using Ct values and the level of target gene expression (2−ΔΔCt) was normalized with respect to the U6 control reference. Plasmid construction and transfection Full-length cDNA of lncRNA SNHG1 and miR-195 was inserted into the pcDNA™ 6.2-GW/EmGFP-miR vector (National Center for Biotechnology Information; http://www.ncbi.nlm.nih.gov/). Then, 2′-O-methyl-anti-lncRNA SNHG1, presented as lncRNA SNHG1 inhibitor, was chemically synthesized by Genechem (Shanghai, China). HepG2 cells were transfected into different groups: mock, mimic control, inhibitor control, SNHG1-mimic, SNHG1-inhibitor, and SNHG1-mimic + miR-195-mimic. After cells were grown in growth medium to 60–70% confluency, HepG2 cells were transfected using Lipofectamine 2000 Reagent (Invitrogen Corp, CA, USA) following the manufacturer’s instruction. Dual-luciferase report assay As suggested by the manufacturer’s protocol, the miR-195 full length 5′ lncRNA SNHG1-binding seed region was inserted into the pGL3 promoter vector (Genscript, Nanjing, China). The sequences in the binding site were mutated using GeneTailor Site-Directed Mutagenesis System (Invitrogen, USA), and then the mutant 3′ UTR was cloned into the same vector. These two constructed vectors were named pGL3-miR-195 and pGL3-miR-195-mut, respectively. A total number of 1×104 HEK293 cells were transfected with 50nM lncRNA SNHG1 and pcDNA6.2-control, and either 100 ng pGL3-miR-195 or 100 ng pGL3-miR-195-mut. Afterwards, cells were seeded into 96-well plates and the Lipofectamine 2000 Reagent was addedfollowing the manufacturer’s instructions. Data collected from the luminometer were normalized by dividing firefly luciferase activity with that of Renilla luciferase vector pRL-SV40, which was also cotransfected into HepG2 cells. Cell proliferation assay Briefly, 3-(4,5)-dimethyl thiahiazo-(-z-y1)-3,5-di-phenytetrazoliumromide (MTT) assay (Sigma, USA) was used to evaluate cell growth status. HepG2 cells were grouped and transfected respectively as mentioned earlier. After 1, 2, and 3 days of transfection, HepG2 cells were incubated with fresh medium containing 20 μL MTT for another four hours. Supernatant was removed and replaced with 150 μL dimethyl sulfoxide. Cells were incubated in a humidified incubator (37°C, 5% CO2). Universal Microplate Spectrophotometer (Bio-Tek Instruments, Inc., Winooski, USA) was used to measure the absorbance value at a wavelength of 490 nm. Transwell assay In vitro, Transwell assay was used to investigate cell migration and invasion status. The experiments used Transwell chambers (24-well, 8 μm pore size, Costar, Corning, Switzerland), uncoated for migration assays, or Matrigel coated for invasion assays. After 48 hours, we trypsin-digested the post-transfected cells with 0.25% trypsin containing EDTA and resuspended the cells in serum-free RPMI 1640 medium. Subsequently, we added 200 μL of the cell suspension into the upper chamber, and 600 μL of medium containing 10% FBS as chemoattractant was added into the lower chamber. After 24-hour incubation in a moist atmosphere (37°C, 5% CO2), we used wet cotton swabs to wipe off non-migrated cells through the pores from the upper face of the filters, while cells adhered to the bottom surface of the inserts were fixed with cold methanol for 10 minutes and then stained with 0.01% crystal violet for 2 minutes. Finally, the entire filters were washed twice in water and observed under an inverted microscope (Olympus, Japan). Wound healing assay The transfected HepG2 cells were seeded in 6-well plates at a cell density of 8×104 and were grown into monolayer cells overnight. A 10-μL micropipette tip was used to scratch the surface of plates to create a “wound” and the suspended cells were mildly washed with PBS. Cells in the plates were cultured in serum-free medium. Wound images were photographed using a phase-contrast microscope on Day 0, 1, and 2. The wound area was scored by the ImageJ software. Statistical analysis We performed statistical analysis using SPSS 17.0 statistical software, and data presented using Graph PAD prism software. The χ2 test was applied to infer the relationship between lncRNA SNHG1 expression and clinicopathological characteristics of HCC patients. All the results obtained from the in vitro experiments were expressed as mean ± standard deviation, and continuous data were analyzed using the double-sided Student’s t-test. A p-value of less than 0.05 was considered statistically significant. Results lncRNA SHNG1 expression correlated with several clinicopathological features Clinicopathological features of 122 HCC patients are summarized in Table 1. The average age of patients was 55.5±10.2 years (56 patients ≤55 years of age and 66 patients >55 years of age). There were 92 males and 30 females in the patient group. As suggested by in situ hybridization, the expression levels of lncRNA SNHG1 were further classified (using mean area of positive staining as the cutoff value) into high expression with lncRNA SNHG1 ≥30% of the tumor section and low expression with lncRNA SNHG1 <30% of the tumor section. The expression of lncRNA SNHG1 in HCC tissues was closely related to several clinicopathological features, including tumor size (p<0.001) and TNM stage (p=0.002). HCC patients with larger tumor size or TNM stage III–IV exhibited relatively higher lncRNA SNHG1 expression levels. However, lncRNA SNHG1 expression was not associated with gender, age, or alcohol consumption (all p>0.05). These results indicated that high expression level of lncRNA SNHG1 might predict the aggravation of HCC. lncRNA SNHG1 showed high-level expression in both HCC tissues and cell lines As revealed by hybridization in situ, lncRNA SNHG1 expression levels were significantly upregulated in HCC tissues compared with matched non-tumor tissues, while miR-195 expression levels were significantly downregulated in HCC tissues (p<0.05; Figure 1A). These results were consistent with the results obtained from RT-PCR (p<0.05; Figure 1B). In addition, lncRNA SNHG1 was significantly upregulated in HepG2 HCC cells compared with L02 normal hepatocyte cells, whereas the expression levels of miR-195 were significantly downregulated as shown in hybridization in situ and RT-PCR (p<0.05; Figure 1C, 1D). We concluded that lncRNA SNHG1 was highly expressed in both HCC tissues and cell lines. Results from HepG2 cell lines were consistent with those from HCC tissues, further indicating that the expression of lncRNA SNHG1 was increased in HCC tissues. lncRNA SNHG1 inhibited miR-195 expression To further explore the relationship between the expression of lncRNA SNHG1 and miR-195 in HepG2 cells, transfection experiments were performed using HepG2 cell lines as previously described. As demonstrated by RT-PCR, miR-195 expression of HepG2 cells transfected with SNHG1-mimic (0.0088±0.0030) was remarkably lower than that of other groups, including the mimic control group (0.2760±0.0562), indicating that overexpression of lncRNA SNHG1 could contribute to a decreased miR-195 expression. In addition, downregulation of lncRNA SNHG1 after transfection of SNHG1-inhibitor significantly increased the expression level of miR-195 (all p<0.05; Figure 2). Interestingly, we noticed that neither upregulation or downregulation of miR-195 had significant effect on lncRNA SNHG1 expression. lncRNA SNHG1 exacerbated cell proliferation, invasion and migration MTT growth assay was conducted over a 3-day period in order to discover the function of lncRNA SNHG1 in cell growth. As demonstrated in Figure 3A, MTT optical density (OD) values of HepG2 cells transfected with SNHG1-mimic on Day 1 (2.52±0.15), Day 2 (3.61±0.27) and Day 3 (4.27±0.30) were significantly higher than those of other groups (p<0.05), indicating that over-expression of lncRNA SNHG1 can significantly enhance cell proliferation. Cells transfected with SNHG1-inhibitor exhibited significantly decreased cell proliferation rates with relatively lower MTT OD values (p<0.05). These results demonstrated that high level of lncRNA SNHG1 expressions were associated with HepG2 cell proliferation. Transwell assay results demonstrated that both the migration cells (137.31±7.15) and the invasion cells (123.72±7.67) in the SNHG1-mimic group were significantly increased compared to other groups, whereas the migration cells (35.88±3.81) and invasion cells (31.71±3.63) in the SNHG1-inhibitor group were significantly reduced (all p<0.05; Figure 3B), suggesting that migration and invasion of HepG2 cells transfected with SNHG1-mimic were significantly enhanced as compared with other groups, while cells transfected with SNHG1-inhibitor exhibited significantly decreased cell migration and invasion. In addition, the wound healing assay showed that the relative wound area of the SNHG1-mimic group on Day 1 (0.183±0.032) and Day 2 (0.061±0.012) were decreased significantly compared to other groups, whereas the relative wound area of the SNHG1-inhibitor group on Day 1 (0.712±0.031) and Day 2 (0.594±0.024) were reduced only slightly (all p<0.05; Figure 3C). These results suggest that high expression levels of lncRNA SNHG1 could facilitate HepG2 cell invasion and migration. miR-195 acted as a direct downstream target of lncRNA SNHG1 We next investigated whether miR-195 was a direct functional target of lncRNA SNHG1. Since a complementary 5′ binding site for the seed sequence of lncRNA SNHG1 (Figure 4A) was predicted through the online database (http://starbase.sysu.edu.cn/mirLncRNA.php), we suspected that miR-195 might act as a potential target of lncRNA SNHG1. Cotransfection with lncRNA SNHG1 and miR-195 wild-type 5′lncRNA SNHG1-binding seed region resulted in a significant reduction in luciferase activity compared with that of the control group (p<0.05; Figure 4B). However, cotransfection with lncRNA SNHG1 and miR-195-mut did not have significant effect on luciferase activity. All of these results indicated that lncRNA SNHG1 directly targeted miR-195. Discussion Unfavorable prognosis of HCC is associated with the invasion and metastasis of cancer cells, and HCC has become a major life-threatening disease in the world [19]. Recent studies have suggested that a number of long non-coding RNAs (lncRNAs) are involved in HCC pathogenesis and progression. Therefore, this study aimed to confirm that the upregulation of lncRNA SNHG1 can directly inhibit miR-195 expression, thereby providing an underlying molecular mechanism which may explain the detailed role of lncRNA SNHG1 in regulating human liver tumorigenesis. Although lncRNAs cannot be translated into protein products, they may participate in a variety of pathobiological processes such as cell proliferation, apoptosis, invasion, and metastasis through regulating protein-coding mRNA expressions [20]. Over the past few years, a class of lncRNAs has been found to be aberrantly expressed in patients with HCC and to play a role in modulating malignant phenotypes, suggesting a new understanding of liver carcinogenesis [11]. Also, it has been shown that lncRNAs may be involved in almost every aspect of gene regulation, including transcription, mRNA splicing, translation, chromosome dosage-compensation, control of imprinting, chromatin modification, and intracellular trafficking [21,22]. Moreover, high expression levels of lncRNA NEAT1 possess a pivotal role in tumorigenesis and metastasis of HCC [23], and lncRNA AOC4P is a tumor suppressor for HCC through which vimentin degradation is promoted and epithelial-mesenchymal transition is suppressed [24]. In addition, J You et al. discovered that lncRNA SNHG1 expression was significantly upregulated in lung cancer cells when compared with normal bronchial epithelial cells [15]. However, to the best of our knowledge, there is limited research explaining the role of lncRNA SNHG1 in HCC. This retrospective study showed that lncRNA SNHG1 was overexpressed in HCC tissues compared with normal liver tissues. Similar results were obtained when we compared the expression of lncRNA SNHG1 between HCC cell lines HepG2 and normal liver cell lines L02. These results were consistent with the the reported function of lncRNA SNHG1 in lung cancer [15]. Furthermore, several clinicopathological features of HCC patients, including tumor size and TNM stage, were closely associated with higher lncRNA SNHG1 expression levels, further implying that lncRNA SNHG1 could contribute to HCC pathogenesis and progression. There is also reported evidence that lncRNAs may regulate miRNAs by acting as competing endogenous RNAs or by being processed into small RNAs [25]. Studies have reported that miR-195 plays an important role in inhibiting the development of various human cancers, including HCC, bladder, cervical, and breast cancers [26–29]. As suggested by previous studies, miR-195 targeted cyclin D1, CDK6, and E2F3 can suppress cell proliferation of human HCC cells [18]. Apart from that, miR-195 targets the TNF-α/NF-κB pathway and downregulates both IκB kinase alpha and TAB3 in HCC [30]. However, current studies have been unable to clarify the exact relationship between lncRNA and miR-195. After transfection with lncRNA SNHG1-mimic, HepG2 cells exhibited downregulated expression of miR-195, while transfection with miR-195 mimic did not significantly influence lncRNA SNHG1 expression. Therefore, we concluded that lncRNA SNHG1 inhibited miR-195 expression in HCC cells. Finally, bioinformatics target gene prediction enabled us to unfold the lncRNA SNHG1 binding sites in the 5′ regions of miR-195, and dual-luciferase report assay was subsequently performed to further verify that miR-195 was a direct target of lncRNA SNHG1. For the purpose of detecting whether lncRNA SNHG1 and miR-195 can influence HCC cell proliferation, invasion and migration status – which are closely related to HCC metastasis – in vitro experiments, including MTT assay, wound healing assay, and Transwell assays, were separately conducted using HepG2 HCC cells. HepG2 cells transfected with lncRNA SNHG1-mimic exhibited increased cell proliferation, invasion, and migration status, while miR-195 overexpression suppressed these cell activities. Therefore, lncRNA SNHG1 may play an important role in regulating human liver tumorigenesis through inhibiting miR-195 expression. This study had several limitations. Despite patients being consecutively selected over a one year period (from 2014 to 2015), we had a small sample size (due to the lack of resources), and selection bias may have occurred. Therefore, mice xenograft models should be carried out in future studies and tumor growth should be observed in vivo to further confirm the relationship between lncRNA SNHG1 and miR-195 in HCC. Conclusions In summary, this study suggests that overexpression of lncRNA SNHG1 can directly suppress miR-195 expression, which contributes to the pathogenesis and progression of HCC. Since HCC has extremely poor prognosis due to the lack of available curative therapeutic approaches [31], we believe that this study contributes to the understanding of the roles of lncRNAs in liver tumorigenesis, and these findings may be important for developing a potential therapeutic candidate for curing HCC. Source of support: This study was funded by the National Clinical Key Specialty Construction Program Figure 1 lncRNA SNHG1 exhibited high expression levels in both HCC tissues and HepG2 cell lines. (A) lncRNA SNHG1 and miR-195 expression was detected in HCC tissues and matched non-tumor tissues from HCC patients using hybridization in situ. (B) RT-PCR was used to measure lncRNA SNHG1 and miR-195 expression in HCC tissues and matched non-tumor tissues. (C) Relative expression levels of lncRNA and miR-195 were detected in HepG2 and L02 cells using hybridization in situ. (D) RT-PCR was used to measure lncRNA SNHG1 and miR-195 expression in HepG2 and L02 cells. lncRNA SNHG1 and miR-195 data were presented in the form of mean ± standard deviation (n=3). Similar results were obtained from three independent experiments. (* p<0.05). Figure 2 lncRNA SNHG1 inhibited miR-195 expression. Expression levels of lncRNA SNHG1 and miR-195 in HepG2 after transfection as mentioned above were detected by RT-PCR. lncRNA SNHG1 and miR-195 data were presented in the form of mean ± standard deviation (n=3). Similar results were obtained from three independent experiments (* P<0.05). Figure 3 lncRNA SNHG1 regulated proliferation, migration and invasion of HCC cells in vitro. (A) HepG2 cell proliferation was measured by MTT assay. (B) HepG2 cell migration and invasion status were restored after transfection as indicated earlier (* P<0.05). (C) HepG2 cells after transfection were performed by wound healing assays with a recovery period of 1–2 days. Wound areas were measured using the Image J software (* P<0.05). Figure 4 miR-195 directly targeted lncRNA SNHG1. (A) The lncRNA SNHG1-binding sequence in the 5′ region of miR-195. A mutation was generated in the miR-195 5′ region sequence in the complementary site for the seed of lncRNA SNHG1. (B) HEK293 cells seeded in 96-well plates were cotransfected with either the wild-type or mutant miR-195-5′ lncRNA SNHG1-binding seed region, together with lncRNA SNHG1 or pcDNA6.2-control. The relative luciferase values were measured and normalized to the values of Renilla luciferase activity in 48 hours. All data were indicated as the mean ± standard deviation (n=3). Similar results were obtained from three independent experiments (* P<0.05). Table 1 Relationship between lncRNA SHNG1 expression and clinicopathological features of 122 HCC patients. Clinicopathological features (n) lncRNA SNHG1(%) P value High expression (n=70) Low expression (n=52) Age (yr) >55 (66) 40 (60.6) 26 (39.4) 0.434 ≤55 (56) 30 (53.6) 26 (46.4) Gender Male (92) 54 (58.7) 38 (41.3) 0.606 Female (30) 16 (53.3) 14 (46.7) Alcohol consumption Low (90) 50 (55.5) 40 (44.5) 0.495 Excessive (32) 20 (62.5) 12 (37.5) Tumor size (cm) ≤5 (82) 38 (46.3) 44 (53.7) <0.001 >5 (40) 32 (80.0) 8 (20.0) Liver cirrhosis Yes (96) 56 (58.3) 40 (41.7) 0.682 No (26) 14 (53.8) 12 (46.2) HBsAg Positive (98) 56 (57.1) 42 (42.9) 0.916 Negative (24) 14 (58.3) 10 (41.7) HCV Ab Positive (22) 12 (54.6) 10 (45.4) 0.767 Negative (100) 58 (58.0) 42 (42.0) Metastasis With (20) 10 (50.0) 10 (50.0) 0.466 Without (102) 60 (58.9) 42 (41.2) TNM stage I–II (42) 16 (38.1) 26 (61.9) 0.002 III–IV (80) 54 (67.5) 26 (32.5) ==== Refs References 1 Roberts LR Sorafenib in liver cancer – just the beginning N Engl J Med 2008 359 420 22 18650519 2 Kuo TY Hsi E Yang IP Computational analysis of mRNA 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==== Front Med Sci MonitMed. Sci. MonitMedical Science MonitorMedical Science Monitor : International Medical Journal of Experimental and Clinical Research1234-10101643-3750International Scientific Literature, Inc. 2813423510.12659/MSM.898131898131Animal StudyEffects of Different Ratio of n-6/n-3 Polyunsaturated Fatty Acids on the PI3K/Akt Pathway in Rats with Reflux Esophagitis Zhuang Jia-Yuan 1BChen Zhi-Yao 2CZhang Tao 3DTang Du-Peng 4EJiang Xiao-Ying 1FZhuang Ze-Hao 5AG1 The School of Nursing, Fujian Medical University, Fuzhou, Fujian, P.R. China2 The Second Affiliated Hospital, Fujian Medical University, Fuzhou, Fujian, P.R. China3 Fuzhou Second Hospital Affiliated to Xiamen University, Fuzhou, Fujian, P.R. China4 People’s Hospital of Fujian Province, Fuzhou, Fujian, P.R. China5 The First Affiliated Hospital, Fujian Medical University, Fuzhou, Fujian, P.R. ChinaCorresponding Author: Ze-Hao Zhuang, e-mail: [email protected] Study Design B Data Collection C Statistical Analysis D Data Interpretation E Manuscript Preparation F Literature Search G Funds Collection 2017 30 1 2017 23 542 547 20 2 2016 23 3 2016 © Med Sci Monit, 20172017This work is licensed under Creative Common Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0)Background We designed this study to investigate the influence of different ratios of n-6/n-3 polyunsaturated fatty acid in the diet of reflux esophagitis (RE) rats’ and the effect on the PI3K/Akt pathway. Material/Methods RE rats were randomly divided into a sham group and modeling groups of different concentrations of n-6/n-3 polyunsaturated fatty acid (PUFA): 12:1 group, 10:1 group, 5:1 group, and 1:1 group. RT-PCR and Western-blot were used to detect the expression of PI3K, Akt, p-Akt, NF-κBp50, and NF-κBp65 proteins in esophageal tissue. Results In the n-6/n-3 PUFAs groups the expression of PI3K, Akt, p-Akt, nf-kbp50, and NF-κBp65 mRNA decreased with the decrease in n-6/n-3 ratios in the diet. The lowest expression of each indicator occurred in the 1:1 n-6/n-3 group compared with other n-6/n-3 groups, the difference was statistically significant (p<0.05). Conclusions The inhibition of n-3 PUFAs in the development of esophageal inflammation in rats with RE was attributed to the function of PI3K/Akt-NF-κB signaling pathway. MeSH Keywords Esophagitis, PepticFatty Acids, Omega-3Rats, Inbred ACI ==== Body Background Domestic as well as overseas scholars perceive that cellular inflammatory factors play important roles in reflux esophagitis (RE); nevertheless, the activation of inflammatory factors relies on the conduction of multiple signaling pathways inside and outside the cells. Some studies have confirmed that the phosphatidylinositol-3-kinase/serine/threonine kinase (PI3K/Akt) pathway is involved in most inflammatory reactions in vivo. The n-3 polyunsaturated fatty acid (PUFA) can modulate the gene expression of inflammatory factors through exerting effects on activation of related transcription factors in inflammatory pathways [1–5]. In recent decades, studies have illustrated that n-3 PUFAs as well as its metabolites (resolvins and protectin D1) are able to inhibit the generation of inflammatory factors and lessen cytokine response by inhibiting NF-κB, subsequently activating its immune regulation function [6,7]. Our study aimed to detect the expression of PI3K/Akt-NF-κB signaling pathway critical target protein in esophageal tissue of rats with reflux esophagitis by feeding reflux esophagitis rats with different ratio of n-6/n-3PUFAs in their diet, exploring its possible intervention mechanism. Material and Methods Experimental animals and forage Experimental animal: sterile male Sprague Dawley (SD) rats weighing 230±20 g were purchased from SLRC Laboratory Animal Center in Shanghai, and kept at 25°C with 12 hour dark/light cycle. There were five rats in each cage. Food: n-6PUFAs was supplied by “Golden Dragon Fish” brand sunflower seed oil; n-3 PUFAs was extracted from deep sea fish oil. The PUFAs were formulated into the pellet food in accordance with standard experimental protocols. Except for the PUFAs, other ingredients of the pellets were the same. The rats were fed at 9 am, as for the proxima luce. Remaining pellets were discarded, and subsequently, new food was added at a rate of 100 g/kg per rat every day. Preparation of animal models Lodophor was used to disinfect rats’ middle abdomen. About 1–2 cm laparotomy was performed at the xiphoid abdominal midline: the fur and abdominal muscles were with layered-cut. The peritoneum in the pylorus duodenal junction was isolated, avoiding blood vessels; loop ligature of nylon cable ties were used to create a predetermined inner diameter after the nylon tie passed through the pylorus duodenal junction. the redundant tie was snipped. The junction between forestomach and glandular stomach was ligated with 5-0 sutures. The stomach and duodenum were checked to ensure there was no hemorrhaging. The abdominal cavity was perfused using 0.5% metronidazole (1 mL) along with gentamicin (20,000 IU) and then closed using 5-0 sutures. Then 75% alcohol was used to disinfect the skin and the wound area. In the sham operation, the abdomen was opened, the stomach and duodenum were dissociated; and the abdominal cavity was closed after 1 minute. All surgery was done using asepsis conditions. The care of the animals conformed to the regulations of friendly treating experimental animals issued by the Ministry of Science and Technology in 2006. Animal grouping Seventy-five SD rats were randomly divided into sham operation and modeling intervention groups. The 15 rats that composed the sham operation group, were raised by common forage diet after receiving a sham operation, while the rest of the 60 rats in modeling intervention group were raised on different ratios of n-6/n-3PUFAs in their forage diet after they underwent modeling. According to different proportions of n-3PUFAs in the forage diet, we divided the 60 rats into four groups: 12: 1n-6/n-3 (12:1), 10:1 n-6/n-3 (10:1), 5: 1 n-6/n-3 (5:1) and 1: 1 n-6/n-3 (1:1) group. There were 15 rats in each group, subsequently they were fed with the forage diet with set ratios, and the esophageal tissue was removed after two weeks for testing. In the common forage diet, the ratio of n-6/n-3PUFAs reached 12: 1; therefore, we selected this group fed by common forage diet as the controls in our study. The quantitative detection of relevant indicators’ protein in esophageal tissue The total protein of 80 μg was extracted and SDS-PAGE gel electrophoresis used to transfer protein to PVDF film, that was then sealed using dried skimmed milk. The first antibody (1:100) was added for overnight incubation at 4°C; then the second antibody incubated for 2 hours at room temperature and subsequently developed using DAB. Quantity One Imaging Analysis Software was used to analyze the strip absorbance value. The detection of correlated indicators’ mRNA in esophageal tissue Total RNA was extracted using the Trizol method according to TaKaRa reverse transcription and amplification kit instructions. Related primer sequences are shown in Table 1. Statistical analysis SPSS 19.0 software was used for the analysis, mean ± standard deviation (χ̄±s) was used to describe the data which were represented by case (n); single factor analysis of variance was used to compare the data between multiple groups; LSD method was used to compare differences between two groups; and rank sum test was used to compare the data. The criterion of the test was a=0.05, p<0.05 indicating the differentiation had statistical significance. Results The expression of PI3K, Akt, p-Akt, NF-κBp50 and NF-κBp65 protein in RE rats’ esophageal tissue with different ratio of n-6/n-3PUFAs in the diet Western blot was utilized to detect the expression of PI3K, Akt, p-Akt, NF-κBp50, and NF-κBp65 protein in inflammation of esophageal mucosa of rats fed different ratio of n-6/n-3 PUFAs in the forage diet. Compared with sham operation group, the expression of PI3K, Akt, p-Akt, NF-κBp50, and NF-κBp65 protein in the modeling groups all were significantly increased with the differences being statistically significant (p<0.05); In the modeling groups with different concentration ratios of n-6/n-3PUFAs in the forage diets, the expression of PI3K, Akt, p-Akt, NF-κBp50, and NF-κBp65 protein kept pace with the proportion of n-6/n-3, in other words, if the ratio of n-6/n-3 decreased, the expression of PI3K, Akt, p-Akt, NF-κBp50, and NF-κBp65 protein decreased. The lowest expression of each indicator occurred when the proportion of n-6/n-3 reached 1: 1 in the forage diet; the difference was statistical significant compared with other ratios of n-6/n-3 in the forage diet of the modeling group (p<0.05) (Figures 1, 2). The expression of PI3K, Akt, NF-κBp50, and NF-κBp65 mRNA in reflux esophagitis rats’ esophageal tissue with different ratio of n-6/n-3PUFAs in the forage diet Compared with the sham operation group, the PI3K, Akt, NF-κBp50, and NF-κBp65 RNA in each modeling group all significantly increased, the difference was statistically significance (p<0.05); in the modeling group with different ratios of n-6/n-3PUFAs in the forage diet, if the ratio of n-6/n-3 decreased, subsequently the amount of PI3K, Akt, NF-κBp50, and NF-κBp65 RNA decreased. When the ratio of n-6/n-3 in the forage diet was 1:1, the expression of each indicator was the lowest. The difference was statistically significant compared with the other ratios in the forage diet (p<0.05) (Figure 3A–3D). Discussion There have been many studies investigating PI3K/Akt signaling pathway in tumorigenesis, tumor development, and metastasis; however, in most neoplastic diseases, the long-term and reduplicated stimulation is the potential element that may accelerate tumorigenesis, tumor development, as well as metastasis, which demonstrates that PI3K/Akt signaling pathway is associated with inflammation. Consequently, there have been more recent reports about the role of PI3K/Akt signaling pathway in inflammatory immune response. Some studies [8–11] have suggested that the inhibition of PI3K/AKT reduced the release of multiple inflammatory factors. Samuel et, al. [12] discovered that the expression of p-Akt and NF-κB in injured pancreatic tissue significantly increased with pancreatic duct ligation to induce animal models to experience acute pancreatitis, while expression of p-Akt and NF-κB significantly decreased after the ligation was loosened. Studies [13,14] on progression of esophageal lesions also reported that Akt was associated with esophageal lesions [15,16], meanwhile, the expression of p-Akt in pancreatic cancer and Barrett’s esophagus with serious dysplasia significantly increased compared with that in esophagus without dysplasia [17–22]. Conclusions Our study illustrated that the expression of the principle target molecules PI3K, Akt, p-Akt, and NF-κB genes and proteins involved in PI3K/Akt-NF-κB signaling pathway significantly increased, implying that the signaling pathway would likely play a lead role in the occurrence and development of reflux esophagitis. Our study also demonstrated through the detection of the critical target protein of PI3K/Akt-NF-κB signaling pathway in esophageal tissue, n-3 PUFAs could inhibit the expression of PI3K, p-Akt, and NF-κB, moreover, with the increasing ratio of n-3PUFAs in the forage diet, its inhibitory effect had the tendency to increase. The result indicated that the inhibition of n-3 PUFAs in the development of esophageal inflammation in rats with reflux esophagitis was attributed to the function of PI3K/Akt-NF-κB signaling pathway. Source of support: Departmental sources Figure 1 The expression of PI3K, Akt, p-Akt, NF-κBp50, and NF-κBp65 in esophageal tissue were tested by Western blot. 1 – The sham operation group; 2 – The 12:1 group; 3 – The 10:1 group; 4 – The 5:1 group; 5 – The 1:1 group. Figure 2 The comparison of different protein (PI3K, Akt, p-Akt, NF-κBp50, and NF-κBp65) expression in esophageal tissues of each group. * Represented p<0.05 vs. sham operation group; # standed for p<0.05 vs. blank control. Figure 3 (A) The comparison of the amount of PI3KmRNA in the esophageal tissue of RE rats with different ratios of n-6/n-3PUFAs in the forage diet. (B) The comparison of the amount of AktmRNA in the esophageal tissue of RE rats with different ratios of n-6/n-3PUFAs in the forage diet. (C) The comparison of the amount of NF-κBp50mRNA in the esophageal tissue of RE rats with different ratios of n-6/n-3PUFAs in the forage diet. (D) The comparison of the amount of NF-κBp65mRNA in the esophageal tissue of RE rats with different ratios of n-6/n-3PUFAs in the forage diet. * Represented p<0.05 vs. sham operation group; # standed for p<0.05 vs. blank control. Table 1 Related primer sequences. Primer Sequence (5′-3′) Product length PI3K Upstream 5′ TGGTTCTTGCGAAGTGAGATAG3′ 117 bp Downstream 5′ CTGCTGCGTGAAGTCCTGTA 3′ Akt Upstream 5′ TAGGCATCCCTTCCTTACAGC 3′ 114 bp Downstream 5′ CGCTCACGAGACAGGTGGA 3′ NF-κBp50 Upstream 5′ GGCAGAAGTCAACGCTCAG 3′ 142 bp Downstream 5′ TGTCGTCCCATCGTAGGT 3′ NF-κBp65 Upstream 5′ AGCGAGACCTGGAGCAAG 3′ 105 bp Downstream 5′ GGACCGCATTCAAGTCATAG 3′ β-actin Upstream 5′ TTCCAGCCTTCCTTCCTG 3′ 102 bp Downstream 5′ GGCATAGAGGTCTTTACGG 3′ ==== Refs References 1 Abliz A Deng W Sun R Wortmannin, PI3K/Akt signaling pathway inhibitor, attenuates thyroid injury associated with severe acute pancreatitis in rats Int J Clin Exp Pathol 2015 8 11 13821 33 26823696 2 Zhou Y Tu C Zhao Y Placental growth factor enhances angiogenesis in human intestinal microvascular endothelial cells via PI3K/Akt pathway: Potential implications of inflammation bowel disease Biochem Biophys Res Commun 2016 470 4 967 74 26775845 3 Liu HB Meng QH Huang C Nephroprotective effects of polydatin against ischemia/reperfusion injury: A role for the PI3K/Akt signal pathway Oxid Med Cell Longev 2015 2015 362158 26576221 4 Malemud CJ The PI3K/Akt/PTEN/mTOR pathway: A fruitful target for inducing cell death in rheumatoid arthritis? Future Med Chem 2015 7 9 1137 47 26132523 5 Zhou LT Wang KJ Li L Pinocembrin inhibits lipopolysaccharide-induced inflammatory mediators production in BV2 microglial cells through suppression of PI3K/Akt/NF-kappaB pathway Eur J Pharmacol 2015 761 211 16 26049009 6 Jones ML Mark PJ Keelan JA Maternal dietary omega-3 fatty acid intake increases resolvin and protectin levels in the rat placenta J Lipid Res 2013 54 8 2247 54 23723388 7 Li Y Wang X Li N Li J The study of n-3PUFAs protecting the intestinal barrier in rat HS/R model Lipids Health Dis 2014 13 146 25200333 8 Stiles BL PI-3-K and AKT: Onto the mitochondria Adv Drug Deliv Rev 2009 61 14 1276 82 19720099 9 Falasca M PI3K/Akt signalling pathway specific inhibitors: A novel strategy to sensitize cancer cells to anti-cancer drugs Curr Pharm Des 2010 16 12 1410 16 20166984 10 Huang JB Ding Y Huang DS Inhibition of the PI3K/AKT pathway reduces tumor necrosis factor-alpha production in the cellular response to wear particles in vitro Artif Organs 2013 37 3 298 307 23330804 11 Saleem M Afaq F Adhami VM Mukhtar H Lupeol modulates NF-kappaB and PI3K/Akt pathways and inhibits skin cancer in CD-1 mice Oncogene 2004 23 30 5203 14 15122342 12 Samuel I Yorek MA Zaheer A Fisher RA Bile-pancreatic juice exclusion promotes Akt/NF-kappaB activation and chemokine production in ligation-induced acute pancreatitis J Gastrointest Surg 2006 10 7 950 59 16843865 13 Beales IL Ogunwobi O Cameron E Activation of Akt is increased in the dysplasia-carcinoma sequence in Barrett’s oesophagus and contributes to increased proliferation and inhibition of apoptosis: A histopathological and functional study BMC Cancer 2007 7 97 17559672 14 Sagatys E Garrett CR Boulware D Activation of the serine/threonine protein kinase Akt during the progression of Barrett neoplasia Hum Pathol 2007 38 10 1526 31 17640711 15 Wang S Du Z Luo J Inhibition of heat shock protein 90 suppresses squamous carcinogenic progression in a mouse model of esophageal cancer J Cancer Res Clin Oncol 2015 141 8 1405 16 25563492 16 Fassan M Realdon S Pizzi M Programmed cell death 4 nuclear loss and miR-21 or activated Akt overexpression in esophageal squamous cell carcinogenesis Dis Esophagus 2012 25 3 263 68 21883657 17 Xiaoping L Xiaowei Z Leizhen Z Weijian G Expression and significance of CD44 and p-AKT in pancreatic head cancer World J Surg Oncol 2015 13 334 26666511 18 Ohtsubo K Yamada T Zhao L Expression of Akt kinase-interacting protein 1, a scaffold protein of the PI3K/PDK1/Akt pathway, in pancreatic cancer Pancreas 2014 43 7 1093 100 25036909 19 Jung KH Yan HH Fang Z HS-104, a PI3K inhibitor, enhances the anticancer efficacy of gemcitabine in pancreatic cancer Int J Oncol 2014 45 1 311 21 24819705 20 Liu J Cheng Sun SH Sun SJ Phosph-Akt1 expression is associated with a favourable prognosis in pancreatic cancer Ann Acad Med Singapore 2010 39 7 548 47 20697673 21 Chadha KS Khoury T Yu J Activated Akt and Erk expression and survival after surgery in pancreatic carcinoma Ann Surg Oncol 2006 13 7 933 39 16788754 22 Ozaki Y Tatebe S Ikeguchi M Molecular mechanism in pathogenesis of pancreatic neoplasms: p-Akt, PTEN Nihon Rinsho 2006 64 Suppl 1 41 43 [in Japanese] 16457218	28134235	PMC5295176	NO-CC CODE	2021-01-05 09:01:57	yes	Med Sci Monit. 2017 Jan 30; 23:542-547
==== Front Med Sci MonitMed. Sci. MonitMedical Science MonitorMedical Science Monitor : International Medical Journal of Experimental and Clinical Research1234-10101643-3750International Scientific Literature, Inc. 2815995610.12659/MSM.898740898740Molecular BiologyMicroRNA-495 Inhibits Gastric Cancer Cell Migration and Invasion Possibly via Targeting High Mobility Group AT-Hook 2 (HMGA2) Wang Huashe 1BCDEFJiang Zhipeng 1BCDFChen Honglei 2BCFWu Xiaobin 1CFXiang Jun 1BFPeng Junsheng 1ADE1 Department of Gastrointestinal Surgery, The Sixth Affiliated Hospital of Sun Yat-Sen University, Guangzhou, Guangdong, P.R. China2 Department of Digestive Endoscopic Center, The Sixth Affiliated Hospital of Sun Yat-Sen University, Guangzhou, Guangdong, P.R. ChinaCorresponding Author: Junsheng Peng, e-mail: [email protected] Study Design B Data Collection C Statistical Analysis D Data Interpretation E Manuscript Preparation F Literature Search G Funds Collection 2017 04 2 2017 23 640 648 27 3 2016 13 6 2016 © Med Sci Monit, 20172017This work is licensed under Creative Common Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0)Background Gastric cancer is one of the most common malignancies, and has a high mortality rate. miR-495 acts as a suppressor in some cancers and HMGA2 (high mobility group AT-hook 2) is a facilitator for cell growth and epithelial-mesenchymal transition (EMT), but little is known about their effect in gastric cancer. This study aimed to investigate the role and mechanism of miR-495 in gastric cancer. Material/Methods miR-495 levels were quantitatively analyzed in gastric cancer tissue and GES-1, SGC-7901, BGC-823, and HGC-27 cell lines by qRT-PCR. Levels of miR-495 and HMGA2 were altered by cell transfection, after which cell migration and invasion were examined by Transwell and E-cadherin (CDH1); vimentin (VIM), and alpha smooth muscle actin (ACTA2) were detected by qRT-PCR and Western blotting. The interaction between miR-495 and HMGA2 was verified by dual-luciferase reporter assay. Results miR-495 was significantly downregulated in cancer tissue and cell lines (p<0.05). Its overexpression inhibited cell migration and invasion, elevated CDH1, and inhibited VIM and ACTA2 levels in BGC-823 and HGC-27 cells. miR-495 directly inhibited HMGA2, which was upregulated in gastric cancer tissue, and promoted cell migration and invasion, inhibited CDH1, and elevated VIM and ACTA2. Conclusions miR-495 acts as a tumor suppressor in gastric cancer by inhibiting cell migration and invasion, which may be associated with its direct inhibition on HMGA2. These results suggest a promising therapeutic strategy for gastric cancer treatment. MeSH Keywords Epithelial-Mesenchymal TransitionHMGA2 ProteinMicroRNAsNeoplasm InvasivenessStomach NeoplasmsTranscellular Cell Migration ==== Body Background Gastric cancer is one of the most common malignancies worldwide and the second most important cause of tumor death after lung cancer, with an average five-year survival rate of less than 20% [1]. The highest incidence rate is found in East Asian countries such as China, Japan, and Korea. Risk factors include Helicobacter pylori infection (which is a predominant risk factor), dietary factors, tobacco use, and obesity [2]. Few specific symptoms can be observed in the earlier stages of gastric cancer, which can often lead to a delayed diagnosis. In addition, genetic complexity and heterogeneity of gastric cancer adds to the difficulty of effective treatment. Recent studies have uncovered blood-related factors [3,4] and possible cellular targets for drug treatment, such as vascular endothelial growth factor and epidermal growth factor receptor [5], but the biology and treatment options of gastric cancer still need further study. Recent studies found evidence that the progression and development of gastric cancer is closely related to the process of epithelial-mesenchymal transition (EMT) [6,7], a process also involved in other diseases [8,9]. Activated EMT endows gastric cancer cells with increased mesenchymal characteristics and promotes cell invasion and metastasis [6]. During EMT, the CDH1 gene-encoded E-cadherin switches to N-cadherin, which is usually expressed in mesenchymal cells [10]. Vimentin (VIM) is a positive regulator of EMT and a potential prerequisite for EMT induction [11]. Alpha smooth muscle actin (ACTA2) is also an early sign of EMT. A previous study indicated that HMGA2 (the high mobility group AT-hook 2) was a promotive factor in many malignant neoplasms, and induced EMT in gastric cancer [12]. However, additional research is required to reveal the complicated mechanisms involved in gastric cancer. MicroRNAs (miRNAs) are a group of small non-coding RNAs that exhibit powerful abilities in gene regulation via repressing translation or degrading mRNAs. Due to their effective regulation of gene expression, miRNAs play vital roles in modulating various diseases, including gastric cancer, through affecting cell cycle, proliferation, migration, invasion, and apoptosis, and further changing tumorigenesis and cancer metastasis [13]. For example, let-7 miRNA modulates an important RNA-binding protein Lin28 and influences the biological behavior of gastric cancer [14]. miR-495 plays a suppressive role in the post-transcription of FOXC1 (forkhead box C1) and thus inhibits endometrial cancer progression [15]. miR-495 has been shown in recent studies to participate in regulating gastric cancer cells via direct interaction with PRL-3 (phosphatase of regenerating liver-3) [16,17]. So miR-495 appears to be active in gastric cancer cells, although the specific mechanisms are as yet unknown. The aim of this study was to analyze the roles and pivotal mechanisms of miR-495 in gastric cancer. The expression pattern of miR-495 was examined in gastric cancer tissue and gastric cell lines of different differentiation degrees. Cell migration and invasion were detected after altering miR-495 levels in BGC-823 and HGC-27 cells. The aforementioned HMGA2 was predicted to be a target for miR-495 and we confirmed this interaction by luciferase reporter assay. The study results suggest a potential therapeutic strategy for gastric cancer treatment and add to the knowledge of the role of miRNAs in modulating gastric cancer. Material and Methods Tissue samples Human gastric cancer tissue and normal gastric tissue were collected from Tianjin Medical University Cancer Institute & Hospital (Tianjin, China). A total of 40 gastric cancer tissue samples from gastric cancer patients and 10 normal tissue samples from patients without gastric cancer were collected during gastrointestinal surgery or gastroscopy with the patients’ informed consent. The patients did not undergo radiotherapy or chemotherapy before their surgery. The 40 gastric cancer tissue samples included high-, moderate-, low-differentiation and un-differentiated tissue, each group containing 10 samples. The gastric cancer stages of the tissue samples were assessed by double-blind protocol by two pathologists according to the TNM staging system proposed by the Union for International Cancer Control/American Joint Committee on Cancer (UICC/AJCC) [18]. No apparent differences existed in the gender, age, or TNM stage between any two groups. This study was approved by a local ethics committee and performed based on the instructions of our institute. Cells Human gastric mucosal cell line GES-1 (Saierbio, Tianjing, China) and human gastric cancer cell lines SGC-7901, BGC-823, and HGC-27 (ATCC, Manassas, VA, USA) were used in this study for in vitro analyses. SGC-7901 had moderate differentiation, BGC-823 had low differentiation, and HGC-27 was undifferentiated [19]. The three cell lines were examined for levels of miR-495 expression. The cell lines were all cultured in Roswell Park Memorial Institute (RPMI)-1640 medium supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, 100 U/mL penicillin, and 100 mg/mL streptomycin (Gibco, Carlsbad, CA, USA) in humidified atmosphere with 5% CO2 at 37°C. The cells were passaged at a confluency of 70%. Cell transfection Cell transfection was performed to alter the level of miR-495 or HMGA2 in cell lines BGC-823 and HGC-27. The miR-495-specific precursor miRNA (pre-miR495), antisense oligo (ASO) and the corresponding controls were synthesized by Ambion (Carlsbad, CA, USA) to alter the miR-495 levels. The HMGA2-specific small interference RNA (siRNA) was synthesized by GenePharma (Shanghai, China) to inhibit the HMGA2 level. The coding sequence of HMGA2 was cloned into pcDNA3.1 vector (Thermo Scientific, Carlsbad, CA, USA) and sequenced to verify the correct ligation, and then pcDNA3.1-HMGA2 was transfected to overexpress HMGA2 and the blank vector was used as a control. Transfection was performed in antibiotic-free medium in 96-well plates with the help of Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Each transfection system contained 5×104 cells, 2 μL Lipofactamine 2000 and pre-miR-495 (10 nM), si-HMGA2 (20 nM), or pcDNA3.1-HMGA2 (200 ng). Further analyses were performed at 48 hours after transfection. Luciferase reporter assay The interaction between HMGA2 3′UTR and miR-495 was predicted by TargetScanHuman 7.0 (www.targetscan.org). To test the binding site of HMGA2 and miR-495, five bases in HMGA2 3′UTR were mutated using QuickChange Multi Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA, USA), and dual-luciferase reporter assay was performed on the wildtype (wt) or mutant (mt) HMGA2 3′UTR in BGC-823 and HGC-27 cells using the kit Dual-Luciferase Reporter Assay (Promega, Madison, WI, USA) according to the manufacturer’s instructions. The intensity of fluorescence signals was detected by a GENios Pro microplate reader (Tecan, Männedorf, Switzerland). Transwell assay Transwell assay was performed to detect the migration and invasion abilities of transfected BGC-823 and HGC-27 cells using Transwell sets (8 μm, BD Biosciences, San Jose, CA, USA). For cell invasion assay, Matrigel (BD Biosciences) was pre-coated to the upper side of the membrane, incubated at 37°C for 30 minutes for gel formation, and hydrated in serum-free medium for one hour before use. The cells were digested, washed in phosphate-buffered saline (PBS), resuspended in serum-free medium, and seeded in the upper chamber at a density of 5×105 cells/mL. The lower chamber was filled with medium containing 20% FBS, and then the set was assembled and incubated at 37°C for 24 hours. After incubation, the membrane was washed in PBS, fixed in methanol for 30 minutes, and dried. The cells remaining on the upper side were wiped away with cotton swabs, and the cells that had crossed the membrane were stained in 0.1% crystal violet (Beyotime, Shanghai, China) for 20 minutes. Stained cells were observed and counted under an optical microscope (Olympus, Tokyo, Japan). For cell migration assay, the same procedures were conducted but without Matrigel on the membrane. qRT-PCR qRT-PCR was performed to detect the relative levels of has-miR-495-3p, HGMA2, CDH1, VIM, and ACTA2 in gastric tissue and cell lines quantitatively. The tissue or cell samples were lysed in TRIzol (Invitrogen, Carlsbad, CA, USA) for total RNA extraction or RNAiso for Small RNA (Takara, Dalian, China) for miRNA extraction. First-strand complementary DNA (cDNA) was synthesized with random primers or the specific reverse transcription primer for hsa-miR-495-3p (5′-CTC AAC TGG TGT CGT GGA GTC GGC AAT TCA GTT GAG AAG AAG TG-3′) under the catalysis of PrimeScript Reverse Transcriptase (Takara). qRT-PCR was conducted on a QuantStudio 6 Flex Realtime PCR system (Applied Biosystems, Carlsbad, CA, USA), with 20 ng of cDNA and the specific primers for miR-495-3p (Fw: 5′-ACA CTC CAG CTG GGA AAC AAA CAT GGT GCA-3′ and Rv: 5′-TGG TGT CGT GGA GTC G-3′), HGMA2 (Fw: 5′-TGG TGC AAG ACT CAG GAG-3′ and Rv: 5′-CAG TCG GAA AGC AAA GG-3′), CDH1 (Fw: 5′-GCA TTG CCA CAT ACA CTC TCT TCT-3′ and Rv: 5′-TCG GTT ACC GTG ATC AAA ATC TC-3′), VIM (Fw: 5′-ACA TTG AGA TTG CCA CCT AC-3′ and Rv: 5′-AAC CGT CTT AAT CAG AAG TGT C-3′) or ACTA2 (Fw: 5′-AGC GTG GCT ATT CCT TCG T-3′ and Rv: 5′-CTC ATT TTC AAA GTC CAG AGC TAC A-3′) in each system. U6 (Fw: 5′-CTC GCT TCG GCA GCA CA-3′ and Rv: 5′-AAC GCT TCA CGA ATT TGC GT-3′) and GAPDH (Fw: 5′-GAA GGT GAA GGT CGG AGT C-3′ and Rv: 5′-GAA GAT GGT GAT GGG ATT TG-3′) were used as the internal references for miR-495 and other genes, respectively. Data were analyzed with the 2−ΔΔCt method. Western blot Total protein from tissue samples and cells were extracted by M-PER Mammalian Protein Extraction Reagent (Thermo Scientific) according to the manufacturer’s instructions. Protein samples (20 μg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes. The membranes were blocked in 5% skim milk at room temperature for two hours and then incubated in the specific primary antibodies for HMGA2 (ab97276, Abcam, Cambridge, UK), CDH1 (ab181860), VIM (ab92547), and ACTA2 (ab32575) at 4°C overnight. GAPDH (ab8245) was used as an internal reference. After washing in PBS, the membranes were incubated in horseradish peroxidase-conjugated secondary antibody at room temperature for one hour, after which positive signals were developed by EasyBlot ECL Kit (Sangon Biotech, Shanghai, China) and the relative density of bands was analyzed by ChemiDoc XRS System (Bio-Rad, Hercules, CA, USA). Statistical analysis All experiments were repeated in triplicate and data were represented as the mean ± standard deviation. Data were first analyzed by F test for homoscedasticity and then t test for significant difference using SPSS 20 (IBM, New York, USA). χ2 test was performed to examine the differences in gender, age, and TNM stages between any two tissue groups. A value of p<0.05 was considered significantly different between groups. Results miR-495 is downregulated in gastric cancer tissue and cell lines The expression level of miR-495 was compared in gastric cancer tissue and normal tissue by qRT-PCR and inhibited miR-495 levels were found in cancer tissue of different differentiation degrees (Figure 1A). Although no significant suppression was detected in the highly differentiated tissue compared to normal tissue, miR-495 was markedly downregulated in tissue samples of moderate differentiation compared to high differentiation (p<0.001), and was further inhibited in the low-differentiation and undifferentiated tissue samples (p<0.001). Its level was also examined in normal gastric cell line GES-1 and three gastric cancer cell lines of different differentiation degrees, and a suppressed miR-495 pattern was also observed. No significant downregulation was detected in moderately-differentiated SGC-7901, but miR-495 was obviously inhibited in low-differentiated BGC-823 and undifferentiated HGC-27 compared to GES-1 (p<0.05, Figure 1B). Taken together, there might be a potential correlation between miR-495 level and gastric cancer differentiation degrees. These results suggest that miR-495 might play a role in gastric cancer progression, which was analyzed in additional experiments. miR-495 inhibits gastric cancer cell migration and invasion Because cell lines BGC-823 and HGC-27 possessed marked miR-495 changes, the two cell lines were used in cell transfection to alter miR-495 levels, and the results showed effective upregulation or downregulation of miR-495 by transfecting pre-miR-495 or miR-495 ASO (p<0.05 and p<0.01, respectively, Figure 2A). Transwell assays showed that miR-495 overexpression decreased migrated and invasive cell numbers in the two cell lines (p<0.05 and p<0.01, respectively, Figure 2B, 2C), and its downregulation increased migrated and invasive cells (p<0.05), indicating that miR-495 might suppress cell migration and invasion in gastric cancer cell lines. Then we examined three EMT-related factors. qRT-PCR showed that the EMT inhibitor CDH1 mRNA was promoted, while the EMT indicators VIM and ACTA2 mRNA levels were inhibited by miR-495 overexpression in BGC-823 cells (p<0.01 and p<0.05, respectively, Figure 2D). Opposite effects were found when miR-495 was inhibited (p<0.05 and p<0.001, respectively). Western blot analysis showed similar changing patterns in the protein levels of these factors (Figure 2E). So miR-495 might regulate CDH1, VIM, and ACTA2, and suppress EMT. HMGA2 is a direct target of miR-495 and has opposite functions against miR-495 As predicted by TargetScanHuman 7.0, HMGA2 mRNA was one target of miR-495, with the sequence “GUUUGUUG” being the predicted binding site. Five bases were mutated (Figure 3A) and both the wildtype and mutant type of HMGA2 3′UTR were applied to dual-luciferase reporter assay. Results showed that in cell lines BGC-823 and HGC-27, the overexpressed miR-495 could only inhibit the luciferase activity of HMGA2 3′UTR-wt (p<0.01 and p<0.05, respectively, Figure 3B), suggesting that HMGA2 3′UTR-wt, rather than the mutant type, could directly interact with miR-495. Then both mRNA and protein levels of HMGA2 were detected in the transfected BGC-823 cells, and results showed that HMGA2 mRNA and protein expression was inhibited by the upregulated miR-495 and promoted by miR-495 ASO (p<0.01 and p<0.05, respectively, Figure 3C–3E). Taken together, this indicates that miR-495 could directly bind to and inhibit HMGA2. In gastric cancer tissue, HMGA2 mRNA and protein levels were significantly upregulated compared to the adjacent normal tissue (Figure 4A, 4B). In addition, miR-495 inhibited HMGA2 in gastric cancer cells as mentioned earlier; we suspected that HMGA2 had the opposite effects from miR-495 on BGC-823 and HGC-27 cells, and thus altered HMGA2 expression in the two cell lines (Figure 4C). Actually, HMGA2 overexpression significantly induced the migration and invasion abilities in both cell lines, and si-HMGA2 suppressed these abilities (Figure 4D, 4E). Besides, HMGA2 inhibited CDH1 and promoted VIM and ACTA2, as shown in qRT-PCR and Western blot results (Figure 4F, 4G). Thus miR-495 and HMGA2 had distinct effects on gastric cancer cells, and HMGA2 might be related to the mechanism of miR-495 in gastric cancer cells. Discussion In the present study, we examined the significantly downregulated level of miR-495 in gastric cancer tissue and cell lines compared to normal tissue and cell lines. The overexpression of miR-495 in cell lines BGC-823 and HGC-27 suppressed cell migration and invasion, and regulated CDH1, VIM, and ACTA2. HMGA2 was a direct target for miR-495 and its overexpression promoted cell migration and invasion in gastric cancer cell lines, which were effects opposite to miR-495. As aforementioned, EMT plays a vital role in the progression and development of gastric cancer, and pivotal factors like CDH1 [20,21], VIM [22], and ACTA2 [23] are involved in this process. In our study, miR-495 overexpression led to the elevation of CDH1 and inhibition of VIM and ACTA2, which might imply a regulated EMT process in the transfected cell lines. A previous study showed that miR-495 inhibits EMT-related factors like Snail and SLUG to suppress EMT [24]. Thus, it is reasonable to speculate that miR-495 may impact EMT in gastric cancer cells, which will be an interesting topic for further investigation. HMGA2 is a nuclear-binding protein that exhibits vital functions in cell growth and differentiation, and its overexpression has been reported in various neoplasms [25,26]. Its regulation on VIM and CDH1 is involved in overexpression-induced EMT [27]. In accordance with these previous findings, the overexpression of HMGA2 in gastric cancer cells led to expression changes in CDH1, VIM, and ACTA2. In addition to the regulation effect of these factors, HMGA2 was upregulated in gastric cancer tissue compared to normal tissue, and its overexpression promoted cell migration and invasion in BGC-823 and HGC-27 cells, suggesting it has a distinct function related to miR-495. Luciferase reporter assay indicated HMGA2 was a direct target for miR-495, which further supports that HMGA2 may be involved in the functional mechanism of miR-495 in gastric cancer cells. In the transfected cells, miR-495 overexpression could inhibit both mRNA and protein levels of HMGA2. Similar results were found in other studies regarding miR-495 regulation of the translation process as well as mRNA stability of its targets [28,29]. However, miR-495 only regulates the translation process of some targets, without affecting their mRNA levels [30]. Studies have found evidence that the sequence signatures, such as A/U content, of target mRNAs are crucial for mRNA degradation induced by miRNAs [31,32]. So based on our results, it is likely that miR-495 inhibits HMGA2 expression via inducing HMGA2 mRNA degradation, or at the same time suppressing the translation progress. miR-495 was been shown to be downregulated in gastric cancer tissue and cell lines, such as BGC-823 and HGC-27, and it suppressed cell migration and invasion in these two cell lines, suggesting its suppressive role in gastric cancer. Similar effects of miR-495 have been found in SGC-7901 cells, where transfection of miR-495 mimics suppressed cell migration and invasion, as revealed by Transwell assay [16]. In addition, earlier studies detected significant downregulated miR-495 in SGC-7901 cells, while our study also suggested that miR-495 was inhibited in SGC-7901 cells and GES-1 cells, although no significant difference was found. We suspect that the disparity may have been generated from the different methods of miR-495 overexpression, which needs further testing. Similar results have also been reported in leukemia [33], non-small cell lung cancer [34], prostate cancer [35], Dicer-deficient acini, and pancreatitis-induced metaplasia [36], where miR-495 functions as a tumor suppressor inhibiting cell migration and invasion. The control of miR-495 level may rely on the methylation status of the miR-495 gene promoter or the regulation by transcription factors like E12/E47, as found in gastric cancer cells and breast cancer stem cells [17,37]. Further research on the functional mechanism and regulation strategies of miR-495 is necessary before applying them to gastric cancer treatment. Conclusions In summary, miR-495 is a tumor suppressor in gastric cancer, suppressing gastric cancer cell migration and invasion, possibly via its direct regulation of HMGA2. This study provides a potential therapeutic strategy focusing on miR-495 for gastric cancer treatment. Future investigations will concentrate on more detailed mechanisms. Source of support: Departmental sources Conflicts of interests There are no conflicts of interest. Figure 1 miR-495 was downregulated in gastric cancer tissue and cell lines. (A) miR-495 was significantly downregulated in gastric cancer tissue compared to normal tissue (n=10). The gastric cancer tissue samples were divided into different groups based on their differentiation degree: highly differentiated (high), moderately differentiated (moderate), lowly differentiated (low) or undifferentiated (un). NS, not significant. *** p<0.001. (B) miR-495 was significantly downregulated in gastric cancer cell lines BGC-823 and HGC-27. NS – not significant compared to GES-1. * p<0.05 compared to GES-1. Figure 2 miR-495 inhibited cell migration and invasion in BGC-823 and HGC-27 cell lines and regulated EMT factors. Pre-miR-495, cells were transfected with miR-495 precursor to overexpress miR-495. miR-495 ASO, cells transfected with miR-495 antisense oligo (ASO) to inhibit miR-495. Pre-ASO control, the corresponding control group of pre-miR-495 or miR-495 ASO. qRT-PCR, Western blot and Transwell assays were performed at 48 hours after transfection. (A) miR-495 was successfully promoted or inhibited by cell transfection in BGC-823 and HGC-27 cells. (B) miR-495 inhibited, and its ASO promoted, cell migration in BGC-823 and HGC-27 cells. (C) miR-495 inhibited, and its ASO promoted, cell invasion in BGC-823 and HGC-27 cells. (D) miR-495 upregulated CDH1 and downregulated VIM and ACTA2 mRNA levels in BGC-823 cells. (E) miR-495 upregulated CDH1 and downregulated VIM and ACTA2 protein levels in BGC-823 cells. GAPDH mRNA and protein were used as internal references for qRT-PCR and Western blot, respectively. * p<0.05. p<0.01. * p<0.001. CDH1 – E-cadherin. VIM – vimentin. ACTA2 – alpha smooth muscle actin. Figure 3 miR-495 directly binds to and inhibits HMGA2. pre-miR-495, cells were transfected with miR-495 precursor to overexpress miR-495. miR-495 ASO, cells transfected with miR-495 antisense oligo (ASO) to inhibit miR-495. Pre-ASO control, the corresponding control group of pre-miR-495 or miR-495 ASO. wt, wildtype. mut, mutant type. (A) A schematic diagram of HMGA2 3′UTR-wt, and HMGA2 3′UTR-mut and their predicted interaction with miR-495. The mutated bases are underlined. (B) Dual-luciferase reporter assay results of altered HMGA2 3′UTR activity by miR-495 in cell lines BGC-823 and HGC-27. (C) qRT-PCR showing relative level of HMGA2 mRNA in transfected BGC-823 cells. (D) Relative HMGA2 protein level in transfected BGC-823 cells based on the Western blot result, as shown in (E). GAPDH mRNA and protein were used as internal references for qRT-PCR and Western blot, respectively. NS – not significant. * p<0.05. ** p<0.01. HMGA2 – high mobility group AT-hook 2. Figure 4 HMGA2 is upregulated in gastric cancer tissue and promotes cell migration and invasion in gastric cancer cell lines. (A) qRT-PCR showing relative HMGA2 mRNA level in gastric cancer tissue and the adjacent normal tissue. (B) Western blot showing HMGA2 protein level in gastric cancer tissue and the adjacent normal tissue. (C) HMGA2 protein level was successfully elevated by HMGA2 overexpression vector (HMGA2) and inhibited by its specific siRNA (si-HMGA2). (D) Transwell assay indicating that HMGA2 increased migrated cells in BGC-823 and HGC-27. (E) Transwell assay indicating that HMGA2 increased invasive cells in BGC-823 and HGC-27. (F) HMGA2 promoted CDH1 and inhibited VIM and ACTA2 mRNA levels in BGC-823 cells. (G) HMGA2 promoted CDH1 and inhibited VIM and ACTA2 protein levels in BGC-823 cells. GAPDH mRNA and protein were used as internal references for qRT-PCR and Western blot, respectively. * p<0.05. p<0.01. * p<0.001. HMGA2, high mobility group AT-hook 2. CDH1 – E-cadherin. VIM – vimentin. 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==== Front Open Access Maced J Med SciOpen Access Maced J Med SciOpen Access Macedonian Journal of Medical Sciences1857-9655ID Design 2012/DOOEL Skopje Republic of Macedonia 28293320OAMJMS-5-06810.3889/oamjms.2017.010StomatologysTNF-R Levels: Apical Periodontitis Linked to Coronary Heart Disease Singhal Rajnish K. 1Rai Balwant 21 Department of Conservative Dentistry and Endodontics, Maharana Pratap College of Dentistry and Research Centre, Gwalior (M.P), India2 World Dental Networks, Denmark, Chief, JBR Society, India; Director, KSI, USA Correspondence: Dr. Rajnish K Singhal, MDS. Prof. and Head, Dept of Conservative Dentistry and Endodontics, Maharana Pratap College of Dentistry and Research Centre, Gwalior (M.P), India. E-mail: [email protected] 2 2017 17 1 2017 5 1 68 71 27 11 2016 28 12 2016 29 12 2016 Copyright: © 2017 Rajnish K. Singhal, Balwant Rai.2017This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License (CC BY-NC 4.0).BACKGROUND: Different studies have implicated the exposure to systemic conditions in the aetiology of cardiovascular diseases like chronic inflammation including chronic periodontitis. AIM: The present study has been conducted to examine whether biomarker sTNF-R was elevated in apical periodontitis as sTNF-R is a systemic marker of inflammation and has been identified as risk factors for cardiovascular diseases. MATERIAL AND METHODS: sTNF-R levels were measured in 52 patients with apical periodontitis (M:F::25:27), aged 20-45 years and in 20 control patients without periodontitis (M:F::10:10, aged 20-48 years). Measurement of sTNF-R1 and sTNF-R2 was carried out in duplicate with standardised, commercially available enzyme immunoassays (R&D Systems Europe, Abingdon, UK). RESULTS: The mean sTNF-R1 and sTNF-R2 levels in periodontitis were 820 (240) pg/ml (413 – 1620 pg/ml) and 1309 (403) pg/ml (540 – 2430 pg/ml), while in normal sTNF-R1 and sTNF – R2 levels were 740 (340) pg/ml (407-1240 pg/ml) and 1283 (414) pg (480 – 2340 pg/ml) respectively. Results indicated a positive high relationship between cardiovascular markers such as sTNF-R1 and sTNF – R2 and apical periodontitis. CONCLUSION: Elevated levels of sTNF-R1 and sTNF – R2 in apical periodontitis patients indicate an increased independent risk of coronary heart disease. sTNF-RApical PeriodontitisSystemic inflammatorycoronary heart disease ==== Body Introduction Periodontal disease is a chronic infection of periodontal tissue characterised by the loss of attachment between tooth and bone, and by the bone loss. Epidemiological associations between periodontitis and cardiovascular disease have been reported [1, 2]. This association could be attributed to the direct action of periodontal pathogens or their products on endothelial cells through transient bacteremia or ultimately due to products of the inflammatory response [3-5]. Advanced stages of dental caries lead to apical periodontitis. Periodontitis and atherosclerosis have complex etiologies, genetic and gender pre-dispositions and might share pathogenic mechanisms as well as general risk factors. Also, increased levels of chronic inflammatory marker CRP, serum LDL-C and t-PA (a parameter of endothelial function) have been related to increased cardiovascular risk [6-8]. Tumour necrosis factor (TNF-alpha) plays a key role in the initiation of the inflammatory response [9]. TNF-alpha has been linked with CVD risk factors, and with carotid intima – media thickness [10]. TNF receptors (sTNFR1 and sTNF-R2) are markers of TNF activity [11]. TNF has also been implicated in the pathogenesis of some cardiovascular diseases, including atherosclerosis, heart failure, myocardial infarction, myocarditis and cardiac allograft rejection, and vascular endothelial cell responses to TNF might underlie the vascular pathology in many of these conditions. This might be because TNFR1 and TNFR2 differentially regulate cardiac responses to TNF. In transgenic mice with TNF-induced cardiomyopathy, ablation of the TNFR2 gene aggravates heart failure and reduces survival, whereas ablation of TNFR1 blunts heart failure and improves survival [12, 13]. In cardiac allografts either TNF receptor is capable of mediating a response that will culminate in graft arterial disease [14]. Patients with chronic inflammatory conditions such as rheumatoid arthritis have the higher incidence of cardiovascular disease. Inflammatory mediators, including TNF, have been concerned with higher cardiovascular risk, and there is some evidence that anti-TNF therapy ameliorates this risk in patients with rheumatoid arthritis [15-20]. A good correlation has been observed between saliva and serum concentrations of biomarkers [21]. Therefore sampling of saliva is advantageous since non-invasive, stress-free, easy and frequent collections are possible [21]. Hence, if periodontal disease is found to be associated with sTNFR1 and sTNF-R2, it might be a potential mediator for the association between apical periodontitis and CVD (Cardiovascular diseases). Our study aimed to assess whether serum sTNFR1 and sTNF-R2 was elevated in apical periodontitis as sTNF-R is a systemic marker of inflammation and has been identified as risk factors for cardiovascular diseases. Material and Methods The total sample of 72 patients was quantified into two groups. Out of 72 patients, fifty-two patients were diagnosed with apical periodontitis (M: F::25:27, aged 20-45 years. Twenty (M: F::10:10, aged 20-48 years) subjects with in normal periodontal condition were selected for control. One or more apical lesions due to dental caries in teeth with non-vital pulp were taken as diagnostic criteria of apical periodontitis [16]. Periodontal parameters such probing depth and clinical loss attachment were measured by William probe. Subjects were excluded from the study if they were the chronic alcoholic or chronic smoker since they are known predisposing factors for periodontitis. None of the subjects selected had any history of a chronic inflammatory disease, diabetes, hypertension or use of steroids or drugs. From all subjects, 10 ml EDTA blood was sampled at before treatment. After cooling centrifugation (10 min at 4°C and 3000 revs/min), the plasma was frozen at -80°C in 250 ml aliquots for up to 30 days. sTNF-R1 and sTNF-R2 were analysed by ELISA kit. Measurement of sTNF-R1 and sTNF-R2 was carried out in duplicate with standardised, commercially available enzyme immunoassays (R&D Systems Europe, Abingdon, UK). The data was statistically analysed using SPSS version 11.0, and Student t-test was applied. Results There was no significant difference in socio-demographic status between two groups (Table 1). Table 1 Socio-demographic characteristics of periodontitis and normal healthy Variables Number in % P value Periodontitis subjects Normal (control) subjects Age in years 0.076 17-25 33 30.1 26-32 29.1 28.9 More than 32 37.9 41 Mean (SD) 29.45 (6.34) 29.34 (5.62) Education Status 0.726 Less than high school 41.6 45.9 High school 31 27.4 More than high school 27.4 26.7 Job status. 0.789 Not employed 90.2 89.2 Employed 9.8 10.8 sTNF-R1 and sTNF–R2 levels and clinical periodontal profile were significantly higher in apical periodontitis patients as compared to normal patients without periodontitis (Table 2). The mean intra-observer agreement was 96.4%, and the mean inter-observer agreement was 94.2%. Table 2 Different clinical parameters of periodontal profile and sTNF-R1 and sTNF-R2 levels in periodontitis and normal healthy control Periodontitis Normal P value Average number of periodontal involved site 9.2 ± 1.2 2.0 ± 1.8 0.001 Average probing pocket Depth (in mm) (William probe) 6.3 ± 1.2 1.3 ± 1.2 0.001 Average clinical loss of attachment (in mm) 5.2 ± 1.3 1.7 ± 1.5 0.001 sTNF-R1 (pg/ml) 820 ± 240 740 ± 340 0.03 sTNF R2 (pg/ml) 1309 ± 403 1283 ± 414 0.04 Data observed that a positive significant relationship between sTNF-R1 and sTNF – R2 cardiovascular disease markers and periodontal disease clinical parameters markers such as an average number of periodontally involved site, probing depth and clinical loss of attachment (Table 3). Table 3 Bivariate correlations between cardiovascular markers and Markers of periodontal in periodontitis patients (after adjusting the age and gender) Variables sTNF-R1 sTNF-R2 Average number of periodontal involved site 0.49 0.42 Average probing pocket (Depth) 0.49 0.43 Average clinical loss of attachment (in mm) 0.69 0.66 Discussion Our study showed the correlation between apical periodontitis and cardiovascular marker TNF receptors. The sTNF-R1 and sTNF-R2 levels were considerably higher in periodontitis patients as compared to normal patients without periodontitis. TNFα is one of the major pro-inflammatory cytokines [10-13]. It’s role in the pathogenesis of chronic inflammatory diseases has been long established, and serves as a source for the novel anti-cytokine therapies lately introduced [10]. An increased TNF secretion without corresponding higher levels in sTNFR shedding advocates a relative deficiency in sTNFR and an increase in the bioavailable TNF. This secretion disproportion between TNF and its soluble receptor had been detected in some chronic inflammatory disorders and had been implicated in their pathogenesis [10-11]. Previous studies reported that no associations between periodontal disease and TNF receptors [10]. This might be due to small sample size or less inflammation observed in the studies. TNF-alpha has been associated with CVD risk factors, and with carotid intima-media thickness [11]. It has been observed that genetic variation at gene locus for TNF-alpha affect the risk of preterm birth independently and as interacting factors [12-15]. Many Studies found that levels of these biomarkers during acute infection revealed levels of sTNF-R ten-fold or greater than those reported in the present study. While differences were statistically significant [13-17], but the clinical significance of these differences was not observable. This could be attributed to the fact that periodontal infection was not so acute and severe as compared to the cardiovascular disease. TNF-α is a potent inflammatory cytokine. The main source of TNF-Tα is activated mononuclear leukocytes, though it is concealed by a broad variety of other immune and nonimmune cell types, including fibroblasts, smooth muscle cells, astrocytes, and neurones [15]. TNF receptor 1 (also known as p55) and TNF receptor 2 (also known as p75) are both soluble receptors discard by different cell types on which they reside [15, 16, 18]. Elevation of TNF-.α and TNF receptor levels occurs in a variety of infectious, autoimmune, inflammatory, and neoplastic diseases. Elevated levels of TNF receptor might be a reflection of the inflammatory mechanisms operative in the atherosclerotic plaque. Macrophages and T-lymphocytes are important in human atheromas, still at the earliest stages of the disease process [16, 17], suggesting that immune processes might play an early role in the development of the lesion in human beings. Our data provides evidence for at least a partial role for activated leukocytes in the chronic process of periodontitis. It has been demonstrated that patients with advanced CHF had increased concentrations of circulating TNF, especially those who were cachectic. Numerous studies have confirmed that CHF is a state of inflammatory cytokine activation [18]. It has been speculated that the association between elevated levels of inflammatory markers and periodontitis reflects chronic subclinical infection, although this hypothesis awaits confirmation. Several observational epidemiological studies [18-20], have suggested an association between chronic infections such as Chlamydia pneumonia and periodontitis and stroke risk or carotid atherosclerosis. Nonetheless, the elevations in TNF receptor levels seen here could also be related to the presence of other noninfectious stimulants of inflammation. Further prospective studies of the relationship between TNF receptors and other inflammatory and infectious markers are needed. While many investigators have examined the relationship between inflammation, infection, periodontitis and atherosclerotic heart disease, these may not reflect the relationship between these processes and stroke. Further study is required on a large scale while considering the risk factors and effect of apical periodontitis treatment on TNF receptor levels. In conclusion, elevated levels of sTNF-R1 and sTNF–R2 in apical periodontitis patients indicate an increased independent risk of coronary heart disease. Funding: This research did not receive any financial support. 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==== Front Int J OncolInt. J. OncolIJOInternational Journal of Oncology1019-64391791-2423D.A. Spandidos 2819764010.3892/ijo.2017.3871ijo-50-03-0893ArticlesSB-T-121205, a next-generation taxane, enhances apoptosis and inhibits migration/invasion in MCF-7/PTX cells Zheng Xiaowei 12Wang Changwei 34Xing Yuanming 5Chen Siying 1Meng Ti 1You Haisheng 1Ojima Iwao 36Dong Yalin 11 Department of Pharmacy, The First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, Shaanxi 7100612 Department of Pharmacy, Xi'an No.1 Hospital, Xi'an, Shaanxi 710002, P.R. China3 Department of Chemistry, Stony Brook University - State University of New York, Stony Brook, NY 11794-3400, USA4 Guangzhou Institute of Biomedicine and Health (GIBH), Chinese Academy of Sciences (CAS), Science Park, Guangzhou, Guangdong 5105305 Hou Zonglian Medical Experimental Class of 2014, Xi'an Jiaotong University, Xi'an, Shaanxi 710061, P.R. China6 Institute of Chemical Biology and Drug Discovery, Stony Brook University - State University of New York, Stony Brook, NY 11794-3400, USACorrespondence to: Professor Yalin Dong, Department of Pharmacy, The First Affiliated Hospital of Xi'an Jiaotong University, No. 277 Yanta West Road, Xi'an, Shaanxi 710061, P.R. China, E-mail: [email protected] Iwao Ojima, Department of Chemistry, Institute of Chemical Biology and Drug Discovery, Stony Brook University-State University of New York, Stony Brook, NY 11794-3400, USA, E-mail: [email protected]* Contributed equally 3 2017 10 2 2017 10 2 2017 50 3 893 902 26 9 2016 23 1 2017 Copyright © 2017, Spandidos Publications2017Breast cancer is the leading cause of cancer death among women. Paclitaxel, a mitotic inhibitor, is highly effective in the treatment of breast cancer. However, development of resistance to paclitaxel limits its clinical use. Identifying new compounds and new strategies that are effective against breast cancer, in particular drug-resistant cancer, is of great importance. The aim of the present study was to explore the potential of a next-generation taxoid, SB-T-121205, in modulating the proliferation, migration and invasion of paclitaxel-resistant human breast cancer cells (MCF-7/PTX) and further evaluate the underlying molecular mechanisms. The results of MTT assay showed that SB-T-121205 has much higher potency to human breast cancer cells (MCF-7/S, MCF-7/PTX and MDA-MB-453 cells) than paclitaxel, while that the non-tumorigenic human bronchial epithelial cells (BEAS-2B) were slightly less sensitive to SB-T-121205 than paclitaxel. Flow cytometry and western blot methods revealed that SB-T-121205 induced cell cycle arrest at the G2/M phase and apoptosis in MCF-7/PTX cells through accelerating mitochondrial apoptotic pathway, resulting in reduction of Bcl-2/Bax ratio, as well as elevation of caspase-3, caspase-9, and poly(ADP-ribose) polymerase (PARP) levels. Moreover, SB-T-121205 changed epithelial-mesenchymal transition (EMT) property, and suppressed migration and invasion abilities of MCF-7/PTX cells. Additionally, SB-T-121205 exerted antitumor activity by inhibiting the transgelin 2 and PI3K/Akt pathway. These findings indicate that SB-T-121205 is a potent antitumor agent that promotes apoptosis and also recedes migration/invasion abilities of MCF-7/PTX cells by restraining the activity of transgelin 2 and PI3K/Akt, as well as mitochondrial apoptotic pathway. Such results suggest a potential clinical value of SB-T-121205 in breast cancer treatment. breast cancertaxaneSB-T-121205apoptosismigrationinvasion ==== Body Introduction Breast cancer is the most frequently diagnosed cancer and the leading cause of cancer-related mortality among females worldwide (1). Currently, standard therapies for breast cancer patients include surgery, radiotherapy and chemotherapy, which plays an irreplaceable role (2). Paclitaxel (PTX; Fig. 1A), a first-line therapeutic agent used clinically to treat breast cancer, exerts its antitumor activity by promoting the polymerization of tubulin and stabilizing the resulting micro-tubules, causing cell cycle arrest at the G2/M phase that leads to apoptotic death of cancer cells (3). However, paclitaxel resistance often occurs after a period of treatment, causing a serious problem in chemotherapy (4). Accumulated studies manifest that there are several major mechanisms for paclitaxel resistance, including point mutations in β-tubulin, alterations in the expression of β-tubulin isotypes, particularly the β-III tubulin isoform, overexpression of ATP-binding cassette transporters and suppression of apoptosis. More importantly, the drug-resistance to chemotherapy may eventually result in mortality of cancer patients due to tumor metastasis (5,6). It is therefore highly desirable to develop novel agents with minimum side-effects and improved activity against various tumors, especially against drug-resistant human breast cancer. In the last decades, a number of taxanes have been designed and synthesized based on the structure of the first-generation taxanes, paclitaxel and docetaxel. Many of these new taxanes exhibited strong antitumor activity against paclitaxel-resistant tumor xenografts. For instance, Roh et al (7) found two of the 3′-N-acyl-paclitaxel analogues, in which the phenyl group of 3′-N-benzoyl was replaced with 1-cyclopentenyl (1k) and 1-cyclohexenyl (1n), displayed seven times higher cytotoxicity than paclitaxel against doxorubicin-resistant breast cancer cells. It was also reported that another taxane, Lx2-32c, was active against paclitaxel-resistant breast cancer cells (MX-1/T) through intrinsic apoptosis signaling pathway, and exhibited efficacy against its tumor xenografts in nude mice, which suggests the potential of Lx2-32c to be a promising drug candidate (8). TPI-287, a new microtubule stabilizer, showed cytotoxicity similar to that of paclitaxel in breast cancer cells and efficacy against primary breast tumor xenografts in an animal model. Also, it was found to significantly decrease metastatic colonization of breast cancer in the brain (9). Moreover, Ojima et al (10) developed a series of novel second-generation taxanes with systematic modifications at the C2, C10, C3′ and C3′N positions. For example, among these new-generation taxanes synthesized and assayed, SB-T-1214 and SB-T-121303, exhibited significantly lower IC50 values, 9.00±0.77 nM and 3.65±0.21 nM, respectively for paclitaxel-resistant ovarian cancer cells than paclitaxel (532.95±3.18 nM). Such results clearly warrant further exploitation of next-generation taxanes with superior potency, efficacy and pharmacological properties against breast cancer. Transgelin 2 is reported to be implicated in tumorigenesis, boosting tumor progression and promoting metastases (11). Additionally, abnormal expression of transgelin 2 was discovered in lung, gastric and colorectal cancer (12–14). We previously reported that transgelin 2 expression was extremely high in paclitaxel-resistant human breast cancer cells (MCF-7/PTX) compared to breast cancer drug-sensitive cells by proteomics analysis (15). Knockdown of transgelin 2 via small interfering RNA sensitized MCF-7/PTX cells to paclitaxel, and suppressed their migration/invasion abilities, suggesting that transgelin 2 might be a new biomarker for breast cancer (16). On the other hand, aberrant activation of the phosphatidylinositol 3 kinase/serine-threonine kinase (PI3K/Akt) pathway contributes to chemo-resistance, tumor metastasis and poor prognosis (17,18). Notably, we reported that the PI3K/Akt pathway was activated in MCF-7/PTX cells and the TAGLN2-knockdown inhibited the PI3K/Akt pathway, suggesting that the PI3K/Akt pathway would be critical to breast cancer progression (19). We confirmed that the MCF-7/PTX cells, developed by our laboratory for the assay used, were highly resistant to paclitaxel and exhibited strong migration/invasion capacities (20,21). In the present study, eight novel next-generation taxanes were screened by MTT assay. Among these taxanes, SB-T-121205 (Fig. 1B) was found to be highly potent against the paclitaxel-resistant MCF-7/PTX cells. Subsequently, the effect of SB-T-121205 on cell apoptosis, epithelial-mesenchymal transition (EMT) property, migration and invasion was assessed in MCF-7/PTX cells, along with the underlying molecular mechanisms. Our data indicated that SB-T-121205 exerted its high potency against MCF-7/PTX cells through activation of the transgelin 2 and PI3K/Akt pathway, which suggests that SB-T-121205 would serve as an efficacious drug candidate for breast cancer treatment. Materials and methods Chemicals and antibodies In the present study, the anticancer activity of SB-T-121205 was assessed using paclitaxel as the positive control. Paclitaxel was purchased from Nanjing Sike Pharmaceutical, Co., Ltd. (Nanjing, China). All taxanes were kindly provided by Dr Changwei Wang, the laboratory of Professor Iwao Ojima at Stony Brook University. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) was obtained from Sigma-Aldrich (St. Louis, MO, USA). Annexin-V-FLUOS staining kit was obtained from Invitrogen (Waltham, MA, USA). The primary rabbit monoclonal antibodies against N-cadherin, and GSK-3β were acquired from Abcam (Cambridge, MA, USA). The primary rabbit monoclonal antibodies against E-cadherin, phosphatase and tensin homologue deleted on chromosome ten (PTEN), Akt, phospho-Akt (p-Akt), p-GSK-3β and Snail were purchased from Cell Signaling Technology (Danvers, MA, USA). The rabbit polyclonal antibodies against caspase-3, caspase-9 and poly(ADP-ribose) polymerase (PARP) were also obtained from Cell Signaling Technology. The rabbit polyclonal antibodies against vimentin and transgelin 2 were from GeneTex, Inc. (Irvine, CA, USA). The rabbit monoclonal antibodies against Bcl-2 and Bax were acquired from Epitomics (Burlingame, CA, USA). The rabbit polyclonal anti-β-actin antibody was obtained from Beijing Bo Aosen Biotechnology Co., Ltd. (Beijing, China). Horseradish-peroxidase-conjugated goat anti-rabbit IgG was from CW Biotech (Beijing, China). Cell lines and cell culture The human breast cancer cell lines, MCF-7/S and MDA-MB-453, and non-tumorigenic human bronchial epithelial cell line (BEAS-2B) were obtained from the Cell Bank of Shanghai, Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. MCF-7/PTX cell line was successfully established as previously described (16) with the concentration of 30 nM paclitaxel. The cells were grown in RPMI-1640 medium (Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific, Waltham, MA, USA) and 1% penicillin (Harbin Pharmaceutical Group, Co., Ltd., Harbin, China)/streptomycin (North China Pharmaceutical Group, Co., Ltd., Shijiazhuang, China) at 37°C in a humidified atmosphere of 5% CO2. Cells in exponential phase growth were observed under inverted light microscope (Olympus Corp., Tokyo, Japan). MTT assay Cells at 5×105/ml density were seeded into 96-well plates (Corning, Inc., Corning, NY, USA) with 100 µl medium for the duration indicated. After 72 h, 20 µl of MTT (5 mg/ml) was added into each well and incubated at 37°C for 4 h. Then, 150 µl of dimethyl sulfoxide (DMSO) was added into each well for dissolving the formazan for 15 min. The absorbance was tested at 490 nm on a microplate reader (BioTek Instruments, Inc., Winooski, VT, USA). The 50% growth inhibitory concentration (IC50) of drug was calculated to evaluate the drug sensitivity. Each experiment was repeated three times. Flow cytometry assay For cell cycle assay, cells were exposed to paclitaxel (600 nM) or SB-T-121205 (10 or 20 nM) for 48 h and then harvested. After three washes with cold phosphate-buffered saline (PBS), cells were fixed with 70% cold ethanol at 4°C overnight. The next day, cells were washed with cold PBS and suspended in a 500 µl staining solution of propidium iodide (PI; Sigma-Aldric) staining solution at 37°C for 30 min without light. The samples were tested using FACSCanto™ II flow cytometry (Becton Dickinson, Franklin Lakes, NJ, USA). For cell apoptosis analysis, cells with different treatments after 48 h were collected, washed and were resuspended with cold PBS. Then, the cells were double stained with Annexin V-FITC and PI at 37°C for 20 min from light with an Annexin-V-FLUOS staining kit in accordance with the manufacturer's instructions. The stained cells were analyzed using FACSCanto™ II flow cytometry. Annexin V+/PI− were regarded as early apoptotic cells and Annexin V+/PI+ were late apoptotic cells. Experiments were repeated in triplicate, independently. Western blot assay Cells with different treatments were lysed in RIPA buffer containing protease inhibitor (Roche, Basel, Switzerland) on ice. Then, equal amount of protein lysates were electrophoretically separated by 10% sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS-PAGE; Beyotime Institute of Biotechnology, Beijing, China) and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). After blocking with 5% non-fat dried milk for 2 h, the membranes were incubated with diluted antibodies N-cadherin (1:1,000, ab76011), E-cadherin (1:1,000, #3195), vimentin (1:500, GTX100619), PTEN (1:1,000, #9188), Akt (1:1,000, #4691), p-Akt (1:800, #4060), GSK-3β (1:1,000, ab32391), p-GSK-3β (1:1,000, #9322), Bcl-2 (1:2,500, #1017-1), Bax (1:2,500, #1063-1), caspase-3 (1:800, #9662), caspase-9 (1:800, #9502), PARP (1:800, #9542), Snail (1:1,000, #3879), transgelin 2 (1:2,000, GTX115082) and β-actin (1:800, bs-0061R) overnight at 4°C. After incubation with a horseradish peroxidase-conjugated secondary antibody (1:25,000, cat. no. CW0103), for 2 h at 37°C, the protein bands were detected using the SuperSignal West Pico kit (Thermo Fisher Scientific). All western blot experiments were repeated at least three times. Mammosphere formation assay Mammosphere culture was carried out in a serum-free DMEM/F-12 (Gibco) supplemented with 2% B27 (Gibco), 20 µg/l human epidermal growth factor (PeproTech, Rocky Hill, NJ, USA), 10 µg/l human basic fibroblast growth factor (PeproTech), and 5 mg/l insulin (Jiangsu Wanbang Biochemical Pharmaceutical, Co., Ltd., Xuzhou, China). Single cells prepared from mechanical and enzymatic dissociation were plated in 6-well ultralow attachment plates (Corning) at 1×104/ml density in culture. Single cell status was confirmed under microscope. After 14 days, the number of mammospheres was counted under an inverted light microscope. Experiments were repeated in triplicate, independently. Wound healing scratch assay Cells (5×105/ml) were seeded in 6-well plate until confluent. Cells were serum-starved overnight and an artificial scratch wound was created. The cells were then maintained in serum-free culture at 37°C in a humidified atmosphere of 5% CO2. Migration photos were captured at 0, 24 and 48 h after scratching. Experiments were repeated in triplicate independently. Percent wound closure was calculated using the following equation: percent wound closure (%) = [1−(Lt/L0)] × 100%. Transwell invasion assay The invasiveness of cells was evaluated by a Boyden chamber method. The polycarbonate filters (8 µm pore size; Corning) were coated with Matrigel Matrix (BD Biosciences, San Jose, CA, USA) and incubated at 37°C for 5 h. Next, 5×105 cells suspended in 200 µl serum-free RPMI-1640 were added into the upper chamber, while 800 µl of complete media was added to the lower chamber. After 48 h, cells migrated through Matrigel and adhered onto the lower chamber was fixed in 4% paraformaldehyde for 30 min, stained with 0.1% crystal violet (Beyotime Institute of Biotechnology) and counted by fluorescent microscopy (Olympus). Each invasion assay was repeated in three independent experiments. Statistical analysis Statistical analysis was performed using one-way ANOVA. All values were expressed as mean ± standard deviation (SD) from triplicate experiments performed in a parallel manner. P<0.05 were considered statistically significant. Results The intrinsic cytotoxicity of SB-T-121205 on cancer and normal cells The intrinsic cytotoxicity of the eight novel taxanes against MCF-7/PTX cells were measured by MTT assay. Treating cells with these taxanes for 72 h significantly inhibited cell growth. Among these taxanes examined, SB-T-121205 showed the highest inhibition. We therefore, chose SB-T-121205 for further study. After 72 h of exposure, SB-T-121205 obviously restrained growth of MCF-7/PTX cells with an IC50 value of 19.01±2.03 nM, which was two orders of magnitude lower than paclitaxel (2290.87±125.18 nM) (16) (Table I and Fig. 1C and D). The molecular formula of SB-T-121205 is C44H56F3NO16 with molecular weight 911.91. Moreover, the effect of SB-T-121205 on the proliferation of parental MCF-7/S cells was examined and the result showed SB-T-121205 inhibited the cell growth with the IC50 value of 2.14±0.32 nM, which was ~10 times lower than that of paclitaxel (20.0±0.9 nM) (16) (Fig. 1C and D). We examined the cell growth inhibitory effect of SB-T-121205 on MDA-MB-453 cells. SB-T-121205 was found to have slightly lower IC50 value (38.67±3.58 nM) than paclitaxel (IC50 54.62±3.28 nM) (Fig. 1E). We also examined the cytotoxicity of paclitaxel and SB-T-121205 against non-tumorigenic BEAS-2B human bronchial epithelial cells. Treating BEAS-2B cells with different concentration of paclitaxel or SB-T-121205 revealed that BEAS-2B cells are slightly less sensitive to SB-T-121205 (IC50 59.80±1.89 nM) than paclitaxel (IC50 54.57±2.10 nM) (Fig. 1F). Thus, for the treatment of MCF7/S and MCF7/PTX and MDA-MB-453 cancer cells, SB-T-121205 has much wider therapeutic index than paclitaxel. SB-T-121205 induces apoptosis and G2/M arrest in MCF-7/PTX cells To confirm whether SB-T-121205 triggered apoptosis of MCF-7/PTX cells, we carried out Annexin V and PI double staining followed by flow cytometric analysis. MCF-7/PTX cells were incubated with paclitaxel alone or SB-T-121205 alone for 48 h. As shown in Fig 2A, paclitaxel (600 nM) and SB-T-121205 (10 or 20 nM) indeed increased cell apoptosis rates that reached 33.48, 31.78 and 39.81%, respectively (Fig. 2A). Since the concentration used for paclitaxel is 30–60 times higher than that of SB-T-121205, SB-T-121205 is far better than paclitaxel in promoting cell apoptosis. In brief, it is confirmed that SB-T-121205 has the ability to induce apoptosis in MCF-7/PTX cells. To further investigate the mechanisms that SB-T-121205 inhibits the cancer cell growth, the MCF-7/PTX cells were exposed to various concentrations of SB-T-121205 for 48 h, and then cell cycle analysis was performed. Compared with the control group, paclitaxel (600 nM) markedly elevated the number of cells in G2/M phase in MCF-7/PTX cells and the proportion of MCF-7/PTX cells in G2/M phase increased from 24.84 to 92.06%. SB-T-121205 at low concentrations (10, 20 and 40 nM) also increased the percentage of cells in G2/M phase in a dose-dependent manner from 26.23% (10 nM) to 52.77% (40 nM) (Fig. 2B). The results confirmed that SB-T-121205 arrests the mitosis of MCF-7/PTX cells at the G2/M phase of the cell cycle in a manner similar to other known taxanes. SB-T-121205 suppresses the EMT property of MCF-7/PTX cells It is well known that EMT is a critical event in the development of cancers (22). To further examine whether SB-T-121205 could alter the EMT property of MCF-7/PTX cells, mammosphere formation and western blot assays were performed. As Fig. 3A shows, SB-T-121205 exposure triggered morphological changes in the MCF-7/PTX cells from elongated shape to cobblestone shape (Fig. 3A), which is a characteristic EMT morphology. After treated with SB-T-121205, the mammosphere forming ability of MCF-7/PTX cells was decreased (Fig. 3B). In addition, a low concentration (20 nM) of SB-T-121205 distinctly increased the expression of epithelial marker E-cadherin, whereas the levels of mesenchymal markers N-cadherin and vimentin were reduced (Fig. 3C). However, the effect of high concentration (600 nM) of paclitaxel was inferior to that of the low concentration of SB-T-121205. Thus, SB-T-121205 possesses much higher potency than paclitaxel for suppression of EMT in MCF-7/PTX cells. SB-T-121205 inhibits migration and invasion in MCF-7/PTX and MDA-MB-453 cells Tumor migration and invasion are major obstacles for successful chemotherapy (23). Accordingly, the effects of SB-T-121205 on the migration and invasion of MCF-7/PTX and MDA-MB-453 cells were evaluated by wound healing scratch and Transwell invasion methods, respectively. MCF-7/PTX cells treated with SB-T-121205 displayed decreased migratory abilities at 24 and 48 h (Fig. 4A). The percent wound closure of four groups at 48 h were 70.59±5.80% (control), 63.43±5.13% (paclitaxel, 600 nM), 43.75±4.52% (SB-T-121205, 10 nM) and 39.39±1.80% (SB-T-121205, 20 nM), respectively. Also, SB-T-121205 (10 and 20 nM) reduced the number of invasive cells in a dose-dependent manner (Fig. 4B), wherein 10 nM of SB-T-121205 showed an equivalent effect to 600 nM of paclitaxel. Similarly, treating MDA-MB-453 cells with paclitaxel (55 nM, a concentration close to the IC50 value) or SB-T-121205 (19.5 nM, a concentration close to the 1/2IC50 value) or SB-T-121205 (39 nM, a concentration close to the IC50 value) inhibited the migration and invasion of MDA-MB-453 cells (Fig. 5A and B). SB-T-121205 exhibited its effects on migration and invasion of MDA-MB-435 cells in a dose-dependent manner, and was clearly more effective than paclitaxel. Thus, we have found that SB-T-121205 not only suppresses the EMT process, but also inhibits migratory and invasive abilities of MCF-7/PTX and MDA-MB-435 human breast cancer cells much more effectively than paclitaxel. SB-T-121205 represses transgelin 2 expression and activation of the PI3K/Akt pathway in MCF-7/PTX cells To understand molecular mechanism by which SB-T-121205 receded cell proliferation and migratory/invasive abilities of MCF-7/PTX cells, the protein levels of transgelin 2 and the PI3K/Akt pathway were analyzed. After the treatment of MCF-7/PTX cells with SB-T-121205, the expression of transgelin 2, p-Akt and p-GSK-3β was downregulated with a concomitant augment expression of PTEN, whereas the levels of Akt and GSK-3β were unaffected (Fig. 6A). Furthermore, the deactivation of Akt signaling pathway augmented the expression of pro-apoptotic factor Bax and the cleavage of caspase-3, caspase-9, PARP significantly, while suppressed pro-survival factor Bcl-2, ultimately leading to apoptosis. On the other hand, the downstream molecule Snail was strongly downregulated (Fig. 6B). In short, these observations suggest that SB-T-121205 is an antitumor agent that affects cell behavior by modulating transgelin 2 and the PI3K/Akt pathway. Discussion Paclitaxel was first approved in April 1992 for the treatment of platinum-resistant breast cancer by the Food and Drug Administration, USA. It exerts vital anticancer effect through its distinct antimitotic mechanism of action (24). However, a number of studies since then have revealed that treatment with paclitaxel brought about undesirable adverse effects, including drug-resistance, hence seriously restricting its therapeutic effects and clinical applications (25). For this reason, it is essential to deal with the problem of paclitaxel resistance to enhance the survival of patients with breast cancer. There are two main ways to solve the problem of drug resistance. The first is to develop resistance reversal agents. Scientists in the past few decades have screened many compounds to identify compounds with multidrug resistance reversal activities against various drug-resistant cell lines. Compounds such as verapamil (26) and metformin (27) were discovered through these efforts and developed. These compounds sensitize tumor cells to chemotherapeutic drugs, and this line of research is continuing. The second is to discover novel compounds with antitumor activities that may have high efficacy against drug-resistant targets and then directly kill cancer cells, which would also provide new insights into prevention and treatment of breast cancer. Considerable efforts have been made to develop new-generation taxoids with outstanding antitumor activity, much better than paclitaxel. For example, Nikolakakis et al (28) reported that 7β-O-glycosylated taxanes (9 and 15) were more potent (IC50 12–15 nM) than docetaxel (IC50 40 nM) and much more potent than paclitaxel (IC50 >5 µM) against adriamycin-resistant human breast cancer cells (MCF-7/ADR). It was also documented that 2′-(N-methylpyridinium acetate) derivative of paclitaxel showed excellent potency in lung cancer cells and breast cancer cells (29). Based on structure-activity relationships (SAR) study, a series of new-generation taxanes have been developed by Ojima et al (10,30), which exhibited 2–3 orders of magnitude higher potency than paclitaxel or docetaxel against multidrug-resistant breast, ovarian, colon, pancreatic and prostate cancer cell lines (31). These new-generation taxanes have modifications at C10, C3′, C3′N and/or C2. A newly developed next-generation taxane, SB-T-121205 possesses a 3-trifluoromethoxylbenzoyl group at C2 on the top of modifications in the new-generation taxanes mentioned above. The present study disclosed, for the first time, the excellent activities of SB-T-121205 in inhibiting the growth of MCF-7/S, MCF-7/PTX and MDA-MB-453 human breast cancer cells. An interesting observation in this study was that BEAS-2B normal human cells were relatively insensitive to SB-T-121205, which means that SB-T-121205 has a good therapeutic index. It was observed that the apoptosis induced at 20 nM SB-T-121205 in MCF-7/PTX cells was more powerful than 600 nM paclitaxel, suggesting SB-T-121205 possesses an extremely strong anti-proliferative activity. SB-T-121205 induced G2/M phase arrest in MCF-7/PTX cells in a manner similar to paclitaxel. In addition, SB-T-121205 changed cell morphology, modulated EMT marker expression and weakened the mammosphere forming ability, then mitigated the EMT process in MCF-7/PTX cells. Importantly, SB-T-121205 exhibited its ability to restrain the migration and invasion capacities of MCF-7/PTX cells and MDA-MB-453 cells. Consequently, as a novel next-generation taxane, SB-T-121205 appears to be a very promising lead compound for drug development. Transgelin 2, located at chromosome 1q21–q25, is an important actin-binding protein responsible for the actin cytoskeleton dynamics (12). Abundant evidence has indicated that transgelin 2 exerts oncogenic activity. Transgelin 2 has been shown to be involved in lymph node metastasis, distant metastasis as well as tumor-lymph node-metastasis (TNM) staging system in colorectal cancer (CRC). Transgelin 2 may serve as a new biomarker for predicting progression and prognosis of CRC (14). Nohata et al (32) revealed that transgelin 2, directly regulated by miR-1, was downregulated by a siRNA and then decreased cell proliferation and invasion in human neck squamous cell carcinoma cells. In our models of paclitaxel-resistant breast cancer, we found that SB-T-121205 suppressed the transgelin 2 protein expression, which can explain the observed altered biological behavior of MCF-7/PTX cells. It has been generally accepted that the PI3K/Akt pathway participates in drug resistance, tumor migration, differentiation and apoptosis. Suppression of the PI3K/Akt pathway has been proven to be an efficient way to attenuate cell growth and migration (33,34). Wang et al (35) verified that the PI3K/Akt pathway was activated in cisplatin-resistant lung cancer cells (A549/CDDP), and deactivation of Akt signaling pathway significantly suppressed Snail expression and subsequently induced a substantial decrease in migratory ability and invasiveness of A549/CDDP cells. Snail, a zinc-finger transcription factor, is known as a crucial regulator in the aggressive phenotype of EMT (36). It was supported that PI3K/Akt signal played an essential function during miR-519a-induced hepatocellular carcinoma cell proliferation and cell cycle progression (37). We found that the downregulation of transgelin 2 by SB-T-121205 caused repression of the PI3K/Akt pathway. Deactivation of Akt signal led to revitalization of the mitochondria apoptosis pathway and downregulation of Snail in MCF-7/PTX cells (Fig. 7), which appears to be an important contributor to the unique mechanism of action of SB-T-121205. In conclusion, we found that SB-T-121205 enhances cell apoptosis, as well as inhibits the migration and invasion abilities of MCF-7/PTX cells, partly by targeting transgelin 2 and the PI3K/Akt pathway. We also found that SB-T-121205 downregulates the transgelin 2 expression. Deactivation of transgelin 2 can be further explored as a basis for new strategies for breast cancer treatment. These findings strongly indicate that SB-T-121205 is a highly promising lead compound for the development of next-generation chemotherapeutic agents in breast cancer treatment. Acknowledgments The present study is supported by grants from the National Natural Science Foundation of China (nos. 81473177, 81502616 and 81672954) and the Science Foundation of Guangdong Province, China (no. 2015B020211012), as well as a grant from the National Institutes of Health, USA (CA103314 to I.O.). Abbreviations PTXpaclitaxel PI3K/Aktphosphatidylinositol 3 kinase/serine-threonine kinase EMTepithelial-mesenchymal transition Figure 1 Effects of paclitaxel and SB-T-121205 on cell viability. The chemical structures of (A) paclitaxel and (B) SB-T-121205. MCF-7/PTX cells and MCF-7/S cells were treated with (C) paclitaxel or (D) SB-T-121205 for 72 h at a range of concentrations, and relative cell viability was determined by MTT assay. In the same manner, (E) MDA-MB-453 cells were treated with paclitaxel or SB-T-121205, and relative cell viability was determined. In the same manner, (F) BEAS-2B cells were treated with paclitaxel or SB-T-121205 and relative cell viability was determined. Figure 2 SB-T-121205-induces cell apoptosis and cell cycle distribution in MCF-7/PTX cells. (A) Treatment with paclitaxel alone (600 nM) or SB-T-121205 (10 and 20 nM) for 48 h, apoptosis in MCF-7/PTX cells was determined by flow cytometry. (B) Cell cycle distribution of MCF-7/PTX cells after treatment with paclitaxel or SB-T-121205 for 48 h was examined. Data were presented as proportion of cells in G1, S and G2/M phases of the cell cycle. Data are shown as mean ± SD from three experiments, *P<0.01 vs. control group. Figure 3 SB-T-121205 inhibits the EMT process in MCF-7/PTX cells. (A) Cell morphology observed by microscopy in MCF-7/PTX cells treated with paclitaxel alone or SB-T-121205 alone (original magnification, ×100). (B) The effect of paclitaxel or SB-T-121205 on mammosphere forming ability of MCF-7/PTX cells was examined (original magnification, ×100). (C) Expression of E-cadherin, N-cadherin and vimentin after treated with paclitaxel or SB-T-121205, was measured by western blot analysis. Data are shown as mean ± SD from three experiments and β-actin was used as loading control, P<0.05, P<0.01 vs. control group. Figure 4 SB-T-121205 inhibits the migration and invasion abilities of MCF-7/PTX cells. (A) Migration of MCF-7/PTX cells treated with paclitaxel or SB-T-121205 was determined by wound healing assay (original magnification, ×100). (B) Invasiveness of MCF-7/PTX cells treated with paclitaxel or SB-T-121205 was detected by Transwell invasion assay (original magnification, ×100). Data are shown as mean ± SD from three experiments, P<0.01 vs. control group. Figure 5 SB-T-121205 inhibits the migration and invasion abilities of MDA-MB-453 cells. (A) Migration of MDA-MB-453 cells treated with paclitaxel alone (55 nM) or SB-T-121205 alone (19.5 and 39 nM) was determined by wound healing assay (original magnification, ×100). (B) Invasiveness of MDA-MB-453 cells treated with paclitaxel or SB-T-121205 was detected by Transwell invasion assay (original magnification, ×100). Data are shown as mean ± SD from three experiments, P<0.05, P<0.01 vs. control group. Figure 6 SB-T-121205 suppresses the PI3K/Akt pathway in MCF-7/PTX cells. (A) The levels of transgelin 2, PTEN, Akt, p-Akt, GSK-3β, and p-GSK-3β were tested in MCF-7/PTX cells treated with paclitaxel alone or SB-T-121205 alone for 48 h. (B) Expression of Bcl-2, Bax, cleaved-caspase-3, cleaved-caspase-9, cleaved-PARP, and Snail was examined in MCF-7/PTX cells treated with paclitaxel or SB-T-121205. Data are presented as mean ± SD from three experiments, and β-actin was used as loading control, *P<0.01 vs. control group. Figure 7 Proposed mechanism of action of SB-T-121205 for inhibition of the growth, migration, and invasion of MCF-7/PTX cells. SB-T-121205 suppresses transgelin 2, then inhibits the PI3K/Akt pathway. Table I The effect of paclitaxel and new-generation taxanes on cell viability in MCF-7/PTX cells.a Taxane IC50 (nM) Taxane IC50 (nM) SB-T-1214 80.50±7.62 SB-T-121303 21.67±2.25 SB-T-101141 66.66±5.59 SB-T-12301 54.59±4.61 SB-T-121205 19.01±2.03 SB-T-121405 34.90±2.97 SB-T-121605 31.43±2.84 SB-T-1230105 119.05±9.68 Paclitaxel 2290.87±125.18 a Cell were treated with various concentrations of taxanes and IC50 values were calculated. 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==== Front 960783520545Mol PsychiatryMol. PsychiatryMolecular psychiatry1359-41841476-55782757387810.1038/mp.2016.139nihpa800494Articlep62 improves AD-like pathology by increasing autophagy Caccamo Antonella Ph.D.1Ferreira Eric M.S.1Branca Caterina Ph.D.1Oddo Salvatore Ph.D.12#1 The Biodesign Neurodegenerative Disease Research Center2 School of Life Sciences, Arizona State University, Tempe, Arizona, 85281# To whom correspondence should be addressed: SALVATORE ODDO, Ph.D., The Biodesign Neurodegenerative Disease Research Center, School of Life Sciences, Arizona State University, 1001 S McAllister Ave, Tempe, AZ 85281, 480-727-3490, [email protected] 8 2016 30 8 2016 6 2017 22 6 2017 22 6 865 873 Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: http://www.nature.com/authors/editorial_policies/license.html#termsThe multifunctional protein p62 is associated with neuropathological inclusions in several neurodegenerative disorders, including frontotemporal lobar degeneration, amyotrophic lateral sclerosis, and Alzheimer’s disease (AD). Strong evidence shows that in AD, p62 immunoreactivity is associated with neurofibrillary tangles and is involved in tau degradation. However, it remains to be determined whether p62 also plays a role in regulating amyloid-β aggregation and degradation. Using a gene therapy approach, here we show that increasing brain p62 expression rescues cognitive deficits in APP/PS1 mice, a widely used animal model of AD. The cognitive improvement was associated with a decrease in amyloid-β levels and plaque load. Using complementary genetic and pharmacologic approaches, we found that the p62-mediated changes in Aβ were due to an increase in autophagy. To this end, we showed that removing the LIR domain of p62, which facilitates p62-mediated selective autophagy, or blocking autophagy with a pharmacological inhibitor, was sufficient to prevent the decrease in Aβ. Overall, these data provide the first direct in vivo evidence showing that p62 regulates Aβ turnover. ==== Body Introduction An imbalance between protein production and degradation contributes to the accumulation of proteinaceous inclusions characteristic of several neurodegenerative disorders, including amyloid-β (Aβ) and tau in Alzheimer’s disease (AD)1, 2. Indeed, several laboratories have shown that increasing protein turnover may have beneficial effects on disease outcome3–6. Autophagy and the ubiquitin-proteasome system represent two major intracellular degradation systems that contribute to the removal of proteinaceous inclusions. Proteasome dysfunction has been linked to AD and other neurodegenerative disorders7–11. Macroautophagy, herein referred to as autophagy, is induced by the formation intracellular autophagosomes that deliver the cargo to be degraded to lysosomes12. The formation of the autophagosome is negatively regulated by the mammalian target of rapamycin (mTOR), and involves sequential reactions carried by several autophagy related proteins13. LC3-I is a cytosolic autophagy-related protein that during autophagosome formation is post-translationally modified to form LC3-II, which is then incorporated into the growing autophagosome14. Notably, the formation of the autophagosome can also be initiated independently of mTOR15. p62 is a multifunctional protein involved in protein turnover, via autophagy and proteasome, oxidative stress, and other cellular functions16, 17. p62 knockout mice show age-dependent accumulation of NFTs and synaptic deficits18, underlying the role of p62 in tau aggregation and degradation. In AD, p62 strongly binds to NFTs19, most likely in an attempt to target them for degradation. Thus, p62 is sequestered in NFTs, which may limit its cellular function by creating reduced levels of available p62 in the cytosol20. p62 has several functional domains, including a ubiquitin-binding domain (UBA) an a LC3-interacting region (LIR). p62 binds to polyubiquitinated proteins, including tau, through the UBA domain and targets them for proteasome degradation21, 22. Through its LIR domain, p62 binds to LC3 to facilitate selective autophagy23, 24. Materials and Methods Mice The generation of the APP/PS1 mice was described previously25. We have backcrossed the APP/PS1 mice for 12 generations into a pure 129/SvJ background. All animal studies were performed with an approved from the Arizona State University Institutional Animal Care and Use Committee. Viral constructs generation and injections AAVs were generated by Vector BioLabs (Malvern, PA). The final titers were 1.0 × 1013 GC/ml for the AAV-GFP and 8.8 × 1012 GC/ml for the AAV-p62-GFP. AAVs were injected bilaterally into the lateral ventricles of newborn, P0 pups, using a 5 µl Hamilton syringe (2 µl of viral suspension per ventricle). Pups were anesthetized by hypothermia. The ventricle were found using the following coordinates: 1.0 mm posterior of bregma, 2.0 mm lateral on each side and 1 mm dorso-ventral from the skull. A plastic stopper was placed in the syringe prior the injection and only 1 mm of the syringe needle was exposed through the stopper. The virus was injected at 1 µl/minute, after which the needle is left in place for 2 additional minutes before it was slowly removed. The experimenter was blinded to the genotype of the mice. Inclusion/exclusion criteria Overall, we infected 20 mice per group, 10 females and 10 males. The sample size was chosen based on our previous experience performing similar experiments with these mice. The behavioral experiments were performed in all 20 mice per group. Eight mice per group were randomly selected to perform the biochemical and histological analyses. Morris water maze The experimental protocol is detailed in26. This test was conducted in a circular pool (diameter = 1 meter) filled with water maintained at 23°C and made opaque by the addition of non-toxic paint. The experimenter was blinded to the group allocation. Brain tissue processing and immunostaining Mice were euthanized by CO2 asphyxiation and their brains removed for analyses. One hemisphere of the brain was post-fixed in 4% paraformaldehyde for 48 hours and used for immunohistochemical evaluation. The other hemisphere was flash-frozen on dry ice and used for biochemical experiments and stored at −80°C. From the fixed tissue, using a sliding vibratome, we obtained 50 µm-thick brain sections. Sections were then stored in 0.02% sodium azide in PBS. The endogenous peroxidase activity was quenched with 3% H2O2 in 10% methanol for 30 minutes. For Aβ1–42 staining tissue was incubated for 7 minutes in 88% formic acid to retrieve the epitope. Tissue was incubated overnight at 4°C with an appropriate primary antibody. Sections were washed to remove excess primary antibody and incubated in the appropriate secondary antibody for 1h at room temperature. Excess secondary antibody was washed and sections were developed with diaminobenzidine substrate using the avidin–biotin horseradish peroxidase system (Vector Labs, Burlingame, CA). The experimenter was blinded to the group allocation. Western blots and ELISA Frozen tissue was homogenized in T-PER solution (Thermo Fisher Scientific, Waltham, MA) containing complete protease inhibitor (Roche, Basel, Switzerland) and phosphatase inhibitor (Life Technologies, Carlsbad, CA). Brain homogenates were ultracentrifuged at 100,000 g for 1 hour at 4 °C. The supernatant was recovered and stored at −80°C until used for western blots and to measure soluble Aβ levels by ELISA. The pellet was resuspended in 70% formic acid, re-homogenized, and centrifuged as described above. The supernatant of this second centrifugation was recovered and stored at −80°C until used as the insoluble fraction for ELISA experiments. Western blots were performed using precast Novex gels (Life Technologies, Carlsbad, CA). Proteins were transferred to nitrocellulose membranes (iBlot, Life Technologies) and were then incubated for 60 minutes in 5% non-fat powdered milk (Great Value) in tris buffered saline with Tween-20 (TBST, 0.1M Tris, 0.15M NaCl, & 0.1% Tween-20). Primary antibodies specific to the experiment were then applied overnight at 4°C in TBST 5% milk. The next day, blots were washed in TBST three times for 10 minutes/wash and then incubated in the appropriate fluorescent secondary antibody(s) for 1 hour at room temperature (RT). The blots were then washed as described above, and imaged/quantified using a LI-COR Oddesy CLx (LI-COR Biosciences, Lincoln, NE). ELISA measurements were conducted using specific Life Technologies kits, and following the manufacturer’s instructions. The experimenter was blinded to the group allocation. Cell culture 7PA2 cells were grown in Dulbecco’s Modified Eagles Media (DMEM, Life Technologies) with 10% fetal bovine serum (FBS, Life Technologies) at 37°C with humidified environment (5% CO2) in 6 well plates (5 × 105 cells/well). Cells were transfected with p62, p62-ΔUBA (a gift from Dr. Wei Ding, Capital Medical University, China), and p62-ΔLIR (a gift from Dr. Zhu, University of Kentucky). Aβ levels were measured 48 hours after transfections. The experimenter was blinded to the group allocation. Hippocampal primary neurons were isolated from APP/PS1 P0 pups and cultured in 6 well plates coated with poly-D-lysine (Sigma-Aldrich, Saint Louis, MO). Neurons were plated in Neurobasal media (Life Technologies) supplemented with 2% B27 (Life Technologies) and 50 U/mL penicillin/streptomycin (Life Technologies). After 24h in culture, neurons were infected with 1µl/ml CamKIIa-p62-GFP AAV (virus titer 8.8×1012 GC/ml). At day 13 post infections, neurons were treated for 14 hours either with 5 mM 3MA (Sigma-Aldrich) or 10 mM MG132 (Thermo Fisher Scientific, Waltham, MA). An untreated plate was used as a control. Aβ levels were measured after this last treatments. The experimenter was blinded to the group allocation. Proteasome activity We used the fluorogenic substrates Bz-VGR-AMC and Suc-LLVY-AMC to measure trypsin-like and chymotrypsin-like activities. Specifically, we incubated 10 µl of brain homogenate with each substrate to probe in a total of 200 µl of assay buffer (25mM HEPES, pH 7.5, 0.5mM EDTA, 0.05% NP-40) using black 96-well plates. We obtained kinetic readings at 37°C every 1.5 minutes for 60 min (excitation 360 nm, emission 460 nm) using the Synergy HT multi-mode microplate reader with the Gen5 software (BioTek, Winooski, VT). Readings were normalized to total protein concentrations assayed via a Coomassie Protein Assay Kit (Bradford, Thermo Scientific, Waltham, MA) following the manufacturer’s instructions. The experimenter was blinded to the group allocation. Antibodies All of the antibodies used in this study were validated by the manufacturers for use in mouse and/or human tissue (see manufacturers webpages). From Cell Signaling: Total p70S6K (dilution 1:1000, catalog number 9202), p70S6K Thr389 (dilution 1:1000, catalog number 9204), β-actin (dilution 1:10000, catalog number 3700), rpS6 (dilution 1:1000, catalog number 5364), Atg3 (dilution 1:1000, catalog number 3415), Atg5 (dilution 1:1000, catalog number 2630), Atg7 (dilution 1:1000, catalog number 2631), Atg12 (dilution 1:1000, catalog number 2010). From Millipore, anti-Aβ42 (dilution 1:200, catalog number AB5078P), p62 (dilution 1:1000, catalog number MABC32), tau t (dilution 1:1000, catalog number 577801). From BioLegend, 6E10 (dilution 1:3000, catalog number SIG-39320). From Calbiochem, CT20 (dilution 1:3000, catalog number 171610). From Novus Biologicals, LC3 (dilution 1:1000, catalog number NB100-2331). CP13 (dilution 1:000) was a gift from Dr. Peter Davies. Statistical analyses Data were analyzed by ANOVAs or student’s t-test as specified in the results and figure legends. When appropriate, post hoc tests were conducted using the Bonferroni correction. These analyses were conducted using GraphPad Prism 5 (GraphPad Software, Inc.). An a priori power analysis was not performed our sample sizes are similar to those reported in previously published papers. Examination of descriptive statistics revealed no violation of any test assumptions that would warrant using statistical test other than the ones used. For all analyses, the variance was approximately the same among groups. Results To study the role of p62 in AD pathogenesis, we generated an adeno-associated virus (AAV) expressing mouse p62 under the CamKIIα promoter. To allow co-expression of the green fluorescent protein (GFP) with p62, we cloned a 2A-like peptide sequence27 between the p62 cDNA and the GFP cDNA (Fig. 1a). We injected new born P0 APP/PS1 and non-transgenic (NonTg) mice with the AAVs expressing p62 or GFP alone (viral titer 8.8 × 1012 and 1.0 × 1013 GC/ml, respectively). The AAVs were injected into the lateral ventricles, 2 µl per each side. Mice that received the p62 virus will be referred to as APP/PS1-p62 and NonTg-p62 mice, while mice that received the GFP virus will be referred to as APP/PS1-GFP and NonTg-GFP mice. Mice were then left to age for seven months, during which all groups gained body weight at a similar rate (Fig. 1b). We first measured p62 levels in the hippocampi of 7-month-old mice and found that they were different among the four groups (p = 0.003; Fig. 1c–d). Specifically, p62 levels were significantly higher in the APP/PS1-p62 and NonTg-p62 mice compared to mice infected with the GFP AAVs. In contrast, p62 levels were similar between APP/PS1-p62 and NonTg-p62 mice. To determine the extent of the viral diffusion, we stained sections from APP/PS1-p62 mice with an anti-GFP antibody. The virus was highly expressed throughout the hippocampus and in to a lesser extent in the cortex (Fig 1f). Consistent with the neuronal specificity of CamKIIα, p62 expression was restricted to neurons and was not found in astrocytes (Fig. 1g–h). Mice received four training trials per day for five consecutive days, in the spatial version of the Morris water maze. For the escape latency there was an effect for days (p < 0.0001) and group (p < 0.0001; Fig. 2a). The day effect indicates that all groups improved across the five days. The group effect indicates that one or more groups learned at a different pace. Specifically, the NonTg-GFP group performed significantly better than the APP/PS1-GFP group at days 4 and 5. In contrast, the APP/PS1-p62 group performed as well as the two NonTg groups throughout the training and significantly better than APP/PS1-GFP on days 3, 4, and 5. We obtained similar results when we analyzed the distance traveled to reach the platform as we found a significant effect for days (p < 0.0001) and group (p = 0.023; Fig. 2b). Specifically, the APP/PS1-GFP group was significantly impaired compared to NonTg-GFP group at days 4 and 5. However, the APP/PS1-p62 group performed significantly better than the APP/PS1-GFP group at day 5 and as well as the two NonTg groups throughout the five days. These results indicate that overexpression of p62 rescued spatial learning deficits in APP/PS1 mice. Twenty-four hours after the last training trial, we conducted a single 60-second probe trial, during which mice were free to swim in the pool without the hidden platform. We measured the time mice spent in the target quadrant and opposite quadrant, the latency to cross over the platform location, and the total number of platform location crosses. The values for these four measurements were different among the four groups (p = 0.017, p < 0.0001, p = 0.002, p = 0.04, respectively; Fig. 2c–f). Specifically, APP/PS1-GFP group performed significantly different from the other three groups in all measurements (Fig. 2c–f). These data indicate that increasing p62 expression rescued spatial memory deficits in APP/PS1 mice as the APP/PS1-p62 group performed as well as the two NonTg groups. At the end of the behavioral tests, we stained sections from transgenic mice with an Aβ42-specific antibody. Aβ immunoreactivity was significantly lower in the hippocampus and cortex of APP/PS1-p62 mice compared to APP/PS1-GFP mice (p = 0.01 and p < 0.05, respectively; Fig. 3a–f). We then measured Aβ levels by sandwich ELISA. Soluble and insoluble Aβ40 levels were significantly reduced in APP/PS1-p62 mice compared to APP/PS1-GFP mice (p = 0.01 and p = 0.001, respectively; Fig. 3g–h). Similarly, soluble and insoluble Aβ42 levels were reduced in APP/PS1-p62 mice compared to APP/PS1-GFP mice (p = 0.02 for both measurements; Fig. 3i–j). Given the reported interaction between p62 and tau, we measured the total and phosphorylated tau levels. Overall, we found that neither total tau nor tau phosphorylated at Ser202 were significantly altered among NonTg-GFP, NonTg-p62, APP/PS1-GFP, and APP/PS1-p62 groups (Supplementary Fig. 1). To begin understanding the mechanisms linking higher p62 levels to reduced Aβ levels, we measured APP processing. APP levels were significantly higher in the APP/PS1-GFP and APP/PS1-p62 mice compared to the two NonTg groups (p < 0.0001; Supplementary Fig. 2a–b). We found similar results for C99 and C83 levels (p < 0.0001 for both measurements; Supplementary Fig. 2c–d). Overall, APP, C99, and C83 levels were unaltered by p62 overexpression as they were similar between APP/PS1-GFP and APP/PS1-p62 mice. To determine the role of protein turnover in the p62-mediated decrease in Aβ levels, we measure proteasome function, given the role of p62 in this process16. Using the fluorogenic substrates Bz-VGR-AMC and Suc-LLVYAMC, we measured trypsin-like and chymotrypsin-like activities, respectively. The chymotrypsin-like activity was significantly different among the four groups, as indicated by the slope of the curve (p = 0.022) and the area under the curve (p = 0.020; Supplementary Fig. 3a–b). These differences were not driven by p62, as the APP/PS1-GFP and the APP/PS1-p62 groups were not statistically significant from each other. The trypsin-like activity was not statistically different among the four groups (Supplementary Fig. 3c–d). p62 also functions by targeting proteins to autophagy for degradation28, 29. To determine the effects of increasing p62 on autophagy, we measured the expression levels of several autophagy related proteins (Atgs), which are routinely used to assess overall autophagy induction12. Atg3, Atg5, and Atg7 levels were different among the four groups (p = 0.001, p = 0.004, p = 0.027, respectively; Fig. 4a–d). For all three proteins, the NonTg-p62 and APP/PS1-p62 groups were significantly different than NonTg-GFP and APP/PS1-p62 groups. In contrast, the levels of Atg12 were not statistically significant among the four groups (Fig. 4a, e). We also found that while LC3-I levels were similar among the four groups (Fig. 4a, f), LC3-II levels were significantly higher in NonTg-p62 and APP/PS1-p62 compared to NonTg-GFP and APP/PS1-GFP groups (p = 0.002; Fig. 4a, g). These data show that increasing p62 expression does not change proteasome activity but facilitates autophagy induction, suggesting a possible mechanism for the p62-mediated changes in Aβ. Once autophagosomes are formed, they deliver their cargo to lysosomes for degradation. Thus, if p62-mediated decrease in Aβ levels are linked to an increase in autophagy, one would expect an increase in Aβ in the lysosomes. We labeled sections from APP/PS1-GFP and APP/PS1-p62 with Lamp2A (a lysosomal marker) and 6E10 (an antibody that recognize Aβ and other Aβ-containing fragments). We found that the amount of co-localization was significantly increased in the APP/PS1-p62 mice (Fig. 4h–I; p = 0.01). These data indicate that p62 increases autophagy induction and facilitates the delivery of Aβ to lysosomes. The mammalian target of rapamycin (mTOR) is a negative regulator of autophagy and it is known to interact with p6213, 30. Further, mTOR hyperactivity is linked to AD pathogenesis31–36. To determine whether the p62-mediated autophagy induction is linked to mTOR, we measured mTOR and its downstream targets S6K1, rpS6, and 4EBP1. While total levels of mTOR and S6K1 were not statistically significant among the four groups (Supplementary Fig. 4a–c), the levels of S6K1 phosphorylated at Thr389 were (p = 0.004; Supplementary Fig. 4a, d). Notably, these changes were independent of p62. Further, we found that the total and the levels of phosphorylated rpS6 and 4EBP1 were similar among the four groups (Supplementary Fig. 4e–h). These data indicate that the p62-mediated increase in autophagy induction is independent of mTOR. p62 has two main functional domains, the UBA and the LIR domains, which regulate protein degradation via the proteasome or autophagy, respectively16. To further dissect the mechanisms underlying the p62-mediated decrease in Aβ levels, we used p62 lacking the LIR domain (p62-ΔLIR37) or the UBA domain (p62-ΔUBA38). We transiently transfected p62-ΔLIR, p62-ΔUBA, or p62 wild type into 7PA2 cells, a widely used cell line known to secrete low molecular weight Aβ oligomers and N-terminal extended peptides39, 40. Notably, all three constructs also expressed a fluorescent tag, which allowed us to determine transfection efficiency (Fig. 5a). We ran a western blot of proteins extracted from transfected 7PA2 cells using a p62 antibody whose epitope is located within the LIR domain. We observed a shift in the molecular weight of p62-ΔUBA, while p62-ΔLIR was not detectable (Fig. 5b). We then measured Aβ levels by sandwich ELISA and found that they were different among the four groups (Fig. 5c, p = 0.001). Notably, transfection of wild type p62 or p62-ΔUBA decreased Aβ levels. In contrast, p62-ΔLIR was unable to significantly reduce Aβ levels, suggesting that the effects of p62 on Aβ are mediated by its LIR domain. Given that 7PA2 cells produce different forms of Aβ, including N-terminal extended peptides, it is possible that the sandwich ELISA used here detected different fragments of Aβ. Therefore, we isolated primary neurons from APP/PS1 mice and infected them with the AAVs expressing p62. Thirteen days after viral infections, neurons were treated with 10 mM MG132 or 5 mM 3MA for 14 hours to inhibit proteasome and autophagy, respectively. To determine infection efficiency, we measured the levels of the GFP tag and p62 (Fig. 5d). GFP levels were not detectable in the uninfected group, and were not statistically different among the other three groups (Fig. 5d–e). Similarly, p62 levels were increased in neurons infected with the p62 virus compared to uninfected neurons (p = 0.002; Fig. 5d, f). Most notably, Aβ42 levels, as measured by sandwich ELISA, were different among the four groups (p < 0.0001; Fig. 5g). Importantly, p62-infected neurons treated with the vehicle had significantly lower levels of Aβ42 than non-infected neurons. This decrease was prevented by the administration of the autophagy inhibitor (3MA) but not by the proteasome inhibitor (MG132; Fig. 5g). Overall, our results show that the p62-mediated decrease in Aβ levels are mediated by autophagy. Discussion Here we provide the first in vivo evidence indicating that p62 plays a role in Aβ degradation. These data underscore a novel mechanism by which Aβ can be targeted for autophagic degradation. p62 is frequently found in protein inclusions of several neurodegenerative disorders41, 42. It has been stipulated that by binding to these inclusions (including NFTs), p62 is sequestered from the cytosol creating a p62 loss-of-function environment16, 20. Furthermore, expression levels of p62 are reduced in AD and this appears to be linked to oxidative damage to its promoter region20, 43. Thus, consistent with the data reported here, increasing p62 levels might be a valid approach to restore neuronal function in proteinopathies. Several aspects of the autophagy-lysosomal systems are impaired in AD1, 2, 44. For example, elegant work by Nixon and colleagues showed accumulation of autophagosomes in postmortem human AD brains, suggesting deficits in autophagy flux45. At the same time, autophagy induction might also be impaired in AD, further highlighting the notion that promoting autophagy clearance might be a valid therapeutic approach for AD46. Consistently, increasing lysosomal function or autophagy induction improves AD-like pathology in transgenic mice3–6. mTOR is a negative regulator of autophagy induction. We and others have shown that decreasing mTOR signaling increases autophagy and improves AD-like pathology in several mouse models of AD5, 31–33, 47, 48. Here we report that the p62-mediated increase in autophagy induction is independent of mTOR activity, indicating that the LIR domain of p62 might engage alternative pathways to induce autophagy. Activation of LC3 by Atg4 can lead to autophagosome formation by a series of reactions mediated by Atg7, Atg3, and Atg5, which culminates into the conversion of LC3-I to LC3-II and the formation of the autophagosome14. Consistently, we found that the mTOR-independent autophagy induction mediated by p62 was linked to increased levels of several of these Atg proteins, including LC3-II. The data presented here, together with reports from the literature, suggest that increasing p62 levels might be a valid therapeutic approach for AD as it facilitates the removal of tau and Aβ by activating autophagy. Given the well-established deficits in autophagosome clearance in AD, the timing at which one would increase autophagy via p62 needs to be very closely considered. Increasing autophagy induction before deficits in autophagosome clearance manifest might be beneficial as it will lead to increased turnover of Aβ and tau. However, increasing autophagy induction after deficits in autophagosome clearance manifest may not have the desired effects on Aβ and tau and may further contribute to the pathological accumulation of autophagosomes. Supplementary Material 1 Conflict of interest The authors declare no conflict of interest Author contributions AC designed and performed the experiments, and analyzed the data. EF performed the in vitro experiments and edited the manuscript. CB performed the proteasome experiments and edited the manuscript. SO designed the experiments, analyzed the data, and wrote the manuscript. We thank Dr. Zhu, University of Kentucky for donating the p62ΔLIR plasmid; Dr. Wei Ding, Capital Medical University, China for donating the wild type and p62ΔUBA plasmids. This work was supported by grants from the Arizona Alzheimer’s Consortium and the National Institutes of Health (R01 AG037637) to SO. Figure 1 p62 gene transfer increases p62 levels in neurons. (a) Diagram depicting the structure of the two adeno associated viruses (AAVs) bilaterally injected into the lateral ventricles of new born APP/PS1 and NonTg mice. (b) Age-dependent change in body weight. (c–d) Western blot and quantitative analysis shows that p62 levels were significantly higher in the two groups infected with the AAV-p62 compared to the two groups infected with the AAV-GFP (n = 8/mice per group). Quantitative analyses were performed by normalizing p62 levels to β-actin, used as a loading control. (e–f) Microphotographs of hippocampal and cortical sections obtained from mice infected with the AAV-p62 virus. Sections were stained with an anti-GFP antibody and show the degree of diffusion of the virus. (g–h) Microphotographs of hippocampal sections co-labeled with GFP (green), and NeuN or GFAP (red). These data indicate that the AAVs almost exclusively infected neurons. Abbreviations: Hp, hippocampous; Cx, cortex. Error bars represent mean ± SEM. Figure 2 p62 overexpression improves cognitive function. (a, b) Learning curves of mice trained in the spatial reference version of the Morris water maze (n = 20/mice per group). The escape latency and the distance traveled to find the hidden platform were plotted against the days of training. Each day represents the average of four consecutive training trials. Both measurements indicate that all four groups significantly improved across the five days. For the escape latency: p < 0.0001 for days and group. Distance traveled: p < 0.0001 and p = 0.023 for days and group. Specifically, the APP/PS1-GFP group was significantly impaired compared to the other three groups at days 3, 4, 5 for the escape latency and day 5 for the distance traveled. (c) The APP/PS1 group spent significantly less time in the target quadrant compared to the other three groups (p = 0.017). (d) The APP/PS1-GFP group spent significantly more time in the opposite quadrant compared to the other three groups (p < 0.0001). (e) The APP/PS1-GFP group needed significantly more time to cross over the platform location compared to the other three groups (p = 0.002). (f) The APP/PS1-GFP group crossed over the platform location significantly fewer times compared to the other three groups (p = 0.04). * indicates that the APP/PS1-p62 group performed significantly better than the APP/PS1-GFP group. # indicates that the APP/PS1-GFP group performed significantly different than the other three groups. Learning data (a–b) were analyzed by two-way ANOVA; probe trials (c–f) were analyzed by one-way ANOVA. Bonferroni’s was used for post hoc tests. Error bars represent mean ± SEM. Figure 3 p62 overexpression reduces Aβ pathology (a, b) Microphotographs of hippocampal sections stained with an Aβ42-specific antibody. (c) Quantitative analysis of the Aβ42 immunoreactivity shows reduced Aβ load in the APP/PS1-p62 mice compared to APP/PS1-GFP mice (p = 0.01). (d, e) Microphotographs of cortical sections stained with an Aβ42-specific antibody. (f) Quantitative analysis of Aβ42 immunoreactivity shows reduced cortical Aβ load in the APP/PS1-p62 mice compared to APP/PS1-GFP mice (p < 0.04). (g–j) Soluble and insoluble Aβ40 and Aβ42 measured by sandwich ELISA. Aβ40 levels were significantly reduced in APP/PS1-p62 mice compared to APP/PS1-GFP mice (p = 0.01 and p = 0.001 for soluble and insoluble, respectively). Aβ42 levels were significantly reduced in APP/PS1-p62 mice compared to APP/PS1-GFP mice (p = 0.02 for both measurements). For all the experiments shown here, n = 8/mice per group. Data were analyzed by student’s t-test and are presented as mean ± SEM. Figure 4 p62 overexpression increases autophagy induction. (a) Western blots of hippocampal proteins probed with the indicated antibodies. (b–d) Atg3, Atg5, and Atg7 levels were different among the four groups (p = 0.001, p = 0.004, p = 0.027, respectively). Post hoc analyses indicated that the two p62 groups were significantly different than the two groups GFP groups. (e–f) Atg12 and LC3-I levels were not statistically different among the four groups. (g) LC3-II levels were different among the four groups (p = 0.002). Post hoc analyses indicated that the two groups infected with p62 were significantly different than the two groups infected with GFP. (h–i) Representative confocal images and quantitative analyses. Sections were labeled with Lam2a (green) and 6E10 (red), to mark lysosomes and Aβ-containing fragments, respectively. The number of yellow pixels was significantly increased in APP/PS1-p62 mice, which indicates higher Aβ levels in lysosomes. For all the experiments shown here, n=8 mice per group. Quantitative analyses of the blots were performed by normalizing the levels of the protein of interest to β-actin, used as a loading control. Data were analyzed by one-way ANOVA and Bonferroni’s post-hoc test and are presented as mean ± SEM. Figure 5 p62 overexpression decreases Aβ by an autophagy-mediated mechanism. (a) Microphotographs of 7PA2 cells transfected with different p62 constructs, as indicated. (b) Western blot from proteins isolated from transfected 7PA2 cells and probed with a p62 antibody, which recognize p62 within the LIR domain. Therefore ΔLIR is not detectable, while there is a clear shift in molecular weight for ΔUBA. (c) Aβ levels obtained from 7PA2 cells and measured by sandwich ELISA were different among the four groups (p = 0.001). Post hoc analyses revealed that transfection of wild type p62 or p62-ΔUBA decreased Aβ42 levels. In contrast, p62-ΔLIR was unable to significantly reduce Aβ levels. (d) Western blot of proteins extracted from APP/PS1 primary neurons infected with the p62 AAVs and treated with compounds to block autophagy (3MA) or proteasome function (MG132), as indicated. (e) GFP levels were similar among the three groups of neurons infected with the p62 AAVs. (f) p62 blot showed that p62 levels were significantly higher in the three groups infected with the p62 AAVs, compared to uninfected neurons. (g) Aβ42 levels obtained from these APP/PS1 neurons and measured by sandwich ELISA, were significantly different among the four groups (p < 0.0001). Post hoc analyses revealed that the p62 and the p62 + MG132 groups had significantly lower Aβ42 levels compared to the other two groups. For all the experiments shown here, n=9 mice per group. Quantitative analyses of the blots were performed by normalizing the levels of the protein of interest to β-actin, used as a loading control. 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==== Front 01007146400Pediatr ResPediatr. Res.Pediatric research0031-39981530-04472835520110.1038/pr.2017.83nihpa860952ArticleSex And Age Differences In Phenylephrine Mechanisms And Outcomes After Piglet Brain Injury Curvello Victor 1Hekierski Hugh 1Riley John 1Vavilala Monica 3Armstead William M 12 1 Department of Anesthesiology and Critical Care, University of Pennsylvania, Philadelphia, PA 19104 2 Department of Pharmacology, University of Pennsylvania, Philadelphia, PA 19104 3 Department of Anesthesiology, University of Washington Seattle, WA 98104Correspondence: William M. Armstead, Ph.D, Department of Anesthesiology and Critical Care, 3620 Hamilton Walk, JM3, University of Pennsylvania, Philadelphia, PA 19104, (215) 573-3674, Fax: (215) 349-5078, [email protected] 3 2017 26 4 2017 7 2017 26 10 2017 82 1 108 113 Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:http://www.nature.com/authors/editorial_policies/license.html#termsBackground Traumatic brain injury (TBI) is the leading cause of injury related death in children, with boys and children under 4 having particularly poor outcomes. Cerebral autoregulation is often impaired after TBI, contributing to poor outcome. In prior studies of newborn pigs, phenylephrine (Phe) preferentially protected cerebral autoregulation in females but not males after TBI. We hypothesized that, in contrast to the newborn, Phe prevents impairment of autoregulation and tissue injury following TBI in both sexes of older pigs. Methods Cerebral autoregulation, CSF ERK and endothelin, and histopathology were determined after moderate fluid percussion brain injury (FPI) in male and female juvenile pigs after Phe. Results Autoregulation was more impaired in male subjects than females. Phe protects autoregulation in both sexes after FPI, blocked ERK and endothelin, and decreased the number of necrotic neurons in males and females after FPI. Conclusions These data indicate that Phe protects autoregulation and limits neuronal necrosis via block of ERK and endothelin after FPI in maleas and females. Together with prior observations in newborn pigs where Phe protected autoregulation in female but not male subjects, these data suggest that use of Phe to improve outcome after TBI is both sex and age dependent. ==== Body Introduction Traumatic brain injury (TBI) is the leading cause of injury related death in children and young adults (1), with boys and children under 4 years having particularly poor outcomes (2). Cerebral perfusion pressure (CPP) is defined as mean arterial pressure [MAP] minus intracranial pressure [ICP]). CPP is low after TBI, causing cerebral ischemia and impaired cerebral autoregulation (1–3). The 2012 Pediatric Guidelines direct clinicians to keep CPP above 40 mm Hg in children after TBI (4). However, strategies that use employment of vasoactive agents to increase MAP and thereby augment CPP following TBI, such as norepinephrine (NE), phenylephrine (Phe), epinephrine (EPI) and dopamine (DA) (5–7) have not been rigorously conducted so as to compare their relative effects on protection of cerebral autoregulation and improvement of ultimate outcome post insult. The cerebral effects of these commonly used drugs in clinical care of TBI patients are not known. By definition, cerebral autoregulation is a means by which to maintain constant CBF over a range of blood pressures. Cerebral autoregulation has been prior studied by us in a newborn pig model of TBI, fluid percussion injury (FPI) (8). Pigs have a gyrencephalic brain containing a white/grey ratio more similar to the human. The latter is important because white matter is more vulnerable to injury following TBI. Previous studies showed that cerebral autoregulation is more impaired in young and male compared to female and older pigs after TBI, which parallels the clinical experience (8–12). From a mechanistic standpoint, our earlier studies have noted a more augmented increase in the phosphorylated form of the extracellular signal-related kinase (ERK) isoform of mitogen activated protein kinase (MAPK) in males compared to females following FPI, which contributed to the equally noted greater impairment of cerebral autoregulation in the former compared to the latter (12). Prior studies have investigated the possibility that choice of vasoactive agent may contribute to differences in outcome in male and female newborn pigs following FPI. For example, Phe protected cerebral autoregulation in newborn females due to blockade of phosphorylation of ERK and release of the spasmogen ET-1 but augmented release of these mediators in newborn males following FPI (13,14). However, DA equally prevented impairment of cerebral autoregulation, which was associated with blockade of ERK phosphorylation in both sexes following FPI (14). In clinical studies, impairment of neurovascular unit (NVU)-mediated autoregulation following TBI appears linked to Glascow Coma Scale (GCS), with greater autoregulatory impairment associated with worse GCS (3). Because we had prior noted that autoregulation was more impaired in newborn compared to juvenile pigs (8), we were interested to determine if the sex based protection of cerebral hemodynamics after FPI with Phe treatment was the same or different in the older juvenile pig and how that related to brain tissue injury post injury. In the present study we therefore investigated whether Phe protects autoregulation and limits histopathology after FPI in male and female juvenile pigs and the role of ERK and ET-1 in that outcome. Materials and Methods Anesthetic regimen, fluid percussion brain injury, and visualization of pial arteries All animal protocols were approved by the University of Pennsylvania Animal Care and Use Committee. Juvenile pigs (4 weeks old, 6.0–7.0 Kg) of either sex were studied. The anesthetic regimen consisted of: pre-medication with dexmedetomidine (20 μg/kg im), induction with isoflurane (2–3%), isoflurane taper to 0% after start of total intravenous anesthesia with midazolam (1mg/kg/hr), fentanyl (100 ug/kg/hr), propofol (2–10 mg/kg/hr), dexmedetomidine (2 μg/kg/hr) and saline (2ml/kg/hr). Blood pressure was monitored via a catheter placed in the femoral artery. The pigs were intubated and ventilated with room air. Temperature was maintained in the normothermic range (37°–39° C), monitored rectally. The closed cranial window technique was used for measurement of pial artery diameter and collection of CSF for ELISA analysis (11–13). ICP was determined with an Integra Camino monitor. A laser Doppler probe was used to measure CBF qualitatively. CBF was measured quantitatively in the cerebral cortex and hippocampus using radioactively labeled microspheres (13. The method used to induce moderate (2 atm) brain FPI has been described previously (13). Protocol Thirty pigs were randomized to one of each experimental intervention group (all n=5): (1) sham control (craniotomy but no injury), (2) FPI, (3) FPI post-treated with Phe. CPP was targeted (65–70 mm Hg per 2012 Pediatric Guidelines) to determine the dose of the iv infusion (typically 0.8–1.3 ug/kg/min iv) of Phe and Phe treatment is started when CPP decreases below 45 mm Hg. Animals in which pial artery reactivity and histopathology were determined were the same, allowing for within animal comparison of outcome. These animals were already being given an infusion of saline (to accommodate for loss during ventilation) and in prior studies an infusion of saline elevated above the latter did not make a significant difference in support of CPP over the prolonged time period of the protocol (4h post FPI) (15,16). Cerebral autoregulation was tested via two techniques. The first quantified the transient hyperemic response ratio (THRR) (16, 17). In the second, hypotension was used as the stimulus and was produced by the rapid withdrawal of either 5–8 or 10–15 ml blood/Kg, yielding moderate or severe hypotension (decreases in MAP of 25 and 45%, respectively). The decrements in blood pressure were maintained constant for 10 min by either additional blood withdrawal or blood reinfusion. The vehicle for all agents was 0.9% saline. In sham control animals, responses to THRR, hypotension (moderate, severe) and papaverine (10−8, 10−6 M) were obtained twice; once and then again 1h later. In drug post-treated animals, drugs were administered after FPI and responses to THRR, hypotension and papaverine and CSF samples collected at 1h post insult. The order of agonist administration was randomized within animal groups. We waited 20 min in between rounds of stimuli each set of stimuli to normalize hemodynamic and biochemical conditions. ELISA Commercially available enzyme-linked immunosorbent assay Kits were used to quantity CSF ERK MAPK and ET-1 (Assay Designs, Farmingdale, NY; Phoenix Belmont, CA) concentration. Histologic Preparation The brains were prepared for histopathology at 4h post FPI using prior published methods (16). We determined mean number of necrotic neurons (± SEM) in CA1 and CA3 hippocampus in vehicle control, FPI, and FPI + Phe, treated pigs with data displayed for the side of the brain contralateral to the site of injury (the side where pial artery reactivity was investigated). Morphologic criteria for a necrotic neuron are: 1. Pyknosis, 2. Granulation of the cytoplasm, and 3. The emergence of an unstained area between the nucleus and the cytoplasm. The investigator was blinded to treatment group. Neuronal pathology scoring was described based on damaged neurons/1.2 mm2 of a specific anatomic region as either mild (1–5), moderate (6–15), or severe (> 15). Statistical analysis Values for pial artery diameter and CSF biochemical values were analyzed using ANOVA for repeated measures. If the value was significant, the data were then analyzed by Fishers protected least significant difference test. An α level of p<0.05 was considered significant in all statistical tests. Values are represented as mean ± SEM of the absolute value or as percentage changes from control value. Using power analysis, we determined shows that a sample size of 5 yielded statistical significance at the p<0.05 level with power of 0.84 for hemodynamic data. Power analysis for histopathology and biochemical indice had powers of 0.82 and 0.85, respectively. Results Phe protects autoregulation in female and male juvenile pigs after FPI The level of injury was equal in male and female juvenile pigs (1.9 ± 0.1 vs 2.0 ± 0.1 atm). We chose CPP based on the 2012 Pediatric Guidelines to determine the dose of the iv infusion (in μg/kg/min) of Phe. The infusion of Phe began when CPP dropped below 45 mm Hg. CPP values for sham, FPI, and FPI +Phe were 70 ± 7, 45 ± 4, and 66 ± 3, respectively in males and 71 ± 7, 45 ± 5, and 70 ± 2 mm Hg, respectively, for sham, FPI, and FPI + Phe in females. ICP increased after TBI, but such elevations were blunted by Phe, resulting in normalized (elevated) CPP. In sham piglets, the THRR was similar in male and female juvenile pigs (Fig 1). During unilateral and bilateral carotid artery compression THRR decreased following FPI to a modestly greater level in male compared to female juvenile pigs (Fig 1). Decreases in THRR values were prevented by Phe following FPI in both males and females (Fig 1). Moderate and severe hypotension (24 ± 1 % and 45 ± 2% decrease in MAP, respectively) produced reproducible increases in pial artery diameter in sham pigs. Pial artery dilation in response to hypotension was similar in male and female juvenile pigs (Fig 2). However, pial artery dilation in response to hypotension was impaired in males and females, but the amount of impairment was significantly larger in the male compared to the female pig (Fig 2). Phe administered following FPI protected pial artery dilatation in response to hypotension in males and females after FPI (Fig 2). Papaverine (10−8,10−6M) produced pial artery dilation that was not affected by FPI and Phe (Fig 3), indicating that alteration of vascular reactivity after FPI was not an epiphenomenon. Phe blocked elevation of CSF ET-1 and ERK MAPK in juvenile male and female pigs after FPI CSF ET-1 and phosphorylated ERK MAPK concentrations were increased more in juvenile males compared to females following FPI (Fig 4). Phe blocked such elevations in CSF ET-1 and ERK MAPK concentration in both males and females (Fig 4). Phe prevented loss of neurons in CA1 and CA3 hippocampus in juvenile males and females after FPI The quantity of necrotic neurons in CA1 and CA3 hippocampus was elevated following FPI, which was blocked by Phe in both juvenile males and females (Fig 5). We observed more necrotic neurons in males compared to females following FPI (Fig 5). Blood Chemistry and temperature There were no statistical differences in blood chemistry values amongst groups. For example, values of 7.44 ± 0.05, 35 ± 4, and 91 ± 11 and 7.43 ± 0.06, 39 ± 5, and 97 ± 12 mm Hg for pH, pCO2, and p O2 were obtained in sham controls. Values of 7.45 ± 0.04, 33 ± 6, and 91 ± 12 and 7.44 ± 0.03, 36 ± 6, and 94 ± 13 mm Hg were obtained in FPI treated animals. These values were obtained at the beginning and at the end of these experiments. Discussion Results of the present study show that Phe prevents impairment of cerebral autoregulation and limits necrosis of hippocampal CA1 and CA neurons following FPI in both male and female juvenile pigs. These observations are distinctly different from that determined to be the case in the younger population of pigs wherein Phe prevented impairment of cerebral autoregulation only in newborn female but not newborn male piglets following FPI (13). Taken together, the present study is the first to demonstrate that there are both sex and age related differences in outcome, with use of Phe to normalize CPP after TBI. Using parameters such as brain water content, suture closure, and others, brain growth curves have been constructed for several species to allow for approximations of human age (9). From the latter approach, newborn and juvenile pigs may approximate the human neonate (6 months- 2yrs old) and child (8–10 yrs old) respectively (9). The present data therefore advocate for use of precision medicine approaches in treatment of younger and older boys and girls following TBI. While we noted some modest differences in outcome between juvenile males and females in this study, such data are insignificant in contrast to the newborn male after TBI where Phe had no ability to limit impairment of cerebral autoregulation post injury. Results of the present study also noted a relationship between ET-1, ERK MAPK and outcome after FPI. For example, impairment of cerebral autoregulation is associated with elevation of the CSF concentration of the spasmogen ET-1 and ERK MAPK. Administration of Phe blocked such upregulation and correspondingly prevented impairment of cerebral autoregulation, suggesting upregulation of both ET-1 and ERK were causally related to impaired cerebral hemodynamics in the juvenile pig. Prior studies conducted in the newborn pig after FPI using pharmacological antagonists of ET-1 and ERK more rigorously demonstrated this cause-effect mechanistic relationship (14). In particular, these studies showed that ET-1 upregulation in the setting of TBI causes release of superoxide, which, in turn, increases the amount of phosphorylated ERK MAPK (14). During hypotension, pial arteries vasodilate to maintain constant CBF (autoregulation) and such vasodilation is dependent on opening of K channels (particularly Katp and Kca) (14). However, the ERK released after TBI impairs K channel function, thereby impairing cerebral autoregulation and producing cerebral ischemia (14). In the present study, by preventing upregulation of ET-1 and ERK MAPK, Phe prevents impairment of cerebral autoregulation presumably by protecting K channel mediated vasodilation. A third important observation in the present study relates to ability of Phe to prevent histopathology in both male and female juvenile pigs. In clinical studies, impairment of autoregulation following TBI is linked to worsening of the GCS (3). Therefore, a provocative conclusion from the present study is that interventions post TBI that are designed to preserve autoregulation might have the value added benefit of also improving cognitive outcome. However, we caution that cognition depends on more than the hippocampus and cognitive testing was not performed in the present studies. Additionally, histology was only assessed at an early time point (4h post injury). Therefore, additional studies will be needed to determine if prevention of loss of NVU integrity durably improves cerebral hemodynamics and cognitive function after pediatric TBI. Results of the present study finally extend prior work supportive of the hypothesis that choice of vasoactive agent has important consequence in determining outcome as a function of sex and age after TBI. Thus far, we have described the actions of three different vasoactive agents: Phe, DA, and NE. The effects of NE on outcome after TBI have been prior studied in two ages. The contribution of ERK MAPK to impairment of cerebral hemodynamics was investigated in all studies. We used Phe in the first study (13) because it is often chosen in treatment of TBI in young children due to its longer duration of action and peak elevation of MAP (10). We observed that both Phe and NE selectively protected cerebral autoregulation in the newborn female though blockade of ERK MAPK phosphorylation. Newborn males were not protected and these pressors actually potentiated phosphorylation of ERK MAPK following FPI (13,15). New data show that Phe prevented impairment of autoregulation in juvenile male and female pigs due to blockade of ERK MAPK phosphorlation, indicating that for this pressor both age and sex will determine outcome. A similar pattern of age and sex dependency in outcome was observed for NE (15,16). However, DA protected cerebral autoregulation equivalently in both male and female newborn pigs due to equal blockade of ERK MAPK phosphorylation (18). These data suggest that use of DA might be preferable in treatment of newborns and either NE or Phe for older children. There are several limitations to this study. ERK MAPK was assayed in CSF and used as an indirect index of what may happen to the cellular concentration within brain parenchyma. We do not feel that this reflects damage or pathology because we have reproducibly detected MAPK in CSF under control conditions and monitored its change with a range of stimuli (13). Changes in CSF concentration therefore reflect intracellular events. Conclusions There are no evidence based guidelines or recommendations regarding choice of vasoactive agent after adult or pediatric TBI. Choice of vasoactive agent across medical centers is variable, and may be related to outcome. The ongoing multiple medical therapies (MMT) project (19) will provide 3,6, and 12 month outcome for patients given various pressors for CPP support. However, this project will not be able to answer cerebral autoregulation or mechanistic questions. Therefore, results of this study inform the downstream interpretation of cerebral hemodynamic findings observed in the MMT project. In conclusion, Phe preserves cerebral autoregulation and limits histopathology after TBI through blockade of ERK MAPK and ET-1 in an age and sex dependent manner. This work was supported by NIH R01 NS090998. Disclosure Statement The authors have nothing to disclose. Figure 1 THRR during unilateral and bilateral carotid artery compression in (A) juvenile male and (B) female pigs before (sham), after FPI, and after FPI treated with Phe, n =5 p<0.05 compared to corresponding sham value, +p<0.05 compared to corresponding FPI alone value, #p<0.05 compared to corresponding female value. Figure 2 Influence of FPI on pial artery diameter during hypotension (moderate, severe) in (A) juvenile male and (B) juvenile female pigs. Conditions are before (sham control), after FPI, and after FPI treated with Phe, n =5. p<0.05 compared to corresponding sham value, +p<0.05 compared to corresponding FPI alone value, #p<0.05 compared to corresponding female value. Figure 3 Influence of papaverine (10−8, 10−6 M) on pial artery diameter in (A) juvenile male and (B) juvenile female pigs. Conditions are before (sham control), after FPI, and after FPI treated with Phe, n =5. Figure 4 Influence of FPI and FPI + Phe on ET-1 amd phosphorylated ERK MAPK (pg/ml) before (0 time) and 4h after FPI in (A, B) juvenile male and (C, D) juvenile female pigs, n=5. p<0.05 compared to corresponding 0 time value, +p<0.05 compared to corresponding FPI alone value, #p<0.05 compared to corresponding female value. Figure 5 (A) Low magnification (40×) typical juvenile male sham control showing both CA1 (#1) and CA3 (#2) hippocampal regions. (B) Higher magnification (100×) typical juvenile male sham control CA3 hippocampus. (C) Typical juvenile male FPI CA3 hippocampus (100×). D) Typical FPI + Phe juvenile male CA3 (100×). (E) Typical juvenile female FPI CA3 hippocampus (100×). (F) Typical FPI + Phe juvenile female CA3 hippocampus (100×), (G) High magnification (600×) typical viable sham control male neuron #3, with intact cytoplasm and darkly stained nucleus and (H) High magnification (600×) typical male necrotic neurons, showing # 4 pyknotic nucleus of small neuron, accompanied by neuronal cytoplasm shrinkage (#5) and granulated eosinophilic characteristics (“red dead” neuron) (#6) associated with cell death. Summary data for mean number of necrotic neurons (I) in CA1 and CA3 hippocampus of juvenile male and female pigs under conditions of sham control, FPI, and FPI + Phe, n=5. *p<0.05 compared to corresponding sham control value, +p<0.05 compared to corresponding FPI alone value, #p<0.05 compared to corresponding female value. ==== Refs 1 Langlois JA Rutland-Brown W Thomas KE The incidence of traumatic brain injury among children in the United States: differences by race J Head Trauma Rehabil 2005 20 229 238 15908823 2 Newacheck PW Inkelas M Kim SE Heath services use and health care expenditures for children with disabilities Pediatrics 2004 114 79 85 15231911 3 Freeman SS Udomphorn Y Armstead WM Fisk DM Vavilala MS Young age as a risk factor for impaired cerebral autoregulation after moderate-severe pediatric brain injury Anesthesiology 2008 108 588 595 18362589 4 Kochanek PM Carney N Adelson PD Guidelines for the acute medical management of severe traumatic brain injury in infants, children, and adolescents-Second Edition Pediatr Crit Care Med 2012 13 Suppl 1 S24 S29 5 Ishikawa S Ito H Yokoyama K Makita K Phenylephrine ameliorates cerebral cytotoxic edema and reduces cerebral infarction volume in a rat model of complete unilateral carotid occlusion with severe hypotension Anesth Analg 2009 108 1631 1637 19372348 6 Sookplung P Siriussawakul A Malakouti A Vasopressor use and effect on blood pressure after severe adult traumatic brain injury NeuroCrit Care 2011 15 46 54 20878264 7 Steiner LA Johnston AJ Czosnyka M Direct comparison of cerebrovascular effects of norepinephrine and dopamine in head injured patients Crit Care Med 2004 32 1049 1054 15071400 8 Armstead WM Age dependent cerebral hemodynamic effects of traumatic brain injury in newborn and juvenile pigs Microcirculation 2000 7 225 235 10963628 9 Dobbing J The later development of the brain and its vulnerability Scientific Foundations of Pediatrics Davis JA Dobbing J London Heineman Medical 1981 744 759 10 Digennaro JL Mack CD Malakouti A Use and effect of vasopressors after pediatric traumatic brain injury Dev Neurosci 2011 32 420 430 11 Armstead WM Vavilala MS Adrenomedullin reduces gender dependent loss of hypotensive cerebrovasodilation after newborn brain injury through activation of ATP-dependent K channels J Cereb Blood Flow Metab 2007 27 1702 1709 17377515 12 Armstead WM Kiessling JW Bdeir K Kofke WA Vavilala MS Adrenomedullin prevents sex dependent impairment of cerebal autoregulation during hypotension after piglet brain injury through inhibition of ERK MAPK upregulation J Neurotrauma 2010 27 391 402 20170313 13 Armstead WM Kiessling JW Kofke WA Vavilala MS Impaired cerebral blood flow autoregulation during post traumatic arterial hypotension after fluid percussion brain injury is prevented by phenylephrine in female but exacerbated in male piglets by ERK MAPK upregulation Crit Care Med 2010 38 1868 1874 20562700 14 Armstead WM Vavilala MS Age and sex differences In hemodynamics in a large animal model of Brain Trauma Vascular Mechanisms in CNS Trauma Lo Eng 269 284 2014 15 Armstead WM Riley J Vavilala MS Preferential protection of cerebral autoregulation and reduction of hippocampal necrosis with norepinephrine after traumatic brain injury in female piglets Pediatr Crit Care Med Mar 2016 17 3 e130 137 16 Armstead WM Riley J Vavilala MS Norepinephrine protects cerebral autoregulation and reduces hippocampal necrosis after traumatic brain injury via block of ERK MAPK and IL-6 in juvenile pigs J Neurotrauma 2016 33 1761 1767 26597684 17 Girling KJ Cavill G Mahajan RP The effects of nitrous oxide and oxygen consumption on transient hyperemic response in human volunteers Anesth Analg 1999 89 175 180 10389799 18 Armstead WM Riley J Vavilala MS Dopamine prevents impairment of autoregulation after TBI in the newborn pig through inhibition of upregulation of ET-1 and ERK MAPK Ped Crit Care Med 2013 14 e103 e111 19 Bell MJ Adelson PD Hutchison JS Multiple medical therapies for pediatric brain injury workgroup. Differences in medical therapy goals for children with severe traumatic brain injury – an International Study Pediatr Crit Care Med 2013 14 811 818 23863819	28355201	PMC5509507	NO-CC CODE	2021-01-05 12:06:37	yes	Pediatr Res. 2017 Jul 26; 82(1):108-113
==== Front 10123197632624Nat Chem BiolNat. Chem. Biol.Nature chemical biology1552-44501552-44692862809410.1038/nchembio.2409nihpa864513ArticleA water-soluble DsbB variant that catalyzes disulfide bond formation in vivo Mizrachi Dario 1Robinson Michael-Paul 1Ren Guoping 2Ke Na 2Berkmen Mehmet 2DeLisa Matthew P. 1* 1 School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY 14853 USA 2 New England Biolabs, 240 County Rd, Ipswich, MA, 01938, USA* Address correspondence to: Matthew P. DeLisa, School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY 14853. Tel: 607-254-8560; Fax: 607-255-9166; [email protected] 3 2017 19 6 2017 9 2017 19 12 2017 13 9 1022 1028 Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: http://www.nature.com/authors/editorial_policies/license.html#termsEscherichia coli DsbB is a transmembrane enzyme that catalyzes the re-oxidation of the periplasmic oxidase DsbA by ubiquinone. Here, we sought to convert membrane-bound DsbB into a water-soluble biocatalyst by leveraging a previously described method for in vivo solubilization of integral membrane proteins (IMPs). When solubilized DsbB variants were co-expressed with an export-defective copy of DsbA in the cytoplasm of wild-type E. coli cells, artificial oxidation pathways were created that efficiently catalyzed de novo disulfide bond formation in a range of substrate proteins and in a manner that depended on both DsbA and quinone. Hence, DsbB solubilization was achieved with preservation of both catalytic activity and substrate specificity. Moreover, given the generality of the solubilization technique, the results presented here should pave the way for unlocking the biocatalytic potential of other membrane-bound enzymes whose utility has been limited by poor stability of IMPs outside of their native lipid bilayer context. ==== Body Introduction Integral membrane proteins (IMPs) account for 25–30% of all open reading frames in sequenced genomes1,2. This class of proteins plays vital roles in diverse cellular functions, many of which involve material and/or information transfer across tightly sealed lipid bilayer membranes. Examples of these functions include molecular transport, energy generation, signal transduction, osmotic regulation, and membrane-associated biochemistry. Owing to their centrality in such a wide variety of cellular processes, it is not surprising that these proteins comprise a majority of known drug targets3–5. From a structural perspective, IMPs are intrinsically hydrophobic and thus have low solubility in aqueous environments. As such, IMPs naturally exist within lipid membranes where they make extensive non-polar contacts with the hydrophobic core of the bilayer6. The poor water solubility of IMPs creates a roadblock to characterizing their structure and function7–9, and also represents one of the most substantial barriers to developing membrane protein technologies10. To overcome this challenge, IMPs can be solubilized using detergents or detergent-like reagents (e.g., protein-based nanodiscs11, peptide-based detergents12, and amphiphilic polymers13) that encircle the protein and provide a lipophilic niche inside a detergent micelle. However, identifying the optimal detergent for a given IMP often involves a time consuming process of trial and error14. Moreover, the technological potential of IMPs solubilized by any of these methods can only be investigated in vitro. A radical alternative for IMP solubilization involves redesigning the protein to dissolve in water without the need for exogenous solubilizing agents. For example, a computational approach has been used to engineer a water-solubilized variant of the bacterial ion channel KcsA by mutating 29 of its lipid-contacting hydrophobic residues to more hydrophilic ones15. Indeed, following expression in Escherichia coli cells, high soluble yields of this designed KcsA variant were recovered in the absence of detergent solubilization. Importantly, the water-soluble variant retained the intended functional and structural properties of the wild-type protein. Along similar lines, we recently described a method for in vivo solubilization of IMPs that retained the correct fold and functional form of the protein without adding detergents or mutations to the IMP sequence16. This technique, called SIMPLEx (solubilization of IMPs with high levels of expression), enables soluble expression of IMPs directly in living cells by genetically modifying an IMP target with a truncated version of human apolipoprotein A-I, which effectively shields the IMP from water and promotes its solubilization. In addition to being amphipathic, truncated ApoAI exhibits substantial structural flexibility that allows it to readily conform to a spectrum of different geometries as needed17. Importantly, because the water-soluble IMPs are expressed inside cells, the SIMPLEx method has the potential to enable in vivo applications (e.g., metabolic pathway engineering) that leverage the activity of solubilized IMPs. To test this notion, we attempted to reconstitute the disulfide bond-promoting activity of the membrane-bound DsbB enzyme in the E. coli cytoplasm. In wild-type E. coli, the oxidation of cysteine pairs to form a disulfide bond occurs in the periplasm18 and is catalyzed by DsbB, which is comprised of four transmembrane helices and two periplasmic loops19, and DsbA, a soluble periplasmic enzyme that donates its own extremely reactive, oxidizing disulfide directly to other proteins as they enter the periplasmic space20. Each periplasmic loop of DsbB contains a pair of conserved cysteines that are required for the re-oxidation of DsbA21. Specifically, the Cys41–Cys44 residues form a disulfide bond in response to quinone reduction21,22. This disulfide is subsequently transferred to Cys104–Cys130, which forms a disulfide that is donated to the active-site cysteines of DsbA23. Hence, DsbB effectively bridges the electron transport chain with oxidative protein folding. To create a cytoplasmic pathway for disulfide bond formation, we used the SIMPLEx method to convert DsbB into a water-soluble biocatalyst that could be functionally expressed in the cytoplasm. Specifically, we designed chimeras in which the C-terminus of DsbB was fused to truncated ApoAI while a highly soluble “decoy” protein was fused to its N-terminus. When these solubilized DsbB chimeras were co-expressed with an export-defective copy of DsbA, the normally periplasmic DsbB-DsbA pathway was completely recompartmentalized in the E. coli cytoplasm where it catalyzed de novo disulfide bond formation in a range of substrate proteins. While IMPs previously solubilized using SIMPLEx retained biological activity, as exemplified by ligand binding in the case of EmrE and stimulation of 17,20-lyase activity in the case of human cytochrome b5 (cyt b5)16, none of these were enzymes. Hence, the functional reconstitution of solubilized DsbB represents the first demonstration of catalytic activity in a solubilized IMP via SIMPLEx. The ability to generate solubilized variants of IMPs that are functional in non-native environments opens the door to harnessing other membrane-bound enzymes whose biocatalytic utility has been heretofore limited by the need for a lipid bilayer or for harsh detergents and/or mutations that serve to stabilize the IMP. Results Engineering a water-soluble DsbB variant Engineering a cytoplasmic DsbB-DsbA pathway (Fig. 1a) required a strategy for making the transmembrane protein DsbB compatible with the E. coli cytoplasm. To this end, we hypothesized that the SIMPLEx technique16 could be used to create water-soluble DsbB variants that retain biocatalytic activity (i.e., re-oxidation of DsbA) in the cytoplasm (Fig. 1b). Previously, we showed that a truncation variant of human ApoAI lacking its 43-residue globular N-terminal domain (hereafter ApoAI) promoted soluble expression of structurally diverse IMPs, which were recovered from the cytoplasm at high titers (~5–10 mg/L of culture) and in functionally relevant conformations16. Here, we designed chimeras in which the C-terminus of DsbB was genetically fused to ApoAI while its N-terminus was fused to a highly soluble ‘decoy’ protein, namely the E. coli maltose-binding protein lacking its N-terminal export signal (cMBP, where c indicates cytoplasmic), to prevent the resulting chimera from becoming inserted in the cytoplasmic membrane (Fig. 2a). Following expression of cMBP-DsbB-ApoAI* (hereafter SIMPLEx-DsbB or SxDsbB) in E. coli BL21(DE3) cells, ~40–50% of the chimera accumulated in the soluble cytoplasmic fraction with the remainder partitioning in the insoluble and detergent-solubilized fractions (Fig. 2b). In stark contrast, wild-type (wt) DsbB or a topologically inverted DsbB variant called H0DsbB1/9b (ref. 24), both of which are membrane-bound enzymes, partitioned exclusively in the detergent-soluble fraction (Fig. 2b). A DsbB-ApoAI* chimera lacking the decoy domain accumulated in the detergent-solubilized and insoluble fractions only, while a cMBP-DsbB fusion without the ApoAI* domain was detected primarily in the insoluble fraction with lesser amounts in the detergent-soluble and soluble fractions (Fig. 2b). It should be pointed out that soluble cMBP-DsbB was extensively aggregated as determined by size-exclusion chromatography (SEC), consistent with the soluble aggregates observed for other decoy-IMP fusions lacking the ApoAI* domain16. Structure-guided optimization of soluble DsbB expression Inspection of the DsbB sequence revealed a GxxxA motif (where × represents any amino acid) in the fourth transmembrane (TM) segment that we speculated could be responsible for folding, solubility, and/or stability of the enzyme (Supplementary Results, Supplementary Fig. 1a). Indeed, GxxxG and GxxxG-like motifs are known to stabilize helix–helix interactions in both membrane and soluble proteins25,26. The GxxxA sequence in particular is overrepresented (21% above that expected at random) in all TM sequences identified to date27. In addition, we predicted that the last 13 amino acids of DsbB, which form a flexible, linker-like segment, could potentially reduce the intimate interaction between the DsbB and ApoAI* domains that appears to be important for IMP solubilization16. Thus, to potentially enhance SxDsbB solubility, we removed the last 13 amino acids of DsbB and added two additional GxxxG motifs along the same face of the fourth transmembrane helix because concatenated GxxxGxxxG motifs known as glycine zippers (GZs) are also overrepresented in TM domains28. The resulting variant was named DsbBΔCGZ (Supplementary Fig. 1a). When expressed in the membrane of E. coli cells lacking native DsbB, DsbBΔCGZ was able to promote disulfide bond formation in E. coli alkaline phosphatase (PhoA) at a level that was indistinguishable from wt DsbB (Supplementary Fig. 1b). Having confirmed the activity of DsbBΔCGZ, this variant was modified with cMBP and ApoAI* to generate a SIMPLEx construct named SxDsbBΔCGZ (Fig. 2a). This new construct exhibited higher levels of soluble cytoplasmic expression compared to SxDsbB (Fig. 2b and c), with purified yields reaching nearly 10 mg/L of culture (Supplementary Fig. 1c). In addition, the GZ motif promoted the formation of soluble SxDsbBΔCGZ tetramers (Supplementary Fig. 1c). Soluble DsbB variants function in the cytoplasm Determining the biological activity of the water-soluble DsbB variants required a specific assay to quantify protein oxidation in the cytoplasm. We investigated whether E. coli PhoA could serve this purpose. The wt PhoA enzyme is secreted by a Sec-specific signal peptide into the periplasmic space where it is rapidly oxidized by the native DsbB-DsbA system to form its two consecutive disulfide bonds that are critical for both stability and phosphatase activity29 (see also Supplementary Fig. 1b). For these studies, we made use of E. coli DHB4(DE3), a K-12 strain in which the genomic copy of phoA is absent and the λDE3 prophage has been site-specifically integrated into the genome, enabling expression of target genes cloned in pET vectors. As expected, expression of periplasmically targeted PhoA (pPhoA) in DHB4(DE3) cells resulted in substantial phosphatase activity in the periplasm and little to no activity in the cytoplasm (Fig. 3a). This activity coincided with the detection of intact PhoA protein in the periplasmic fraction (Fig. 3b). To confirm that this activity was due to the DsbB-DsbA pathway, we performed similar experiments using strain DHB4(DE3) ΔdsbAB, a derivative of DHB4(DE3) in which the dsbA and dsbB genes have been insertionally inactivated. When pPhoA was expressed in this strain, the periplasmic phosphatase activity was diminished almost to background levels (Fig. 3a). Next, we created a cytoplasmic PhoA variant (cPhoA) by removal of its native N-terminal signal peptide. Consistent with earlier findings29, expression of cPhoA in DHB4(DE3) ΔdsbAB cells resulted in no measurable activity above background in either the cytoplasm or periplasm (Fig. 3a) and no detectable bands in the corresponding immunoblot (Fig. 3b). This result was attributed to the lack of conformational stability of cPhoA in the absence of its structural disulfides, which cannot form in the reducing cytoplasm. As a positive control, cPhoA was expressed in the cytoplasm of SHuffle T7 Express cells (hereafter SHuffle), a strain whose cytoplasmic reductive pathways are diminished, thereby allowing for the formation of disulfide bonds in the cytoplasm30. As expected, SHuffle cells produced high levels of phosphatase activity in the cytoplasm (Fig. 3a) and the stability of cPhoA in this compartment was dramatically enhanced (Fig. 3b). Taken together, these results confirmed cPhoA as a reliable reporter of cytoplasmic disulfide bond-forming activity. To evaluate the activity of water-soluble DsbB chimeras, each was co-expressed with the cPhoA reporter and an export-defective version of DsbA in which the N-terminal signal peptide was removed (cDsbA). Importantly, both SxDsbB and SxDsbBΔCGZ catalyzed efficient oxidation of cPhoA in the presence of cDsbA in the cytoplasm of DHB4(DE3) ΔdsbAB cells (Fig. 3a,b). This substantial gain in cPhoA activity and stability was on par with that measured following co-expression of cPhoA, cDsbA and the inverted DsbB variant (Fig. 3a,3b), which has its active site located on the cytoplasmic side of the membrane and functions with inverted topology24. Notably, similar results were obtained using a B strain of E. coli, namely BL21(DE3) (Supplementary Fig. 2), indicating that the water-soluble DsbB-DsbA pathway can be readily reconstituted in the cytoplasm of an unrelated host strain with retention of activity. Interestingly, when the pathway was transferred to SHuffle cells, the effects on cPhoA activity were not additive but instead inhibitory (Supplementary Fig. 2), suggesting that the two cytoplasmic oxidation mechanisms may negatively interact. To investigate the redox state of cPhoA in these strains, we performed 4-acetamido-4′-maleimidylstilbene-2,2′-disulfonic acid (AMS) alkylation to quantify in vivo protein oxidation30. AMS alkylates any free thiol group found in the side chains of cysteine residues, covalently adding 500 daltons and causing a discernable mobility shift in SDS-PAGE analysis. As expected, cPhoA was completely reduced in the cytoplasm of DHB4(DE3) ΔdsbAB cells and completely oxidized in the cytoplasm of SHuffle cells (Supplementary Fig. 3). When expressed in the presence of the water-soluble DsbB-DsbA pathway in DHB4(DE3) ΔdsbAB cells, ~90% of cPhoA was oxidized (Supplementary Fig. 3). For comparison, DHB4(DE3) ΔdsbAB cells equipped with inverted DsbB and cDsbA oxidized ~80% of cPhoA (Supplementary Fig. 3). These results unequivocally confirm that the water-soluble DsbB-DsbA pathway inserts disulfide bonds into cPhoA with equal or better efficiency than alternative mechanisms. Mechanism of water-soluble DsbB The oxidation of cPhoA depended on the presence of solubilized DsbB, as activity was completely abrogated when cPhoA was expressed in the presence of cDsbA only (Fig. 3a). Moreover, cPhoA activity was abolished when essential catalytic residues in either cDsbA or SxDsbBΔCGZ were mutated21,31, namely the active-site Cys30-Pro-His-Cys33 motif of cDsbA changed to Ala-Pro-His-Ala, and Cys44 of DsbB substituted with Ala in SxDsbBΔCGZ (Fig. 3a). Given that cPhoA oxidation failed in the presence of the catalytically inactive cDsbA(APHA) construct, we next investigated whether a specific interaction between SxDsbBΔCGZ and cDsbA was a key feature of this activity. In the native DsbB-DsbA system, hetero-dimerization of the DsbB and DsbA components enables thiol–disulfide exchange19. While the DsbB-DsbA heterodimer exists only transiently as a reaction intermediate, a stable complex that mimics a reaction intermediate can be formed with a Cys130 mutant of DsbB and a Cys33 mutant of DsbA, which are held together by a Cys30 (DsbA)–Cys104 (DsbB) intermolecular disulfide bond19. Accordingly, we produced a C130S mutant of SxDsbBΔCGZ and a C33A mutant of cDsbA. When an oxidized mixture of these purified proteins was subjected to an amylose affinity resin, both co-eluted in the same fraction, indicating the formation of a SxDsbBΔCGZ(C130S)–cDsbA(C33A) complex (Supplementary Fig. 4a). When complex formation was interrogated by SEC, an interaction was clearly observed for the oxidized mixture of SxDsbBΔCGZ(C130S) and cDsbA(C33A), with the proteins co-eluting in the early fraction and no material eluting in the later fraction (Supplementary Fig. 4b,c). We next determined whether SxDsbBΔCGZ transmitted electrons from cDsbA to components of the membrane electron-transport system, namely ubiquinone (UQ) and menaquinone (MK), as is the case with membrane-bound DsbB21,22. To this end, we assayed cPhoA activity in the cytoplasm of E. coli strain BL21(DE3) ΔmenA ΔmalF::kan…ubiA420, which is deficient in MK and UQ biosynthesis32. The topologically inverted DsbB variant previously failed to catalyze disulfide bond formation in a similar strain background24, and that result was replicated here (Supplementary Fig. 5). When the water-soluble DsbB-DsbA pathway was tested in this quinone-deficient strain, activity was severely attenuated although still above background (Supplementary Fig. 5), suggesting that cPhoA oxidation by solubilized DsbB is primarily catalytic, requiring an intact electron transport chain. Taken together, these results confirm that the catalytic activity and substrate (both DsbA and quinone) specificity of the enzyme is preserved following solubilization. Structural characterization of water-soluble DsbB To structurally characterize SxDsbBΔCGZ and its complex with cDsbA, we used biological small-angle X-ray scattering (SAXS)33,34. Reconstructions of the molecular envelopes of cDsbA(C33A) and SxDsbBΔCGZ(C130S) were computed ab initio using DAMMIF software35, and the average of 20 bead models was determined for each protein and protein complex (Fig. 4 and Supplementary Fig. 6). No symmetry was imposed in the reconstruction algorithm for cDsbA(C33A), while a p222 symmetry (tetramer) was imposed on SxDsbBΔCGZ(C130S) and its complex with cDsbA(C33A). All 20 models were similar based on mean normalized spatial discrepancy (NSD) values, which were smaller than 0.9 in each case (monomeric DsbA, NSD±SD = 0.551±0.066; dimeric DsbA, NSD±SD = 0.492±0.053; SxDsbBΔCGZ(C130S), NSD±SD = 0.799±0.058; and SxDsbBΔCGZ(C130S) crosslinked to cDsbA(C33A), NSD±SD = 0.817±0.099). As expected, the radius of gyration (Rg), an indicator of how the mass of a particle is distributed around its center of mass, was greater for dimeric cDsbA(C33A) compared to its monomeric counterpart (Fig. 4a). In support of a complex between solubilized DsbB and DsbA, the Rg value for crosslinked SxDsbBΔCGZ(C130S)–cDsbA(C33A) was greater than that measured for SxDsbBΔCGZ(C130S) alone (Fig. 4b). Further analysis of the reconstructed envelopes revealed that both maximum particle size (Dmax) and the volume of the SxDsbBΔCGZ(C130S)–cDsbA(C33A) complex were increased relative to the uncomplexed SxDsbBΔCGZ(C130S) (Fig. 4b). The “pear” shape of the SxDsbBΔCGZ(C130S) envelope can be explained by a model whereby the tetrameric SxDsbBΔCGZ(C130S) proteins are all in a parallel bundle (Fig. 4b). A similar model for the SxDsbBΔCGZ(C130S)–cDsbA(C33A) complex reveals how binding of each cDsbA protein occurs at the top of the pear, effectively increasing envelope height and volume (Fig. 4b). Oxidation of diverse substrates by water-soluble DsbB PhoA contains two disulfide bonds that are consecutive in the primary structure. To determine whether more challenging targets could be oxidized by solubilized DsbB, we investigated additional substrate proteins including: (1) phytase (AppA), an E. coli periplasmic protein that has similar activity to PhoA but contains four disulfide bonds, one of which is non-consecutive; and (2) murine urokinase-type plasminogen activator (uPA) that contains 12 disulfide bonds, most of which are non-consecutive. The cAppA and c-uPA variants were generated by removing the native N-terminal signal peptides from each. When cAppA was expressed in the cytoplasm of DHB4(DE3) ΔdsbAB cells, little to no enzyme activity was detected (Fig. 5a). The detection of cAppA protein in cell lysates under these conditions (Fig. 5b) indicated that the lack of activity was attributable to misoxidation/misfolding of the expressed protein. In contrast, expression in the cytoplasm of SHuffle cells resulted in a substantial level of activity (Fig. 5a) suggesting that an oxidizing cytoplasm was sufficient for proper folding. A similarly high level of activity was observed when cAppA was co-produced with SxDsbB or SxDsbBΔCGZ, along with an expression-enhanced fusion comprised of cDsbA and glutathione S-transferase (GST-cDsbA, which was used because unfused cDsbA from pET28a used in these experiments resulted in weak expression). Notably, the expression and activity achieved with water-soluble DsbB was on par with that observed in cells co-expressing inverted DsbB and GST-cDsbA (Fig. 5a,b). For both cAppA and cPhoA, a DHB4(DE3) strain background was used because it produces little to no endogenous phosphatase activity owing to knockout of the native phoA gene. However, non-phosphatase target substrates do not require a phoA-deficient host. Thus, for c-uPA (and all additional targets tested below), we used BL21(DE3) because this strain is capable of high-level protein expression due to the lack of Lon and OmpT proteases. Expression of c-uPA in the cytoplasm of BL21(DE3) cells resulted in no measurable activity above background unless the water-soluble DsbB-DsbA pathway was also co-produced (Fig. 5c). As with cAppA, the c-uPA expression and activity was on par with that measured in SHuffle or in BL21(DE3) cells that co-expressed inverted DsbB along with GST-cDsbA (Fig. 5c,d). We also investigated whether solubilized DsbB could promote cytoplasmic folding of antibodies including: (1) scFv13, a single-chain Fv (scFv) antibody fragment that specifically binds E. coli β-galactosidase (β-gal) and requires 4 disulfide bonds for stability and activity36; and (2) anti-HAG cyclonal, a full-length immunoglobulin (IgG) specific for influenza virus hemagglutinin (HAG) that has been engineered for cytoplasmic expression (i.e., removal of N-terminal signal peptides from both heavy and light chains)37 and requires 16 disulfide bonds for folding and activity. It is firmly established that full-length antibodies and antibody fragments fail to fold properly when expressed in the cytoplasm of wt E. coli because essential disulfide bonds do not form in this reducing environment. Indeed, expression of scFv13 in the cytoplasm of wt BL21(DE3) cells resulted in no measurable antigen-binding activity above background and no detectable accumulation of the protein (Supplementary Fig. 7a,b). However, expression of scFv13 in SHuffle cells resulted in a large increase in antigen-binding activity that correlated with substantial accumulation of the protein in the cytoplasm (Supplementary Fig. 7a,b). Importantly, when scFv13 was co-expressed with GST-cDsbA and either SxDsbB or SxDsbBΔCGZ in the cytoplasm of BL21(DE3) cells, antigen-binding activity and soluble accumulation were nearly identical with the levels observed in SHuffle cells or in BL21(DE3) cells co-expressing GST-cDsbA and inverted DsbB (Supplementary Fig. 7a,b). Analogously, co-expression of the anti-HAG cyclonal along with SxDsbB or SxDsbBΔCGZ in the cytoplasm of BL21(DE3) cells resulted in strong antigen-binding activity that compared favorably to that measured for SHuffle cells (Supplementary Fig. 8a). Under the conditions tested here, the topologically inverted DsbB variant was incapable of oxidizing the anti-HAG cyclonal. To determine whether the solubilized DsbB variants catalyzed the formation of fully assembled IgG molecules, SDS-PAGE analysis of Protein A-purified IgGs was performed. The heterotetrameric assembly efficiency of anti-HAG cyclonal produced in the presence of SxDsbB and SxDsbBΔCGZ was estimated to be 51 and 57%, respectively (Supplementary Fig. 8b), compared to ~90% for anti-HAG cyclonal assembly in SHuffle cells reported previously37. This difference is likely due to the fact that SHuffle cells carry a genomic copy of cytoplasmic DsbC, a disulfide bond isomerase that assists in the formation of correctly folded multi-disulfide bonded proteins. Nonetheless, these data reveal that solubilized DsbB is a remarkably flexible biocatalyst that promotes disulfide bond formation in a wide array of structurally diverse substrate proteins. Discussion In this work, we successfully converted membrane-bound DsbB into a water-soluble protein that could be expressed cytoplasmically at relatively high levels and with retention of function. This feat was achieved by leveraging a method called SIMPLEx, which enables in vivo solubilization of IMPs in structurally and functionally relevant conformations without the need for potentially inactivating detergents, lipid reconstitutions, or mutations to the IMP itself16. The SIMPLEx technique was previously shown to be generally useful for solubilizing an array of structurally diverse IMPs, leading to the accumulation of non-aggregated, water-soluble IMPs at high titers (~5–10 mg/L of culture) that retained biological activity, namely ligand binding in the case of EmrE and stimulation of 17,20-lyase activity in the case of human cytochrome b5 (cyt b5)16. Here, we demonstrate for the first time in vivo catalysis with a SIMPLEx-solubilized enzyme in the context of a multi-enzyme pathway, opening the door to a wide array of in vivo applications involving solubilized IMPs. A catalytic mechanism for solubilized DsbB is supported by the observation that both DsbA and quinone substrate dependence was preserved in the engineered DsbB chimeras. Analogous to the native Dsb pathway, the engineered pathway transferred oxidizing potential to cPhoA in a process that involved known catalytic residues, namely the active-site Cys30-Pro-His-Cys33 motif in cDsbA and the Cys44 residue in solubilized DsbB. In further support of a catalytic mechanism, the water-soluble DsbB-DsbA pathway was extremely efficient with ~90% of cPhoA accumulating in an oxidized state. Further, re-oxidation of solubilized DsbB appeared to involve UQ or MK based on the observation that cPhoA oxidation was severely attenuated in a quinone-deficient strain background. Interestingly, a low level of cPhoA oxidation still occurred in this strain, consistent with the observation that in the absence of quinones, DsbB can oxidize ~40% of DsbA in a 1:1 stoichiometric reaction38. However, alternative origins for this low level of quinone-independent activity, such as an unknown cytoplasmic factor reoxidizing solubilized DsbB, cannot be ruled out at this point. Interestingly, the ability of the ApoAI* domain not only to promote solubilization of DsbB but also to accommodate interactions with its biological partner DsbA suggests a remarkable structural plasticity of this unique amphipathic domain. Along similar lines, SIMPLEx-solubilized human cyt b5 stimulated the 17,20-lyase activity of CYP17A116, an activity that is known to involve transmembrane helix–helix interactions between cyt b5 and CYP17A1. Hence, these results collectively reveal that the ApoAI* domain is sufficiently flexible to allow protein–protein interactions that are necessary to promote proper function. Additionally, these data justify the use of SIMPLEx as a tool for studying IMPs and their soluble partners. The formation of disulfide bonds in the cytoplasm has long been possible using trxB gor suppressor strains such as FÅ11339 or SHuffle30. Compared to these mutant strains, an advantage of our engineered pathway is that it can be readily used in any strain background simply by transforming the host of interest with a plasmid encoding the SxDsbB/cDsbA (or SxDsbBΔCGZ/cDsbA) genes. Indeed, we showed that the SxDsbB/cDsbA and SxDsbBΔCGZ/cDsbA could be directly transferred between unrelated strain backgrounds without loss of function. An alternative approach to cytoplasmic protein oxidation involves inverting the membrane topology of DsbB24. While this construct can also be readily shuttled between strain backgrounds, a drawback is that it relies on membrane protein expression, which often imposes a burden on the host that is deleterious to cell viability40. By directing the expression of DsbB to the cytoplasm of E. coli, we take advantage of this compartment’s ability to support recombinant product yields exceeding 50% of the total cellular protein41 while eliminating the energy-intensive process of membrane integration. In light of these advantages, we envision that our strategy could be used to redirect a wide range of biological pathways involving IMPs to the cytoplasmic compartment. Moreover, the generality of the method could also unlock previously intractable membrane-bound biocatalysts for stand-alone or pathway-mediated applications in cellular, or alternatively, cell-free reaction environments. Online Methods Bacterial strains and plasmids The following E. coli strains were used in this study: DHB4 (MC1000 ΔphoA(PvuII) phoR ΔmalF3 F’[lac+(lacIQ) pro]), SHuffle T7 Express (New England Biolabs), and BL21(DE3) (Novagen). DHB4 ΔdsbAB strain was derived from DHB4 by insertional inactivation of the dsbA and dsbB genes with the kanamycin resistance gene as described previously42. Briefly, this was accomplished by P1 phage transduction using the BW25113 dsbA::kan and BW25113 dsbB::kan strains from the Keio collection as donors and plasmid pCP20 to remove the Kan marker as needed. The DHB4(DE3) and DHB4(DE3) ΔdsbAB strains were generated using a λDE3 lysogenization kit (Novagen) according to manufacturer’s instructions and positive clones identified based on the ability to produce active GFP from plasmid pET-GFP43. Strain BL21(DE3) ΔmenA ΔmalF::kan…ubiA420 was constructed exactly as described previously32, with the nonessential menA gene disrupted by P1 phage transduction and the ubiA gene conditionally knocked out by introducing the ΔmalF::kan…ubiA420 allele into the BL21(DE3) ΔmenA strain. The mutant has a negligible UbiA activity. However, when supplied with 1 mM hydroxybenzoic acid (HBA), this mutant strain can make ~20% amount of the UQ as the wild-type strain; this quinone level is sufficient for the growth of E. coli cells32. All plasmids used in this study are listed in Supplementary Table 1. Plasmids constructed here were made using standard cloning procedures and all plasmid sequences were confirmed by sequencing. For expression and purification of different DsbB chimeras, the plasmid pET21d (Novagen) was used. Specifically, the ΔspMBP-DsbB-ApoAI* gene fusion (defined here as SxDsbB) was PCR-amplified from pET28-ΔspMBP-DsbB-ApoAI16 and subsequently cloned in plasmid pET21d. This new plasmid, pET21-SxDsbB, served as the template for creating plasmids pET21-SxDsbBΔCGZ, pET21-SxDsbBΔCGZ(C44A), and pET21-SxDsbBΔCGZ(C130S), which were all created by standard site-directed mutagenesis. In addition, removal of the genes encoding cMBP or ApoAI, or both, from pET21-SxDsbB or pET21-SxDsbBΔCGZ gave rise to the following plasmids: pET21-DsbB, pET21-DsbBΔCGZ, pET21-cMBP-DsbB, pET21-cMBP-DsbBΔCGZ, pET21-DsbB-ApoAI, and pET21-DsbBΔCGZ-ApoAI. For cDsbA expression and purification, we used pET39b (Novagen), which carries the full-length E. coli dsbA gene. The cDsbA(C33A) mutant was generated using site-directed mutagenesis with pET39b as template. For H0DsbB1/9b expression and purification, plasmid pFH265 was used24. For PhoA expression in the periplasm, plasmid pBAD-pPhoA was generated by cloning the gene encoding full-length E. coli phoA into plasmid pBAD18-Cm44. For PhoA expression in the cytoplasm, pFH-cPhoA was constructed by excising the gene encoding cDsbA from plasmid pFH27324. For co-expression of PhoA and DsbA in the cytoplasm, we used pFH27324, a pLysSBAD derivative that enables bicistronic expression of the genes encoding mature PhoA lacking its N-terminal signal peptide (cPhoA; residues R22-K471) and mature DsbA lacking its N-terminal signal peptide (cDsbA; residues A20-K208) and also carrying an N-terminal 6x-His tag. Plasmid pFH273mut was generated whereby the active-site Cys30-Pro-His-Cys33 motif of cDsbA was mutated to Ala-Pro-His-Ala, which completely inactivates the enzyme31. Cytoplasmic PhoA expression was also performed using pET24-cPhoA, which was created by cloning mature PhoA (residues R22-K471) in plasmid pET24b. Plasmids for expressing the other substrates, namely cAppA, c-uPA, scFv13, and anti-HAG cyclonal IgG, are listed in Supplementary Table 1. While working with cAppA, which carried a C-terminal 6x-His tag, we observed conflicting bands in Western blots using the anti-6x-His antibody due to the fact that SxDsbB or SxDsbBΔCGZ, which were also 6x-His tagged, migrated similarly. Therefore, we generated a new cAppA expression plasmid in which the C-terminal 6x-His tag was replaced with a FLAG epitope tag in plasmid pHIS. These substrate expression plasmids were each paired with a bicistronic expression plasmid encoding a genetic fusion between E. coli GST and cDsbA, and one of the DsbB variants. These bicistronic expression plasmids were constructed as follows: first, a genetic fusion between the genes encoding GST and cDsbA was inserted between NheI-XhoI in the bicistronic expression plasmid pET28-BicExp, which has the following multiple cloning site: NcoI-HindIII-RBS2-NheI-XhoI (where RBS = second ribosome binding site). Next, the gene encoding SxDsbB was inserted between NcoI and HindIII, yielding pET28-SxDsbB::GST-cDsbA. Plasmid pET28-H0DsbB1/9b::GST-cDsbA was generated similarly, while plasmids pET28-SxDsbBΔCGZ::GST-cDsbA, pET28-SxDsbBΔCGZ::GST-cDsbA(APHA), and pET28-SxDsbBΔCGZ(C44A)::GST-cDsbA were generated by site-directed mutagenesis using pET28-SxDsbB::GST-cDsbA as template. Protein expression and purification For small cultures, single colonies of the strain of interest carrying the plasmid(s) of interest were grown at 30°C in 5 mL of Luria-Bertani (LB; 10 g/L tryptone, 5 g/L yeast extract, 5 g/L NaCl, pH 7.2) supplemented with the corresponding antibiotics, and grown until optical density at 600 nm (OD600) reached a value of 1.0. At this point isopropyl-β-D-thiogalactopyranoside (IPTG) or arabinose was added to a final concentration of 1 mM or 0.5 % arabinose, respectively. Protein expression was allowed to proceed overnight at 25°C. For the expression and purification of SxDsbBΔCGZ(C130S), wt cDsbA, and cDsbA(C33A), a single colony of BL21(DE3) cells carrying the corresponding plasmid was inoculated into 5 mL of LB supplemented with 50 μg/mL of kanamycin and grown overnight at 30°C. The next day, 500 mL of freshly prepared Terrific Broth (TB) supplemented with 100 μg/mL kanamycin was inoculated 1/100 with the overnight culture and cells were grown at 30°C until reaching OD600 of 0.8. Protein expression was induced by the addition of 0.25 mM IPTG, after which cells were incubated an additional 18 h at 16°C. Cells were harvested by centrifugation before preparation of lysates or purification of protein targets. Protein purification of SxDsbBΔCGZ(C130S) was carried out with amylose resin (New England Biolabs) according to manufacturer’s specifications, followed by SEC using an ÄKTA Explorer FPLC system (GE Healthcare). Ni-NTA purification of wt cDsbA and cDsbA(C33A) was followed by SEC. SEC was performed using a Superdex 200 10/300 GL column. Standards used to calibrate the SEC column were a lyophilized mix of thyroglobulin, bovine γ-globulin, chicken ovalbumin, equine myoglobin and vitamin B12, MW 1,350–670,000, pI 4.5–6.9 (Bio-Rad). Proteins were stored at a final concentration of 1 mg/ml in SEC buffer (20 mM Tris pH 7.5, 50 mM NaCl, 1 mM EDTA pH 8.0) at 4°C. Full-length anti-HAG IgG was purified using Protein-A. Briefly, protein-A agarose resin was equilibrated with 10 ml PBS and then mixed with the filtered soluble lysate fraction. The resin-soluble lysate fraction mixture was incubated at room temperature for 2 h. After settling in a poly-propylene column, the protein-A agarose was washed extensively with PBS. Anti-HAG IgGs were eluted from the column with 0.1 M glycine-HCl (pH 3.0) in 1-ml fractions and immediately neutralized with 100 μl of 1 M Tris (pH 9.0). Purified fractions were resolved by SDS-PAGE under non-reducing conditions and visualized by staining with Coomassie Blue G-250. Subcellular fractionation Following protein expression, 5 ml of cells expressing the various substrate targets were harvested. Cultures were normalized by OD600 and culture aliquots were pelleted via centrifugation for 10 min at 4°C and 4,000 × g. Cells were then resuspended in lysis buffer containing 30 mM Tris pH 8.0, 500 mM NaCl, and 40 mM imidazole pH 8.0 and lysed using a sonicator. To separate soluble proteins from membranes, the homogenate was ultracentrifuged (100,000 × g) for 1 h at 4°C and the supernatant was collected as the soluble fraction. Detergent soluble fractions were obtained by treating the pellets resulting from the previous step with 1 ml of lysis buffer containing 2% n-dodecyl-β-D-maltoside (DDM; Anatrace). Pellets were resuspended by douncing. Partitioning of membrane proteins into the DDM-containing lysis buffer was achieved by rotating the lysate at 4°C for 2 h. Following centrifugation (50,000 × g) for 30 min at 4°C, the supernatant represented the detergent-solubilized fraction and the pellet represented the insoluble fraction. IMPs in the various fractions were separated by SDS-PAGE using 10% polyacrylamide gels (Bio-Rad) and subsequently detected by Western blotting according to standard protocols using a 1:5,000-diluted monoclonal anti-6x-His horseradish peroxidase (HRP)-conjugated antibody (Abcam). For experiments that involved the isolation of periplasmic fractions, cells were initially resuspended in 20% sucrose, 30 mM Tris-HCl pH 8.5, 1 mM EDTA and 1 g/L lysozyme and incubated at room temperature for 10 min. Following centrifugation (10 min at room temperature and 10,000 × g), cell pellets were fractionated according to standard ice-cold osmotic shock. The supernatant resulting from the centrifugation step (10 min at 4°C and 15,000 × g) was taken as the periplasmic fraction, while the remaining pellet was used to prepare the soluble cytoplasmic fraction as described above. IMPs in the various fractions were separated by SDS-PAGE using 10% polyacrylamide gels (Bio-Rad) and subsequently detected by Western blotting according to standard protocols using a 1:5,000-diluted monoclonal anti-6x-His HRP-conjugated antibody (Abcam) or 1:5,000-diluted monoclonal anti-FLAG HRP-conjugated antibody (Abcam) or 1:5,000-diluted monoclonal anti-PhoA-HRP conjugated (New England Biolabs). For loading controls in SDS-PAGE experiments 1:50,000-diluted polyclonal rabbit anti-GroEL (Sigma) and a secondary antibody goat anti-HRP (Abcam) diluted 1:10,000 were used. Enzyme activity assays For measuring alkaline phosphatase activity, harvested cells were resuspended in subcellular fractionation buffer supplemented with 50 mM N-ethyl maleimide (Sigma) to prevent the spontaneous oxidation of disulfide bonds. Alkaline phosphatase activity was measured in a continuous assay by monitoring absorbance at 410 nm (Abs410) upon the hydrolysis of 4-nitrophenyl phosphate (Sigma) [0.1% (w/v) in 1 M Tris (pH 8.0)] with a plate reader using 5 μl of the fraction to be assayed (periplasmic or cytoplasmic) and 195 μl of substrate in a 96-well plate at room temperature. Measured phosphatase activity was normalized to total protein amount as determined by standard Bradford assay. Phytase activity was quantified as described earlier45 with slight modifications. Assays were performed in 96-well plates with 20 μl of soluble protein. Reaction was stopped with 50 μl 5 M NaOH. Phytase activity was measured at Abs410 and normalized to total protein amount as determined by standard Bradford assay. Urokinase activity was quantified using a 96-well plate at room temperature. A 50-μl aliquot of soluble protein was added to wells containing 50 mM Tris pH 8, 60 mM 6-aminohexanoic acid (Sigma), 0.1 mg/ml Bovine Plasminogen (American Diagnostica) and 0.4 mM Spectrozyme PL (American Diagnostica) to a final volume of 150 μl. The plate was incubated at 37°C and Abs405 was monitored for 2–3 h until reaching plateau. Activity was measured at Abs405 in a linear range and normalized to total protein amount as determined by standard Bradford assay. AMS alkylation Cultures were prepared as described above for the determination of PhoA activity. After induction overnight, 1 mL of culture (normalized by OD600) was used to prepare protein samples. Initially, each sample was subjected to trichloroacetic acid (TCA) precipitation at room temperature for 20 min. The supernatant was discarded after centrifuging at 10,000 rpm for 10 min. Pellets were washed with 1 mL of cold acetone, vortexed, centrifuged again at 10,000 rpm for 10 min), and the supernatant discarded. Pellets were air dried for 30 min at room temperature and then resuspended in 80 μl of alkylation buffer (100 mM Tris·Cl, pH 6.8, 1% SDS) with or without freshly dissolved AMS (Molecular Probes). Samples were incubated for 30 min at 37°C. Fully oxidized and reduced controls were generated using aliquots from cells expressing cPhoA. Specifically, pellets washed with acetone and air dried were treated with 50 μL of 3.6 mM (in water) 4,4′-dithiodipyridine (4-DPS) (Sigma) for oxidation or 100 mM dithiothreitol (DTT) (Sigma) for reduction, and incubated at 30°C for an additional 30 min prior to AMS alkylation. For Western blot analysis, samples were boiled and loaded in BioRad 7.5% Mini-PROTEAN TGX Precast Protein Gels. To ensure sufficient separation of the reduced and oxidized cPhoA species, gels were run at 200 mV for 40 min or until the 25-kDa band of the marker exited the gel. The entire AMS alkylation experiment was repeated in triplicate, with all replicates showing similar results. Enzyme-linked immunosorbent assay Cell lysates derived from strains expressing scFv13 or anti-HAG IgG were analyzed by enzyme-linked immunosorbent assay (ELISA). Costar 96-well ELISA plates (Corning) were coated overnight at 4°C with 50 μl of 4–10 μg/ml antigen in 0.05 M sodium carbonate buffer (pH 9.6). For scFv13, plates were coated with 50 μl of 10 μg/ml β-gal (Sigma) in PBS whereas for anti-HAG, plates were coated with recombinant GST fused to hemagglutinin (GST-HAG) produced in-house37. Following two 5-min washes with PBS, a 3% non-fat milk blocking solution was applied to each well and incubated with shaking for 3 h at room temperature. Subsequently, samples were applied to each well and incubated with shaking for 2 h at room temperature. Extensive washing (four 5-min washes) was performed prior to the addition of one of the following primary antibodies: anti-6x-His HRP-conjugated antibody (Abcam) for scFv13 and anti-human Fc HRP-conjugated antibody (Thermo Scientific) for anti-HAG IgG. Immunodetection was performed by adding 50 μl of blocking solution containing a 1:5,000 dilution of the appropriate antibody to each well followed by incubation for 1 h at 4°C. This was followed by six 5-min washes with cold washing solution (0.5%Tween-20 in PBS). Detection of bound proteins was carried out using SigmaFAST o-phenylenediamine (OPD) tablets followed by monitoring at Abs492. Crosslinking of SxDsbBΔCGZ(C130S) and cDsbA(C33A) Following purification of SxDsbBΔCGZ(C130S) and cDsbA(C33A), as described above, proteins were mixed in an equimolar ratio. The mixture was oxidized on ice for 1 h with 5 mM K3[Fe(CN)6] and stored in SEC buffer for downstream applications. SAXS analysis SAXS data were collected at the Cornell High Energy Synchrotron Source (CHESS) G1 station in Ithaca, New York. Protein samples of SxDsbBΔCGZ(C130S), wt cDsbA and cDsbA(C33A) and crosslinked SEC fractions of SxDsbBΔCGZ(C130S) and cDsbA(C33A) were exposed with a 250 × 250 μm beam of 9.968 keV X-ray. Sample preparation included centrifugation at 30,000 × g for 30 min and filtration to remove any aggregates. Samples (30 μl) in a range of concentrations from 1 to 10 mg/mL were loaded and oscillated in the beam using an automated system with a plastic chip-based sample cell (2-mm path) and polystyrene X-ray transparent windows. The sample cell and X-ray flight path were placed under vacuum to reduce background scattering. Scattering patterns were captured on a Pilatus 100K-S detector (Dectris, Baden, Switzerland) at 1,504-mm distance. The exposure time was 5 s for each image and 10 images were recorded for each sample. All mathematical manipulations of the data (azimuthal integration, normalization, averaging and buffer subtraction) as well as error propagation were carried out using RAW software46. The range of momentum transfer was calculated to be 0.0068<q=4∏ sin(θ)/λ<0.28 Å–1, where 2θ is the scattering angle and λ=1.257 Å is the X-ray wavelength. Molecular weight estimated from a lysozyme standard (3.5 mg/ml, 50 mM NaOAc, 50 mM NaCl pH 4.0) agreed with our expectations within error. Rg was calculated using both Guinier approximation47 and the inverse Fourier transform method as implemented in the GNOM-ATSAS 2.6.1 package by D. Svergun EMBL-Hamburg. The pair distance distribution function P(r) was calculated using the GNOM program47. Dmax was estimated based on the goodness of the data fit and smoothness of the decaying tail. The GNOM output file for the dimer was used as input to DAMMIF35 to perform ab initio shape reconstruction without imposing any symmetry. The 20 reconstructed bead models were superimposed and averaged using DAMAVER35 in the automatic mode. The mean NSD ± SD values were <1 for all envelopes calculated, indicating close agreement between different reconstructed models. The creation of the atomistic models for both SxDsbBΔCGZ(C130S) and SxDsbBΔCGZ(C130S) crosslinked to cDsbA(C33A) were created using PyMOL and the following crystal structures: ΔspMBP crystal structure (pdb ID: 1NL5), ApoAI lipid-free crystal structure (pdb ID: 2A01), DsbA monomer (pdb ID:1a23), DsbA dimer (pdb ID: 1u3a), monomeric DsbB (pdb ID: 2k74), and DsbB in complex with DsbA (pdb ID: 2zup, 2hi7, 3e9j, 2leg). The computation of SAXS profiles of the models constructed for SxDsbBΔCGZ(C130S) (NSD=0.799) and SxDsbBΔCGZ(C130S) crosslinked to cDsbA(C33A) and their fit to experimental profiles was carried out using the Fast X-ray scattering web server (https://modbase.compbio.ucsf.edu/foxs/)48,49 Data Availability All data generated or analyzed during this study are included in this published article (and its supplementary information files). Supplementary Material 1 We thank Professor Lloyd Ruddock (University of Oulu, Finland) for providing plasmids used in this study. We thank Dr. Carolyn Sevier (Cornell University) for helpful discussions of the manuscript. This work is based upon research conducted at the Cornell High Energy Synchrotron Source (CHESS), which is supported by the NSF and the NIH/NIGMS under NSF award DMR-1332208, using the Macromolecular Diffraction at CHESS (MacCHESS) facility, which is supported by award GM-103485 from the NIH/NIGMS. This work also made use of the Cornell Center for Materials Research Shared Facilities, which are supported through the NSF MRSEC program (Grant # DMR-1120296). This work is based upon work supported by NIH Grants # R21DA031409-01 (to M.P.D.), NSF Grants # CBET 1159581 and CBET 1264701 (both to M.P.D.), a Ford Foundation Predoctoral Fellowship (to M.-P.R.), and a National Science Foundation Graduate Research Fellowship (to M.-P.R.). Author Contributions. D.M. designed research, performed all research, analyzed all data and wrote the paper. M.-P.R. performed experiments and analyzed data related to antibody expression. G.R. and N.K. performed experiments and analyzed data related to AMS alkylation, quinone-deficient cPhoA activity, and c-uPA activity. M.B. designed research and analyzed data. M.P.D. designed research, analyzed data and wrote the paper. Competing Financial Interests. The authors declare no competing financial interests. Figure 1 A water-soluble DsbB variant that catalyzes disulfide bond formation in vivo (a) Schematic of the native E. coli disulfide bond formation pathway, which involves the endogenous transmembrane enzyme DsbB. DsbB is located in the inner membrane and interacts with its soluble periplasmic partner DsbA, which is localized to the periplasmic compartment by virtue of an N-terminal signal peptide specific for the cotranslational signal recognition particle (SRP) pathway. Electron transport is represented by the black arrows. DsbB obtains its electrons directly from quinones (Q). (b) Expression of DsbB as a soluble biocatalyst in the E. coli cytoplasm is accomplished using the SIMPLEx technology, which renders IMPs water-soluble by introduction of a ‘decoy’ domain (cMBP) and a ‘shield’ domain (ApoAI). DsbA is redirected to the cytoplasm by removal of its native signal peptide. Following co-expression, solubilized SxDsbB and export-defective DsbA (cDsbA) effectively transform the cytoplasm into a disulfide bond formation compartment. If needed, cDsbA expression can be improved by fusion to E. coli GST, a resident cytoplasmic protein that promotes solubility of its fusion partners. Figure 2 In vivo solubilization of DsbB using the SIMPLEx strategy (a) Molecular architecture of SIMPLEx constructs SxDsbB and SxDsbBΔCGZ used in this study. Each construct included N-terminal cMBP as decoy protein (orange), C-terminal ApoAI as shield domain (green), and intervening flexible linkers (L, grey) that connected cMBP and ApoAI* to the DsbB domain. The DsbB domains tested were wt DsbB (top) and DsbBΔCGZ (bottom), the latter of which is a C-terminally truncated DsbB variant harboring an engineered glycine zipper (GZ, red). (b) Western blot analysis of soluble (s), detergent solubilized (d), and insoluble (i) fractions prepared from E. coli strain BL21(DE3) carrying pET21-based plasmids encoding one of the following DsbB constructs: topologically inverted DsbB (H0DsbB1/9b), wt DsbB, DsbB-ApoAI, cMBP-DsbB, and cMBP-DsbB-ApoAI (SxDsbB) as indicated. Blot was probed with anti-6x-His antibody. Molecular weight (MW) markers are shown on the left. (c) Western blot analysis identical to panel (b) except with DsbBΔCGZ in place of wt DsbB in all constructs as indicated. See Supplementary Figure 9 for uncropped versions of the blot images. Figure 3 Solubilized DsbB variants promote folding of alkaline phosphatase (a) Schematic showing disulfide bond connectivity for E. coli alkaline phosphatase, PhoA (2 disulfide bonds depicted by yellow circles connected by yellow lines). Alkaline phosphatase activity measured in periplasmic (white bars) and cytoplasmic (gray bars) fractions derived from the following strains: wt DHB4(DE3) cells (lacking the native phoA gene) carrying no plasmid (empty) or pBAD18-pPhoA (pPhoA); DHB4(DE3) ΔdsbAB cells carrying no plasmid (empty), pBAD18-pPhoA (pPhoA), pFH273 (cPhoA + cDsbA), or pFH273mut (cPhoA + cDsbA(APHA)), along with a pET21d derivative containing SxDsbB, SxDsbBΔCGZ, SxDsbBΔCGZ(C44A), or H0DsbB1/9b as indicated; and SHuffle cells carrying no plasmid (empty) or pFH-cPhoA (cPhoA). Data is the mean of biological triplicates and the error bars represent the standard error of the mean (SEM). (b) Western blot analysis of the same periplasmic (p) and cytoplasmic (c) fractions assayed in (a) as indicated. Blots were probed with anti-PhoA antibody to detect PhoA (top panel) and anti-GroEL antibody to detect GroEL (bottom panel), which served as a cytoplasmic fractionation marker and loading control. Molecular weight (MW) markers are shown on the left. See Supplementary Figure 10 for uncropped versions of the blot images. Figure 4 Structural characterization of solubilized DsbB by biological SAXS Reconstructed particle envelopes calculated ab initio from the SAXS data for cDsbA(C33A) as a monomer (yellow) or dimer (purple) (a) and tetrameric SxDsbBΔCGZ(C130S) (blue) and tetrameric SxDsbBΔCGZ(C130S) crosslinked to cDsbA(C33A) (red) (b). Radius of gyration (Rg) and maximum particle size (Dmax) are reported for each. Below each envelope is the corresponding crystal structure (PDB 1a23 for monomeric DsbA, PDB 1u3a for dimeric DsbA, PDB 2k74 for monomeric DsbB, PDB 2leg for the complex DsbB(C130S) and DsbA(C33A), PDB 2a01 for ApoAI and 1NL5 for MBP). For each construct, the mathematical representation of the fit for the corresponding crystal structure and its calculated envelope (log intensity, I(q), versus q) is plotted in Supplementary Fig. 6. Figure 5 Proper folding of complex substrate proteins by solubilized DsbB variants (a) Disulfide bond connectivity for E. coli phytase, AppA (4 disulfide bonds depicted by yellow circles connected by yellow lines). Phytase activity (measured as absorbance at 410 nm) in cytoplasmic fractions derived from: E. coli strain DHB4(DE3) ΔdsbAB carrying no plasmids (empty) or pHIS-cAppA (cAppA) along with pET28-SxDsbB::GST-cDsbA, pET28-SxDsbBΔCGZ::GST-cDsbA, or pET28-H0DsbB1/9b::GST-cDsbA as indicated; and SHuffle cells carrying no plasmid (empty) or pHIS-cAppA (cAppA). (b) Western blot analysis of same cytoplasmic fractions assayed in (a). (c) Disulfide bond connectivity for murine urokinase, uPA (12 disulfide bonds depicted by yellow circles connected by yellow lines). Urokinase activity (measured as absorbance at 405 nm) in cytoplasmic fractions derived from E. coli strain BL21(DE3) carrying no plasmids (empty) or pET24b-urokinase along with pET28-SxDsbB::GST-cDsbA, pET28-SxDsbBΔCGZ::GST-cDsbA, or pET28-H0DsbB1/9b::GST-cDsbA as indicated; and SHuffle cells carrying no plasmid (empty) or pET24b-urokinase. (d) Western blot analysis of same cytoplasmic fractions assayed in (c). Blots were probed with anti-FLAG antibody to detect cAppA and c-uPA (top panels) and anti-GroEL antibody to detect GroEL (bottom panel), which served as a cytoplasmic fractionation marker and loading control. Molecular weight (MW) markers are shown on the left. Activity data is the mean of biological triplicates and the error bars represent the standard error of the mean (SEM). 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==== Front Nat CommunNat CommunNature Communications2041-1723Nature Publishing Group ncomms644610.1038/ncomms644625399761ArticleA photon thermal diode Chen Zhen 1Wong Carlaton 2Lubner Sean 1Yee Shannon 1Miller John 2Jang Wanyoung 1Hardin Corey 2Fong Anthony 2Garay Javier E. 2Dames Chris a131 Department of Mechanical Engineering, University of California, Berkeley, California 94720, USA2 Department of Mechanical Engineering, University of California, Riverside, California 92521, USA3 Division of Materials Sciences, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USAa [email protected] 11 2014 2014 5 544602 05 2014 01 10 2014 Copyright © 2014, Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved.2014Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved.A thermal diode is a two-terminal nonlinear device that rectifies energy carriers (for example, photons, phonons and electrons) in the thermal domain, the heat transfer analogue to the familiar electrical diode. Effective thermal rectifiers could have an impact on diverse applications ranging from heat engines to refrigeration, thermal regulation of buildings and thermal logic. However, experimental demonstrations have lagged far behind theoretical proposals. Here we present the first experimental results for a photon thermal diode. The device is based on asymmetric scattering of ballistic energy carriers by pyramidal reflectors. Recent theoretical work has predicted that this ballistic mechanism also requires a nonlinearity in order to yield asymmetric thermal transport, a requirement of all thermal diodes arising from the second Law of Thermodynamics, and realized here using an ‘inelastic thermal collimator’ element. Experiments confirm both effects: with pyramids and collimator the thermal rectification is 10.9±0.8%, while without the collimator no rectification is detectable (<0.3%). A thermal diode is the heat transfer analogue of an electrical diode: it favours the flow of energy carriers such as photons, phonons or electrons in one direction. Here, the authors demonstrate a photon thermal diode that uses pyramidal reflectors to asymmetrically scatter the photons. ==== Body Energy is one of the dominant issues of modern times, and the large majority of it passes through the thermal domain1. Traditional thermal engineering is based on the paradigm of linear thermal elements, such as thermal resistors (R) and capacitors (C), and in this sense lags many decades behind electrical engineering, which has benefited greatly from ingenious applications of nonlinear elements. The most basic such element would be a thermal rectifier. Following one common convention23, we define the thermal rectification as where QFwd and QRev are the heat transfers under the same thermal bias ΔT=TH−TC but different directions: Forward (Fwd) and reverse (Rev). Regardless of the type of energy carrier, all thermal rectifiers require two key ingredients (Fig. 1): asymmetry and nonlinearity456789. Classical thermal diodes include natural convection in a gravitational field (molecular gases) and a Fourier Law mechanism exploiting temperature-dependent thermal conductivities (electrons and phonons)710. Additional thermal rectification mechanisms, including atomistically based proposals45611, have been summarized in refs 7, 12. Within radiation heat transfer (photons), to our knowledge, rectification has not been experimentally established by any mechanism, classical or otherwise, although recent theoretical work has suggested far-field12 and near-field1314 mechanisms exploiting temperature-dependent total emissivity, ε(T). Here we present a new mechanism of photon thermal rectification, inspired by a pioneering ballistic electrical diode15 and previous explorations of ballistic phonon thermal diode89161718. Figure 1 shows the basic concept, described in more detail in the Supplementary Notes 1–4. Owing to the taper of the pyramidal mirrors, photons of near-normal incidence (θ≈0°) have higher transmission for trajectories from bottom to top than from top to bottom. However, the trend is opposite for trajectories entering the test section at highly oblique incidence (θ approaching 90°). At equilibrium, these effects must exactly cancel, a fundamental requirement of the second Law of Thermodynamics (equivalently, conservation of optical etendue19). As recently noted by ourselves9 and others8, this further implies that the asymmetric structure of Fig. 1a alone1617 cannot show rectification even far away from equilibrium. A nonlinearity is also mandatory456789, which we achieve through an ‘inelastic thermal collimator’ whose angle-dependent emission distorts as a function of the net thermal flux (see also Supplementary Notes 3 and 4). Here we experimentally demonstrate both the rectification effect and the necessity of the collimator. Results Experimental design Figure 2a shows the experimental concept. Photons are emitted from a graphite blackbody cavity (BBC) at TBBC=573 K, which offers a well-defined boundary condition with a nearly Lambertian emission (for design details see Supplementary Note 1). The cold side of the radiation field is maintained at T∞=283 K using a water-cooled black plate. For asymmetric scatterers we use highly polished copper pyramids, chosen for their high infrared reflectivity. As in Fig. 1a, photons incident towards the pyramids’ peaks have higher transmission for normal incidence, while photons incident towards their bases have higher transmission for oblique incidence (see Supplementary Figs 2 and 3 for further verification). The second key ingredient is the nonlinearity, realized here through an ‘inelastic thermal collimator’ (see also Supplementary Notes 3 and 4). This differs quite fundamentally from a conventional optical collimator. The inelastic collimator must absorb a substantial portion of the incident photons and re-emit them at a lower energy, an inelastic interaction that provides bias-dependent angular weighting of emission as depicted in Figs 1b and 2a. In contrast, a conventional optical collimator ideally interacts with the photon field only by reflection and/or refraction, which as purely elastic processes are incapable of providing the needed nonlinearity89. To realize an inelastic thermal collimator in practice, we created a perforated graphite plate coated with gold film as an infrared reflector on the side facing the BBC (Fig. 2a). The collimator is mounted to the BBC using weak thermal links. These features ensure that, whenever the diode experiences substantial thermal bias, the temperature of the collimator floats to an intermediate value, which is significantly lower than that of the BBC (for example, Tcol~473 K when TBBC=573 K and T∞=283 K). Then, owing to the open holes in the collimator, for angles close to θ≈0° many of the photons entering the pyramid test section were emitted from the BBC core at TBBC. However, photons entering at oblique angles are much more likely to have been emitted from the internal cylindrical surfaces of the collimator holes, with lower intensity since Tcol<TBBC. The greater the thermal bias, the larger the difference between Tcol and TBBC, and thus the greater the angular distortion of the intensity incident on the pyramids. The configuration shown in Fig. 2a is Fwd-biased: when the BBC is hot, photons emitted normally have more intensity and are relatively easy to transmit through the pyramidal test section. Similarly, flipping the test section upside down creates Rev bias: the collimator emission is still weighted towards normal incidence; however, this is now relatively difficult to transmit through the test section. The result is thermal rectification. The experimental design ensures that convection and conduction losses from the BBC are below 0.1% (see Supplementary Note 1). To minimize radiation heat losses from the BBC exterior (<1%), we surround it with five concentric radiation shields and an active guard heater. A kinematic coupling between test section and BBC ensures positioning accuracy and repeatability when flipping the test section between polarities. The BBC and its guard each have two independent heater loops (Supplementary Fig. 1b), each typically stable to within ±0.5 K, with maximum differences of 1.5 K among these four heater zones as measured by six thermocouples. This experiment requires highly accurate measurements of the heat transfer through the test section, Qdiode, under Fwd and Rev biases. Direct evaluation of the steady-state BBC power from the heaters’ current I and voltage V was found to be impractical because of the highly oscillatory nature of PWM-switched AC electricity. Instead we implemented the lumped cooling method20 depicted in Fig. 2b,c. First, the BBC and guard are both stabilized at 573 K. Then for times t>0, the BBC heaters are turned off (PBBC=0), while the guard is maintained at its original temperature. From the time-dependent cooling curve (Fig. 2c) we extract Qdiode as follows. The excellent shielding between BBC and guard ensures QBBC-G≪Qdiode. This approaches the perfect-shield limit (see Supplementary Note 6), whereby the BBC energy balance of Fig. 2b simplifies to where CBBC is the thermal capacitance of the BBC (with SI units J K−1). In our experimental regime, the perfect-shield approximation of equation (2) agrees with exact numerical solutions to within 0.1% (see Supplementary Note 6), and also brings several experimental advantages. Most importantly, the cooling curve becomes independent of TG and RBBC-G; therefore, the final results are insensitive to drifts or fluctuations in these quantities. In addition, Qdiode comes directly from the cooling rate, \|dTBBC/dt\|, which is measured with much better accuracy than the electrical power PBBC=[IV]BBC. Furthermore, although quantifying Qdiode requires an estimate for CBBC based on the BBC’s geometry and specific heat capacity21, CBBC is a common parameter for both Fwd and Rev biases, and thus cancels out in equation (1). Thus, rectification is calculated directly from the cooling curves using where both derivatives are evaluated numerically over the same temperature range. This differential expression is also advantageous because many possible errors are common-mode and thus would cancel out. Thermal transport results We performed two types of experiments to verify the key predictions89 that both asymmetry and nonlinearity are required in a ballistic thermal rectifier. First, we performed six control experiments with the test section but without the collimator. These tests quantify the repeatability of the experiment and rule out potential artifacts, and check the theoretical prediction that asymmetry alone is insufficient for rectification. Between every trial, the test section is manually removed and replaced to capture effects of possible misalignment errors. The open points in Fig. 3a show two such control experiments, with the test section in Fwd and Rev biases. The BBC heater is turned off at 573 K, and we allow a 4-K buffer before defining t=0 to ensure the system has fully transitioned to its free-cooling trajectory. To better detect any differences between the curves, in Fig. 3b we plot the cooling rate, \|dTBBC/dt\|, evaluated numerically over a 10-point (10 min) moving average. Both panels confirm that these two control experiments are virtually indistinguishable from each other. To conveniently describe each trial with a single number, we average the cooling rate from 569 to 553 K and calculate the corresponding heat flow through the diode using equation (2). The results for three trials each of Fwd and Rev biases are plotted in Fig. 3c using the unfilled columns. Comparing the Fwd bias and Rev bias trials among themselves shows that the repeatability of these experiments is better than 0.8% (s.d.). Having quantified the noise floor of this apparatus, we next compare the average QDiode,Fwd to the average QDiode,Rev in these six control experiments. The difference of 0.3% is well within experimental uncertainty. Thus, as expected89, this is the first experimental confirmation that ballistic thermal rectification is not possible through asymmetric scattering alone. Finally, we measure the complete device with pyramidal test section and inelastic thermal collimator. The collimator geometry was guided by a simple model (see Supplementary Note 5), with a pore aspect ratio H/D=1 and areal porosity chosen to be as large as practical (here 0.55). As summarized in Fig. 3a,b, with this collimator in place the measurements now show that the Fwd bias configuration (red filled squares) now clearly cools faster than the Rev bias case (blue filled squares). For example, at a BBC temperature of 563 K, Fig. 3b shows that the BBC cooled at 0.352 K min−1 in Fwd bias, but 0.314 K min−1 in Rev bias. Repeatability was confirmed after the Rev bias test by flipping the test section again and re-measuring Fwd bias, with very good consistency (red filled circles). Thus, as summarized in Fig. 3c using the filled columns, this ballistic thermal diode showed a rectification γ=10.9%, far above the uncertainty floor of 0.8%. To further verify the effect, the experiment was repeated using a second collimator with the same thickness and areal porosity but holes of half the diameter (H/D=2). A simple model of the inelastic thermal collimator (Supplementary Note 5) predicts that this should degrade the overall rectification performance. This was confirmed by experiment: the striped columns in Fig. 3c show that the resulting heat transfer was again asymmetric and highly repeatable among four trials, and as expected the rectification was significantly reduced (γ=4.1%). Lastly, Fig. 1b and Supplementary Fig. 4 qualitatively suggest that the distorting effect of the inelastic thermal collimator is degraded at lower thermal bias. This trend is also seen in the simple collimator model (Supplementary Fig. 8) and confirmed by the experiment. As shown in Fig. 4, for Collimator 1 the rectification decreases from 10.9% at TBBC=573 K to 6.6% at TBBC=473 K, while keeping T∞ fixed at 283 K. Similar trends are observed for Collimator 2, with rectification decreasing from 4.1% at 573 K to below the noise threshold at 425 K. Discussion The results in Fig. 3 represent the first experimental demonstration of a photon thermal diode using any mechanism. This is also the first demonstration of the two-terminal ballistic thermal rectification mechanism891617 for any carrier type, and in principle is generalizable to other ballistic carriers including electrons and phonons, for example, using pyramidal quantum dots22, sawtooth nanowires161723 or microfabricated structures (see Supplementary Note 7). Highly effective thermal rectifiers could find numerous applications in thermal engineering. Many arise from electrical analogues24. For example, in solar-thermal power, a temperature doubler (analogous to a voltage doubler) can exploit nighttime cold temperatures as well as daytime highs to increase the average temperature difference driving a heat engine, increasing both efficiency and power output925. A thermal diode’s clamping functionality could be useful for thermal regulation of building envelopes26, as well as thermal protection of delicate components in electrical hardware, spacecraft thermal shielding and satellite radiators. Thermal diodes are also a fundamental element for logic operations; besides the suggestion of thermal information processing27 this can also benefit power generation, since a suitable array of thermal diodes can automatically pick out only the hottest of sources and coldest of sinks. Additional information How to cite this article: Chen, Z. et al. A photon thermal diode. Nat. Commun. 5:5446 doi: 10.1038/ncomms6446 (2014). Supplementary Material Supplementary Information Supplementary Figures 1-12, Supplementary Notes 1-7 and Supplementary References This work was supported in part by a DARPA YFA (Grant No. N66001-09-1-2098) and an AFOSR MURI (Grant No. FA9550-08-1-0407). The authors declare no competing financial interests. Author contributions Z.C., C.W. and C.D. designed the experiments. C.D. conceived the experiments. C.H., A.F. and J.G. helped to design the pyramidal test section. Z.C., S.L. and S.Y. set up the experiment. Z.C. performed the experiment, and Z.C. and C.D. interpreted the results. J.M. developed the ray tracing simulation. W.J. conducted the phonon experiment. Z.C. and C.D. wrote the paper. All authors commented on the manuscript. 08/21/2017 This paper has been retracted at the request of the authors. Figure 1 The two key ingredients of a photon thermal diode: asymmetry and nonlinearity. (a) Asymmetry arises from angle-dependent transmission through the test section containing pyramidal reflectors: from bottom to top, transmission is higher for energy carriers with normal incidence, while from top to bottom, transmission is favoured for carriers of oblique incidence. (b) The nonlinearity arises because the emission from the hot reservoir (not shown) has an angular weighting that is also bias-dependent due to the ‘inelastic thermal collimator’. At zero thermal bias (ΔT=0), the angular weighting is nearly uniform (that is, Lambertian), while for non-zero thermal bias (ΔT>0), the emission becomes increasingly forward-peaked. (c) The combined effect is thermal rectification. See also Supplementary Notes 1–4. Figure 2 Experimental concept. (a) A hot BBC (its guard heater and shields omitted for clarity) generates photons with a Lambertian distribution. The essential diode components are the inelastic thermal collimator and the test section with pyramidal mirrors. The depicted configuration is Fwd-biased, with Rev bias obtained by flipping the test section. (b) Energy balance applied to the BBC (dashed line): changes in stored thermal energy are balanced by the electrical heater (PBBC) and heat transfers through the diode (QDiode) and to/from the guard (QBBC-G). (c) Lumped cooling scheme: after stabilizing the BBC and guard at TBBC=TG=573 K, the BBC power is shut down, while TG held constant. QDiode is extracted from the resulting BBC cooling curve. Figure 3 Experimental results. (a) Cooling curves for representative experiments without (open points: controls) and with (filled points) the inelastic thermal collimator. (b) Cooling rates calculated from a using a 10-point moving average. Symbols as in a. The two control experiments (shaded in grey) are virtually indistinguishable from each other, while the three experiments with the collimator (shaded in orange) are clearly separated. (c) Diode heat transfers calculated from equation (2) by averaging the cooling rate in b from 569 to 553 K, and including additional trials. These key results demonstrate how thermal rectification requires both asymmetry and nonlinearity. Another collimator (Col. 2: striped bars) with narrower holes shows similar results, but as expected degrades the rectification (Col. 2 data omitted from a,b for clarity). Figure 4 Bias-dependent thermal rectification. 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==== Front Nat CommunNat CommunNature Communications2041-1723Nature Publishing Group ncomms496110.1038/ncomms496124892923ArticleCapturing carbon dioxide as a polymer from natural gas Hwang Chih-Chau 1Tour Josiah J. 1Kittrell Carter 2Espinal Laura 3Alemany Lawrence B. 124Tour James M. a1251 Department of Chemistry, Rice University, 6100 Main Street, Houston, Texas 77005, USA2 The Richard E. Smalley Institute for Nanoscale Science and Technology, Rice University, 6100 Main Street, Houston, Texas 77005, USA3 Material Measurement Laboratory, National Institute of Standards and Technology, Gaithersburg, Maryland 20899, USA4 Shared Equipment Authority, Rice University, 6100 Main Street, Houston, Texas 77005, USA5 The Department of Materials Science and NanoEngineering, Rice University, 6100 Main Street, Houston, Texas 77005, USAa [email protected] 06 2014 2014 5 396113 02 2014 25 04 2014 Copyright © 2014, Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved.2014Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved.Natural gas is considered the cleanest and recently the most abundant fossil fuel source, yet when it is extracted from wells, it often contains 10–20 mol% carbon dioxide (20–40 wt%), which is generally vented to the atmosphere. Efforts are underway to contain this carbon dioxide at the well-head using inexpensive and non-corrosive methods. Here we report nucleophilic porous carbons are synthesized from simple and inexpensive carbon–sulphur and carbon–nitrogen precursors. Infrared, Raman and 13C nuclear magnetic resonance signatures substantiate carbon dioxide fixation by polymerization in the carbon channels to form poly(CO2) under much lower pressures than previously required. This growing chemisorbed sulphur- or nitrogen-atom-initiated poly(CO2) chain further displaces physisorbed hydrocarbon, providing a continuous carbon dioxide selectivity. Once returned to ambient conditions, the poly(CO2) spontaneously depolymerizes, leading to a sorbent that can be easily regenerated without the thermal energy input that is required for traditional sorbents. Natural gas is a widely used fossil fuel, but its extraction results in the venting of carbon dioxide to the atmosphere. Here, the authors demonstrate that nucleophilic porous carbons can store this carbon dioxide as a polymer, and that the polymerization requires lower pressures than previously observed. ==== Body An automobile operating on natural gas produces ~30% lower carbon dioxide (CO2) emissions than when operating on gasoline1. Provided technological efficiencies are maintained to minimize direct natural gas leakage to the atmosphere, there is a significant lowering of greenhouse gas emissions when using natural gas rather than liquid hydrocarbons23. At the same time, natural gas wells have typical CO2 concentrations of 10–20 mol%, and that can rise to as high as 70 mol% in some locations3. The CO2 is generally vented into the atmosphere at natural gas collection stations, significantly offsetting the environmental advantages of using natural gas as a fuel. Typically, aqueous amine scrubbers are used to remove CO2 from natural gas, but aqueous amines are corrosive, and the CO2-containing liquid requires heating to 125–140 °C to liberate the CO2 from the amine carbonate. This heating demands a high energy input and the scrubbers are not easily amenable to offshore CO2 capture due to their size and weight45. Long and other groups extensively reviewed aqueous amines and other methods of CO2 capture that include metal oxide frameworks, zeolites, ionic liquids, cryogenic distillation, membranes and metal oxides6789. Some of these have hydrolytic instabilities and/or low densities leading to low volumetric efficiencies, or sometimes poor selectivity relative to methane, but most often there are synthesis constraints or energy costs associated with these technologies that lessen their suitability for on-site CO2 capture from natural gas. Here we report new materials from simple and inexpensive carbon–sulphur or carbon–nitrogen solid sorbents, which separate CO2 from natural gas, with 0.82 g CO2 per g of sorbent (82 wt%) captured at 30 bar. A mechanism is described where CO2 is polymerized in the channels of the support, as initiated by the sulphur or nitrogen atoms that are part of the carbon framework. No temperature swing is needed; it proceeds at ambient temperature. Eventually, heat transfer between cylinders during the exothermic sorption and endothermic desorption might provide the requisite thermodynamic exchanges. The process uses the inherent natural gas-well pressure of 200–300 bar as a driving force during the polymerization. By lowering the pressure back to ambient conditions after CO2 uptake, the poly(CO2) then depolymerizes where it can be off-loaded, or pumped back downhole into the structures that had held it for geological timeframes. Results Synthesis and characterization of porous carbons Sulphur- and nitrogen-containing porous carbons (SPC and NPC, respectively) were prepared by treating bulk precursor polymers with potassium hydroxide (KOH) at 600 °C (Fig. 1a; refs 10, 11). The resulting products were solid porous carbon materials with homogeneously distributed sulphur or nitrogen atoms incorporated into the carbon framework. They exhibited pores and channel structures as well as high surface areas of 2,500 and 1,490 m2 g−1 (N2, Brunauer–Emmett–Teller) for the SPC and the NPC, respectively, with pore volumes of 1.01 cm3 g−1 and 1.40 cm3 g−1, respectively. The scanning electron microscopy and transmission electron microscopy (TEM) images are shown in Fig. 1b–d, and the X-ray photoelectron spectroscopy (XPS) analyses are shown in Supplementary Fig. 1. CO2 uptake measurements Although CO2 has no liquid state at ambient conditions, it can easily liquefy when the system pressure is higher, which can cause a serious analytical error during the measurements1213. Since there are presently no accepted standards for CO2 uptake measurements14, further verification was sought: the same samples were analysed using different volumetric analysis instruments at Rice University and at the National Institute of Standards and Technology (NIST), and these were further confirmed with gravimetric measurements15. Figure 2 shows the pressure-dependent CO2 excess uptake for the SPC sorbent at different temperatures peaking at 18.6 mmol CO2 per g of sorbent (82 wt%) when at 22 °C and 30 bar. The sorption results measured by volumetric and gravimetric analyses were comparable, as were those measurements on the two volumetric instruments. We chose 30 bar as the upper pressure limit in our experiments because a 300-bar well-head pressure at 10 mol% CO2 concentration would have a CO2 partial pressure of 30 bar. Figure 2b–d shows three consecutive CO2 sorption–desorption cycles on SPC over a pressure range from 0 to 30 bar, which indicates that the SPC could be regenerated using a pressure swing process while retaining its original CO2 sorption capacity. In the case of microporous materials with negligible external surface area, total uptake is often used as an approximation for absolute uptake, and the two values here are within 10% of each other; for example, the absolute CO2 uptake of the SPC was 20.1 and 13.9 mmol g−1 under 30 bar at 22 °C and 50 °C, respectively (Supplementary Figs 2 and 3 and Supplementary Note 1). Similarly, although absolute adsorption isotherms can be used to determine the heat of sorption, excess adsorption isotherms are more often used to calculate the heat of CO2 sorption (QCO2) before the critical point of the gas1617. Thus, the excess CO2 sorption isotherms measured at two different temperatures, 23 °C and 50 °C (Fig. 2e), were input into the Clausius–Clapeyron equation18 (see Supplementary Note 2). At lower surface coverage (≤1 bar), which could be expected to be more indicative of the sorbate–sorbent interaction, the SPC exhibits a heat of CO2 sorption of 57.8 kJ mol−1. Likewise, the maximum QCO2 values for nucleophile-free porous materials, such as activated carbon, Zeolite 5 A and zeolitic imidazolate framework (ZIF-8, a class of the metal oxide frameworks) were measured to be 28.4, 31.2 and 25.6 kJ mol−1, respectively, at low surface coverage (see Supplementary Table 1). Based on this data, the SPC possesses the highest CO2 sorption enthalpy among these complementary sorbents measured at low surface coverage. Discussion In order to better assess the sorption mechanism during the CO2 uptake, attenuated total reflectance infrared spectroscopy (ATR-IR) was used to characterize the properties of the sorbents before and after the CO2 uptake. A sample vial with ~100 mg of the SPC was loaded into a 0.8 l stainless steel autoclave equipped with a pressure gauge and valves. Before the autoclave was sealed, the chamber was flushed with CO2 (99.99%) to remove residual air, and the system was pressurized to 10 bar (line pressure limitation). The sorbent was therefore isobarically exposed to CO2 in the closed system at 23 °C. After 15 min, the system was vented to nitrogen at ambient pressure and the sorbent vial was immediately removed from the chamber and the sorbent underwent ATR-IR and Raman analyses in air. Figure 3a,b shows the ATR-IR spectra of the SPC before (black line) and after exposure to 10 bar of CO2 followed by ambient conditions for the indicated times. The two regions that appear in the ATR-IR spectra (outlined by the dashed-line boxes) after the CO2 sorption are of interest. The first IR peak, located at 2,345 cm−1, is assigned to the anti-symmetric CO2 stretch, confirming that CO2 was physisorbed and evolving from the SPC sorbent. The other IR band, centred at 1,730 cm−1, is attributed to the C=O symmetric stretch from the poly(CO2) on the SPC. Interestingly, this carbonyl peak is only observed with the porous heteroatom-doped carbon, such as the SPC and NPC. Other porous sorbents without nucleophilic species, such as ZIF-8 and activated carbon, only showed the physisorbed or evolving CO2 peak (~2,345 cm−1) (Supplementary Figs 4 and 5). Once the CO2-filled SPC returned to ambient pressure, the key IR peaks attenuated over time and disappeared after 20 min. Based on this data, the ATR-IR study confirmed the poly(CO2) formation. Raman spectroscopy was further used to probe individual chemical bond vibrations as shown in Fig. 3c. The carbonaceous graphitic G-band and defect-derived diamondoid D-band were at 1,590 and 1,350 cm−1 (refs 19, 20). The peak at 798 cm−1 can be attributed to the symmetric stretch of the C–O–C bonds2122, which was not observed for the other nucleophile-free porous materials, suggesting that the poly(CO2), with the –(O–C(=O))n– moiety, was formed. The monothiocarbonate and carbamate anions within the channels of the SPC and NPC, respectively, were the likely initiation points for the CO2 polymerization since no poly(CO2) was seen in activated carbon (Supplementary Fig. 5). Furthermore, 13C NMR also confirms the presence of the poly(CO2) formation. The sorbent gives a broad signal characteristic of aromatic carbon (Fig. 3d, bottom). After exposure to CO2, a relatively sharp signal on top of the broad sorbent signal appears at 130.6 p.p.m., which can be assigned to the CO2 that is evolving from the support. A sharp signal also appears at 166.5 p.p.m. (Fig. 3d, middle), which is characteristic of the carbonyl resonance for poly(CO2). Both of these signals are gone 19 h later (Fig. 3d, top), see Supplementary Note 3. Compared with secondary amine-based CO2 sorbents where maximum capture efficiency is 0.5 mol CO2 per mol N (2 RNH2+CO2→RNH3+ −O2CNHR), the SPC and NPC demonstrate a unique mechanism during the CO2 uptake process resulting in their remarkably higher CO2 capacities versus S or N content (8.1 atomic % of S and 6.2 atomic % of N in the SPC and NPC, respectively, by XPS analysis). Figure 3e,f shows a mechanism to illustrate this CO2 fixation by polymerization. Dimeric CO2 uptake has been crystallographically observed in metal complexes22, and polymeric CO2 has been detected previously but only at extremely high pressures of ~15,000 bar (ref. 21). The spectroscopic determination here confirms poly(CO2) formation at much lower pressures than formerly observed. A series of porous materials with and without the nucleophilic heteroatoms were tested to compare their CO2 capture performance up to 30 bar at 30 °C (Fig. 4a). The SPC had the highest CO2 capacity; the NPC, activated carbon, zeolite 5A and ZIF-8 had lower capacities. Although NPC had significantly lower CO2 capacity than SPC, its uptake performance could be improved by 21 wt% after H2 reduction at 600 °C, producing reduced-NPC (R-NPC) with secondary amine groups (Fig. 1a). Even though the surface area of R-NPC (1,450 m2 g−1) is only slightly greater than that of the activated carbon (1,430 m2 g−1), the presence of the amine groups induces the formation of the poly(CO2) under pressure, promoting the CO2 sorption efficiency of the R-NPC. The pore volume of R-NPC is 1.43 cm3 g−1. Purification of natural gas from wells relies upon a highly CO2-selective sorbent, especially in a CH4-rich environment. Thus, CH4 uptake experiments were carried out on three different types of porous materials, SPC, activated carbon and ZIF-8. Figure 4b–d compares CO2 and CH4 sorption over a pressure range from 0 to 30 bar at 23 °C. In contrast to the CO2 sorption, the CH4 isotherms for these three sorbents reached equilibrium while the system pressure was approaching 30 bar. The order of the CH4 uptake capacities was correlated to the surface area of the sorbents. Comparing these sorbents, the observed molecular ratio of sorbed CO2 to CH4 (nCO2/nCH4) for the SPC (2.6) was greater than that for the activated carbon (1.5) and ZIF-8 (1.9). In addition, the density of the SPC calculated using volumetric analysis is sixfold higher than in the ZIF-8 (2.21 versus 0.35 g cm−3) and threefold higher than the zeolite 5A (2.21 versus 0.67 g cm−3). The high CO2 capacity and high density observed for SPC greatly increase the volume efficiency, which would reduce the volume of the sorption material for a given CO2 uptake production rate. In order to mimic a gas well environment and further characterize the SPC’s selectivity to CO2, a premixed gas (85 mol% CH4, 10 mol% CO2, 3 mol% C2H6 and 2 mol% C3H8) was used with quadrupole mass spectrometry (MS) detection. The MS inlet was connected to the gas uptake system so that it could monitor the gas effluent from the SPC throughout the sorption–desorption experiment. Supplementary Fig. 6 shows the mass spectrum recorded during the sorption process. The peaks at 15 and 16 amu correspond to fragment and molecular ions from CH4, while the peaks at 28 and 44 amu are from CO2 in the premixed gas. Other minor peaks can be assigned to fragment ions from C2H6 and C3H8. Although the peak at 44 amu can also come from C3H8 ions, the contribution is negligible because of the lower C3H8 concentration in the mixed gas, and it is distinguishable by the fragmentation ratios in the MS (C3H8: m/z=29 (100), 44 (30); CO2: m/z=44(100), 28(11)). The observed intensity ratio of two peaks at 16 and 44 amu (I16/I44=9.1) indicates the abundance of CH4 versus CO2 during the sorption and also reflects the relative amount of CH4 and CO2 in the premixed gas. Once the sorption reached equilibrium under 30 bar, the desorption process was induced by slowly venting into the MS system. The I16/I44 ratio reduced to ~0.7. The SPC has been shown to have 2.6-fold higher CO2 than CH4 affinity at 30 bar when using pure CO2 and CH4 as feed gases (Fig. 4b). If the binding energy of CH4 and CO2 were assumed to be similar, and the partial pressure of CH4 versus CO2 in the premixed gas is considered (PCH4/PCO2=8.5), then the number of sorbed CH4 should be ~3.3-times more than that of the sorbed CO2. Typically, CO2-selective materials have selective sites and once the CO2 occupies those sites, the selectivity significantly decreases and the materials behave as a physisorbent with lower selectivities at higher pressures. On the contrary, here the SPC demonstrates much higher CO2 selectivity than expected since the chemisorbed sulphur-initiated poly(CO2) chain displaces physisorbed gas. Under the mechanism described here for CO2 polymerization in the channels of inexpensive nucleophilic porous carbons, these new materials have continuous selectivity toward CO2, limited only by the available pore space and pressure. Through development of these enhanced stationary phase sorbents, capture and reinjection of CO2 at the natural gas sites could be realized, thereby leading to greatly reduced CO2 emissions from natural gas streams. CO2 fixation through polymerization is disclosed here as a major advance for future capture and possibly storage of this greenhouse gas. Methods Instrumentation at Rice University An automated Sieverts instrument (Setaram PCTPro) was adopted to measure gas (CO2, CH4 or premixed gas) sorption properties of materials. Typically, ~70 mg of sorbent was packed into a ~1.3 ml of stainless steel sample cell. The sample was pretreated under vacuum (~3 mm Hg) at 130 °C for 6 h and the sample volume was further determined by helium before the uptake experiment. At each step of the measurement, testing gas was expanded from the reference reservoir into the sample cell until the system pressure reached equilibrium. A quadrupole mass spectrometer (Setaram RGA200) was connected to the Sieverts instrument so that it could monitor the gas effluent from the sorbent throughout the entire sorption–desorption experiment. With the assistance of a hybrid turbomolecular drag pump, the background pressure of the MS can be controlled lower than 5 × 10−8 Torr. All material densities were determined using volumetric analysis on this same instrument. XPS was performed using a PHI Quantera SXM Scanning X-ray Microprobe with a base pressure of 5 × 10−9 Torr. Survey spectra were recorded in 0.5 eV step size and a pass energy of 140 eV. Elemental spectra were recorded in 0.1 eV step size and a pass energy of 26 eV. All spectra were standardized using C1s peak (284.5 eV) as a reference. The ATR-IR experiment was conducted using a Fourier transform infrared spectrometer (Nicolet Nexus 670) equipped with an attenuated total reflectance system (Nicolet, Smart Golden Gate) and a MCT-A detector. Raman spectra were measured using a Renishaw inVia Raman Microscope with a 514 nm excitation argon laser. Scanning electron microscope images were taken at 15 KeV using a JEOL-6500 F field emission microscope. High-resolution TEM images were obtained with a JEOL 2100 F field emission gun TEM. An automated BET surface analyser (Quantachrome Autosorb-3b) was used for measurements of sorbents’ surface areas and pore volumes based on N2 adsorption–desorption. Typically, a ~100 mg of sample was loaded into a quartz tube and pretreated at 130 °C under vacuum (~5 mm Hg) in order to remove sorbates before the measurement. MAS NMR spectra were recorded on a Bruker Avance III 4.7 T spectrometer with a standard MAS probe for 4 mm outer diameter rotors. Instrumentation at NIST Volumetric CO2 sorption measurements were carried out on computer-controlled custom-built volumetric sorption equipment previously described in detail23, with an estimated reproducibility within 0.5% and isotherm data error bar of less than 2% compared with other commercial instruments. An amount of ~79 mg of sample was used for the experiments. Sample degassing, before the CO2 sorption experiment, was done at 130 °C under vacuum for 12 h. Gravimetric CO2 sorption measurements were performed on a high pressure thermal gravimetric equipment (Model: TGA-HP50) from TA Instruments. An amount of ~15 mg of sample was used for the experiments. Sample degassing, before CO2 sorption experiment, was done at 130 °C under vacuum for 12 h. Synthesis of SPC Poly[(2-hydroxymethyl)thiophene] (PTh) (Sigma-Aldrich) was prepared using FeCl3 (ref. 24) In a typical synthesis, 2-thiophenemethanol (1.5 g, 13.1 mmol) in CH3CN (10 ml) was slowly added under vigorous stirring to a slurry of FeCl3 (14.5 g, 89.4 mmol) in CH3CN (50 ml). The mixture was stirred at room temperature for 24 h. The polymer (PTh) was separated by filtration over a sintered glass funnel, washed with distilled water (~1 l) and then with acetone (~200 ml). The polymer was dried at 100 °C for 12 h to afford 1.21 g (96% yield) of the desired compound. The PTh was activated by grinding PTh (500 mg) with KOH (1 g, 17.8 mmol) with a mortar and pestle and then heated under Ar at 600 °C in a tube furnace for 1 h. The Ar flow rate was 500 sccm. After cooling, the activated sample was thoroughly washed 3 × with 1.2 M HCl (1 l) and then with distilled water until the filtrate attained pH 7. The SPC sample was dried in an oven at 100 °C to afford 240 mg of the black solid SPC. The BET surface area and pore volume were 2,500 m2 g−1 and 1.01 cm3 g−1, respectively. Synthesis of NPC Commercial polyacrylonitrile (PAN, 500 mg, average Mw 150,000, Sigma-Aldrich) powder and KOH (1,500 mg, 26.8 mmol) were ground to a homogeneous mixture in a mortar. The mixture was subsequently carbonized by heating to 600 °C under Ar (500 sccm) in a tube furnace for 1 h. The carbonized material was washed 3 × with 1.2 M HCl (1 l) and then with distilled water until the filtrate attained pH 7. Finally, the carbon sample was dried in an oven at 100 °C to afford 340 mg of the solid black NPC. To produce R-NPC, the activated material (270 mg) was further reduced by 10% H2 (H2:Ar=50:450 sccm) at 600 °C for 1 h to provide 255 mg of the final material. The BET surface area and pore volume were 1,450 m2 g−1 and 1.43 cm3 g−1, respectively. Additional information How to cite this article: Hwang, C.-C. et al. Capturing carbon dioxide as a polymer from natural gas. Nat. Commun. 5:3961 doi: 10.1038/ncomms4961 (2014). Supplementary Material Supplementary Information Supplementary Figures 1-6, Supplementary Table 1, Supplementary Notes 1-3 and Supplementary References We thank G. Ruan, C. Xiang and Z. Peng for imaging. The work at Rice University was funded by Apache Corporation. The work at NIST was funded by NIST. Certain commercial materials, equipment, or processes are identified in the paper only to facilitate understanding. In no case does identification imply recommendation by NIST nor does it imply that the material, equipment, or process identified is necessarily the best available for this purpose. Rice University has filed patent applications (PCT/US2013/021239, 61/839,567, 61/865,296, 614/865,323) on the carbon stationary phases and the mechanisms of fixation described here for CO2 capture from natural gas. Apache Corp. has licensed the intellectual property (agreement no. 0112011). None of the authors own rights to the technology described here and none hold stock in Apache Corp., aside from what might be held in broad mutual funds. Author contributions C.-C.H. designed and performed the experiments and wrote the manuscript. J.J.T. conducted some of the experiments. C.K. helped with the uptake analyses at Rice University. L.E. performed the uptake experiments at NIST. L.B.A. performed the solid state NMR and analyses. J.M.T. oversaw all phases of the research, suggested many of the experiments and revised the manuscript. All authors discussed and commented on the manuscript. 11/01/2016 This paper has been retracted at the request of the authors. Figure 1 The synthetic schemes and micrographic images. (a) The synthesis of SPC or NPC by treating poly[(2-hydroxymethyl)thiophene] or poly(acrylonitrile) with KOH at 600 °C and then washing with dilute HCl and water until the extracts are neutral. The NPC is further reduced using 10% H2 at 600 °C to form R-NPC, see Methods. (b) Scanning electron microscopy (SEM) image of NPC. Scale bar, 100 μm. (c) SEM image of SPC. Scale bar, 500 nm. (d) TEM image of the SPC. Scale bar, 25 nm. Figure 2 CO2 uptake measurements. (a) Volumetric and gravimetric uptake of CO2 on SPC at different temperatures and pressures. Those designated with ‘*’ were recorded volumetrically at Rice University. That designated with ‘§’ was performed volumetrically at NIST. That designated ‘+’ was measured gravimetrically at NIST. All gravimetric measurements were corrected for buoyancy. (b–d) Three consecutive CO2 sorption–desorption cycles on the SPC over a pressure range from 0 to 30 bar at 30 °C. All solid circles indicate CO2 sorption, while the open circles designate the desorption process. (e) Volumetric SPC CO2 sorption isotherms at 23 °C and 50 °C over a pressure range from 0 to 1 bar. Figure 3 Spectral changes before and after sorption–desorption and polymerization mechanism. (a,b) ATR-IR, (c) Raman and (d) 50.3 MHz 13C MAS NMR spectra before and after CO2 sorption at 10 bar and room temperature. All spectra were recorded at the elapsed times indicated on the graphs after the SPC sorbent was returned to ambient pressure. In the NMR experiments, the rotor containing the SPC was tightly capped during the analyses. For the 19 h NMR experiment the same material was left under ambient conditions for 19 h before being repacked in the rotor to obtain the final spectrum. Each NMR spectrum took 80 min to record. (e,f) A mechanism that illustrates the poly(CO2) formation in SPC or NPC, respectively, in a higher pressure CO2 environment. With the assistance of the nucleophile, such as S or N, the CO2 polymerization reaction is initiated under pressure, and the polymer is further likely stabilized by the van der Waals interactions with the carbon surfaces in the pores. Figure 4 Volumetric gas uptake data. (a) Volumetric CO2 uptake performance at 30 °C of SPC, NPC, R-NPC and traditional sorbents: activated carbon, ZIF-8, and zeolite 5A. Al foil was used as a reference to ensure no CO2 condensation was occurring in the system at this temperature and pressure. Volumetric CO2 and CH4 uptake tests at 23 °C on (b) SPC, (c) activated carbon and (d) ZIF-8 sorbents. ==== Refs US Dept. of Energy. Argonne National Laboratory Report: A Full Fuel-Cycle Analysis of Energy and Emissions Impacts of Transportation Fuels Produced from Natural Gas. Available at http://www.transportation.anl.gov/pdfs/TA/13.pdf (1999 ). Alvarez R. A. , Pacala S. W. , Winebrake J. J. , Chameides W. L. & Hamburg S. P. 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==== Front Nat CommunNat CommunNature Communications2041-1723Nature Publishing Group ncomms747310.1038/ncomms747325751743ArticleBmi1 limits dilated cardiomyopathy and heart failure by inhibiting cardiac senescence Gonzalez-Valdes I. 1Hidalgo I. 1Bujarrabal A. 1Lara-Pezzi E. 2Padron-Barthe L. 23Garcia-Pavia P. 3Gómez-del Arco Pablo 45Redondo J.M. 4Ruiz-Cabello J.M. 6Jimenez-Borreguero L.J. 6Enriquez J.A. 7de la Pompa J.L. 8Hidalgo A. 9Gonzalez S. a11 Stem Cell Aging Group, Centro Nacional de Investigaciones Cardiovasculares (CNIC), E-28029 Madrid, Spain2 Molecular Regulation of Heart Development and Disease Group, Centro Nacional de Investigaciones Cardiovasculares (CNIC), E-28029 Madrid, Spain3 Heart Failure and Inherited Cardiac Diseases Unit, Hospital Universitario Puerta de Hierro Majadahonda, Manuel de Falla, 1, E-28222 Madrid, Spain4 Gene Regulation in Cardiovascular Remodelling and Inflammation Group, Centro Nacional de Investigaciones Cardiovasculares (CNIC), E-28029 Madrid, Spain5 Department of Molecular Biology, Universidad Autonoma de Madrid, E28049 Madrid, Spain6 Advanced Imaging Unit, Ciber de Enfermedades respiratorias and UCM, Centro Nacional de Investigaciones Cardiovasculares (CNIC), E-28029 Madrid, Spain7 Functional Genetics of the Oxidative Phosphorylation System, Centro Nacional de Investigaciones Cardiovasculares (CNIC), E-28029 Madrid, Spain8 Intercellular Signaling In Cardiovascular Development and Disease Group, Centro Nacional de Investigaciones Cardiovasculares (CNIC), E-28029 Madrid, Spain9 Imaging the Cardiovascular Inflammation and the Immune Response, Centro Nacional de Investigaciones Cardiovasculares (CNIC), E-28029 Madrid, Spaina [email protected] 09 03 2015 2015 6 647326 08 2014 30 01 2015 Copyright © 2015, Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved.2015Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved.Dilated cardiomyopathy (DCM) is the most frequent cause of heart failure and the leading indication for heart transplantation. Here we show that epigenetic regulator and central transcriptional instructor in adult stem cells, Bmi1, protects against DCM by repressing cardiac senescence. Cardiac-specific Bmi1 deletion induces the development of DCM, which progresses to lung congestion and heart failure. In contrast, Bmi1 overexpression in the heart protects from hypertrophic stimuli. Transcriptome analysis of mouse and human DCM samples indicates that p16INK4a derepression, accompanied by a senescence-associated secretory phenotype (SASP), is linked to severely impaired ventricular dimensions and contractility. Genetic reduction of p16INK4a levels reverses the pathology of Bmi1-deficient hearts. In parabiosis assays, the paracrine senescence response underlying the DCM phenotype does not transmit to healthy mice. As senescence is implicated in tissue repair and the loss of regenerative potential in aging tissues, these findings suggest a source for cardiac rejuvenation. The epigenetic factor Bmi1 regulates self-renewal of many adult stem cells, but its role in heart function is unknown. Here the authors show that Bmi1 prevents cardiac senescence by inhibiting the tumor suppressor protein p16INK4a in adult mice, protecting them from dilated cardiomyopathy and heart failure. ==== Body Dilated cardiomyopathy (DCM) is the commonest form of non-ischaemic cardiomyopathy and can lead to sudden cardiac death and heart failure1. The health burden associated with DCM is a major contribution to health-care costs; however, the mechanisms underlying the regulation of noninherited DCM remain unexplored2. DCM is defined by the presence of dilated and poorly functioning left ventricle in the absence of abnormal loading conditions, such as valve defect or hypertension or ischaemic heart failure sufficient to induce global systolic impairment. A large number of cardiac and systemic diseases can cause systolic impairment and left ventricular dilatation; however, in the majority of patients no identifiable cause is found (hence, idiopathic DCM). Therefore, understanding the aetiology of idiopathic DCM is of great clinical relevance in view of the devastating consequences of this disease in a large patient population. The epigenetic signature establishes a cell-type-specific chromatin pattern that is of paramount importance for cell commitment and regeneration during development and adulthood345. Epigenetic regulation offers a critical means for governing cardiac gene expression under different physiological and pathological states6. In fact, the nature of cardiomyopathy and heart failure intensely links with irregular cardiac gene expression. One of the causes of congenital heart disease is deficiency for specific epigenetic instructors during cardiogenesis5, such as the histone methyltransferases Ezh2 (ref. 7) and MLL2 (ref. 8), or transcription factors Tbx5 and Nkx2-5 that require histone-modifying enzymes to regulate gene expression5. Essential epigenetic changes at developmental cardiac-specific genes are also required for reprogramming of cardiac fibroblasts (FBs) into cardiomyocytes (CMs) and for cardiac lineage commitment910. However, the role of critical epigenetic modifications in adult cardiac function has received comparatively little attention. A good example of that is the well-established role of epigenetic Bmi1 factor in the maintenance of adult stem cell populations11; however, its direct or indirect implication in regulating the cardiac function is unknown. Senescence protects organisms against damaged cells by inducing a stable growth arrest not only in tumour suppression12 but also in noncancer pathologies13. Growth arrest is achieved, at least in part, through activation of the p16INK4a/Retinoblastoma tumour-suppressor pathways1415. Concomitant with this arrest, senescent cells communicate with their environment, secreting a complex mixture of factors called the senescence-associated secretory phenotype (SASP) or senescence-messaging secretome1617. Here we reasoned that the epigenetic factor Bmi1 might be involved in the maintenance of adult cardiac function. Using a combination of conditional knockout models, biochemical analysis, parabiotic assays and characterization of the DCM transcriptional signature in humans and mice, this study reveals the importance of a genuinely protective role of the epigenetic Bmi1 in cardiac adult pathophysiology, namely protection of adult heart from cellular senescence. Therefore, controlling cardiac gene expression by epigenetic-regulating factors gives a promising approach to the treatment of human cardiomyopathy. Results Bmi1 expression is not required for cardiogenesis Several epigenetic Polycomb regulators are differently regulated during embryonic development and adult aging18, prompting us to analyse endogenous cardiac Bmi1 expression in the mouse. The amount of Bmi1 increased progressively throughout cardiogenesis, stabilizing in adulthood, and then declining in the hearts of aged mice (Supplementary Fig. 1a). This profile correlated inversely with the levels of p16INK4a (Supplementary Fig. 1b), as noted in other studies with adult stem cells192021222324. To analyse the implication of Bmi1 in heart failure, we induced hypertrophy in wild-type mouse hearts, either surgically with a transverse aortic constriction (TAC) or by infusion with isoproterenol (Supplementary Fig. 1c). We additionally analysed the left ventricular myocardium from eight non-failing and eight failing human hearts. In humans and mice, analysis of cardiac Bmi1 expression showed a marked reduction in failing hearts, levels inversely correlated with p16INK4a expression (Supplementary Fig. 1c,d). These results indicate a possible association of Bmi1 downregulation with the development of cardiac hypertrophy. To explore the implication of Bmi1 in cardiogenesis, we crossed Bmi1fl/fl mice with Nkx2.5-Cretg/+ mice25 to induce cardiac-specific Bmi1 deletion from embryonic day (E) 7.5 onwards. Bmi1f/f;Nkx-Cretg/+ mice were born at normal Mendelian frequency and were able to support pregnancy. Histologic examination of Bmi1f/f;Nkx-Cretg/+ embryos at E14.5−E18.5 did not reveal any cardiac abnormality (Fig. 1a). Expression of Bmi1 mRNA on post-natal day 2 (P2) was significantly lower in Bmi1f/f;Nkx-Cretg/+ hearts than in controls and correlated with below-normal levels of the epigenetic mark H3K9me3 (Fig. 1b; Supplementary Fig. 1e). Interestingly, Bmi1f/f;Nkx-Cretg/+ hearts also had a significantly higher heart-to-body-weight (HW/BW) ratio, and expressed higher levels of fetal cardiac genes than littermate controls (Fig. 1c; Supplementary Fig. 1f). Histological comparison with Bmi1f/f;Nkx-Cre+/+ controls at 22 weeks after birth showed that Bmi1-null hearts were enlarged and presented fibrosis and CMs with the above-normal cross-sectional area (Fig. 1d; Supplementary Fig. 1g). Two-dimensional echocardiography revealed the hearts of Bmi1f/f;Nkx-Cretg/+ mice to be dilated and poorly contractile (Fig. 1e). These hearts, moreover, had a markedly enlarged left ventricular internal diameter at diastole and severely depressed left ventricular mass and fractional shortening, a direct measure of cardiac contractile function, consistent with progressive DCM (Fig. 1e,f). Lung weight in 22-week-old Bmi1f/f;Nkx-Cretg/+ mice was significantly increased (Fig. 1g; Supplementary Fig. 1h), suggesting the development of lung oedema secondary to the LV dysfunction. This was confirmed by serial pulmonary magnetic resonance imaging (MRI), which revealed significant pulmonary congestion and oedema (Fig. 1h) coinciding with the impaired cardiac contractile function and maximal left ventricular dilatation for Bmi1-null hearts (Fig. 1f). Perl’s Prussian blue staining on lung sections revealed impaired permeability of the alveolar septal barrier in Bmi1f/f;Nkx-Cretg/+ mice, indicated by the accumulation of siderophages (hemosiderin-laden macrophages) in the perivascular regions of Bmi1f/f;Nkx-Cretg/+ lungs (Fig. 1i). All Bmi1f/f;Nkx-Cretg/+ mice died between 20 and 30 weeks, whereas Bmi1f/f; Nkx-Cre+/+ had a normal lifespan (Fig. 1j). These data thus suggest that cardiac Bmi1 deletion during embryogenesis does not affect cardiogenesis but causes DCM in adulthood. Adult Bmi1 deficiency causes DCM To further test the contribution of Bmi1 deficiency to heart disease, we engineered CM-specific Bmi1 deletion in adulthood by crossing Bmi1fl/fl mice24 with the α-myosin heavy chain (αMHC)-Cretg/+ strain26. Conditional mutants occurred at a normal Mendelian ratio, indicating an absence of embryonic lethality. Examination of 5-week-old Bmi1f/f;αMHC-Cretg/+ mice confirmed cardiac Bmi1 deletion (Supplementary Fig. 2a). At 12 weeks of age, Bmi1f/f;αMHC-Cretg/+ mice showed typical features of DCM: impaired cardiac ejection fraction, a high HW/BW ratio, enlarged CMs, fibrosis and larger-than-normal ventricular chambers (Fig. 2a,b; Supplementary Fig. 2b). These cardiac defects were accompanied by pulmonary oedema and the presence of siderophages (Fig. 2c–e). Consistent with this phenotype, adult Bmi1f/f;αMHC-Cretg/+ mice developed full-blown DCM and suffered heart failure at 15–20 weeks of age (Fig. 3f). To test whether heart failure was sensitive to the Bmi1 dosage, we analysed the hearts of Bmifl/+;αMHC-Cretg/+ and Bmifl/+;αMHC-Cre+/+ mice. Young Bmi1 heterozygotes showed no noticeable cardiac defects (Supplementary Fig. 2c); however, at ~6 months, they developed characteristics of DCM, including a severely depressed ejection fraction (Supplementary Fig. 2c), a trend towards a high HW/BW ratio (Supplementary Fig. 2d) and interstitial fibrosis (Supplementary Fig. 2e). These mice invariably died by 7 months after birth (Supplementary Fig. 2f), indicating that Bmi1 is critical to maintain adult heart function. To confirm the requirement of Bmi1 in the adult heart, we used a tamoxifen (TM)-inducible α-myosin heavy chain (αMHC)TM-Cre line27 to delete Bmi1 in the hearts of 5-week-old adult mice (Fig. 3a,b). Heart weight and function in Bmi1f/f;αMHCTM-Cretg/+ conditional knockouts and Bmi1f/f;αMHCTM-Cre+/+ and Bmi1+/+;αMHCTM-Cretg/+ control mice were monitored using histology and echocardiography (Fig. 3c–e). At 12 weeks post-TM treatment, average left ventricular internal diameter at diastole was similar in control and conditional knockout mice; however, dilatation and loss of contractility subsequently became evident in the mutants (Fig. 3d,e). At 22 weeks post induction, ventricular diameter in Bmi1f/f;αMHCTM-Cretg/+ mice had increased from 3.8 mm at baseline to 4.59 mm, correlating with a fractional shortening of 17% (Fig. 3d,e). MRI revealed significant pulmonary congestion and oedema, which correlated with a 54% increase in lung weight, compared with TM-treated control littermates (Fig. 3f,g). Iron overload in the lung, detected by Perl’s staining, was also evident (Fig. 3h). TM-treated Bmi1f/f;αMHCTM-Cretg/+ mice died between 28 and 32 weeks (23 and 27 weeks post induction), compared with only 1% of TM-treated control littermates (Fig. 3i). Thus, Bmi1 regulates CM function and maintenance throughout adult life. Bmi1 overexpression in the heart safeguarded the hypertrophic stage To assess the antihypertrophic effect of Bmi1, we crossed the doxycycline-inducible MLC2-rRTAtg/+ mouse line28 with tetO-Bmi1tg/tg mice, generating iBmi1tg/tg;MLC2tg/+ mice and iBmi1tg/tg;MLC2+/+ control littermates (hereafter iBmi1tg;MLC2 and iBmitg control mice). Inclusion of doxycycline in the drinking water of 8-week-old iBmi1tg/tg;MLC2tg/+ mice induced cardiac-specific overexpression of Bmi1 (Fig. 4a; Supplementary Fig. 3a). Doxycycline treatment did not induce overt cardiac abnormalities in Bmi1 transgenic mice or control counterparts (Fig. 4b–d). As in wild-type mice, TAC surgery in doxycycline-treated iBmi1tg/tg;MLC2+/+ controls triggered massive cardiac hypertrophy, an increase in CM cross-sectional area and fibrosis (Fig. 4b–e; Supplementary Fig. 3b). In contrast, TAC surgery induced none of these symptoms in the hearts of doxycycline-treated iBmi1tg/tg;MLC2tg/+ mice (Fig. 4e), indicating that Bmi1 overexpression blocks pathological ventricular remodelling. Regulation of a cardiac-specific transcriptional programme by Bmi1 To explore the primary cause of DCM in Bmi1-null mice, we set out to identify differentially expressed genes by massively parallel sequencing of heart samples from DCM-diagnosed patients who had undergone heart transplant (hDCM) and from Bmi1f/f;αMHCTM-Cretg/+ mice (17-week post induction; mDCM). Pairwise comparisons (with corresponding controls) identified 649 and 2,435 genes differentially expressed in mDCM and hDCM samples, respectively (Fig. 5a; Supplementary Data set). Differentially upregulated genes common to mDCM and hDCM samples included those associated with the fetal cardiac gene programme29, such as Acta2, Myh7 and Myl1 and genes encoding extracellular matrix components and regulators, such as Tgfβ, collagens, chemokines and metalloproteinases (Fig. 5a). Significantly upregulated senescence genes were the critical senescence regulator p16INK4a (Cdkn2a)193031 and essential components of the SASP (chemokines and Tgfβ family ligands; Fig. 5a). Consistent with the proliferative arrest that accompanies senescence, we found downregulated genes related to cell-cycle progression such as CDKN3, CyclinD2 and CyclinA2 (CCNA2). There was, moreover, robust upregulation of genes encoding pro-inflammatory factors associated with the SASP, including interleukin (IL)-6, IL-7, granulocyte macrophage colony-stimulating factor and certain insulin-like growth factor-binding proteins32. These findings are suggestive of an important role of the senescence response mediated by the Bmi1/p16INK4a axis in cardiac pathophysiology, consistent with the role of Bmi1 in preventing senescence in other contexts19. To assess the epigenetic signature on the promoter regions of Bmi1 target genes, chromatin silencing was examined by measuring ubiquitination of histone H2A at the Lys119 residue (H2AK119ub), a histone modification mediated by PRC1 (ref. 3), and by Bmi1-specific binding using mDCM and control hearts (Fig. 5b). Additional Ezh1/Ezh2-dependent repressive H3K27me3 mark30, and the chromatin active H3K4me3 mark were also analysed3 (Fig. 5b). Absence of Bmi1 led to an important increase in H3K27me3 mark bound and significantly enriched for the H2AK119ub signal in the promoter regions of the downregulated CyclinA2, Gbe1 and Gmnn genes, but not in the promoters of upregulated p16INK4a, Il1 and Tgfβ3 genes in Bmi1f/f;αMHC-Cretg/+ hearts (Fig. 5b). However, Bmi1 binding was located at the promoters of downregulated genes in the Bmi1f/f;αMHC-Cre+/+ control hearts. Interestingly, mDCM heart cells showed a stronger association with H3K4me3 at the RD domain in the p16INK4a/ARF locus, which has been demonstrated to be the main Bmi1-binding site in this locus3334. In contrast, specific binding of H3K4me3 to the promoter regions of CyclinA2, Gbe1 and Gmnn was lower in the absence of Bmi1 (Fig. 5b). These gene expression data strongly correlate with the cardiac phenotype of Bmi1-null mice and suggest the implication of the senescence response in the progression of DCM. CM senescence is increased in a Bmi1-specific manner Sections from 12-week-old Bmi1f/f;αMHC-Cretg/+ hearts, but not controls, stained positive for senescence-associated β-galactosidase (SA-β-gal) marker (Fig. 6a). In contrast, apoptotic-mediated caspase 3 expression was not detected in Bmi1f/f;αMHC-Cretg/+ or control hearts (Fig. 6a). We next assessed proliferation rate and cardiac SA-β-gal activity35 in CMs isolated by the Langendorff perfusion method36 and nonmyocyte subpopulations of FBs (CD3−/CD31−/Thy1+) and endothelial cells (ECs; CD3−/CD31+/Thy1−)37. Bmi1 expression in Bmi1f/f;αMHC-Cretg/+ CMs was ~10% of the level detected in control CMs, whereas FBs and ECs showed no significant differences between the two genotypes (Fig. 6b), as expected26. Analysis of the cell-cycle status by 5-bromodeoxyuridine (BrdU) incorporation indicated that total cardiac cells and the FB and EC subpopulations cycled similarly in mutant mice and control littermates (Fig. 6c). In Bmi1fl;αMHCCre hearts, ~31% of CMs were positive for SA-β-gal activity, compared with 18.4% in control hearts (Fig. 6d,e). Surprisingly, ~21% of FBs in Bmi1-null hearts were also positive, compared with 13% in control hearts (Fig. 6d,e), suggesting that CM-expressed Bmi1 might directly control the expression of paracrine factors that spread the senescence response to surrounding populations. To test this, we assessed the contribution of Tgfβ family ligands17 to DCM in Bmi1-null mice. Injection of Tgfβ-neutralizing antibodies into Bmi1f/f;αMHC-Cretg/+ mice significantly reduced maximum left ventricular wall thickness and reversed the fractional shortening (Supplementary Fig. 3c,d). Moreover, Tgfβ blockade significantly reduced CM cross-sectional area (Supplementary Fig. 3e). Together, these results indicate that senescence originating in CMs plays an important role in the development of DCM in Bmi1-null hearts. Bmi1 limits the senescence response in the heart by repressing Cdkn2a To further define the involvement of senescence in the DCM of cardiac Bmi1-deficient mice, we eliminated p16INK4a expression in the αMHCCre-mediated Bmi1-null background, generating Bmi1fl/fl;αMHC-Cretg/+;p16INK4a−/− mice and Bmi1fl/fl;αMHC-Cre+/+;p16INK4a−/− littermates. Deletion of p16INK4a (p16−/−), with p19ARF expression unaffected38, was neither accompanied by compensatory upregulation of other Polycomb members nor by increase in p53 and p21 senescence markers (Fig. 6f). Moreover, p16−/− mice with intact Bmi1 (Bmi1fl/fl;p16−/−) showed no noticeable adult cardiac abnormalities over the period of study (40 weeks; Fig. 6g–k). Bmi1fl/fl; αMHC-Cretg/+;p16−/− mice had normal expression levels of the hypertrophy marker Myh7 and the profibrosis factor Tgfβ (Fig. 6f), and their hearts were of near normal size (Fig. 6g,h) and contained CMs of normal cross-sectional area, with no gross evidence of fibrosis (Fig. 6g,i). Bmi1fl/fl;αMHC-Cretg/+;p16−/− mice also had normal left ventricular wall thickness (Fig. 6j) and significantly improved FS capacity (Fig. 6k). Notably, the deletion of p16 almost completely averted the senescence effect of cardiac Bmi1 deletion, as shown by the normal proportion of CMs exhibiting SA-β-gal activity (14.4% in Bmi1fl/fl;αMHC-Cretg/+;p16−/− mice and 15.2% in Bmi1fl/flp16−/− controls; Fig. 6l–n). Moreover, p16 deletion significantly improved survival of mice lacking Bmi1 (Fig. 6n), suggesting that p16INK4a is required for the senescence effect in Bmi1-null hearts. Exposure of an old mouse to the circulation of a young mouse by parabiosis can reverse major effects of age-related cardiac hypertrophy39. Moreover, senescent cells can communicate with their environment in a paracrine manner17. These observations prompted us to examine the impact of the systemic environment on senescence by surgically conjoining the circulation of Bmi1f/f;αMHC-Cretg/+ and control littermate mice in parabiotic pairs (Fig. 7a). Efficiency of parabiosis was evaluated by using congenic markers to distinguish blood cells in parabiotic pairs, in which one partner was CD45.1+ (Supplementary Fig. 3f). The effect of a wild-type circulation on DCM hearts (parabiotic no-DCM/DCM pairs) after 1 month was readily apparent on cardiac histologic sections (Fig. 7b). Masson’s trichrome staining showed a reduction in interstitial cardiac fibrosis in the DCM heart, accompanied by reduced CM size (Fig. 7b). Accordingly, parabiosis reduced the HW/BW ratio and CM cross-sectional area in the DCM heart, whereas isochronic parabiosis (no-DCM/no-DCM or DCM/DCM pairs) had no effect (Fig. 7c,d). Parabiotic exposure of DCM mice to the circulation of healthy non-DCM littermates for 4–5 weeks rescued FS from initial values of 16.0±1.1–24.0±2.4% (Fig. 7e), indicating that the wild-type circulation restores cardiac function of the DCM partner in no-DCM/DCM pairs. These data indicate that factors in the circulation could modify discrete molecular pathways associated with ventricular remodelling and DCM, allowing reversal of the dilated cardiac function. Remarkably, however, DCM-related paracrine senescence factors were not able to transmit the DCM phenotype to healthy mice. Discussion Here we propose a mechanistic model of how Bmi1 protects from DCM by limiting heart senescence. Absence of cardiac Bmi1 profoundly compromises expression of the senescence marker p16INK4a in CMs, leading to a cardiac-senescent response caused likely by SASP, which mobilizes the systemic and local tissue milieu for tissue repair and aging1240. The detection of senescence factors by neighbouring healthy cells might further drive cellular senescence, thus contributing to a spiral of increasing inflammation and dysfunction that increases the original features of the DCM. The increasing burden of senescent cells might contribute to the early aetiology of DCM and accelerate progression of these age-related cardiac diseases (Fig. 8a). Remarkably, this paracrine nonproliferative senescence phenotype is not propagated between organisms via parabiosis, whereas a healthy circulation is able to reverse CM hypertrophy in mice with DCM. This effect is gender-independent and the reduction in CM size translates into a reduction in global cardiac mass. Factors present in the circulation of healthy mice are thus to some extent able to reverse critical structural and molecular aspects of cardiac aging. Although the full cast of factors involved in the rejuvenation of DCM are yet to be identified, our data provide proof of principle that signals from the systemic environment can override age-related, intrinsic changes in DCM, suggesting that these changes are predominantly epigenetic. In fact, in recent years, emerging evidence indicates that there are factors within the blood of young animals that have the ability to restore youthful characteristics to a number of organ systems in older animals3941. These studies offer compelling evidence that effects of aged-associated disease can be reversed. Risk assessment in DCM patients is currently limited to echocardiography studies, measurement of haemodynamic parameters and cardiopulmonary exercise performance. Understanding the role of epigenetic regulators in adult cardiac senescence provides a possible route towards more accurate profiling of cardiac dysfunction in dilated cardiomyopathies. Modulation of cardiac senescence response may further provide a novel strategy for treating heart failure, with Bmi1 serving as an attractive target. Methods Mice ckoBmi1fl/+ mice24 were backcrossed to the C57BL/6 background. Mice used in this study included αMHC-Cretg/+ (ref. 26), αMHCTM-Cretg/+ (ref. 27), Nkx2.5-Cretg/+ (ref. 25), MLC2-rRTAtg/+ (ref. 28) and p16INK4a −/− (ref. 38). The αMHC-Cretg/+ and αMHCTM-Cretg/+ lines were kindly provided by Dr Redondo, Nkx2.5-Cretg/+ by Dr de la Pompa, MLC2-rRTAtg/+ by Dr Lara-Pezzi and p16INK4a−/− by Dr Berns. For the generation of tetO-Bmi1tg/+ mice, Bmi1 cDNA was cloned into the pBS31 vector that included the minimal tetracycline-independent promoter. This Bmi1 vector was coelectroporated together with the vector harbouring FLPe recombinase under the control of the CAGGS promoter into KH2 ES cells (Open Biosystems). All genotyping primers are listed in Supplementary Table 1. All strains were on the C57BL/6J background; male and female mice aged 5–12 weeks were used, in the cases of not indicated. For iBmi1tg/tg;MLC2tg/+ and control mice, doxycycline (2 mg ml−1, supplemented with sucrose at 5 mg ml−1) was administrated to mice in drinking water for 2 weeks). The αMHCTM-Cre line and their control littermates were injected with 1 mg TM (Sigma) in corn coil on five consecutive days. All mice were bred in-house in a pathogen-free environment and were provided with standard care and nutrition according to the EU guidelines. All animal study proposals were approved by the Centro Nacional de Investigaciones Cardiovasculares (CNIC). For cardiogenesis studies, hearts at various embryonic stages or post-natal (P) days were collected for RNA/protein analysis or for histology. Echocardiography and MRI Echocardiograms were performed on mice anaesthetized with 2.0% isoflurane, using a Vevo 770 High-Resolution In Vivo Micro-Imaging System and RMV 707B Scanhead (VisualSonics Inc). Scans were conducted by two experienced researchers blinded to the mouse genotype. Measurements of left parasternal long and short axes and M-mode (left parasternal short axis) images were obtained at a heart rate of 500–550 bpm. LV end-diastolic diameter (LVEDD), LV end-systolic diameter (LVESD) and wall thickness were measured from M-mode tracings, and the average of three consecutive cardiac cycles is reported. The LV fractional shortening percentage was calculated as ([LVEDD−LVESD]/LVEDD) × 100 MRI of lung was performed with a 7-T Agilent scanner (Agilent, Santa Clara, USA) equipped with a DD2 console and an actively shielded gradient set (205/120 insert of maximum 130 mT m−1 gradient strength). For image acquisition, we employed a combination of volume coil/surface coil coil to enhance signal-to-noise ratio formed by a 72-mm inner diameter quadrature birdcage TX coil (Rapid Biomedical GmBH, Germany) and an actively detuning 30-mm flexible customized surface RX coil (Neos Biotec, Pamplona, Spain). Following a tripilot gradient-echo image, a gradient-echo sequence without gating was used to acquire oblique coronal slices (one to two slices) and axial slices (7–10 slices covering the entire lung, 72-s acquisition time per slice) using the following parameters: TR/TE=6.7/2.2 ms, flip angle=10 degree, bandwidth=100 kHz, field of view=3 × 3 cm, matrix=256 × 128, slice thickness=1 mm and number average experiment (Number average EXperiments=60). Mice were anaesthetized with 2% isoflurane and oxygen and positioned on a thermoregulated (38.7 °C) mouse bed. Ophthalmic gel was placed in their eyes to prevent retinal drying. Human DCM samples The study population comprised six patients with medically refractory DCM, excluding specifically those patients with the evidence of ischaemic cardiomyopathy or myocarditis. Before cardiac transplantation, patients received a two-stage drug regime designed to avoid deterioration (β1/β2-blocker (carvedilol), angiotensin-converting enzyme inhibitor (lisinopril), angiotensin receptor I antagonist (losartan) and aldosterone antagonist (spironolactone)). Patients were subsequently switched to a selective β1 blocker (bisoprolol) and given the β2-agonist, clenbuterol, with a view to stimulate hypertrophy and improve cardiac function. Echocardiography and exercise testing were performed regularly during treatment before transplantation to monitor the recovery process. In addition, four ventricular samples were available for mRNA analysis from donor organs used for transplantation (n=2), and donor organs unsuitable for transplantation (n=2). Data collected from these RNA samples were used in gene expression analyses. Histology and immunohistochemical analyses For histological analysis, embryos or heart tissues were fixed in 10% paraformaldehyde (PFA) at the stages indicated in each figure legend, dehydrated and embedded in paraffin for preparation of 5- or 10-μm histological sections. Rehydrated slides were stained with haematoxylin and eosin and Masson’s trichrome. For determination of CM cross-sectional area, deparaffinized and rehydrated heart sections were incubated for 1 h at room temperature with fluorescein isothiocyanate (FITC)-labelled wheatgerm agglutinin (Sigma-Aldrich) to visualize myocyte membranes. Regions that included the circular shapes of capillaries were selected from the epicardial side of the LV-free walls. The mean cross-sectional area of CMs was determined from 60 to 80 cells. For fluorescence detection of SA-β-gal activity, cells (107 cell ml−1) were incubated with C12FDG (fluorescein di-β-D-galactopyranoside; 33 μM; Sigma), a β-galactosidase substrate that generates a fluorescent product upon cleavage, for 60 min at 37 °C (ref. 35). Cytochemical detection of senescent cells in vitro was determined in cells and fixed tissues with the Senescence β-Galactosidase Staining Kit (Cell Signaling). Chromatin immunoprecipitation (ChIP) assays For ChIP analysis, we used sorted 106 cells per condition and added formaldehyde directly to the tissue culture medium to a final concentration of 1% and incubated cultures for 10 min at room temperature on a shaking platform. We stopped the crosslinking by adding glycine to a final concentration of 0.125 M. We washed crosslinked cells twice with cold phosphate-buffered saline and lysed them at a density of 5 × 106 cells ml−1 for 10 min at 4 °C in 1% SDS, 50 mM Tris-HCl (pH 8.0) and 10 mM EDTA-containing protease inhibitors. We sonicated lysates to obtain chromatin fragments <1 kb and centrifuged them for 15 min in a microfuge at room temperature. We diluted 400 μl of lysate 1:10 with 1.1% Triton-X100, 2 mM EDTA, 150 mM NaCl and 20 mM Tris-HCl (pH 8.0) containing protease inhibitors, precleared with 50% salmon sperm DNA and protein A agarose slurry (Upstate). Antibodies used for ChIP assays42 were rabbit polyclonal anti-Bmi1, H3K27me3, H2AK119 and H3K4me3 (all from Upstate; 1:2,500). We then added salmon sperm DNA and protein A agarose beads (60 μl) and incubated for 1 h. We washed the immunoprecipitated pellets with 0.1% SDS, 1% Triton-X100, 2 mM EDTA, 20 mM Tris-HCl (pH 8.0) and 150 mM NaCl (one wash); 0.1% SDS, 1% Triton-X100, 2 mM EDTA, 20 mM Tris-HCl (pH 8.0) and 500 mM NaCl (one wash); 0.25 M LiCl, 1% Nonidet P-40, 1% sodium deoxycholate, 1 mM EDTA and 10 mM Tris-HCl, pH 8.0 (one wash); and 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (two washes). We then eluted the chromatin from the beads twice by incubation with 250 μl 1% SDS and 0.1 M NaHCO3 during 15 min at room temperature with rotation. After adding 20 μl of 5 M NaCl, we reversed the crosslinks for 4 h at 65 °C. Samples were supplemented with 20 μl of 1 M Tris-HCl (pH 6.5), 10 μl of 0.5 M EDTA, 20 μg of RNase A and 40 μg of proteinase K and incubated for 1 h at 45 °C. DNA from precipitated complexes was amplified using reverse transcription–polymerase chain reaction (RT–PCR). RT–PCR amplifications were performed in triplicate with multiple dilutions, and primer sequences are included in the Supplementary Table 1. RNA extraction and real-time PCR Heart tissue samples were stored in RNAlater RNA stabilization reagent (QIAGEN) at 4 °C. Total RNA was extracted using the RNeasy Fibrous Tissue Mini Kit (QIAGEN). First-strand cDNA synthesis was performed with 1 μg of total RNA, random hexamers and SuperScript III Reverse Transcriptase (Invitrogen). Real-time PCR was performed using a QuantiTect SYBR Green PCR kit (QIAGEN) in a Light-Cycler (Roche). The expression level of each gene was normalized to that of 18S rRNA, which served as an endogenous internal control. The primer sequences are available at the Supplementary Table 1. Cardiac hypertrophy induction Isoproterenol (60 mg per kg body weight) was administered to 9-week-old mice for 14 days using subdermally implanted ALZET osmotic minipumps. PBS-filled minipumps were used as a control. For TAC surgery, mice (8–10 weeks old, 21–24 g body weight) were anaesthetized by intraperitoneal injection of a mixture of xylazine (5 mg kg−1) and ketamine (100 mg kg−1). The animals were then placed in a supine position, an endotracheal tube was inserted and the animals were ventilated using a volume-cycled rodent ventilator with a tidal volume of 0.4 ml room air and a respiratory rate of 110 breaths per minute. The chest cavity was exposed by cutting open the proximal portion of the sternum. The aortic arch between the innominate and left common carotid arteries was isolated and was constricted with a 7–0 nylon suture tied firmly three times against a 25-gauge blunted needle for LI-TAC or a 27-gauge needle for HI-TAC. Sham-operated mice underwent the same surgical procedure, including isolation of the aorta, but without placement of the suture. Tgf-β blocking activity assay DCM mice (Bmi1flαMHC-Cretg/+) received intraperitoneal injections of Tgf-β antibody (10 mg per kg body weight in PBS; AB-100-NA; R&D Systems) every 3 days for 2 weeks before being killed. Control mice were treated with identical doses of rabbit IgG (R&D Systems). Protein analysis Antibodies used for western blots were as follows: anti-Bmi1 (Millipore), anti-Ezh2 (Active Motif), anti-aMHC (Millipore), anti-H3K9me3 (Upstate), anti-murine p16 (Santa Cruz Biotechnology), anti-human p16 (Cell Signaling) and anti-β-actin (Sigma; these antibodies were used at 1:500). Secondary antibody was the horseradish peroxidase-linked anti-mouse IgG (Dako; 1:2,500). Isolation of CMs and cardiac FBs Cells were isolated from Langendorff heart preparations followed by enzymatic digestion43. For isolation of nonmyocyte-enriched cells, hearts were dissected free of vessels and atria, washed in ice-cold modified Krebs–Henseleit bicarbonate buffer (pH 7.2; Sigma-Aldrich) and rapidly cut into pieces. The heart pieces were incubated in 5 ml of digesting solution (0.25 mg ml−1 Liberase TH (Roche) and 10 mM HEPES in balanced salt solution-containing calcium and magnesium) for 8 min at 37 °C with vigorous stirring. The supernatant was then added to 10 ml of ice-cold Krebs–Henseleit bicarbonate. Five millilitres of fresh digesting solution were added to the remaining tissue fragments, and the digestion and sampling steps were repeated until all the tissues were dissolved. The collected cells were filtered through a 35-μm nylon mesh (BD Falcon) and then used for flow cytometry. Different cellular subsets were incubated with phycoerythrin-conjugated anti-Thy1 antibody (eBioscience), FITC-conjugated anti-CD31 antibody (BD Biosciences) and allophycocyanin-conjugated CD3 antibody (eBioscience), after which they were analysed and sorted on a FACSAria II flow cytometer (BD Biosciences) using the FlowJo softwarem (these antibodies were used at 1:100). For analysis of SA-β-gal expression, cells were stained with the fluorogenic β-galactosidase substrate fluorescein di-β-D-galactopyranoside (1:250). Cell proliferation BrdU (Sigma-Aldrich) was injected daily into the peritoneum (100 mg per kg body weight in PBS) for 3 days, with the last injection 2 h before harvesting the hearts. The hearts were fixed in 4% PFA, paraffin-embedded and sectioned. BrdU-positive nuclei were detected with mouse monoclonal anti-BrdU primary antibody (Dako; 1:250) and immunofluorescent staining as described above. BrdU- or Ki67-labelled cells and 4',6-diamidino-2-phenylindole (DAPI)-stained nuclei were counted in nonmyocyte cells from 5 to 10 fields ( × 400 magnification) with focal fibrosis and/or expanded interstitium (identified with wheatgerm agglutinin staining) and in areas with preserved myocardial architecture (for example, absence of both focal fibrosis and expanded interstitium) in 9–15 sections per heart. Myocytes were identified with fluorescent microscopy visualization of sarcomeres. Nuclei of sarcomere-negative cells within interstitial regions were designated as nonmyocyte nuclei. Nuclei were quantified using the ImageJ nucleus counter software. The percentage of proliferating cells was calculated as the number of positive BrdU- or Ki67-labelled nuclei divided by the number of DAPI-stained nuclei. Parabiosis Anaesthetized mice were shaved at the corresponding lateral aspects and matching skin incisions were made from the olecranon to the knee joint of each mouse, and the subcutaneous fascia was bluntly dissected to create ~0.5 cm of free skin. The olecranon and knee joints were attached by a single 5–0 polypropylene suture and tie, and the dorsal and ventral skins were approximated by continuous suture. A single dose of flunixin meglumine (Schering-Plough, 1 mg kg−1) was injected subcutaneously into each partner at the end of the surgical procedure. One month after surgery, blood samples were obtained from each of the partners for analysis of haematopoietic progenitors. Statistical analysis All data are expressed as the mean±s.d. Paired data were evaluated using Student’s t-test. Survival rates among mice were analysed using the long-rank test. Differences were considered statistically significant at P<0.05. Additional information Accession codes: The RNAseq data from mDCM and hDCM samples have been deposited in the Gene Expression Omnibus under accession code GSE64391 and GSE65447, respectively How to cite this article: Gonzalez-Valdes, I. et al. Bmi1 limits dilated cardiomyopathy and heart failure by inhibiting cardiac senescence. Nat. Commun. 6:6473 doi: 10.1038/ncomms7473 (2015). Supplementary Material Supplementary Figures and Supplementary Table Supplementary Figures 1-3 and Supplementary Table 1 Supplementary Data 1 The supplementary dataset shows 2435 genes differentially expressed between heart samples of dilated cardiomyopathy-diagnosed patients (who had undergone heart transplant; hDCM) and healthy control patients, as well as 649 genes differentially expressed from heart samples of Bmi1f/f;aMHCTM-Cretg/+ and controls mice (17 weeks postinduction; mDCM). We thank Miguel Torres and Jose Antonio Enriquez for helpful discussions; Rebeca Diges for excellent technical assistance; and Simon Bartlett for text editing. S.G. is funded by the Human Frontiers Science Program Organization and the Spanish Ministries of Economy and Competitiveness (SAF2010-15386 and SAF2013-42252-R). The CNIC is supported by the Ministery of Economy and Competitiveness and the Pro-CNIC Foundation. The authors declare no competing financial interests. Author contributions I.G.-V. has performed most of the experiments; I.H., L.P.-B., P.G., J.M.R. and A.B. have contributed to data analysis and discussion of the paper; J.M.R.-C., L.J.J.-B. have performed the imaging data; P.G.-P. and E.L.-P. have contributed with the samples of DCM patients; J.A.E., J.L.d.l.P. and A.H. have supervised the data analysis; S.G. designed and supervised the study and wrote the paper. 03/07/2017 This paper has been retracted at the request of the authors. 05/14/2015 In the original version of this Article, the last name of the author Pablo Gómez-del Arco was incorrectly given as Gomez, and the affiliation details were incorrect. This has now been corrected in both the PDF and HTML versions of the Article. Figure 1 Bmi1 is not required for normal cardiac development. (a) Representative heart sections from Bmi1f/f;Nkx-Cretg/+ (Bmi1fl;NkxCre) and Bmi1f/f;Nkx-Cre+/+ (Bmi1f/) E14.5, E16.5 and E18.5 embryos (scale bar, 200 μm). Rv, right ventricle; lv, left ventricle. (b) Quantitative RT–PCR (qRT–PCR) analysis of Bmi1, p16INK4a, ARF and p15INK4b mRNA expression in total heart cells from Bmi1fl;NkxCre (Bmi1-knock out) mice. Data are standardized to β-actin levels and are expressed relative to Bmi1f/ (Control) mice (means±s.d.; n=12, P<0.05; Student’s t-test). (c) HW/BW ratio in 22-week-old Bmi1fl;NkxCre mice and Bmi1f/ controls (means±s.d.; n=12, P<0.05; Student’s t-test). (d) Gross cardiac phenotype of 22-week-old Bmi1fl;NkxCre mice and Bmi1f/ controls. Representative views are shown of external anatomy (top row; bars, 0.5 cm) and haematoxylin and eosin (H&E) staining on sections in adult hearts (second row; bars, 1 mm), Masson’s trichrome staining to detect fibrosis (third row; bars, 40 μm) and left ventricular muscle sections stained with wheatgerm agglutinin (WGA; bottom row; bars, 10 μm). (e,f) Trans-thoracic M-mode echocardiographic and physiological analyses of Bmi1fl;NkxCre and Bmi1f/ mice. Panel i shows representative traces from Bmi1fl;NkxCre and Bmi1f/ mice at 7, 15 and 22 weeks of age. IVSd, diastolic interventricular septal wall thickness; LVDd, diastolic left ventricular internal dimension; LVDs, systolic left ventricular internal dimension; LVPWd, diastolic left ventricular posterior wall thickness; FS, fractional shortening of left ventricle dimension; EF, ejection fraction; LVmass, left ventricular mass. Data are means±s.d. (n=12, *P<0.001, P<0.05; Student’s t-test). (g) Representative images of whole lungs from 12-week-old Bmi1fl;NkxCre mice and Bmi1f/ littermates. Scale bars, 5 mm. (h) Thoracic magnetic resonance MRI of similar mice in transverse view, showing both heart and lungs (left), and in the coronal view (right). Scale bars, 2.5 mm. (i) Representative Perls iron staining of lung sections from mice as in g, h (bars, 30 μm). (j) Kaplan–Meier survival curve for Bmi1fl;NkxCre mice and Bmi1f/ littermates (means±s.d., P<0.001; Student’s t-test). Figure 2 Bmi1 activation blocks development of cardiac hypertrophy. (a) Gross cardiac phenotype of Bmi1fl;αMHCCre mice and Bmi1fl controls. Representative views are shown of external anatomy and H&E staining on sections in adult hearts (12-week-old; top two rows; bars, 50 mm), Masson’s trichrome staining to detect fibrosis (third row; bars, 40 μm) and left ventricular muscle sections stained with WGA to detect cardiomyocyte borders (bottom row; bars, 10 μm). (b) M-mode echocardiographic analysis of Bmi1fl;αMHCCre and Bmi1fl mice. IVSd, diastolic interventricular septal wall thickness; LVDd, diastolic left ventricular internal dimension; LVDs, systolic left ventricular internal dimension; LVPWd, diastolic left ventricular posterior wall thickness; FS, fractional shortening of left ventricle dimension; EF, ejection fraction; LVmass, left ventricular mass. Data are means±s.d. (n=13, *P<0.001, P<0.05; Student’s t-test). (c) Thoracic MRI of Bmi1fl;αMHCCre and Bmi1fl mice in transverse view, showing both heart and lungs (top), and in coronal view (bottom). Scale bars, 2.5 mm. (d) Representative Perls iron staining of lung sections from representative 12-week-old Bmi1fl;αMHCCre and Bmi1fl littermates (bars, 30 μm). (e) Lung weight in 12-week-old Bmi1f/f;αMHCTM-Cretg/+ and Bmi1+/+;αMHCTM-Cretg/+ mice (means±s.d.; n=10, P<0.05; Student’s t-test). (f) Kaplan–Meier survival curve for Bmi1fl;αMHCCre mice and Bmi1fl littermates (means±s.d.P<0.001; Student’s t-test). Figure 3 Cardiac dysfunction in tamoxifen-treated Bmi1f/f;αMHCTM-Cretg/+mice. (a) qRT–PCR analysis of the mRNA expression of Bmi1, p16INK4a, ARF and p15INKb in total heart cells from Bmi1fl;αMHCCreTM (Bmi1-KO) and αMHCCreTM (Control) mice. Data are standardized to β-actin levels and are expressed relative to Control mice (means±s.d.; n=9, P<0.05; Student’s t-test). (b) HW/BW ratio in Bmi1fl;αMHCCreTM mice and αMHCCreTM controls mice at 22 weeks post induction (27 weeks old; means±s.d.; n=10, P<0.05; Student’s t-test). (c) Gross cardiac phenotype of Bmi1fl;αMHCCreTM mice and αMHCCreTM controls at 22 weeks post induction (27 weeks old). Representative views are shown of external anatomy (bar, 50 mm), Masson’s trichrome staining to detect fibrosis (bars, 40 μm), left ventricular muscle sections stained with WGA to detect cardiomyocyte borders (bars, 10 μm). (d,e) Trans-thoracic M-mode echocardiographic and physiological analyses of Bmi1fl;αMHCCreTM and αMHCCreTM mice at 8, 12 and 22 weeks post induction. Panel e shows representative traces. Panel g shows echocardiographic measurements and physiological parameters. IVSd, diastolic interventricle septal wall thickness; LVDd, diastolic left ventricle internal dimension; LVDs, systolic left ventricle internal dimension; LVPWd, diastolic left ventricle posterior wall thickness; FS, fractional shortening of left ventricle dimension; EF, ejection fraction; LVmass, left ventricle mass. Data are means±s.d.; n=12, P<0.001, P<0.05; Student’s t-test. (f) Thoracic MRI of representative 12-week-old Bmi1fl;αMHCCre mice and Bmi1f/f littermates in transverse view, showing both heart and lungs (top), and in coronal view (bottom). Scale bars, 2.5 mm. (g) Lung weight in Bmi1fl;αMHCCreTM mice and αMHCCreTM controls at 22 weeks post induction (27 weeks old; means±s.d.; n=10, P<0.05; Student’s t-test). (h) Representative Perls iron staining of lung sections from representative 12-week-old Bmi1fl;αMHCCre mice and Bmi1f/f littermates (bars, 30 μm). (i) Kaplan–Meier survival curve for Bmi1fl;αMHCCre mice and Bmi1f/f littermates (means±s.d. P<0.001; Student’s t-test). Figure 4 Bmi1 activation blocks development of cardiac hypertrophy. (a) mRNA levels of Bmi1 in heart samples from nontransgenic control mice (iBmi1tg/tg) and Bmi1 transgenic mice (iBmi1tg/tg;MLC2tg/+; means±s.d.; n=8, P<0.05; Student’s t-test). (b) HW/BW ratios in iBmi1tg;MLC2 mice and iBmitg littermates 4 weeks after the TAC surgery or sham operation. Data are means±s.d.; n=8 mice per group (means±s.d.; n=6, P<0.05; Student’s t-test). (c,d) Left ventricular wall thickness and fractional shortening measured using echocardiography in the same hearts as in b. (e) Representative low-magnification views of H&E-stained cross-sections at the midventricle from nontransgenic and Bmi1 transgenic mice subjected to sham or TAC treatment and stained with WGA (top; scale bars, 10 μm) or Masson’s trichrome to detect fibrosis (bottom; scale bars, 40 μm). Figure 5 Control of cardiac-specific profile by Bmi1. (a) Representative heat maps show the expression of genes with fetal cardiac programme-related genes, senescence-associated and cell-cycle functional annotations that are significantly down- and upregulated in Bmi1f/f;αMHCTM-Cretg/+ (mDCM) and control heart cells (mControl), and hDCM and hControl. The rows correspond to genes and the columns to samples. Gene expression values (relative to the mean expression in control cells) are indicated on a log2 scale according to a colour scheme shown. (b) ChIP analyses of the promoter regions in the indicated genes from total heart cells from Bmi1f/f;αMHCTM-Cretg/+ mice and littermate controls. Chromatin-bound DNA was probed with antibodies to Bmi1, H3K27me3, H2AK119 and H3K4me3. Percentages of input DNA are shown as the means±s.d. of triplicate independent experiments (P<0.05; Student’s t-test; Student’s t-test). Figure 6 Bmi1-mediated cardiomyocyte senescence. (a) Cytochemical staining of SA-β-gal activity and immunostaining of Caspase 3 in paraffin sections of hearts from 15-week-old Bmi1f/f;αMHC-Cretg/+ mice and Bmi1f/ controls. Bars, 50 μm. (b) qRT–PCR analysis of Bmi1 mRNA expression in sorted populations of CMs, endothelial cells (ECs) and FBs from Bmi1f/f;αMHC-Cretg/+ and Bmi1f/ mice. Expression is standardized to β-actin and is expressed relative to the level in Bmi1f/ mice (means±s.d.; n=12, P<0.05; Student’s t-test). (c) Proliferation rate of CM, EC and FB subpopulations measured by in vivo BrdU incorporation over 1 week. Values are means±s.d. (n=5). (d,e) Representative histograms for C12-fluorescein show the relative levels of SA-β-gal in CM, EC and FB subsets from Bmi1f;αMHCCre and control mice (d); the values above the peaks are the median fluorescence intensities of the respective populations. (e) Percentage of SA-β-gal-positive cells in CM, EC and FB subpopulations. Values are means±s.d. (n=6, P<0.05; Student’s t-test). (f) qRT–PCR analysis of the expression of Bmi1, p16INK4a, Ezh2, Myh7, Tgfβ, p53 and p21 mRNA in total heart cells from Bmi1fl/fl;αMHC-Cretg/+;p16INK4a−/− mice and Bmi1fl/fl;αMHC-Cre+/+;p16INK4a−/− mice. Values are means±s.d.; n=10 (*P<0.001, P<0.05; Student’s t-test). (g) The gross cardiac phenotype of 12-week-old Bmi1fl/fl;αMHC-Cre+/+;p16INK4a−/− (Bmi1fl;p16−/−), Bmi1fl/fl;αMHC-Cretg/+;p16INK4a+/+ (Bmi1fl;αMHCCre) and Bmi1fl/fl;αMHC-Cretg/+;p16INK4a−/− (Bmi1fl;αMHCCre;p16−/−) mice. Representative views are shown of H&E-stained heart cross-sections at the midventricle (bars, 1 mm), Masson’s trichrome staining of left ventricle to detect fibrosis (bars, 40 μm) and WGA staining to outline cardiomyocytes (bars, 10 μm). (h–k) HW/BW ratios (h), myocyte cross-sectional area (i), left ventricular wall thickness (j) and fractional shortening (k) in 12-week-old Bmi1fl;p16−/−, Bmi1fl;αMHCCre and Bmi1fl;αMHCCre;p16−/− mice. Data are means±s.d. n=8 mice per group (P<0.05; Student’s t-test). (l) Cytochemical staining of SA-β-gal activity in paraffin sections of hearts from Bmi1fl;p16−/−), Bmi1fl;αMHCCre) and Bmi1fl;αMHCCre;p16−/− mice. Scale bars, 50 μm. (m,n) Representative fluorescence histograms show the relative levels of SA-β-gal in CM, EC and FB subsets from Bmi1fl;p16−/− and Bmi1fl;αMHCCre;p16−/− hearts (m); the values above the peaks are the median fluorescence intensities of the respective populations. Percentage of SA-β-gal-positive cells in CM, EC and FB subpopulations (n). Values are means±s.d. (n=6; Student’s t-test). (o) Kaplan–Meier survival curves for Bmi1fl;p16−/−, Bmi1fl;αMHCCre and Bmi1fl;αMHC-Cre;p16−/− littermates (means±s.d., P<0.001; Student’s t-test). Figure 7 Paracrine senescence response of Bmi1-related DCM does not transmit to healthy mice. (a) Parabiotic pairings; no-DCM as Bmi1fl mice and DCMhigh as Bmi1fl/fl;αMHC-Cre mice. Comparisons were always made with littermate pairs (no-DCM to no-DCM or DCMhigh to DCMhigh). (b) The gross cardiac phenotype of 12-week-old mice after 4 weeks of parabiosis as indicated. Representative views are shown of H&E-stained cross-sections at the midventricle (bars, 1 mm), Masson’s trichrome staining of left ventricle to detect fibrosis (bars, 40 μm), and WGA staining to outline cardiomyocites (bars, 10 μm). (c–e) HW/BW ratio (c), myocyte cross-sectional area (d) and fractional shortening (e) after 4 weeks of parabiosis as indicated. Data are means±s.d.; n=8 mice per group (P<0.05; Student’s t-test). Figure 8 A model for the impact of cardiac-specific Bmi1 action. (a) As aging poses the largest risk for cardiovascular disease, the cardiac Bmi1 action could be determinant to limit the heart senescence response. Our data establish the idea that the nonproliferative cardiomyocyte-related senescence phenotype can be locally propagated through the SASP. ==== Refs McNally E. M. , Golbus J. R. & Puckelwartz M. J. Genetic mutations and mechanisms in dilated cardiomyopathy . 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==== Front Nat CommunNat CommunNature Communications2041-1723Nature Publishing Group ncomms476110.1038/ncomms476124781844ArticleCarbon enters silica forming a cristobalite-type CO2–SiO2 solid solution Santoro Mario a12Gorelli Federico A. 12Bini Roberto 23Salamat Ashkan 4Garbarino Gaston 4Levelut Claire 5Cambon Olivier 6Haines Julien b61 Istituto Nazionale di Ottica, Consiglio Nazionale delle Ricerche (INO-CNR), Sesto Fiorentino 50019, Italy2 European Laboratory for Non-Linear Spectroscopy (LENS), Sesto Fiorentino 50019, Italy3 Dipartimento di Chimica dell’Università di Firenze, Sesto Fiorentino 50019, Italy4 European Synchrotron Radiation Facility, 38043 Grenoble CEDEX 9, France5 Laboratoire Charles Coulomb, UMR 5221, Centre National de la Recherche Scientifique (CNRS), Département Colloïdes, Verres et Nanomatériaux (CVN), Université Montpellier 2, 34095 Montpellier CEDEX 5, France6 Institut Charles Gerhardt Montpellier, UMR 5253, Centre National de la Recherche Scientifique (CNRS), Equipe C2M, Université Montpellier 2, 34095 Montpellier CEDEX 5, Francea [email protected] [email protected] 04 2014 2014 5 376120 12 2013 31 03 2014 Copyright © 2014, Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved.2014Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved.Extreme conditions permit unique materials to be synthesized and can significantly update our view of the periodic table. In the case of group IV elements, carbon was always considered to be distinct with respect to its heavier homologues in forming oxides. Here we report the synthesis of a crystalline CO2–SiO2 solid solution by reacting carbon dioxide and silica in a laser-heated diamond anvil cell (P=16–22 GPa, T>4,000 K), showing that carbon enters silica. Remarkably, this material is recovered to ambient conditions. X-ray diffraction shows that the crystal adopts a densely packed α-cristobalite structure (P41212) with carbon and silicon in fourfold coordination to oxygen at pressures where silica normally adopts a sixfold coordinated rutile-type stishovite structure. An average formula of C0.6(1)Si0.4(1)O2 is consistent with X-ray diffraction and Raman spectroscopy results. These findings may modify our view on oxide chemistry, which is of great interest for materials science, as well as Earth and planetary sciences. Novel materials synthesized under extreme conditions can challenge long-held views of fundamental chemistry. Santoro et al. combine fluid CO2 and solid SiO2 to create a new crystalline compound, via experimentation at ultra-high pressures and temperatures, which is stable at ambient conditions. ==== Body Extreme conditions lead to the development of new chemistry123. Studies are increasingly showing that the known extensive chemical homogeneity among elements of the same group in the periodic table can be extended, in particular, to the lightest elements of the group, which often behave differently under ambient conditions. Until recently, the potential analogy between C and Si in forming extended oxides was very limited. On one hand, there is CO2, which is an archetypical gas under normal conditions with carbon in twofold coordination, and the molecule is held together by two strong, double bonds. Molecular CO2 is the main greenhouse gas. It is one of the main components in the atmosphere of Earth-like planets and it has an important role in volcanic and seismic activity. It is found in ice form in the outer planets and asteroids. It is also used as a supercritical solvent in chemical reactions. Alternatively, SiO2 is a solid material with Si in either four- or sixfold coordination (Fig. 1). Silica exhibits both amorphous forms and crystalline phases, many of which are important minerals. Silica is also one of the most used technological materials as silica glass and quartz. In many applications, such as in optical waveguides, SiO2 is also used in solid solutions with GeO2, its heavier analogue. Although traditionally perceived as being incompatible, a molecular gas (CO2) and a network solid (SiO2), recent high-pressure experiments have radically altered this view. In fact, CO2 is found to form non-molecular, amorphous and crystalline silica-like solids above 25–30 GPa composed of CO4 tetrahedra4567891011121314. The transformation is linked to the pressure-induced destabilization of the π bonds in CO2. However, to date, none of these materials have been recovered at ambient pressure. The possibility that CO2–SiO2 compounds could exist was recently predicted1516. The synthesis of such compounds would remarkably add to the chemistry of oxides and would provide an updated view of the periodic table, as well as practical applications. The main challenge in synthesizing such materials is overcoming the incompatibility of the coordination environments of C and Si in solid oxides under any given P-T conditions. The sixfold coordinated phase of SiO2, stishovite, is already thermodynamically stable above 7–14 GPa, depending on the temperature17, whereas CO2 is well within the pressure range of stable molecular solids. High pressure and high temperature are mandatory for the formation of carbon in fourfold coordination. Phases with fourfold coordination in CO2 are only found above 25–30 GPa (see refs 11, 18, 19, 20 and references therein). On the other hand, working pressures cannot be much higher than the transition pressure from coesite to stishovite, 7–14 GPa, in order to limit the chances of forming silicon in sixfold coordination. In order to facilitate the reaction, we used silicalite, a fourfold coordinated SiO2 zeolite, as the starting phase for SiO2, with 5.5 Å diameter micropores (see ref. 21 and references therein) that can readily be filled by atoms and small molecules such as Ar, CO2 and C2H4 under pressure22232425. The mixture of silicalite and nano-confined CO2 has a huge interaction surface, and we recently reported a disordered, silicon carbonate phase obtained from these reactants at 18–26 GPa and 600–980 K (ref. 26). In silicon carbonate, carbon is threefold coordinated by oxygen and the CO3 ion has planar structure. Here we present, in contrast to the former material, the synthesis of a CO2–SiO2 solid solution, which is a covalent compound where both Si and C are fourfold coordinated by oxygen. The solid solution is obtained by reacting CO2 and silica, initially in the form of silicalite, at 16–22 GPa and temperatures in excess of 4,000 K and is characterized by X-ray diffraction (XRD) and Raman spectroscopy. The new phase is recovered at ambient conditions. Results Synthesis and XRD characterization We loaded the diamond anvil cell (DAC) with a mixture of silicalite and CO2 and directly heated with a CO2 laser (λ=10.6 μm). The sample was thermally insulated by coating the diamond culets with a few micrometre-thick NaCl layers (Methods). We heated the porous silicalite/CO2 mixture for 30 s at temperatures in excess of 4,000 K at a starting pressure of 21.7 GPa using a laser power density of about 50 KW mm−2. Under these P-T conditions, both pure SiO2 and CO2 are liquid, and the reaction most likely occurs in the liquid state (Fig. 1). After heating, the sample was quenched at room temperature and the pressure was found to be 16 GPa. XRD data were collected at this pressure, at 7 GPa (Fig. 2) and at room pressure on decompression. For all three pressures, in addition to the diffraction peaks of NaCl, a small amount of remaining pure CO2 (phase I) and Re from the gasket, a series of new strong characteristic sharp diffraction peaks were observed. These diffraction peaks could readily be indexed using a tetragonal unit cell with a c/a ratio between 1.283 at 16 GPa and 1.303 at 0 GPa (Fig. 2 and Table 1). Unfortunately, the diffracted intensities were not reliable as they do not correspond to a randomly oriented powder, but to only several well-crystallized particles formed at the very high temperature used to obtain this new material. We were thus only able to refine the unit cell parameters. The observed systematic absences are consistent with the P41212 space group of the α-cristobalite structure, and the indexation of the strongest, low-angle diffraction peaks is the same as that of α-cristobalite SiO2 (ref. 27). This space group implies that the Si and C atoms are randomly distributed on the 4a Wyckoff positions in a disordered manner and corresponds to the statistically average structure of the solid solution. In the present case, the c/a values are lower than those of pure SiO2 which decrease from 1.391 at room pressure to 1.3329 at 9.1 GPa (refs 28, 29), owing to the more compact structure of the new material. The unit cell volume of this new α-cristobalite-type phase is 6% lower than that of pure SiO2 at 7 GPa and 26% lower at ambient pressure. This is entirely consistent with the incorporation of much smaller carbon atoms, which replace silicon in the α-cristobalite-type structure, leading to a collapse of the oxygen sublattice. If one interpolates between the ambient pressure experimental unit cell volume of SiO2 (V=171.42 Å3) (ref. 28) and the theoretical volume of metastable α-cristobalite-type CO2 (V=88.04 Å3) (ref. 7) using Vegard’s law, the chemical composition of the solid solution (V=127.61 Å3) can be estimated to be ∼C0.5Si0.502. The c/a values for this new phase are lower than those of pure SiO2 α-cristobalite at high pressure after significant collapse of the pure SiO2 structure, and are essentially equivalent to that of the rutile-type oxygen sublattice, which can be described using the same unit cell with a c/a ratio of 1.288 (ref. 30). This collapse to a dense oxygen sublattice can explain the very small variation in volume with pressure observed for this phase. Although the data points are not sufficient to be fitted to an equation of state and the remaining molecular CO2 is a soft solid and not truly hydrostatic (maximum pressure variations of ±7% were observed over the whole sample surface; note that the pressure was measured at the same spot as the diffraction pattern with the 2-μm diameter X-ray beam to minimize errors), a bulk modulus of the order of 240 GPa is consistent with the experimental data. If one describes rutile-type silica, stishovite, using the same unit cell, the c/a ratio varies from 1.276 at ambient pressure31 to 1.298 at 55 GPa (ref. 32). The present result is also consistent with theoretical studies of the α-cristobalite—stishovite phase transition mechanism3334, which predict that the transition occurs in two steps: first the α-cristobalite-type structure collapses such that the oxygen sublattice is equivalent to that in the rutile-type structure, and then the cations migrate to octahedral sites. The present material corresponds at high pressure to end of the first step and it is most probably retained owing to the significant amount of carbon in the structure, which does not have a tendency to pass into octahedral coordination. In the case of the CO2–SiO2 solid solution, an α-cristobalite structure is obtained rather than a collapsed I2d cristobalite structure, as in CO2 phase V, due to the presence of larger Si4+ cations. The collapsed cristobalite structure is characteristic of materials containing small cations, typically B3+, C4+ and P5+ (refs 13, 30, 35, 36). Raman spectroscopy characterization Raman spectroscopy was essential to determine the nature of the chemical bonds in the solid solution and to check for other possible materials present in the sample region. In addition to signatures of the solid solution (Fig. 3) and of unreacted, molecular CO2 (see also XRD), we found some microcrystalline/amorphous graphite with broad peaks in the 1,300–1,600 cm−1 frequency range, which is consistent with the blackish aspect of part of the sample and confirms that some of the initial CO2 precursor was indeed dissociated at high temperature. Dissociation of CO2 under high P-T conditions was reported by other authors (ref. 19 and references therein). The spectrum of the CxSi1-xO2 solid solution is dominated by a sharp peak with fine structure at around 540 cm−1, which we easily assign to deformation modes of the tetrahedra. In fact, this peak lies in between the analogous, strong peaks of pure α-cristobalite SiO2 (ref. 37) and cristobalite-like CO2 (refs 11, 13, 18), respectively. This mode displays higher frequency for the solid solution than for α-cristobalite SiO2 because of the smaller reduced mode mass and the stronger bond strength with respect to that of pure SiO2. On the other hand, the frequency is lower than in non-molecular CO2, where the reduced mode mass is smaller and the bond strength is stronger. It has been recently found that amorphous silicon oxycarbide materials of potential interest for planetary interiors can be synthesized as a result of carbon substitution for oxygen in silicates38. This kind of material includes Si–C chemical bonds, which typically exhibit intense Raman peaks at frequencies >750–800 cm−1 (ref. 39). As we do not observe similar peaks, carbon substitution for oxygen rather than for silicon is ruled out in our solid solution. We also rule out the presence of silicon carbonate26 in the present material, which would exhibit much higher frequencies than those reported in our Raman spectrum. In addition, silicon carbonate, in contrast to our solid solution, was not recovered to ambient conditions. A remarkable insight now comes from the analysis of the fine structure of the peak (Fig. 3b and Table 2). This peak was fit to five slightly asymmetric components that we assign to the same vibrational mode of unit cells containing different numbers of C and Si atoms, respectively, that is, unit cell content: CnSi4−nO8, where n=0, 1, 2,…4. Therefore, we have the lowest frequency component that corresponds to unit cells with 0 C and 4 Si atoms, up to the highest frequency component corresponding to unit cells with 4 C and 0 Si atoms. The number of components in the Raman peak, five, arises from individual cells with the five different possible contents, and the relative intensities are because of the statistical amount of each type of unit cell content linked to the chemical composition of the solid solution. Therefore, the statistical average of the n index, naverage, will not be an integer, but rather a real number ranging between 0 and 4. The relative intensities of the five Raman components (Table 2) can then be used to estimate the stoichiometry of the CxSi1−xO2 solid solution. This result can be compared with the analogous value obtained based on the unit cell volume from the XRD analysis. The statistical distribution of the number n obtained in this way yields: naverage=2.8(8), where the statistical error is given by the s.d. of the distribution. The spatial dependence of naverage was also checked over the whole sample surface, and the changes in this quantity have been found to be negligible with respect to the statistical error estimated at each single point. Therefore, as we have four cations in the generic unit cell, the average value for the composition x will be: xaverage=naverage/4=0.7(2), giving a chemical formula for the solid solution of C0.7(2)Si0.3(2)O2. This result is consistent with the chemical formula obtained from the XRD analysis, although higher in C content, and an average chemical formula of C0.6(1)Si0.4(1)O2 agrees with both the XRD and the Raman results. We note that the high carbon content identified by the combination of these two methods is consistent with the strong decrease in c/a lattice parameter ratio with respect to pure SiO2 at a volume of about 130 Å3 in the relationship between c/a and unit cell volume for α-cristobalite-type structures (Fig. 2b). The small size of carbon results in a collapse of the structure. The decrease in c/a is of a similar order of magnitude as that between GeO2 and SiO2. Discussion The present study using a combination of XRD and Raman spectroscopy clearly shows that a solid solution between CO2 and SiO2 with an α-cristobalite-type structure can be obtained under high P-T conditions. The P-T (P=16–22 GPa, T>4,000 K) conditions for the formation of such a solid solution correspond to a compromise between the respective stabilities of three- and fourfold coordination in CO2 and four- and sixfold coordination in SiO2. The formation of the solid solution enables one to retain a metastable hard solid based on CO4 tetrahedra down to ambient conditions owing to the incorporation of a quantity of silicon atoms. A new oxide chemistry is now possible in which silicon atoms can be replaced by carbon in silica and even silicates. This chemistry can give rise to a unique class of materials with novel physical properties, a class of hard, light, carbon-rich oxides, metastable at ambient conditions, with high thermal conductivity and application-tailored index of refraction. Such carbon-substituted materials may also be relevant for Earth and planetary sciences. Methods Powder XRD methods Angle-dispersive powder XRD patterns were obtained on the ID27 beam line at the European Synchrotron Radiation Facility (ESRF) using a monochromatic beam (λ=0.3738 Å) and a MAR165 CCD detector. The nominal size of the X-ray focal spot was 2 μm. The diffraction patterns were analysed and integrated using the FIT2D programme40. The Le Bail fit to the XRD data was performed using the programme Fullprof41. Raman spectroscopy methods Raman spectra (2–3-μm focal spot) were measured using the 647.1 nm line of a Kr+ laser as the excitation source, and the setup is described elsewhere13. The spatial uniformity of the sample was checked using 4 μm step meshes, and the typical spectrum of the solid solution (Fig. 3) was found over about 50% of the sample surface. Synthesis and high-pressure methods Hydrophobic silicalite-1-F was obtained from SOMEZ. A DAC was used for the high-pressure experiments. CO2 was loaded in the DAC (IIa diamonds were used, as they are transparent for the CO2 laser) in the liquid phase at −20 °C and 30 bar together with powdered silicalite. The diameter (thickness) of the rhenium gasket used as a sample chamber after DAC loading was 80 μm (40 μm). Diamond culets were coated by a few micrometre-thick NaCl layers in order to thermally insulate the sample during the laser heating. Laser heating was performed for 30–60 s in different points of the sample with a CO2 laser (λ=10.6 μm), and temperature was measured by optical pyrometry42. The maximum laser power and beam spot were of 150 W and 30–40 μm, respectively. The NaCl-coating layers were also used for measuring the pressure during the XRD experiment based on the equation of state of NaCl43. The pressure shift of the Raman diamond peak was used for measuring the pressure during the Raman measurements44. Additional information How to cite this article: Santoro, M. et al. Carbon enters silica forming a cristobalite-type CO2–SiO2 solid solution. Nat. Commun. 5:3761 doi: 10.1038/ncomms4761 (2014). We acknowledge the ESRF for provision of beam time at ID27 and M. Mezouar (ESRF), J.A. Montoya (University of Cartagena, Colombia) and S. Scandolo (International Centre for Theoretical Physics (ICTP), Trieste, Italy) for very useful discussions. We also thank the support from the European Union (European Laboratory for Non-Linear Spectroscopy (LENS) contract G.A. no. 284464 LASERLABEUROPE), the Ente Cassa di Risparmio di Firenze, the Deep Carbon Observatory initiative (Grant from the Alfred P. Sloan Foundation for the project entitled ‘Physics and Chemistry of Carbon at Extreme Conditions’), and the Agence Nationale de la Recherche (contract ANR-09-BLAN-0018-01). M.S. thanks the ‘Pôle Chimie Balard’ of Montpellier for having supported his research as an Invited Professor at the Institut Charles Gerhardt in 2013 in the framework of the Total chair. The authors declare no competing financial interests. Author contributions M.S., F.A.G. and J.H. proposed the research, did the project planning, the high-pressure synthesis of the CO2–SiO2 solid solution and performed the measurements. M.S. and J.H. performed data analysis and interpretation and wrote the paper. R.B., O.C., C.L., A.S. and G.G. contributed to the XRD measurements and to revising the paper. A.S. and G.G. were the beam line scientists during the experiment. 11/29/2016 This paper has been retracted at the request of the authors. Figure 1 Pure SiO2 and CO2 phase diagrams and P-T path. Black lines: SiO2 phase boundaries17. Light magenta, continuous and dotted lines: CO2 phase boundaries; light magenta, dashed lines: kinetic boundaries for CO211181920. Blue dashed arrows and orange ellipse: P-T path followed in this study. All five solid phases of CO2 shown are molecular crystals. Non-molecular CO2 phases are formed above 25–30 GPa. Crist, cristobalite; Tryd, trydimite. Figure 2 XRD pattern and structure and unit cell volume dependence of the c/a ratio of the solid solution. (a) Experimental (dots) and calculated (Le Bail fit—red line) XRD patterns at 7 GPa. Vertical bars: diffraction angles of the α-cristobalite (P41212) solid solution, CO2-I from unreacted CO2, NaCl, used for thermally insulating the sample, and Re from the gasket. Inset: structure of the solid solution (C/Si atoms in blue and O atoms in red). (b) c/a ratio as a function of the unit cell volume for the α-cristobalite-type solid solution and pure α-cristobalite-type SiO22829 and GeO245, respectively. The value of c/a=1.288 corresponds to a rutile-type oxygen sublattice. Figure 3 Selected Raman spectrum of the solid solution. (a) Full view, with frequency positions observed for similar tetrahedral structures of pure SiO2 (α-cristobalite, extrapolated to 7.5 GPa37) and non-molecular CO2 (experimental collapsed β-cristobalite like111318). (b) Zoom of the fine structure of the peak (dots) fitted to five peaks (violet for the single components and red for the sum), which we assign to unit cells containing different numbers of C and Si atoms, respectively, that is, unit cell content: CnSi4−nO8, where n=0, 1, 2,…4. Table 1 Lattice parameters (Å) and c/a ratio for the solid solution with the P41212 structure. P (GPa) a (Å) c (Å) c / a 0 4.609 (1) 6.007 (3) 1.303 7 4.594 (1) 5.938 (2) 1.293 16 4.540 (1) 5.826 (1) 1.283 Table 2 Relevant parameters of the peaks fitted to the Raman spectrum of the solid solution. Peak label Peak frequency (cm−1) Relative intensity Number ( n) , of C atoms in a unit cell with content: C n Si 4− n O 8 Peak 1 517.6 (7) 0.040 (6) 0 Peak 2 523.5 (1) 0.103 (7) 1 Peak 3 531.5 (1) 0.27 (1) 2 Peak 4 538.9 (2) 0.44 (2) 3 Peak 5 541.5 (2) 0.147 (7) 4 Frequency, relative intensity and assignment are reported for each labelled peak. Intensity values are relative to the total intensity of the fine structured peak. ==== Refs Eremets M. I. , Gavriliuk A. G. , Trojan I. A. , Dzivenko D. A. & Boehler R. Single-bonded cubic form of nitrogen . Nat. Mater. 3 , 558 –563 (2004 ).15235595 Ma Y. et al. Transparent dense sodium . Nature 458 , 182 –186 (2009 ).19279632 Lundegaard L. F. , Weck G. , McMahon M. I. , Desgreniers S. & Loubeyre P. 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==== Front Jpn J Cancer ResJpn. J. Cancer Res10.1111/(ISSN)1349-7006aCASJapanese Journal of Cancer Research : Gann0910-50501876-4673Blackwell Publishing Ltd Oxford, UK 1137655810.1111/j.1349-7006.2001.tb01122.xCAE499ArticleStrain‐dependency of Chromosomal Abnormalities in Lymphomas Developed in Eμ‐myc Transgenic Mice Akagi Kiwamu 1Yamamura Ken‐ichi 21 Saitama Cancer Center Research Institute, 818 Komuro, Ina, Kitaadachigun, Saitama 362‐08062 Department of Developmental Genetics, Institute of Molecular Embryology and Genetics, Kumamoto University School of Medicine, 4‐24‐1 Kuhonji, Kumamoto 862‐0976* To whom correspondence should be addressed. E‐mail: [email protected] 2001 92 5 10.1111/cas.2001.92.issue-5499 505 (Received December 26, 2000/Revised March 5, 2001/Accepted March 8, 2001)We previously showed that B and T cell lymphoma development in Eμ (immunoglobulin heavy chain enhancer)‐mye transgenic mice is dependent on the mouse strain. To determine whether any non‐random chromosomal abnormality that was present was caused by variations in the lymphoma cell type or by a different genetic background, we crossed C3H transgenic mice with other inbred strains of mice, C57BL/6 or BALB/c. Cytogenetic analysis showed a high frequency of non‐random chromosomal aberrations, namely, duplication or amplification of part of chromosome 5 containing the transgene and trisomy of chromosome 1, 6, or 12 in the genetic background of C3HXC57BL/6 mouse and C3HxBALB/c mouse, respectively, regardless of cell type of lymphoma. These results suggest that non‐random chromosomal abnormalities in lymphoma cells are dependent on the genetic background of mouse, not on the tumor cell type in Eμ‐myc transgenic Genetic instabilityChromosomal abnormalityLymphomaGenetic background source-schema-version-number2.0cover-dateMay 2001details-of-publishers-convertorConverter:WILEY_ML3GV2_TO_NLMPMC version:4.6.9 mode:remove_FC converted:04.11.2015 ==== Refs Reference 1 Leder , P. , Battey , J. , Lenoir , G. , Moulding , C. , Murphy , W. , Potter , H. , Stewart , T. and Taub , R. Translocations among antibody genes in human cancer . Science , 222 , 765 – 771 ( 1983 ). 6356357 2 Tujimoto , Y. , Finger , L. R. , Yunis , J. , Nowell , P. C. and Croce , C. M. Cloning of the chromosome breakpoint of neoplastic B cells with the t(14;18) chromosome translocation . Science , 226 , 1097 – 1099 ( 1984 ). 6093263 3 Rowley , J. D. 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==== Front ACS Cent SciACS Cent SciocacsciiACS Central Science2374-79432374-7951American Chemical Society 2980600310.1021/acscentsci.8b00050Research ArticleOvercoming Ovarian Cancer Drug Resistance with a Cold Responsive Nanomaterial Wang Hai †‡§Agarwal Pranay ‡§Zhao Gang ∥Ji Guang ⊥Jewell Christopher M. †#∇○◆Fisher John P. †Lu Xiongbin ●He Xiaoming †‡§#∇†Fischell Department of Bioengineering and #Robert E. Fischell Institute for Biomedical Devices, University of Maryland, College Park, Maryland 20742, United States‡Department of Biomedical Engineering and §Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio 43210, United States∥ Center for Biomedical Engineering, Department of Electronic Science and Technology, University of Science and Technology of China, Hefei, Anhui 230027, China⊥ Institute of Digestive Diseases, Longhua Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai 200032, China∇ Marlene and Stewart Greenebaum Comprehensive Cancer Center, University of Maryland, Baltimore, Maryland 21201, United States○ Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, Maryland 21201, United States◆ United States Department of Veterans Affairs, Maryland VA Health Care System, Baltimore, Maryland 21201, United States● Department of Medical and Molecular Genetics and Melvin and Bren Simon Cancer Center, Indiana University School of Medicine, Indianapolis, Indiana 46202, United States E-mail: [email protected] 04 2018 23 05 2018 4 5 567 581 18 01 2018 Copyright © 2018 American Chemical Society2018American Chemical SocietyThis is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes. Drug resistance due to overexpression of membrane transporters in cancer cells and the existence of cancer stem cells (CSCs) is a major hurdle to effective and safe cancer chemotherapy. Nanoparticles have been explored to overcome cancer drug resistance. However, drug slowly released from nanoparticles can still be efficiently pumped out of drug-resistant cells. Here, a hybrid nanoparticle of phospholipid and polymers is developed to achieve cold-triggered burst release of encapsulated drug. With ice cooling to below ∼12 °C for both burst drug release and reduced membrane transporter activity, binding of the drug with its target in drug-resistant cells is evident, while it is minimal in the cells kept at 37 °C. Moreover, targeted drug delivery with the cold-responsive nanoparticles in combination with ice cooling not only can effectively kill drug-resistant ovarian cancer cells and their CSCs in vitro but also destroy both subcutaneous and orthotopic ovarian tumors in vivo with no evident systemic toxicity. A cold-responsive hybrid nanoparticle of phospholipid and polymers is developed to overcome the multidrug resistance of cancer stem cells with ice cooling. document-id-old-9oc8b00050document-id-new-14oc-2018-00050hccc-price ==== Body Introduction Development of drug resistance in cancer cells is a major challenge to cancer chemotherapy.1−3 Research on the mechanisms of drug resistance is usually focused on drug metabolism including its uptake, efflux, and detoxification.1,4 A major advance in the understanding of drug metabolism is the identification of the membrane transporter P-glycoprotein (P-gp) that could pump free drug out of cancer cells.5−7 However, it is worth noting that multiple mechanisms contribute to cancer drug resistance.8,9 A critical advance in this aspect is the finding of the subpopulations of cancer cells that are highly tumorigenic and drug resistant. These cancer cells are usually referred to as cancer stem cells (CSCs) or tumor initiating/reinitiating cells.10−12 There is mounting evidence showing that the CSCs are responsible for cancer metastasis and tumor recurrence or relapse associated with conventional chemo-, radio-, and hormone therapies.13−15 Several properties of the CSCs contribute to their high resistance to chemotherapeutic drugs including the overexpression of drug efflux pumps, enhanced DNA repair ability, overexpressed antiapoptotic proteins, and dormancy.11,16,17 Therefore, it is important to account for the multiple mechanisms responsible for the drug resistance of cancer when developing strategies for effective cancer therapy. Nanoparticle-based drug delivery systems have been explored for reducing the side effect of chemotherapeutic drugs as well as overcoming drug efflux pump-associated drug resistance.18−22 The latter is because nanoparticles can be actively taken up by drug-resistant cancer cells via endocytosis instead of passive diffusion across the plasma membrane. Consequently, the drug encapsulated in nanoparticles can bypass the efflux pumps on the cell plasma membrane and enter the inner cytoplasm.23 However, the function of the drug efflux pumps is not compromised during the uptake of the nanoparticles, and they can still pump out the drug slowly released from the nanoparticles in cells. Therefore, it may be important to achieve burst release (i.e., release in a short time of seconds or minutes) of a large amount of free drug inside cancer cells, so that a significant amount of free drug could bind with its target (e.g., DNA, RNA, or proteins) before its depletion by the efflux pumps, for overcoming drug resistance. Stimuli-responsive nanoparticles hold great promise for controlling drug release inside cells.24,25 However, drug release from most existing stimuli-responsive nanoparticles (mainly pH- and heat-responsive ones) may still occur over hours to days.26,27 This slowly released drug could be easily depleted by the efflux pumps before it binds with its target in cells. Moreover, no study has been conducted to test the existing stimuli-responsive nanoparticles for overcoming drug resistance using CSCs. More recently, cold and freezing-temperature treatments (e.g., cryosurgery, cryotherapy, cryoablation, and hypothermia) have been widely studied and used for treating various diseases including cancer.28−34 Nonetheless, nanoparticles that are responsive to cold (i.e., lower than room temperatures) have never been reported, although there are studies on nanoparticles responsive to temperatures higher than room temperature but blow body temperature.35−37 Furthermore, the use of cold and nanoparticle drug delivery for overcoming cancer drug resistance has never been explored in the literature. In this work, we developed a cold-responsive nanoparticle that quickly disassembles upon ice cooling, leading to burst release of most of the encapsulated chemotherapeutic drug (doxorubicin hydrochloride or DOX) in seconds. Moreover, the NCI/RES-ADR multidrug-resistant cancer cells (human ovarian cancer cells that were called MCF-7/ADR cells in early studies) and their CSCs together with A2780ADR drug resistant ovarian cancer cells, were used in this study to demonstrate the capability of overcoming cancer drug resistance with the cold-responsive nanoparticle in vitro and in vivo. Results Preparation and Characterization of Nanoparticles As shown in Figure 1a, the cold-responsive nanoparticles were prepared with the double-emulsion method using dipalmitoylphosphatidylcholine (DPPC) and four different polymers including Pluronic F127 (PF127), poly(N-isopropylacrylamide-co-butyl acrylate) (PNIPAM-B, NIPAM:B = 8:1, Mn = 30,000), chitosan-modified PF127 (PF127-chitosan), and hyaluronic acid (HA). All four polymers have been widely used for various biomedical applications and are considered as biocompatible biomaterials, and PF127, chitosan, and HA have been approved by the Food and Drug Administration (FDA) for medical use.38−41 PF127 is an amphiphilic polymer consisting of hydrophilic polyethylene glycol (PEG) blocks and more-hydrophobic polypropylene glycol (PPG) blocks.35 The PNIPAM-B is a thermally responsive polymer with a lower critical solution temperature (LCST) of 14–16 °C, which means the polymer is insoluble in water (hydrophobic) at room temperature (∼22 °C), while it is highly soluble in water (hydrophilic) at or below 14 °C.42 DPPC is used to improve the biocompatibility of the nanoparticles.43,44 First, aqueous solution containing DOX was emulsified with organic solvent containing PF127, PNIPAM-B, and DPPC. This leads to the formation of a water-in-oil structure to encapsulate DOX in the hydrophilic core dispersed in organic solvent (Figure 1a). For second emulsion, the product of the first emulsion is emulsified with the aqueous solution of chitosan-PF127 and HA. The hydrophobic part of chitosan-PF127 could integrate into the hydrophobic inner layer, while the hydrophilic part of chitosan-PF127 could bind with HA at the outermost layer of the resultant nanoparticles.43,45 The reason to decorate HA on the surface of the nanoparticles is that HA is a natural ligand of the variant CD44 commonly overexpressed on many types of cancer cells and particularly CSCs.46−48 Therefore, the surface of the nanoparticles consists mainly of PEG and HA, which makes the nanoparticles highly dispersible in aqueous solutions as both PEG and HA are hydrophilic. The nanoparticles can be collected by centrifugation after removing organic solvent by rotary evaporation under a vacuum. Figure 1 Synthesis and characterization of cold-responsive nanoparticle. (a) Hyaluronic acid (HA or H), lipid (dipalmitoylphosphatidylcholine or DPPC in this study, L), Pluronic F127 (PF127, P), poly(N-isopropylacrylamide-co-butyl acrylate) (PNIPAM-B or N), and chitosan (C)-modified Pluronic F127 (PF127-chitosan) were used to prepare the doxorubicin (DOX, D) laden HCLPN-D nanoparticles using the double-emulsion method. (b) TEM images showing the HCLPN-D nanoparticles are spherical with a multicore–shell configuration. (c) The thermally induced phase transition behavior of PNIPAM-B from being water-insoluble to highly water-soluble, which can cause disassembly of the HCLPN-D nanoparticles upon cooling to below room temperature. This can result in burst release of the encapsulated drug. (d) TEM images showing the HCLPN-D nanoparticles become completely disassembled after 3 min incubation at 10 °C. (e) An extensive network of polymer fibers rather than nanoparticles is observable after warming back to 22 °C. (f) Photographs of the aqueous samples of HCLPN-D nanoparticles at various temperatures before and after shining a red laser beam through them in the dark. As a result of the Tyndall effect (i.e., scattering of laser beam by nanoparticles in solution), a bright white track of light is visible in the dark in the solutions of HCLPN-D nanoparticles above 10 °C. However, it is not clearly observable at or below 10 °C and after warming back to 22 or 37 °C, indicating the HCLPN-D nanoparticles disassemble upon cooling to 10 °C (or a lower temperature), and the disassembling process is not reversible. (g) Size distribution of HCLPN-D nanoparticles measured by dynamic light scattering (DLS) at different temperatures. The results show a narrow size distribution of the HCLPN-D nanoparticles at 37, 22, and 15 °C. An additional peak of large particles is seen at 12 °C, probably due to aggregation of polymers. No stable peak of nanoparticles can be detected when the temperature is decreased to 10 and 6 °C. Typical transmission electron microscopy (TEM) image of the resultant nanoparticles (HCLPN-D, H for HA, C for chitosan, L for the lipid DPPC, P for PF127, N for PNIPAM-B, and D for DOX) is shown in Figure 1b. The nanoparticles are ∼100 nm in diameter with a spherical morphology, and interestingly, they have a multicore–shell structure. The multicore–shell structure is probably mainly due to the presence of lipid during the first emulsion when the lipid together with PNIPAM-B and PF127 may form small sized water-in-oil (W-in-O) structures. A few of them together may form the final nanoparticles after the second (W-in-O)-in-W emulsion. This is because the multicore–shell structure is not obvious for nanoparticles prepared in the same way without lipid (Figure S1). Moreover, few nanoparticles could be collected by centrifugation if PF127 and DPPC (PF127 + DPPC) were used for synthesizing nanoparticles using the same procedure (Figure S2a). Although more nanoparticles could be collected if PNIPAM-B and DPPC (PNIPAM-B+DPPC) were used, the drug encapsulation efficiency (EE) is lower than that of HCLPN nanoparticles (Figure S2b, 4.7 ± 2.5% for PF127 + DPPC, 14.5 ± 2.2% for PNIPAM-B+DPPC, and 59.7 ± 4.3% for HCLPN nanoparticles). This can be confirmed visually by the redness (the color of DOX) of the three different samples after removing nonencapsulated DOX by centrifugation and resuspending in deionized (DI) water (Figure S3a). When a red laser beam was shined through the three samples, the light track is weak in the PF127 + DPPC sample while it is evident in the PNIPAM-B + DPPC and HCLPN-D samples (Figure S3b) as a result of the Tyndall effect due to light scattering by nanoparticles. The TEM image of the PF127+DPPC sample shows that most of the DPPC form liposomes during the preparing process (Figure S3c). Although core–shell nanoparticles are formed in the PNIPAM-B + DPPC sample, their size is not uniform, and some can be as big as ∼400 nm in diameter (Figure S3d). Cold-Responsiveness of HCLPN-D Nanoparticles We next investigated the cold-responsive property of the HCLPN-D nanoparticles. As aforementioned, the PNIPAM-B polymer for making the nanoparticles is hydrophobic at room temperature and has an LCST of 14–16 °C. Once the temperature is lower than the LCST, the PNIPAM-B polymer becomes water-soluble. This may cause disassembly of the nanoparticles in aqueous solutions (Figure 1c). Indeed, almost all the HCLPN-D nanoparticles were disassembled after incubating at 10 °C for 3 min (Figure 1d). Extensive polymer aggregates formed when the temperature was increased back to room temperature (22 °C, Figure 1e). This is further confirmed by shining a red laser beam through the aqueous samples of the HCLPN-D nanoparticles at different temperatures. A light track in the sample indicates the presence of nanoparticles. As shown in Figure 1f, the aqueous solution of the nanoparticles appears homogeneous at both 37 and 22 °C before cooling. This is attributed to the high dispensability of the nanoparticles in aqueous solutions. When the temperature decreases to 10 or 6 °C, the solution of HCLPN-D nanoparticles becomes transparent, and no evident light track could be observed, suggesting disassembly of the nanoparticles and dissolution of polymers in the nanoparticles in the aqueous solution. More importantly, many visible polymer aggregates formed when the temperature was increased back to room or body temperatures (22 and 37 °C respectively, Figure 1f), suggesting the disassembly is irreversible. We further checked the size distribution of HCLPN-D nanoparticles at different temperatures by dynamic light scattering (DLS). The HCLPN-D nanoparticles have a narrow size distribution with one single peak at temperatures of 37, 22, and 15 °C (82.3 ± 4.6, 96.7 ± 3.4, and 110.3 ± 2.5 nm in diameter, respectively), but a second peak of much larger particles/aggregates appears at 12 °C (Figure 1g). Moreover, no stable measurement could be made at 10 and 6 °C, probably because nearly all the nanoparticles are dissembled at the cold temperatures. The stability of HCLPN-D nanoparticles in acidic solution (pH 6.5) was further checked as the tumor microenvironment is often acidic with a pH value of ∼6.5.49 As shown in Figure S4, the size distribution measured by DLS suggested the HCLPN-D nanoparticles are stable at pH 6.5. However, the nanoparticles are not stable at pH 5.0 (the pH value of late endosomes and lysosomes) as their size increases and even form some aggregates of ∼2.5 μm in pH 5.0 solution. Lastly, the HCLPN-D nanoparticles are stable at room temperature for at least 49 days according to the DLS size analyses (Figure S5). We further checked the ultraviolet–visible (UV–vis) absorbance of the HCLPN-D nanoparticles at different temperatures. As shown in Figure 2a, the absorbance peak (arrow) of DOX at 486 nm is not obvious at or above 12 °C due to the strong absorbance of the HCLPN nanoparticles at the same wavelength. However, it shows up more clearly at 10 or 6 °C because of dissolution of polymers in the nanoparticles. When the temperature increased back to 22 °C, the absorbance is strong at all wavelength. This suggests the polymer aggregates may block the light to result in the strong absorbance. To confirm this, the drug release profile of HCLPN-D nanoparticles was determined by using ice to cool the samples. The drug release from HCLPN nanoparticles is slow at 37 °C (∼2.2% in 5 h), while more than 70% of DOX can be released from the nanoparticles after cooling on ice for 5 min (Figure 2b). In contrast, the drug release is less than 5% if the nanoparticles are kept at low pH (5.0) for 5 min. These drug release data indicate that cold is an effective stimulus for triggering drug release from the HCLPN nanoparticles, and it is much more efficient than pH excursion. Figure 2 Cold-triggered burst drug release from HCLPN-D nanoparticles. (a) UV–vis absorbance of HCLPN-D nanoparticles at different temperatures showing the cold-responsiveness of HCLPN-D nanoparticles. Arrows indicate the absorbance peaks of DOX. (b) A comparison of the release of DOX from HCLPN-D nanoparticles under pH 7.4, acidic pH (5.0, 5 min), and ice cooling (5 min), showing the cold temperature is much more effective than low pH in triggering drug release from the HCLPN-D nanoparticles. Error bars represent ± standard deviation (SD, n = 3). Overcoming Drug Resistance and Cancer Targeting in Vitro Both two-dimensional (2D) cultured NCI/RES-ADR multidrug-resistant cancer cells and three-dimensional (3D)-cultured CSC-enriched spheres (obtained by suspension culture of the NCI/RES-ADR cells in CSC medium in ultralow attachment plates) were used in this study. We confirmed the resistance to free DOX of the 2D cultured NCI/RES-ADR cells by incubating the cells with free DOX (10 μg/mL) for 3 h. As shown in the first row of Figures 3a and S6, no red fluorescence of DOX is observable in the 2D cultured NCI/RES-ADR cells. In stark contrast, red fluorescence of DOX is observable in the cells when they are incubated with the HCLPN-D nanoparticles (Figures 3a and S7). However, DOX is predominantly distributed in the cytosol, and it is barely observable in the nuclei of the NCI/RES-ADR cells incubated with the HCLPN-D nanoparticles. Since DOX has to enter the nuclei for cytotoxicity, the data suggest that simply using nanoparticles for uncontrolled drug release may not be able to efficiently overcome the drug resistance of the NCI/RES-ADR cells. To check if cold-triggered burst drug release could overcome the drug resistance, the cells treated with free DOX or HCLPN-D nanoparticles for 3 h were further cooled with ice for 5 or 10 min. For the cells treated with free DOX, the red fluorescence is still not observable (Figure 3a and Figure S6). Interestingly, DOX is located in part of the nuclei of the cells treated with HCLPN-D nanoparticles after cooling for 5 min, and DOX overlaps with nearly all the nuclei after 10 min of cooling (Figure 3a and Figure S7). In contrast, almost all DOX is still distributed in the cytosol if the cells are continuously cultured at 37 °C, indicating the cold-triggered burst drug release indeed can overcome the drug-resistant capacity of the NCI/RES-ADR cancer cells. Figure 3 Overcoming cancer drug resistance with cold-triggered burst drug release form HCLPN-D nanoparticles. (a) Confocal micrographs of 2D cultured NCI/RES-ADR multidrug-resistant cancer cells after incubating them with either free DOX or HCLPN-D nanoparticles for 3 h at 37 °C, followed by either continued culturing in incubator (37 °C) or ice cooling (+I) for 5 or 10 min. (b) Confocal images of CSC-enriched spheres derived from the multidrug-resistant cancer cells after incubating them with HCLPN-D nanoparticles for 3 h at 37 °C, followed by either continued culturing in incubator (37 °C) or ice cooling (+I) for 5 or 10 min. DOX could enter the cell nuclei only when treated with both HCLPN-D nanoparticles and ice cooling, indicating the cold-triggered burst drug release from the HCLPN-D nanoparticles could be used to overcome the drug resistance of the 2D cultured cancer cells and their CSCs. In order to confirm the temperature drop to below 12 °C in the cells with ice cooling, FLIR (Wilsonville, Oregon, USA) near-infrared thermography was used to determine the temperature of the samples. As illustrated in Figure S8, the temperature of samples treated with free DOX or HCLPN-D nanoparticles is ∼37 °C in incubator, decreases to ∼4–0 °C (within ∼3 min) after ice cooling for 5 or 10 min, and returns to ∼37 °C after warming in incubator for 5 min. This is consistent with the medium temperature in the sample measured with thermocouples (Figure S9). It is also noticeable that the overall fluorescence intensity of DOX inside cells after ice cooling increases compared with cells kept at 37 °C, probably due to the following three reasons. First, the fluorescence intensity of DOX decreases after encapsulated inside the nanoparticles due to self-quenching (Figure S10). After ice cooling, the free DOX released from the HCLPN-D nanoparticles results in higher fluorescence intensity. Second, the fluorescence intensity of DOX decreases slightly in acidic solution (Figure S10). HCLPN-D nanoparticles are taken up by cells via endocytosis and locate inside the endo-/lysosomes (pH ≈ 5.0). The fluorescence intensity of DOX should increase after the DOX is released from the nanoparticles and enter the nuclei with a pH value of ∼7.0. Third, the high binding affinity of DOX with cell nuclei may lead to accumulation of DOX in the cell nuclei, which should result in increased fluorescence.50 CSC-enriched spheres were also treated in the same way as that aforementioned for 2D cultured cells. Similarly, the CSC-enriched spheres are resistant to free DOX, but can take up the HCLPN-D nanoparticles (Figures S11 and 3b). Importantly, most of the DOX can enter the cell nuclei after cooling the spheres with ice for 5 or 10 min. Further quantitative analyses show that the intensity of DOX in cell nuclei is significantly increased after ice-cooling (Figure S12). Next, we investigated the cellular uptake of different concentration (5, 10, and 25 μg/mL) of free DOX or HCLPN-D nanoparticles with or without ice cooling for 10 min. As shown in Figures S13–16 (Figures S13–14 for free DOX and Figures S15–16 for HCLPN-D nanoparticles), no red fluorescence of DOX is observable in the multidrug-resistant cells treated with free DOX, while more DOX could be observed in cells treated with high concentration of HCLPN-D nanoparticles. After ice cooling, stronger fluorescence is observable in the cells treated with HCLPN-D nanoparticles, particularly at high concentrations. This is not observable for free DOX treated cells. Similarly, quantitative analysis of DOX in the cell nuclei reveals that cells treated with HCLPN-D nanoparticles and ice cooling have higher DOX concentration in their nuclei (Figure S17). Since the pH value of the tumor microenvironment is ∼6.5, we checked the capacity of overcoming drug resistance by the HCLPN-D nanoparticles at pH 6.5. As shown in Figure S18, most of the DOX released from the HCLPN-D nanoparticles as a result of ice cooling enters the nuclei of cancer cells both under 2D culture and in CSC-enriched spheres, suggesting the HCLPN-D nanoparticles can still be used to overcome cancer drug resistance in the acidic tumor microenvironment. As the HA modified on the surface of HCLPN-D nanoparticles is used to target CD44, we first studied the expression of CD44 on the 2D cultured NCI/RES-ADR cells and cells in the CSC-enriched spheres by using flow cytometry. As shown in Figure S19a,b, the expression of CD44 on the NCI/RES-ADR cells is positive, and it is similar to that on MDA-MB-231 cancer cells that are considered as CD44 positive.51,52 Moreover, the expression of CD44 on cells in the CSC-enriched spheres is significantly higher than that on the 2D cultured NCI/RES-ADR cells, suggesting the HCLPN-D nanoparticles can be used for targeting both the drug-resistant cancer cells and their CSCs. The data also demonstrate that CSCs are enriched in the 3D cultured NCI/RES-ADR spheres because CD44 is a common CSC marker.47,48,53 Furthermore, the binding between the nanoparticles with HA on their surface and CD44 is confirmed using confocal fluorescence microscopy. As shown in Figure S20, most of the CD44 in control group is located on the surface of the cell membrane. Moreover, many of them are internalized into the cytoplasm after incubated with the HCLPN-D nanoparticles, suggesting the nanoparticles bind with CD44 and are then taken up by the cells. Interestingly, some line structures with CD44 are observable after ice cooling. This is probably because polymers bound with CD44 on the surface of the HCLPN-D nanoparticles could form fibers after cooling-induced disassembly of the nanoparticles (Figure 1e). The targeting capability of HCLPN-D nanoparticles was further confirmed by treating the drug resistance cells using nanoparticles without HA modification on their surface (LPN-D). As shown in Figure S21a, more DOX could be delivered inside the cells with HCLPN than LPN nanoparticles due to the targeting capability of HA on the HCLPN nanoparticles. Therefore, after cooling with ice, the HCLPN-D nanoparticles can be used to deliver more DOX into the nuclei than LPN-D nanoparticles (Figure S21b). Enhanced Anticancer Capacity in Vitro via Overcoming Drug Resistance To investigate the anticancer capacity of HCLPN-D nanoparticles, both the 2D drug-resistant cancer cells and 3D CSC-enriched spheres were treated with HCLPN nanoparticles (without DOX), free DOX, and HCLPN-D nanoparticles at various concentrations without or with ice (+I) cooling for 5 or 10 min. The total treatment time for all the drug formulations is either 24 or 48 h. For treatments with ice cooling, it was conducted after incubating cells with the various formulations at 37 °C for 12 h, and the cells were put back in 37 °C incubator after the cooling treatment to further culture for 12 or 36 h. FLIR thermographs indicate that cells cultured in a 96-well plate can be efficiently cooled to ∼4–0 °C with ice for 5 or 10 min (Figure S22a), which is consistent with the medium temperature in the sample measured with thermocouples (Figure S22b). The safety of ice treatment is confirmed by checking the viability of cells incubated with ice for 5 or 10 min. As shown in Figure S23, the ice treatment alone has no effects on the cell viability at both 24 and 48 h. According to the viability data of both 2D cultured NCI/RES-ADR cells (Figure 4a) and CSC-enriched spheres (Figure 4b), blank HCLPN nanoparticles with or without ice cooling are also not harmful to the cells for both the 24 and 48-h treatments. Interestingly, HCLPN-D nanoparticles show higher cytotoxicity than free DOX only at high drug concentrations (50, 25, and 10 μg/mL for 2D cells; 50 μg/mL for CSC-enriched spheres, p < 0.05), probably due to the capability of the membrane transporter to pump out drug slowly released from the nanoparticles. However, the cytotoxicity of DOX is not significantly affected if it is simply mixed with blank HCLPN nanoparticles with or without ice cooling. In addition, ice cooling for 10 min could further significantly decrease the viability of 2D cells or spheres treated with free DOX at 50 μg/mL (p < 0.05, for both 2D cultured NCI/RES-ADR cells and CSC-enriched spheres). This is probably due to the reduced activity of membrane transporters in the cells cooled with ice for 10 min, which may allow some free DOX to enter the cells treated with free DOX at the high concentration. To confirm this, we further checked the uptake of free DOX at high concentrations (50, 80, and 160 μg/mL) under the same thermal treatments for obtaining the data in Figure 3. As shown in Figure S24, although some free DOX could enter the drug-resistant cells at 50 μg/mL, it is minimal in the cell nuclei. This may explain the high viability of the cells treated with 50 μg/mL free DOX (Figure 4a). The fluorescence intensity gradually increases at higher drug concentrations. Although ice cooling for 5 min does not seem to significantly improve the intracellular DOX at all the three concentrations (Figure S24), significantly more DOX could enter the cells with ice cooling for 10 min. The latter is probably because the membrane transporter activity of the cells is significantly decreased after 10 min of ice cooling. Figure 4 Enhanced in vitro anticancer capacity by HCLPN-D nanoparticles with ice cooling for overcoming drug resistance. Viability of (a) 2D cultured NCI/RES-ADR multidrug-resistant cancer cells and (b) CSC-enriched spheres derived from the multidrug-resistant cancer cells after treating them with blank nanoparticles (HCLPN), free DOX, and HCLPN-D nanoparticles without or with ice cooling for 5 or 10 min. The viability of control cells cultured in pure medium is 100%. Error bars represent SD (n = 3). : p < 0.05 (Kruskal–Wallis H test), which indicates cells treated with HCLPN-D nanoparticles and ice cooling for 10 min is significantly lower than other treatments with the same drug concentration. (c) TEM images of the NCI/RES-ADR cancer cells treated with saline, HCLPN-D nanoparticles with or without ice cooling for 10 min. The endo-/lysosomes in HCLPN-D treated cells light up due to the existence of intact (without ice cooling) or disassembled (with ice cooling) HCLPN-D nanoparticles. The insets are the endo-/lysosomes indicated by the arrows with either intact or disassembled HCPN-CG nanoparticles. (d) A schematic illustration of the combination of the HCLPN-D nanoparticle and ice cooling for overcoming the multidrug resistance to enhance cancer destruction, in comparison to the HCLPN-D nanoparticle alone and free drug. The combination can overcome the drug resistance in cancer cells by (1) cold-triggered burst drug release from the HCLPN-D nanoparticles and (2) the cold-induced low activity of the membrane transporters to pump out the released drug. It is worth noting that most of the DOX remains in the cytoplasm after entering the drug-resistant cells incubated with the free DOX at the usually high concentrations, although free DOX usually enter the nuclei of non-drug-resistant cancer cells. This is further confirmed by incubating the cancer cells with free DOX on ice for 1 h. As shown in Figure S25, more free DOX could enter the cells compared to 5 or 10 min of ice cooling (Figure 3a). This suggests cold could decrease the activity of the efflux pump (i.e., temperature-dependent activity of the pump), but it requires more than 10 min for the activity to be sufficiently compromised for free DOX to enter the drug-resistant cells. Nonetheless, most of the free DOX stays in the cytoplasm after the 1 h of ice cooling, which is similar to the observation after incubating the cells with free DOX at high concentrations (50–160 μg/mL) on ice for 5–10 min (Figure S24). This may explain the cell viability data shown in Figure S26 because DOX must enter the cell nuclei to induce cytotoxicity. Although the toxicity of free DOX at 160 μg/mL to the drug-resistant cells with 10 min of ice cooling is significantly higher than that to the cells kept at 37 °C or with 5 min of ice cooling, more than 60% of the cells could still survive at such an unusually high concentration. These observations suggest the diffusion of free DOX through the plasma membrane into the drug-resistant cells may activate some protective mechanism to prevent the drug from entering the nuclei, in addition to trying to pump out the free drug with the membrane transporters. In stark contrast, the cytotoxicity of HCLPN-D nanoparticles can be significantly enhanced after ice cooling (particularly for 10 min) at both low and high concentrations (Figure 4a,b), which is in accordance with the data on cellular uptake and DOX distribution in the cells (Figures 3 and S12). A higher cytotoxicity (∼40 versus ∼60%) could be achieved by using HCLPN-D+I (10 min) treatment with very low drug concentration compared with free DOX treatment (32 times lower, 5 versus 160 μg/mL). The cold-responsive capacity of HCLPN-D nanoparticles inside cells is further confirmed by cell TEM imaging. As shown in Figure 4c, although endo-/lysosomes are not easily identifiable in saline treated cells, they are lit up as white dots in cancer cells treated with the HCLPN-D nanoparticles. This is probably a result of the white core–shell structure of the nanoparticles under TEM (Figure 1b). Moreover, almost all the HCLPN-D nanoparticles in the endo-/lysosomes become disassembled after ice cooling, suggesting the cold-responsive capacity of the HCLPN-D nanoparticles retains inside cells. It is worth noting that disassembly of HCLPN-D nanoparticles with ice cooling partially disrupts the structure of endo-/lysosomes according to the TEM images. This could facilitate the DOX released from the nanoparticles to further escape from the endo-/lysosomes into the cytoplasm. These data indicate that the aforementioned challenges associated with free DOX can be efficiently resolved by using the cold-responsive HCLPN-D nanoparticles for intracellular delivery of the drug (Figures 3 and 4). We also incubated both 2D cultured cells and 3D CSC-enriched spheres with free DOX and loperamide, a substrate of P-gp and can be used as the P-gp inhibitor.54 As shown in Figure S27, by mixing with P-gp inhibitor, free DOX indeed is significantly more toxic than the free drug alone (Figure 4a) to the 2D cultured cells after 24 h of incubation, and the cytotoxicity further increases at 48 h. For 3D CSC-enriched spheres, free DOX mixed with loperamide shows higher cytotoxicity than the free drug alone (see Figure 4b) only after 48 h of incubation. Moreover, the overall cytotoxicity of free DOX mixed with loperamide is still less than that of HCLPN-D nanoparticles with ice cooling (Figure 4a,b). This is probably because most of the free DOX and sustained/slowly released DOX from HCLPN-D nanoparticles (without ice cooling) may still stay in the cytoplasm after entering the cancer cells in the presence of loperamide to block the efflux pump. This hypothesis is confirmed with confocal images of cells treated with free DOX and HCLPN-D nanoparticles in the absence or presence of loperamide, as shown in Figure S28. A schematic illustration of the combination of the HCLPN-D nanoparticles and ice cooling for overcoming the drug resistance to enhance cancer destruction, in comparison to the HCLPN-D nanoparticle alone and free drug, is given in Figure 4d. Free drug can be quickly pumped out of cells after its diffusion into cells due to its close proximity to the membrane transporters (or immobilized in the cytoplasm by possible additional mechanism when the drug concentration is unusually high). Using nanoparticles alone for slow drug release, the nanoparticles may be taken up by cancer cells via CD44 receptor-mediated endocytosis (Figure S20), which may also be mediated by clathrin or caveolin.55,56 However, most of the slowly released drug may be still pumped out of the cells before it binds with its targets in the cells. This is because the activity of transmembrane transporters is intact, and the concentration of drug in cells is not high enough to outperform the pumping capacity of the transporters. Importantly, the HCLPN-D nanoparticles could quickly disassemble to release most of the encapsulated drug when cooled with ice and disrupt the structure of endo/lysosomes. Due to its small size, the released free DOX can diffuse out of the damaged endo-/lysosomes driven by its concentration gradient without the need of any metabolic energy. Although some of the released drug may be pumped out of cells during the cooling process, most of the drug can enter and bind with the nuclei due to the burst release-induced high drug concentration in the cytoplasm and the compromised pumping capacity of the transporters at cold temperature (Figure 4d). In Vivo Tumor Targeting and Enhanced Capacity of Destroying Drug Resistant Tumors Next, we investigated the biodistribution of the HCLPN nanoparticles in drug-resistant tumor-bearing mice by encapsulating indocyanine green (ICG or G) to obtain ICG-laden nanoparticles (HCLPN-G). Tumors were produced by subcutaneous injection into the upper hindlimb of 20, 000 CSC-enriched sphere cells per animal (7 week-old female nude mouse). As shown in Figure 5a, the ICG fluorescence was detectable over almost the whole animal body for both HCLPN nanoparticles and free ICG at 1 h after intravenous injection. More importantly, the HCLPN-G nanoparticles treated mice have stronger fluorescence in their tumors, suggesting preferential accumulation of the HCLPN nanoparticles in the tumors. The accumulation of HCLPN-G nanoparticles in tumor further increases after 3 and 6 h of injection. In contrast, the fluorescence in free ICG-treated mice decreases at 3 h and almost disappears at 6 h after injection (Figure 5a). To confirm the observations from whole animal imaging, various organs were harvested for ex vivo imaging to check the distribution of ICG fluorescence after sacrificing the mice at 9 h. As shown in Figure 5b, only the tumors from mice treated with HCLPN-G nanoparticles has ICG fluorescence with an exposure time of 3 s, which is consistent with the whole-animal imaging data. This is probably due to the enhanced permeability and retention (EPR) effect of tumor vasculature compared to that of normal organs, together with the capability of HA on the surface of the HCLPN-D nanoparticles in targeting CD44 overexpressed on the cancer cells and CSCs.57,58 Figure 5 In vivo tumor targeting capacity of HCLPN-D nanoparticles. (a) In vivo whole animal imaging of ICG fluorescence at different times after intravenous injection of free ICG and ICG-laden HCLPN-G nanoparticles via the tail vein. Arrows indicate the locations of tumors in mice. (b) Ex vivo imaging of ICG fluorescence in tumor and five critical organs collected after sacrificing the mice at 9 h. (c) Imaging of total ICG fluorescence of free ICG and ICG-laden HCLPN-G nanoparticles in three samples prepared in the same way as the solutions used for injection into mice. The images were taken under the same condition as that for both the in vivo and ex vivo imaging. (d) Quantitative analysis of the distribution of HCLPN-G and free ICG in tumor and five critical organs collected from free ICG and HCLPN-D nanoparticles treated mice. The data show that the HCLPN-G nanoparticles could accumulate in tumor much more efficiently than free ICG. NCI/RES-ADR cells detached (with trypsin) from CSC-enriched spheres were used to obtain xenografts of multidrug-resistant tumors for imaging. To quantify the biodistribution of ICG fluorescence in the various organs including tumors, the total ICG fluorescence of free ICG and ICG-laden HCLPN-G nanoparticles used for injection into each mouse were obtained. This was done by diluting the 100 μL of free ICG and ICG-laden HCLPN-G nanoparticles (prepared in the same way as the 100 μL of samples used for injection into each mouse) into 400 μL (to prevent fluorescence overflow) in a centrifuge tube and imaging in the same way as that for both in vivo and ex vivo imaging. This experiment was conducted in triplicates (Figure 5c). Interestingly, the average total fluorescence of HCLPN-G nanoparticles used for injection into each animal is ∼99% of that of free ICG, indicating minimal quenching of the ICG fluorescence in the nanoparticles. The total fluorescence in each organ shown in Figure 5b was subtracted with the total fluorescence of the corresponding organ from the saline group (to correct any auto fluorescence from tissue). The corrected total fluorescence of ICG in each organ from the free ICG (or HCLPN-G nanoparticles) group shown in Figure 5b was then divided by the average total fluorescence of free ICG (or HCLPN-G nanoparticles) used for injection into each animal (Figure 5c) and averaged, to obtain the percentage of ICG distribution in each organ. As shown in Figure 5d, the accumulation of ICG in tumors is significantly improved by ∼50 times (∼15% versus ∼0.3%) with the nanoparticle encapsulation. This further confirms the observations from the qualitative in vivo and ex vivo imaging. Lastly, we treated the drug-resistant tumor-bearing mice with different drug formulations to understand the safety and efficacy of the HCLPN-D nanoparticles in combination with 10 min of ice cooling for overcoming cancer drug resistance. To assess the cooling effect in vivo, ice was applied through the skin over the tumor area. FLIR near-infrared thermography was used to determine the temperature immediately after ice cooling (Figure 6a). After 10 min of cooling, the temperature in the tumor area decreases to 4–7 °C. The ice cooling treatment is also efficient on human (hand, Figure 6a). The drug-resistant tumor-bearing mice were randomly divided into six groups: saline, blank nanoparticles (HCLPN), free DOX without or with ice cooling (DOX or DOX+I), and HCLPN-D nanoparticles without or with ice treatment (HCLPN-D or HCLPN-D+I). Mice were treated with the various formations at a total DOX dose of 3 mg/kg body weight via intravenous injection when the tumor reached ∼100 mm3 on day 1, 8, 15, 22, and 29. After 12 h of each of the injections, tumors were cooled with ice for 10 min. No mice died during the course of the 59 days of treatment and observation. Figure 6 In vivo antitumor capacity of HCLPN-D nanoparticles with ice cooling studied using subcutaneous tumor model. (a) Near infrared thermographs of whole animal and human hand before and after ice cooling for 10 min (+I), showing temperature in the region with cooling can be effectively decreased to ∼0 °C. (b) Typical photographs showing the size of tumors (indicated by arrows) on day 59 in mice with six different treatments. (c) Tumor growth curves for the six different treatments. Error bars represent SD (n = 5). The red arrow heads indicate the times of conducting injections. : p < 0.05, : p < 0.01 (Kruskal–Wallis H test). (d) Weight of the tumors collected after sacrificing the mice on day 59. Error bars represent SD (n = 5). : p < 0.01 (Kruskal–Wallis H test). (e) Representative histology (H&E) images of the tumors collected on day 59. The HCLPN-D+I treated tumors are more necrotic than tumors with the other five treatments. (f) Immunofluorescent staining of CD44 and CD133 in tumor showing diminished expression of both CD44 and CD133 after the treatment with HCLPN-D+I. (g) Body weight and (h) representative micrographs of H&E staining of four important organs with various treatments showing the minimized systemic toxicity of HCLPN-D+I compared to treatments with free DOX (DOX and DOX+I). As shown in Figure 6b,c, tumor growth for treatments with blank HCLPN nanoparticles and free DOX is similar to that of saline control. This is not surprising as the tumors are generated with multidrug-resistant and CSC-enriched cancer cells. In addition, ice cooling does not affect the antitumor ability of free DOX. The tumor volume for the treatment with HCLPN-D is slightly reduced compared to saline control although the difference is not significant. Importantly, the HCLPN-D nanoparticles with ice cooling (HCLPN-D+I) exhibit excellent antitumor capacity and significantly inhibit the tumor growth compared to all the other five treatments. The size and weight of the tumors for the HCLPN-D+I treatment are significantly less than that from all the other five treatments (Figure 6d and Figure S29). Moreover, histological examination (hematoxylin&eosin or H&E stain) reveals extensive necrosis in the tumors from the HCLPN-D+I treatment group while tumors from all the other groups are more viable (Figure 6e and Figure S30). CD44 and CD133 are two surface markers involved in many cell functions and believed to be associated with tumorigenicity.47,48,53 Both have been commonly used as the surface markers of various CSCs.47,48,53 We checked the expression of both markers in 2D cultured NCI/RES-ADR cells and 3D-cultured CSC-enriched spheres first. As shown in Figures S31 (for 2D cultured NCI/RES-ADR cells) and S32 (for the CSC-enriched spheres), the expression of CD44 and CD133 in the CSC-enriched spheres is increased compared with 2D cultured NCI/RES-ADR cells. After treatment with HCLPN-D nanoparticles for 12 h without ice cooling, the expression of CD44 and CD 133 is decreased only slightly. Importantly, after ice cooling and further incubation for 12 h at 37 °C, the expression of both markers is minimized in both 2D cultured NCI/RES-ADR cells and 3D cultured CSC-enriched spheres (Figure S31–32). In order to check the anti-CSC ability of the HCLPN-D nanoparticles with 10 min of ice cooling in vivo, the expression of the variant CD44 and CD133 was further studied in the in vivo tumors after the various treatments. Typical micrographs of immunofluorescent staining of the two CSC markers are shown in Figure 6f. The expression of the two markers in tumors treated with saline, HCLPN nanoparticles, DOX, DOX+I, and HCLPN-D nanoparticles is high. In contrast, their expression in tumors with the HCLPN-D+I treatment is negligible. These observations from the immunofluorescent staining were further confirmed by immunohistochemistry staining of the two markers in tumors from the various treatments (Figure S33), which suggests that the HCLPN-D nanoparticles combined with 10 min of ice cooling are effective to kill the CSCs in vivo. Collectively, these data demonstrate the enhanced in vivo antitumor efficacy of the HCLPN-D nanoparticles combined with ice cooling. Equally important, we did not notice any obvious sign of side effects for the HCLPN-D+I treatment. Although during the ice treatment, normal tissue around tumor might be affected by ice cooling and DOX released from nanoparticles, neither death nor significant drop of body weight was noted for saline, HCLPN nanoparticles, HCLPN-D, and HCLPN-D+I treatments (Figure 6g). In contrast, the body weight of mice treated with free DOX and DOX+I were significantly reduced during the treatments, indicating significant systemic toxicity of the treatments with the free drug. To further confirm this, various critical organs including liver, kidney, heart, and lung from saline, DOX, and HCLPN-D+I treatments were harvested, fixed, and assessed by histology (H&E stain). As shown in Figure 6h and Figure S34, free DOX treatment results in hepatic damage including macro- and microvesicular steatosis and bile stasis. Slight nephrotoxicity and cellular damage (vacuolization) of cardiac muscle were also observable for the free DOX treatment. Severe pulmonary damage with markedly reduced alveolar surface area (honeycomb lung) was obvious in the lung of mice with free DOX treatment. However, no obvious damage to these organs was observable in the H&E stained tissue slices for the mice with HCLPN-D+I treatment (Figure 6h and Figure S34). In order to further confirm the antitumor capacity and safety of the HCLPN-D nanoparticles with ice cooling, an orthotopic metastasis model of ovarian cancer was established by intraperitoneal injection of the NCI/RES-ADR cells into the peritoneal cavity of mice. The mice were then treated with saline, free DOX, and HCLPN-D nanoparticles at a total DOX dose of 3 mg/kg body weight via intraperitoneal injection on day 7, 14, and 21. After 12 h of each of the injections, tumors (if any) were cooled by applying ice for 10 min on the skin outside the peritoneal cavity on the ventral side. As shown in Figure 7a, the temperature decreased to 6–10 °C at the ice treated area after the 10 min of ice cooling. All the mice were sacrificed on day 32, and tumors can only be found in the saline and free DOX treated mice (Figure 7b-d). The tumors are further confirmed by H&E staining data (Figure 7e). This is not surprising as the NCI/RES-ADR cells are resistant to free DOX, which can be overcome by using HCLPN-D nanoparticles with ice cooling. The body weight of mice treated with free DOX is also reduced and significantly different from that of mice in the saline and HCLPN-D groups on day 21 (Figure 7f). Figure 7 In vivo antitumor capacity of HCLPN-D nanoparticles with ice cooling studied using orthotopic metastasis model of ovarian cancer. (a) Near infrared thermographs of whole animal on the ventral side before and after ice cooling for 10 min. The data show that temperature on the skin with cooling on the ventral side of the peritoneal cavity can be effectively decreased to ∼6–10 °C. (b) Photographs showing the typical in situ locations of tumors (indicated by arrows and circles) from mice treated with saline and free DOX. (c) Photograph showing the size of tumors collected after sacrificing the mice on day 32 with three different treatments. (d) Weight of the tumors collected on day 32. Error bars represent SD (n = 3). (e) Representative histology (H&E) images of the tumors collected on day 32. (f–g) Body weight (f) and representative micrographs of H&E staining of five critical organs (g) with various treatments. The data show reduced systemic toxicity of the treatment of HCLPN-D nanoparticles with ice cooling for 10 min (HCLPN-D+I) compared with the free DOX+I treatment. *: p < 0.05 (Kruskal–Wallis H test). H&E staining of various critical organs (kidney, spleen, liver, heart, and lung) indicates that the free DOX treatment causes severe kidney injury together with some hepatic and cardiac damage (Figure 7g and Figure S35). The severe renal injury may be because of the intraperitoneal injection free DOX that could diffuse into the organ. Importantly, these side effects of free DOX can be minimized by encapsulating the drug inside HCLPN nanoparticles for delivery via intraperitoneal injection. To further support this, we incubated noncancerous human umbilical vein endothelial cells (HUVECs) with both free DOX and HCLPN-D nanoparticles. As shown in Figure S36, free DOX can easily enter HUVECs but not the NCI/RES-ADR cells (top panels). In contrast, uptake of HCLPN-D nanoparticles by the HUVECs is minimal compared to the NCI/RES-ADR cells either without (middle panels) or with (bottom panels) ice cooling. These data indicate the HCLPN nanoparticles are capable of not only significantly enhancing the efficacy of DOX in destroying drug-resistant tumors when combined with ice cooling, but also minimizing its systemic toxicity via intraperitoneal or intravenous injection. Discussion In this study, we developed a cold responsive nanoparticle for overcoming the drug resistance of NCI/RES-ADR cells and reducing the potential side effects associated with chemotherapy drug (DOX). As schematically illustrated in Figure 8, there are two possible mechanisms for this strategy to overcome cancer drug resistance. The first mechanism is the ice cooling induced burst release of drug from the HCLPN-D nanoparticles. As shown in Figure 2b, more than 70% of DOX can be released from the HCLPN-D nanoparticles during ice cooling for 5 min. The released free DOX can diffuse out of the endo-/lysosomes driven by its concentration gradient without the need of any metabolic energy. At the same time, the structure of endo-/lysosomes may be damaged during the disassembly of the HCLPN-D nanoparticles as a result of ice cooling (Figure 4c), to further facilitate the escape of the released DOX from the endo/lysosomes. With a large amount of free DOX being released into cytoplasm in a short time, a significant amount of free DOX could bind with its target before being pumped out of the cells by the efflux pumps on the cell membrane (Figures 3, S7, S12, S15–S18, S21, S31, and S32). In contrast, only ∼2.2% of the drug encapsulated in the nanoparticles could be released in 5 h at 37 °C (Figure 2b). This slowly released drug from the nanoparticles could be easily pumped out of the cells by the efflux pump before entering the nuclei (Figures 3, S7, S12, S15–S18, S21, S31, and S32). This mechanism is further supported by the cell viability data showing that ice cooling could greatly and significantly improve the toxicity of the HCLPN-D nanoparticles to the multidrug-resistant NCI/RES-ADR cells (Figure 4a,b). The second mechanism is that the activity of efflux pumps can be reduced during the ice cooling process. This is supported by the data on cell uptake of free DOX without and with ice cooling (Figures S24 and S25) and the viability data of cells treated by free DOX at high concentrations (50–160 μg/mL) without and with ice cooling (Figures 4a,b and S26). These high concentrations of DOX are relevant because the intracellular concentration of DOX delivered with the HCLPN-D nanoparticles (extracellular DOX: 10 μg/mL) could be this high according to the intracellular fluorescence intensity of DOX (Figure S24 versus Figure 3a). This mechanism is due to the minimized metabolic activity of cells at ice-cold temperature to deprive the energy supply to the efflux pumps that work against the concentration gradient of the chemotherapy drug across the cell membrane (i.e., the drug concentration outside the multidrug-resistant cells is higher than that inside the cells). Furthermore, the HCLPN-D nanoparticles could efficiently and selectively accumulate in tumors compared with free drug (Figure 5), which could carry the chemotherapy drug selectively into tumor. This together with the minimal release of drug from the nanoparticles in normal tissue (always at 37 °C, Figure 2b) can reduce the potential side effects of chemotherapy drug to normal organs in vivo (Figures 6 and 7). We tested this strategy for overcoming cancer multidrug resistance using both the 2D cultured NCI/RES-ADR multidrug-resistant cancer cells and their spheres enriched with CSCs. Figure 8 A Schematic illustration of overcoming drug resistance with HCLPN-D nanoparticles and ice cooling for enhanced cancer therapy. In vivo accumulation of HCLPN-D nanoparticles in tumor through the enhanced permeability and retention (EPR) effect of tumor vasculature could minimize the side effects associated with free DOX. Moreover, the HCLPN-D nanoparticles can specifically target cancer stem cells (CSCs) via the HA-CD44 interaction to facilitate their uptake by the CSCs. Although drug slowly released from nanoparticles at 37 °C (or mild hyperthermic temperatures) could be still pumped out of the multidrug-resistant cancer cells, the cold-triggered burst drug release together with the compromised pumping activity of membrane transporters in the multidrug-resistant cancer cells under cold temperature could efficiently overcome their drug-resistant capacity. As a result, the cold-responsive nanoparticle in combination with ice cooling could efficiently inhibit the growth of multidrug-resistant tumor in vivo. The aforementioned two mechanisms for overcoming cancer drug resistance with the HCLPN-D nanoparticles and ice cooling are further supported by data obtained with the drug-resistant A2780ADR ovarian cancer cells. As shown in Figure S37, no red fluorescence of DOX is observable in the A2780ADR cells after incubating them with free DOX (10 μg/mL) at 37 °C for 3 h, followed by either continuously incubating at 37 °C or cooling with ice for 10 min. In stark contrast, red fluorescence of DOX is observable in the cells incubated with the HCLPN-D nanoparticles (containing 10 μg/mL DOX) at 37 °C for 3 h, although the DOX fluorescence is predominantly distributed in the cytosol. Importantly, DOX is located in the nuclei of the cells treated with HCLPN-D nanoparticles with 10 min of cooling with ice after the 3 h of incubation at 37 °C, while almost all DOX is still distributed in the cytosol if the cells are continuously cultured at 37 °C for 10 min more. Quantitative analyses indicate that the intensity of DOX in the cell nuclei is significantly increased after ice cooling, as shown in Figure S38. We further investigated the cell viability of the A2780ADR cells treated with free DOX and HCLPN-D nanoparticles with or without ice cooling for 10 min. As shown in Figure S39, the cytotoxicity of HCLPN-D nanoparticles is significantly enhanced after ice cooling, which is in accordance with the data on cellular uptake shown in Figures S37 and S38 and discussed above. We also noticed that ice cooling for 10 min could further significantly decrease the viability of A2780ADR cells treated with free DOX at 50 μg/mL (p < 0.05), which is consistent with the viability data of the NCI/RES-ADR cells with the same treatment (Figure 4a). This is probably due to the second mechanism via reducing the activity of the membrane transporters, as aforementioned for the NCI/RES-ADR cells (Figures S24–S26). This is further confirmed by incubating the A2780ADR cells with free DOX (10 μg/mL) on ice for 1 h. As shown in Figure S40, more free DOX could enter the cells compared with 10 min of ice cooling (Figure S37). This supports that ice cooling could decrease the activity of the efflux pump (i.e., temperature-dependent activity of the pump), which could minimize the amount of drug (burst-released from the HCLPN-D nanoparticles inside the drug-resistant cells in response to ice cooling) to be pumped out of the cells before it enters the cell nuclei. Since the HCLPN-D nanoparticles disassemble at ∼10 °C, and biological tissues and cells may become frozen (i.e., form solid ice) with minimized diffusion coefficient for small molecules including chemotherapy drugs below ∼0 °C, the therapeutic temperature with the HCLPN-D nanoparticles for burst drug release to destroy tumors is suggested to be ∼0–10 °C. This can be conveniently achieved by cooling with ice that is readily available in nearly all clinic settings, without the need of a complex refrigeration system to achieve subzero or freezing temperatures. It is worth noting the HCLPN-D nanoparticles are also applicable for applications requiring subzero temperatures. This is because they may disassemble to induce burst drug release during cooling or warming between the subzero temperatures and 37 °C (body temperature). In fact, the temperature is not constant (i.e., decreases with time from 37 °C to ∼4–0 °C) during ice cooling in this study (Figures S8, S9, and S22). Therefore, we compared the viability and DOX distribution in cells treated with free DOX and HCLPN-D nanoparticles under two conditions: at body temperature (37 °C) and with ice cooling to cold temperature (below 10 °C). Our data show the combination of the HCLPN-D nanoparticles and ice cooling could overcome cancer resistance (Figure 3) to effectively kill the multidrug-resistant cells in vitro (Figure 4) and in vivo (Figures 6 and 7). It is worth noting that the CSC-enriched spheres are derived from NCI/RES-ADR cells and can be maintained only in in vitro culture using ultra low attachment plate and CSC medium. After injection in vivo, some of the cells from the spheres may differentiate, and some of them may remain undifferentiated. In other words, we have no control of the stemness of the cells from the CSC-enriched spheres in vivo. Therefore, the difference between NCI/RES-ADR cells and CSC-enriched spheres with different treatments was studied in vitro, and overall the CSC-enriched spheres are more resistant to drug than 2D cultured NCI/RES-ADR cells (Figure 4a,b). In view of the latter, we used cells from the CSC-enriched spheres to produce tumors for our in vivo studies to investigate if the HCLPN-D nanoparticles with ice cooling could overcome cancer drug resistance in vivo. We do not intend to compare the NCI/RES-ADR cells and CSC-enriched spheres in vivo since we could not control the stemness of the cells in vivo. We tested the strategy of overcoming cancer drug resistance with the HCLPN-D nanoparticles and ice cooling using both subcutaneous and orthotopic metastasis model of ovarian cancer. For the orthotopic metastasis model, the cells from the CSC-enriched spheres were injected into the peritoneal cavity (where ovarian cancer cells usually metastasize to)59 of mice via intraperitoneal injection. Consistent with the clinical practice of treating ovarian cancer metastasis,60 we used intraperitoneal injection to deliver the HCLPN-D nanoparticles for treating the orthotopic metastasis model in this study. In other words, the nanoparticles are delivered into the peritoneal cavity where the tumors are. This may be the major factor that contributes to the excellent therapeutic outcome of the treatment with the HCLPN-D nanoparticles and ice cooling for the orthotopic metastasis model (no tumor was identifiable, Figure 7c), compared with the subcutaneous model (small tumors could be seen, Figure S29). For the latter, the nanoparticles are injected intravenously through the tail vein that is away from the subcutaneous tumors and the nanoparticles are diluted in blood before reaching the subcutaneous tumors. In this study, the potential of the proposed strategy of using the cold-responsive nanoparticles for overcoming cancer drug resistance is demonstrated by ice cooling of subcutaneous and intraperitoneal tumors in small animals. Admittedly, ice cooling may be difficult to apply for tumors in deep internal organs (e.g., kidney and liver). Nonetheless, catheters with lumens perfused with cold saline have been widely used for local delivery of cold into deep organs to achieve local hypothermia with the aid of minimally invasive surgical technologies such as thoracoscopy, laparoscopy, and endoscopy.61−63 Therefore, the local delivery of cold required for the nanoparticle system developed in this study is not a hurdle to its future potential applications for destroying tumors in deep organs. Since techniques using cold and freezing temperatures have been widely studied and used for treating various diseases including cancer in the clinic (known as cryosurgery, cryotherapy, cryoablation, and hypothermia),28−34 our cold responsive nanoparticle-mediated drug delivery may be combined with these techniques to further improve their safety and efficacy of treating various diseases including cancer. Conclusions We developed a cold-responsive hybrid HCLPN-D nanoparticle composed of HA, chitosan, DPPC, PNIPAM-B, and PF127 for targeted delivery of chemotherapeutics (DOX) into multidrug-resistant cancer cells and their CSCs in vitro and multidrug-resistant tumors in vivo. The HCLPN-D nanoparticles could significantly improve drug delivery into tumors through the EPR effect of tumor vasculature after intravenous injection into the tail vein (Figure 8). Moreover, the HCLPN-D nanoparticles could quickly and irreversibly disassemble at cold temperatures (<12 °C), which can induce burst release of most encapsulated drug from the nanoparticles. Moreover, the cold-triggered burst release of DOX together with the cold temperature per se (to reduce the activity of membrane transporters) can efficiently overcome the multidrug-resistant capacity of NCI/RES-ADR cells (Figure 8). Furthermore, CSCs enriched spheres derived from the multidrug-resistant cancer cells were used to account for the multifaceted mechanisms of cancer drug resistance. Our extensive in vitro studies with both 2D cultured multidrug-resistant cells and 3D microscale tumors (i.e., spheres) enriched with multidrug-resistant CSCs as well as in vivo studies using the CSC-derived tumors grown in mice, demonstrate the great potential of the HCLPN-D nanoparticles with ice cooling for overcoming different mechanisms associated with cancer multidrug resistance for effective and safe cancer therapy. Supporting Information Available The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acscentsci.8b00050.Experimental details of preparation of nanoparticles, drug encapsulation and release, in vitro cell imaging and cell viability, flow cytometry, TEM imaging of cell, in vivo animal study and immunohistochemical staining. Supporting Figures S1–S40 (PDF) Supplementary Material oc8b00050_si_001.pdf This work was partially supported by American Cancer Society (#120936-RSG-11-109-01-CDD) and NIH (R01CA206366). CMJ is an employee of the Maryland Veterans Affairs (VA) Health Care Systems at the Baltimore VA Medcial Center. The authors declare the following competing financial interest(s): The views reported in this paper do not reflect the views of the Department of Veterans Affairs or the United States Government. CMJ has an equity position in Cellth LLC. All other authors declare no competing financial interest. ==== Refs References Gottesman M. M. ; Fojo T. ; Bates S. E. Multidrug resistance in cancer: role of ATP-dependent transporters . Nat. Rev. Cancer 2002 , 2 , 48 –58 . 10.1038/nrc706 .11902585 Szakács G. ; Paterson J. K. ; Ludwig J. A. ; Booth-Genthe C. ; Gottesman M. M. Targeting multidrug resistance in cancer . Nat. Rev. Drug Discovery 2006 , 5 , 219 –234 . 10.1038/nrd1984 .16518375 Rubbia-Brandt L. ; Audard V. ; Sartoretti P. ; Roth A. ; Brezault C. ; Le Charpentier M. ; Dousset B. ; Morel P. ; Soubrane O. ; Chaussade S. 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==== Front ACS OmegaACS OmegaaoacsodfACS Omega2470-1343American Chemical Society 3002376410.1021/acsomega.7b00838ArticleSoft Nanotubes Derivatized with Short PEG Chains for Thermally Controllable Extraction and Separation of Peptides Kameta Naohiro Ding Wuxiao Dong Jiuchao Nanomaterials Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Central 5, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8565, Japan E-mail: [email protected]. Tel: +81-29-861-4478. Fax: +81-29-861-4545.26 09 2017 30 09 2017 2 9 6143 6150 22 06 2017 13 09 2017 Copyright © 2017 American Chemical Society2017American Chemical SocietyThis is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes. By means of a two-step self-assembly process involving three components, including short poly(ethylene glycol) (PEG) chains, we produced two different types of molecular monolayer nanotubes: nanotubes densely functionalized with PEG chains on the outer surface and nanotubes densely functionalized with PEG chains in the nanochannel. Turbidity measurements and fluorescence spectroscopy with an environmentally responsive probe suggested that the PEG chains underwent dehydration when the nanotubes were heated above 44–57 °C and rehydration when they were cooled back to 25 °C. Dehydration of the exterior or interior PEG chains rendered them hydrophobic and thus able to effectively extract hydrophobic amino acids from the bulk solution. Rehydration of the PEG chains restored their hydrophilicity, thus allowing the extracted amino acids to be squeezed out into the bulk solutions. The nanotubes with exterior PEG chains exhibited selectivity for all of the hydrophobic amino acids, whereas the interior PEG chains were selective for hydrophobic amino acids with an aliphatic side chain over hydrophobic amino acids with an aromatic side chain. The higher selectivity of the latter system is attributable that the extraction and back-extraction processes involve encapsulation and transportation of the amino acids in the nanotube channel. As the result, the latter system was useful for separation of peptides that differed by only a single amino acid, whereas the former system showed no such separation ability. document-id-old-9ao7b00838document-id-new-14ao-2017-00838jccc-price ==== Body Introduction Poly(ethylene glycol)s (PEGs) have attracted much attention for biological and medical applications involving peptides and proteins. Specifically, owing to the high water solubility, low toxicity and antigenicity, and thermal responsivity of PEGs, they have been widely used to increase the water solubility of peptides and proteins,1−3 improve their cellular internalization,4 prolong their blood circulation time,5,6 facilitate their condensation and separation7−10 and crystallization,11,12 control their adsorption,13−16 suppress their aggregation,17,18 and accelerate their refolding.19−22 However, only polydisperse PEGs23,24 with relatively high molecular weights have been used to date. Recent studies have suggested that the physicochemical properties of PEGs depend strongly not only on their molecular weights and topology25−30 but also on whether or not they are confined in nanospaces.31,32 Noncovalently bonded polymer nanotubes with controllable cavity sizes and functionalizable surfaces33−35 are important materials for encapsulation, storage, transport, and release of peptides and proteins.36−38 For example, nanotubes formed by self-assembly of rationally designed amphiphilic molecules in water are useful for qualitative and quantitative analyses of proteins,39−41 stabilization of proteins under harsh conditions,42−44 acceleration of protein refolding,45−47 and mimicking of proteins.48 Herein, we report the selective introduction of a dense layer of short PEG chains to the outer surface or the nanochannels of nanotubes. Thermal dehydration and rehydration of the PEG chains enabled us not only to extract hydrophobic amino acids and peptides from bulk solution but also to back-extract them. We discovered that nanotubes with interior PEG chains, but not nanotubes with exterior PEG chains, were useful for separation of peptides that differed by a single amino acid. Results and Discussion Construction of Nanotubes Functionalized with Short PEG Chains As previously reported,49 self-assembly of rationally designed asymmetric amphiphiles with a different head group at each end produces molecular monolayer nanotubes with different inner and outer surfaces. In this study, we synthesized an asymmetric amphiphile designated lipid 1 and two PEG derivatives (PEG8Ste and GlyGlyPEG8) as components for the construction of two types of nanotubes functionalized with short PEG chains. In one type, the PEG chains were located on the outer surface of the nanotubes, and in the other type, the chains were located on the inner surface (i.e., in the nanochannel) of the nanotubes (Figure 1). Figure 1 Molecular monolayer nanotubes: (left panel) exPEG8-nanotubes composed of lipid 1, GlyGlyEtOH, and PEG8Ste and (right panel) inPEG8-nanotubes composed of lipid 1, GlyGlyPEG8, and lipid 2. The yellow and green bands in the chemical structures shown at the top of each panel indicate areas of intermolecular hydrogen bonding and hydrophobic interactions, respectively. The graphic at the bottom of each panel illustrates the thermal dehydration and rehydration behavior of the nanotubes. First, we prepared binary self-assemblies of lipid 1 and either GlyGlyEtOH or GlyGlyPEG8 by dispersing a mixture of lipid 1 (10 μmol) and GlyGlyEtOH (10 μmol) or GlyGlyPEG8 (10 μmol) in pure water (10 mL) under reflux conditions and then gradually cooling the hot solution to room temperature. Transmission electron microscopy (TEM) revealed that this process exclusively produced nanotubes—designated GlyGlyEtOH-nanotubes and GlyGlyPEG8-nanotubes—with an inner diameter of 7–9 nm and a wall thickness of 3–4 nm (Figure S1, Supporting Information). Although self-assembly of GlyGlyEtOH or GlyGlyPEG8 alone gave micelles (Figure S2, Supporting Information), we did not observe these morphologies in either of the binary self-assembly systems. Second, we prepared nanotubes functionalized with short PEG chains on the outer surface, designated exPEG8-nanotubes, by heating the GlyGlyEtOH-nanotubes (composed of 10 μmol each of lipid 1 and GlyGlyEtOH) with PEG8Ste (10 μmol) at about 50 °C in 1:1 (v/v) water/methanol (10 mL). In addition, we prepared nanotubes with short PEG chains on the inner surface, designated inPEG8-nanotubes, by heating GlyGlyPEG8-nanotubes (composed of 10 μmol each of lipid 1 and GlyGlyPEG8) with lipid 2 (10 μmol) under the same conditions. After cooling the hot solutions, we used TEM to confirm that the morphology of the nanotubes had not changed and that no other structures had formed (Figure 2a,b). We note that lipid 2 alone self-assembles in 1:1 (v/v) water/methanol to form bilayer nanotubes with an inner diameter of about 70 nm and a wall thickness of about 70 nm,50 and PEG8Ste alone formed nanofibers with widths of 100–500 nm under the same conditions (Figure S2, Supporting Information). On the other hand, coassembly (one-step self-assembly) of the three components gave mixtures including the desirable nanotubes, the intermediate nanotubes, and the self-assembled structures of each component (Figure S3, Supporting Information). Figure 2 Transmission electron microscopy images of (a) exPEG8-nanotubes and (b) inPEG8-nanotubes. The nanotube channels were visualized by means of negative staining with 2 wt % phosphotungstate. Length distributions of (c) exPEG8-nanotubes and (d) inPEG8-nanotubes. Photographs of the aqueous dispersions of (e) exPEG8-nanotubes and (f) inPEG8-nanotubes. Variable-temperature circular dichroism spectroscopy enabled us to estimate the gel-to-liquid crystalline phase transition temperature (Tg–l) of the nanotube monolayer membrane formed by chiral molecular packing51 derived from the chirality of the d-glucose moieties in lipid 1 and lipid 2 (Figure S4, Supporting Information). The two-component nanotubes, that is, the GlyGlyEtOH-nanotubes and GlyGlyPEG8-nanotubes, had relatively low Tg–l values (around 45–55 °C) in water, which we ascribed to void spaces in the molecular packing arising from the lack of long alkyl chains in GlyGlyEtOH and GlyGlyPEG8 for hydrophobic intermolecular interactions.52 In contrast, the Tg–l values of the three-component nanotubes, that is, the exPEG8-nanotubes and inPEG8-nanotubes, exceeded 100 °C, the suggestion being that the PEG8Ste and lipid 2 molecules, with their long alkyl chains, filled the void spaces within the molecular packing structure of the GlyGlyEtOH-nanotubes and GlyGlyPEG8-nanotubes, respectively (Figure 1). Elemental analysis conducted by gas chromatography–mass spectroscopy showed that the lipid 1/GlyGlyEtOH/PEG8Ste and lipid 1/GlyGlyPEG8/lipid 2 molar ratios of the three-component nanotubes were 1.00:0.95:0.95 and 1.00:0.95:0.98, respectively. IR spectroscopy confirmed the molecular packing of the three components within the monolayer membrane of the nanotubes. GlyGlyEtOH or GlyGlyPEG8 formed a polyglycine-II-type hydrogen bond network with the digylcine moiety of lipid 1 (Figure S5, Supporting Information). Neither PEG8Ste nor lipid 2 disordered the lateral chain packing of the oligomethylene spacer of lipid 1; the packing was assignable to a triclinic parallel (T∥) type, which was easily distinguishable from the orthorhombic perpendicular (O⊥) type that was observed for the PEG8Ste-nanofibers and the bilayer nanotubes self-assembled from lipid 2 (Figures S5 and S6, Supporting Information). To support the location of the PEG chains functionalized in the nanotubes, we carried out time-lapse fluorescence microscopic observations for the single exPEG8-nanotube and inPEG8-nanotube bearing a fluorescent dye partly immobilized on the exterior PEG chains or the interior PEG chains upon addition of a quencher dye. The quenching based on fluorescence resonance energy transfer from the fluorescent dye of the exPEG8-nanotube to the quencher dye randomly and quickly occurred compared to that in the inPEG8-nanotube system (Figure S7, Supporting Information). This result indicates that the quencher dye easily accesses the fluorescent dye, proving that the PEG chains are located on the outer surface of the nanotubes. On the other hand, the quenching in the case of the inPEG8-nanotube started from both the open ends of the nanotube and gradually moved toward the center part (Figure S8, Supporting Information). This result indicates that the quencher dye penetrates into the nanotube from the outside and gradually quenches the fluorescent dye with flowing in the nanotube channel, proving that the PEG chains are located on the inner surface of the nanotubes. The fact that TEM observations indicated that the inner diameters and membrane wall thicknesses of the two types of three-component nanotubes (Figure 2a,b) were similar was ascribable to the low contrast of the images of the hydrated PEG chains.53 The peaks of the length distributions of the exPEG8-nanotubes and inPEG8-nanotubes estimated from those TEM images were around 350 and 200 nm, respectively (Figure 2c,d), which are comparable to the size distributions of both the nanotubes in the aqueous dispersions estimated from dynamic light scattering measurements (Figure S9, Supporting Information). Therefore, both the nanotubes well dispersed in water (Figure 2e,f) should keep the length distributions. Thermal Dehydration and Rehydration of Exterior and Interior PEG Chains Long linear PEG chains in water are known to undergo dehydration in response to elevated temperatures, as indicated by conformational changes of the C–C bonds from the gauche form at low temperatures to the anti form at high temperatures.54−59 As a result of these changes, the solubility of the PEG chains decreases and the aqueous solutions become cloudy, although there are a few reports that concern such thermal dehydration; the examples were short linear PEG chains and oligo(ethylene glycol) chains.60 By measuring the turbidity of aqueous solutions of the exPEG8-nanotubes, we were able to estimate that the dehydration temperature of the exterior PEG chains was 57 °C (Figure S10, Supporting Information). When the solution was cooled, the turbidity cleared, indicating that the solubility of the exterior chains was increased by rehydration. The rehydration temperature, 43 °C, was slightly lower than the dehydration temperature. The thermal dehydration/rehydration cycle could be repeated several times. Similar turbidity measurements for the PEG8Ste-fibers showed that the PEG chains undergo dehydration at 85 °C, whereas the dehydrated PEG chains never undergo rehydration (Figure S11, Supporting Information). To investigate the dehydration/rehydration behavior of the inPEG8-nanotubes, we encapsulated an environmentally responsive probe, 8-anilinonaphthalene-1-sulfonate (1,8-ANS), in the nanotubes to detect dehydration and rehydration of the PEG chains lining the nanochannels.32 We found that raising the temperature produced a remarkable increase in the intensity of the blue-shifted fluorescence band of the 1,8-ANS encapsulated in the inPEG8-nanotubes (Figure S10, Supporting Information). Drastic spectral changes were observed at 44 °C, the implication being that the environment of the nanochannels became relatively hydrophobic at that temperature. The enhancement of the hydrophobicity of the nanochannel is ascribable to thermal dehydration of the interior PEG chains.32 Because Tg–l of the inPEG8-nanotubes was high (>100 °C), we could exclude the possibility that 1,8-ANS was embedded in the hydrophobic membrane wall.61,62 Lowering the temperature restored the original fluorescence spectrum, indicating that the nanochannels regained their hydrophilicity upon rehydration of the interior PEG chains. The rehydration temperature was estimated to be 38 °C. The fluorescence spectroscopy also revealed that there is no evidence of thermal dehydration of the GlyGlyPEG8-micelles (Figure S11, Supporting Information). The dehydration and rehydration temperatures of the inPEG8-nanotubes differed from those of the exPEG8-nanotubes, even though the PEG chains of the two types of nanotubes have similar molecular weights and chemical structures. This difference must be related to (1) the apparent higher modification density of the interior PEG chains as a result of the larger curvature of the inner surface and (2) the presence of confined water molecules with specific physical properties, such as higher viscosity and lower polarity, in the nanotube channels.42,63 Peptide Separation by Extraction and Back-Extraction We utilized the thermal dehydration/rehydration behavior of the PEG chains of the exPEG8-nanotubes and inPEG8-nanotubes for extraction and back-extraction of amino acids. All 20 amino acids (10 μmol each) were separately added to aqueous dispersions (10 mL) of exPEG8-nanotubes (one of the compositions, PEG8Ste: 10 μmol) or inPEG8-nanotubes (one the compositions, GlyGlyPEG8: 10 μmol). The dispersion abilities of the exPEG8-nanotubes and inPEG8-nanotubes were independent of additions of the amino acids, based on no electrostatic attraction of the nonionic glucose OH groups and the PEG chains on the outer surface of the nanotubes with the amino acids. To dehydrate the PEG chains, we heated the mixtures at 70 °C for 30 min, which was long enough to reach extraction equilibrium. Then, the mixtures were quickly filtered through polycarbonate membranes with a pore size of 0.2 μm. The amounts of unextracted amino acids in the filtrates were determined by means of fluorescence spectroscopy with the labeling reagent 4-fluoro-7-nitrobenzofurazan.64 Extraction ratios were estimated by subtraction of the concentrations determined by fluorescence spectroscopy from the initial concentrations. After extraction and filtration, the nanotubes were redispersed in water (10 mL) at 25 °C for rehydration of the PEG chains. Membrane filtration at various intervals and subsequent fluorescence spectroscopy of the filtrates enabled us to estimate release ratios, that is, ratios for back-extraction of the amino acids from the nanotubes into the bulk solution. Finally, destruction of the nanotubes with dimethyl sulfoxide (DMSO) released any remaining amino acids. We confirmed that total recovery ratios for the above-described procedure were 100 ± 5%. At elevated temperature, the exPEG8-nanotubes extracted substantial amounts of Trp, Phe, Pro, Met, Ile, Leu, Val, Ala, Gly, and Tyr, all of which are classified as hydrophobic or neutral amino acids (Figure 3a). Because the exPEG8-nanotubes showed no extraction ability for any of the amino acids unless the temperature was elevated, we contend that the driving force for the extraction was hydrophobic interactions between the hydrophobic amino acids and the dehydrated PEG chains on the outer surface of the nanotubes. Figure 3 Amino acid extraction ratios obtained with (a) exPEG8-nanotubes and (b) inPEG8-nanotubes. The inPEG8-nanotubes also extracted several of the hydrophobic amino acids upon thermal dehydration of the PEG chains in the nanotube channels, but all of the extraction ratios were lower than the corresponding ratios for the exPEG8-nanotubes (Figure 3b). The inPEG8-nanotube selectively extracted amino acids with aliphatic side chains, such as Met, Ile, Leu, Val, and Ala, over amino acids with aromatic or cyclic aliphatic side chains, such as Trp, Phe, and Pro. The hydrophobic interactions between the interior PEG chains and the amino acids occurred via encapsulation of the amino acids from the bulk solution and subsequent transport of the encapsulated amino acids into the nanotube channel, whereas the exterior PEG chains on the exPEG8-nanotubes were in direct contact with the amino acids in the bulk solution. In the former case, the encapsulation and transportation processes influenced not only extraction efficiency but also extraction selectivity. Lowering the temperature of the nanotube dispersions released the amino acids, allowing them to be back-extracted into the bulk solution (Figure 4). Back-extraction was likely induced by disruption of the hydrophobic interactions between the PEG chains of the nanotubes and the amino acids as a result of rehydration of the PEG chains. The back-extraction rates in the exPEG8-nanotube system were remarkably higher than the rates in the inPEG8-nanotube system, and this difference reflects the different locations of the interactions between the amino acids and the nanotubes, that is, the nanotube outer surface versus the nanotube channel. The surface is entirely exposed to the bulk solution, whereas the channel is exposed to the bulk solution only at the two open ends of the nanotube. In the exPEG8-nanotube system, the back-extraction profiles for all of the amino acids were similar (Figure 4a), whereas in the inPEG8-nanotube system, the profiles strongly depended on the amino acid side chain (Figure 4b). In the inPEG8-nanotube system, the back-extraction rates of the amino acids increased in the order Pro < Trp < Gly < Ala < Met < Phe < Leu < Val < Ile, which corresponds well to the order of their hydropathy indexes.65 The more hydrophobic amino acids were preferentially squeezed out of the hydrophilic nanotube channels upon thermal rehydration of the interior PEG chains. Figure 4 Time dependence of amino acid back-extraction ratios obtained with (a) exPEG8-nanotubes and (b) inPEG8-nanotubes. We expected that the extraction and back-extraction abilities of the PEG-derivatized nanotubes would make them useful for separation of peptides that shared similar amino acid sequences. To evaluate this possibility, we chose three angiotensin analogues as model peptides: Asp-Arg-Val-Tyr-Ile-His-Pro-Phe (angiotensin II), Asp-Arg-Val-Tyr-Val-His-Pro-Phe (Val5-angiotensin II), and Ala-Arg-Val-Tyr-Ile-His-Pro-Phe (angiotensin A), hereafter abbreviated as Pep(Asp-Ile), Pep(Asp-Val), and Pep(Ala-Ile), respectively, to emphasize the variations in their amino acid sequences. All three peptides (10 μmol each) were added to an aqueous dispersion (10 mL) of the exPEG8-nanotubes (one of the compositions, PEG8Ste: 300 μmol) or inPEG8-nanotubes (one of the compositions: GlyGlyPEG8: 300 μmol). The mixtures were heated at 70 °C for 30 min and then quickly filtered through 0.2 μm pore size polycarbonate membranes. We carried out one and three back-extraction batch processes for the exPEG8-nanotube and inPEG8-nanotube systems, respectively. In batch I, the nanotubes collected on the membrane were redispersed in water (10 mL) and then the dispersion was allowed to stand at 25 °C for 120 min. A sample of the dispersion was subjected to electrospray ionization-mass spectrometry (ESI-MS) for the qualitative analysis of the peptides. The remainder of the dispersion was used for two additional back-extraction processes (batches II and III, respectively), which involved repetition of the membrane filtration and redispersion steps (Figure S12, Supporting Information). ESI-MS was also carried out for samples from batches II and III. In the exPEG8-nanotube system, the mass spectrum of the batch I sample clearly exhibits signals corresponding to each of the three peptides (Figure 5a); the spectrum is consistent with the similar back-extraction profiles for the three peptides (Figure S12, Supporting Information). These results indicate that the exPEG8-nanotubes showed no ability to separate the peptides. In contrast, in the inPEG8-nanotube system, the mass spectra of the batch II and III samples indicated that they consisted mainly of Pep(Asp-Ile) and Pep(Asp-Val), respectively (Figure 5b), whereas the spectrum of the batch I sample showed signals for both Pep(Ala-Ile) and Pep(Asp-Ile). The order in which the peptides appeared in the spectra was the same as the order of the back-extraction rates, Pep(Ala-Ile) > Pep(Asp-Ile) > Pep(Asp-Val), which was in turn consistent with the order of the hydropathy indexes of the amino acids that were varied in the three peptides (Ile > Ala ≫ hydrophilic Asp; Figure S12, Supporting Information). The inPEG8-nanotubes were able to separate these three peptides even though they differed by just one amino acid. This level of selectivity has not previously been reported for peptides, although conventional polymers, such as inverse micelles and amphiphilic monodisperse PEGs, have been used as extraction agents for group separation of peptides.66,67 The system described herein can be expected to be applicable to sample pretreatments for mass spectrometric analysis of peptides, which usually requires the extraction and condensation of target analytes coexisting with large amounts of bioimpurities.68,69 Figure 5 Positive-mode electrospray ionization mass spectra obtained after back-extraction of peptides with (a) exPEG8-nanotubes and (b) inPEG8-nanotubes. Conclusions Herein, we have reported that PEG chains on the exterior surface of nanotubes and in nanotube channels can be thermally dehydrated at 57 and 44 °C, respectively. The resulting dehydrated nanotubes can be used to extract hydrophobic amino acids by means of hydrophobic interactions with the PEG chains. Cooling the nanotube dispersions to 25 °C led to release of the extracted amino acids from the nanotubes to the bulk solutions by disruption of the hydrophobic interactions as a result of rehydration of the PEG chains. Nanotubes with interior PEG chains were superior to nanotubes with exterior PEG chains in terms of extraction selectivity, owing to the encapsulation and transport of the amino acids in the nanotube channel. The extraction and back-extraction abilities of the interior PEG chains allowed us to separate peptides on the basis of a difference in a single amino acid in their sequences. This system can be expected to be widely applicable for pretreatments of peptide samples for peptidomics and proteomics analysis. Experimental Section Synthesis of GlyGlyEtOH Z-GlyGly-OSu (Bachem) was condensed with ethanol amine in methanol at room temperature. After evaporation of the solvent, the residue was washed with 5% aqueous citric acid and 10% aqueous NaHCO3. Hydrogenation over Pd/C in methanol removed the benzyloxycarbonyl (Z) protecting group. Condensation of the resulting compound (NH2GlyGlyNHCH2CH2OH) and acetyl chloride in methanol and subsequent recrystallization from water gave the target compound (68% overall yield). 1H NMR (500 MHz, DMSO-d6, δ): 8.11 (t, 1H, NH), 8.05 (t, 1H, NH), 7.76 (t, 1H, NH), 4.67 (t, 1H, OH), 3.68 (d, 2H, −NHCH2C=O), 3.66 (d, 2H, −NHCH2C=O), 3.38 (q, 2H, −NHCH2CH2OH), 3.12 (q, 2H, −NHCH2CH2OH), 2.12 (s, 3H, −CH3). ESI-MS (m/z): 218.1 [M + H]+. Anal. calcd for C8H15N3O4: C 44.23, H 6.96, N 19.34. Found: C 44.28, H 6.95, N 19.37. Synthesis of Lipid 1 18-[(2,3,4,6-Tetra-O-acetyl-N-β-d-glucopyranosyl)carbamoyl]octadecanoic acid70 was coupled with NH2GlyGlyNHCH2CH2OH in the presence of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl morpholinium chloride in methanol at room temperature, and the coupled product was purified by recrystallization from ethanol. Removal of the acetyl protecting groups on the sugar moiety by methanolysis with NaOMe gave lipid 1 (45% overall yield). 1H NMR (500 MHz, DMSO-d6, δ): 8.11 (t, 1H, NH), 8.09 (d, 1H, NH), 7.80 (t, 1H, NH), 7.51 (t, 1H, NH), 4.96 (d, 1H, OH-4), 4.87 (d, 1H, OH-3), 4.81 (d, 1H, OH-2), 4.69 (t, 1H, H-1), 4.66 (br, 1H, OH), 4.47 (t, 1H, OH-6), 3.69 (d, 2H, −NHCH2C=O), 3.65 (d, 2H, −NHCH2C=O), 3.63 (m, 1H, H-6), 3.40 (m, 1H, H-6), 3.39 (q, 2H, −NHCH2CH2OH), 3.16 (m, 1H, H-4), 3.13 (q, 2H, −NHCH2CH2OH), 3.0 (m, 3H, H-2, H-3, H-5), 2.04 (m, 4H, −CH2C=O), 1.47 (m, 4H, −CH2CH2C=O), 1.24 (m, 28H, −CH2−). ESI-MS (m/z): 661.4 [M + H]+. Anal. calcd for C32H60N4O10: C 58.16, H 9.15, N 8.48. Found: C 58.18, H 9.10, N 8.47. Synthesis of PEG8Ste PEG8Ste was synthesized in 98% yield by a coupling reaction between stearoyl chloride and m-dPEG8-amine (Quanta Biodesign). 1H NMR (500 MHz, DMSO-d6, δ): 7.88 (t, 1H, NH), 3.59 (m, 24H, −OCH2CH2O−), 3.47 (m, 2H, −CH2−), 3.37 (m, 2H, −CH2−), 3.28 (s, 3H, −OCH3), 3.18 (m, 2H, −CH2−), 3.10 (m, 2H, −CH2−), 2.04 (m, 2H, −CH2C=O), 1.46 (m, 2H, −CH2−), 1.23 (m, 28H, −CH2CH2C=O), 0.85 (t, 3H, −CH3). ESI-MS (m/z): 650.5 [M + H]+. Anal. calcd for C35H71NO9: C 64.68, H 11.01, N 2.16. Found: C 64.58, H 11.10, N 2.07. Syntheses of Lipid 2 and GlyGlyPEG8 Lipid 2 and GlyGlyPEG8 were synthesized as reported previously.32,50 Morphological Observations Aqueous dispersions of the self-assembled structures were dropped onto a carbon grid, negatively stained with a phosphotungstate solution (2 wt %, pH adjusted to 7 with NaOH), and observed with a transmission electron microscope (H-7000; Hitachi) at 75 kV. Molecular Packing Analysis The self-assembled structures were lyophilized and analyzed with a Fourier transform IR spectrometer (FT-620; JASCO) operated at 4 cm–1 resolution and equipped with an unpolarized beam, an attenuated total reflection accessory system (Diamond MIRacle, horizontal attenuated total reflection accessory with a diamond crystal prism; PIKE Technologies), and a mercury cadmium telluride detector. The X-ray diffraction patterns of the structures were measured with a Rigaku diffractometer (type 4037) using graded d-space elliptical side-by-side multilayer optics, monochromated Cu Kx3b1; radiation (40 kV, 30 mA), and an imaging plate (R-Axis IV). The exposure time was 5 min with a 150 mm camera length. Circular dichroism spectra of aqueous dispersions of the nanotubes were measured with a spectropolarimeter (J-820; JASCO) equipped with a temperature control unit (PTC-423L; JASCO). Preparation of inPEG8-Nanotubes Encapsulating 1,8-ANS Lyophilized inPEG8-nanotubes prepared from 10 μmol each of lipid 1, GlyGlyPEG8, and lipid 2 were added to an aqueous solution of 1,8-ANS (50 μmol). After aging overnight, the mixture was filtered through a polycarbonate membrane with a pore size of 200 nm. The inPEG8-nanotubes on the membrane were washed several times with water to remove any 1,8-ANS on the outside of the nanotubes. After complete destruction of the inPEG8-nanotubes by heating in dimethyl sulfoxide, measurement of UV–vis spectra with a spectrophotometer (U-3300; Hitachi) equipped with a temperature control unit (BU150A; Yamato) allowed us to calculate the amount (2.7 μmol) of encapsulated 1,8-ANS. The fluorescence spectrum of the encapsulated 1,8-ANS was recorded with a spectrophotometer (F-4500; Hitachi) equipped with a DCI temperature control unit (Haake). Supporting Information Available The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.7b00838.Morphological observation of the self-assembled structures; molecular packing and size distribution analysis of the nanotubes; evaluation of the thermal dehydration and rehydration of the PEG chains; monitoring of the back-extraction of the peptides (PDF) Supplementary Material ao7b00838_si_001.pdf The authors declare no competing financial interest. Acknowledgments This work was supported by JSPS KAKENHI no. JP17H02726. ==== Refs References Review: Veronese F. M. Peptide and Protein PEGylation: A Review of Problems and Solutions . Biomaterials 2001 , 22 , 405 –417 . 10.1016/S0142-9612(00)00193-9 .11214751 Review: Pasut G. ; Veronese F. M. Polymer–Drug Conjugation, Recent Achievements and General Strategies . Prog. Polym. Sci. 2007 , 32 , 933 –961 . 10.1016/j.progpolymsci.2007.05.008 . Review: Veronese F. M. ; Mero A. 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==== Front MoleculesMoleculesmoleculesMolecules : A Journal of Synthetic Chemistry and Natural Product Chemistry1420-3049MDPI 1800737810.3390/10091126molecules-10-01126ArticleNew Multi-1,2,3-Selenadiazole Aromatic Derivatives Al-Smadi Mousa Ratrout Samer Department of Applied Chemical Sciences, Jordan University of Science and Technology, P.O. Box 3030, Irbid 22110, Jordan Author to whom correspondence should be addressed; e-mail: [email protected] 9 2005 9 2005 10 9 1126 1134 23 4 2005 © 2005 by MDPI (http://www.mdpi.org).2005Reproduction is permitted for noncommercial purposes.The aromatic polyketones 3a-d are versatile compounds for the synthesis of the multi-1,2,3-selenadiazole aromatic derivatives 1a-d. The preparation starts with the reaction between the multi-bromomethylene benzene derivatives 2a-d and 4-hydroxy-acetophenone to give compounds 3a-d which are transformed through the reaction with semicarbazide hydrochloride or ethyl hydrazine carboxylate into the corresponding semicarbazones derivatives 4a-d or hydrazones 5a-d. The reaction with selenium dioxide leads to regiospecific ring closure of semicarbazones or hydrazones to give the multi-1,2,3-selenadiazole aromatic derivatives in high yield. 1,2,3-Selenadiazolessemicarbazonesaromatic derivatives ==== Body Introduction Heterocyclic systems with multi-arm 1,2,3-thiadiazoles were recently prepared by Meier et. al [1,2] and heterocyclic systems containing two 1,2,3-selenadiazole rings were also recently prepared by Reddy et. al [3,4], but multi-arm 1,2,3-selenadiazoles are still unknown. Therefore depending on the previous experience of the principal investigator in synthesizing multi-arm 1,2,3-thiadiazoles, the analogous multi-arm selenadiazoles were prepared following the method that was first reported by Lalezari et. al [5,6,7], through reaction in the presence of acetic acid of selenium dioxide with α-ketomethylene semicarbazones or hydrazones which contain aminocarbonyl or ethoxycarbonyl groups as good leaving groups. NOE measurements showed that the (E)-configuration largely predominated around the CN double bond. Selenium containing heterocycles are of increasing interest because of their interesting chemical properties [8,9,10,11,12,13] and varied biological activities [14,15,16]. Remarkable differences are known to exist between Se- and S-containing compounds. Due to the larger size of the Se-atom, selenium compounds show an increased polarizability and therefore they are, in general, less stable than the corresponding S-analogues [17,18,19,20]. We report herein on our efforts to generate the multi-branched benzene derivatives 1a, 1b, 1c and 1d, in which the 1,2,3-selenadiazole rings are linked to the central benzene core via phenoxymethylene spacers. molecules-10-01126-sch001_Scheme 1Scheme 1 Results and Discussion Our synthetic procedure (see Scheme 1) started from the commercially available bromomethyl-benzene derivatives 2a-2d. Multiple substitution with 4-hydroxyacetophenone gave the corresponding polyketones 3a-3d, which were transformed into the target compounds 1a-1d by the reaction of the corresponding semicarbazones 4a-4d or ethoxycarbonyl hydrazones 5a-5d, essentially as described by Lalezari et. al. [5,6,7]. The yields of all three steps were optimized, so that the total overall yields for the sequences 2a → 1a, 2b → 1b, 2c → 1c and 2d → 1d amounted to 90%, 85%, 89% and 85%, respectively (Table 1). Experimental General The solvents were purified by standard procedures. The melting points (m.p.) were determined on an Electrothermal digital melting point apparatus and are uncorrected. Infrared (IR) spectra of pure substances were recorded as KBr-pellets using a Nicolet 410 FT-IR spectrometer (ν in cm-1). The 1H- and 13C-NMR spectra were recorded on Bruker AM400 and AC200 spectrometers in CDCl3 or DMSO-d6 using TMS as internal standard. The spectral data are reported in delta (δ) units relative to the TMS reference peak. The mass spectra were recorded using a Finnigan MAT95 field desorption (FD, 5 kV ionizing energy) instrument. The signals are given as m/z with the relative intensity between brackets. Elemental analyses were performed in the Analytical Laboratory of the Institute of Organic Chemistry of University of Mainz, Germany. Bromo compounds 2a-2d (1,2,3,4,5,6-hexakis-, 1,2,4,5-tetrakis-, 1,3,5-tri- and 1,4-dibromomethylbenzene, respectively), ethyl hydrazine carboxylate, semicarbazide hydrochloride and sodium acetate were obtained from Aldrich. General Procedure for the Preparation of Multi-Ketones 3a-d [1] A mixture of 4-hydroxyacetophenone (1 equivalent) and 2a (0.14 equivalents), 2b (0.21 equivalents), 2c (0.3 equivalents) or 2d (0.45 equivalents), potassium carbonate (1 equivalent) and potassium iodide (in the same equivalent amount as the bromo compound used) plus a few drops of Aliquat 336 were refluxed in dry acetone (100 mL) for 48 hours. The reaction was followed by TLC (eluent: chloroform) till completion. After cooling, the reaction mixture was diluted with water (50 mL) and extracted with dichloromethane (3 × 40 mL). The combined organic layers were dried over magnesium sulphate. The solvent was evaporated under vacuum and the residual solid was washed with diethyl ether. When necessary, a recrystalization from acetone or chloroform was performed. 1-{4-[2,3,4,5,6-Penta(4-acetylphenoxymethyl)benzyloxy]phenyl}-1-ethanone (3a). Colorless powder (87% yield), m.p. 234-235°C; IR: ν 1675, 1604, 1508, 1239, 1002, 832 cm-1; 1H-NMR (CDCl3): 2.50 (s, 3H, CH3), 5.26 (s, 2H, CH2O), 6.89, 7.83 (d, d, 2H, 2H, AA′BB′); 13C-NMR (CDCl3): 26.20 (CH3), 63.60 (CH2O), 137.70 (central benzene ring Cq), 114.21 and 130.70 (side chain benzene CH), 131.30, 161.80 (side chain benzene Cq), 196.40 (C=O); MS: 967 (M+, 100); Anal. % Calcd. for C60H54O12: C, 74.52; H, 5.63. Found: C, 74.27; H, 5.54. 1-{4-[2,4,5-Tri(4-acetylphenoxymethyl)benzyloxy]phenyl}-1-ethanone (3b). Colorless powder (90% yield); m.p. 224-226°C; IR: ν 1665, 1590, 1502, 1250, 1170, 830 cm-1; 1H-NMR (CDCl3): 2.53 (s, 3H, CH3), 5.23 (s, 2H, CH2O), 6.94, 7.89 (d, d, 2H, 2H, AA′BB′), 7.65 (s, benzene ring central CH); 13C-NMR (CDCl3): 26.30 (CH3), 67.70 (CH2O), 129.80 (central benzene ring CH), 135.10 (central benzene ring Cq), 114.41 and 130.60 (side chain benzene CH), 131.10, 162.10 (side chain benzene Cq), 196.50 (C=O); MS: 670 (M+, 100); Anal. % Calcd. for C42H38O8: C, 75.21; H, 5.71. Found: C, 75.13; H, 5.59. 1-{4-[3,5-Di(4-acetylphenoxymethyl)benzyloxy]phenyl}-1-ethanone (3c). Pale yellow powder (100% yield); m.p. 82-83°C; IR: ν 2910, 1670, 1591, 1500, 1250, 1172, 836 cm-1. 1H-NMR (DMSO-d6): 2.49 (-s, 3H, CH3), 5.22 (s, 2H, CH2O), 7.09, 7.90 (d, d, 2H, 2H, AA′BB′), 7.53 (s, benzene ring central CH); 13C-NMR (DMSO-d6): 26.30 (CH3), 69.20 (CH2O), 126.60 (central benzene ring CH), 137.20 (central benzene ring Cq), 114.61 and 130.50 (side chain benzene CH), 130.10, 162.00 (side chain benzene Cq), 196.30 (C=O); MS: 523 (M+, 100); Anal. % Calcd. For C33H30O6: C, 75.84; H, 5.79. Found: C, 75.81; H, 5.68. 1-{4-[4-Mono-(4-acetylphenoxymethyl) benzyloxy] phenyl}-1-ethanone (3d). Colorless crystals (89% yield); m.p. 181-182°C; IR: ν 1668, 1591, 1239, 996, 823 cm-1; 1H-NMR (CDCl3): 2.53 (s, 3H, CH3), 5.12 (s, 2H, CH2O), 7.00, 7.90 (d, d, 2H, 2H, AA′BB′), 7.44 (d, benzene ring central CH); 13C-NMR (CDCl3): 26.40 (CH3), 69.70 (CH2O), 127.80 (central benzene ring CH), 136.20 (central benzene ring Cq), 114.50 and 130.60 (side chain benzene CH), 130.60, 162.40 (side chain benzene Cq), 196.70 (C=O); MS: 374 (M+, 100); Anal. % Calcd. for C24H22O4: C, 76.99; H, 5.92. Found: C, 76.78; H, 5.81. General procedure for the preparation of multiple semicarbazones 4a-d A mixture of semicarbazide hydrochloride (1 equivalent) and sodium acetate (1 equivalent) was dissolved in absolute ethanol (40 mL). The mixture was heated for 15 min under reflux, then filtered while hot to remove precipitated sodium chloride. The filtrate was mixed with ketone 3a (0.14 equivalents), ketone 3b (0.21 equivalents), ketone 3c (0.30 equivalents) or ketone 3d (0.45 equivalents), respectively. The reaction mixture was heated to reflux then two drops of concentrated hydrochloric acid were added. The mixture was heated under reflux for overnight with continuously removal of generated water. After that the solvent was removed under vacuum and the residue was washed with diethyl ether. 1-{4-[2,3,4,5,6-Penta(4-acetylphenoxymethyl) benzyloxy]phenyl}-1-ethanone-N-aminocarbonyl semi-carbazone (4a). White powder (91% yield), m.p. 300°C (dec.); IR: ν 3417, 3212, 1687, 1597, 1431, 1239, 1014, 829 cm-1; 1H-NMR (DMSO-d6): 2.08 (s, 3H, CH3-C=N)), 5.37 (s, 2H, CH2O), 6.41 (s, 2H, NH2), 6.93, 7.70 (d, d, 2H, 2H, AA′BB′), 9.18 (s, 1H, N-H); 13C-NMR (DMSO-d6): 13.29 (CH3-C=N), 63.90 (CH2O), 137.60 (central benzene ring Cq), 114.30 and 127.50 (side chain benzene CH), 130.40, 159.60 (side chain benzene Cq), 143.90 (C=N), 157.40 (C=O); MS: 1309.4 (M+, 100); Anal. % Calcd. for C66H72N18O12: C, 60.54; H, 5.54; N, 19.25, Found: C, 60.35; H, 5.54; N, 19.30. 1-{4-[2,4,5-Tri(4-acetylphenoxymethyl)benzyloxy]phenyl}-1-ethanone-N-aminocarbonylsemi-carbazone (4b). White powder (81% yield); m.p. 250°C (dec.); IR: ν 3429, 3180, 1670, 1599, 1418, 1258, 836 cm-1; 1H-NMR (DMSO-d6): 2.12 (s, 3H, CH3-C=N)), 5.34 (s, 2H, CH2O), 5.99 (s, 2H, NH2), 7.06, 7.86 (d, d, 2H, 2H, AA′BB′), 7.71 (s, central benzene ring CH), 9.19 (s, 1H, N-H); 13C-NMR (DMSO-d6): 13.38 (CH3-C=N), 67.08 (CH2O), 129.10 (central benzene ring CH), 134.80 (central benzene ring Cq), 114.70 and 127.50 (side chain benzene CH), 130.50, 159.70 (side chain benzene Cq), 144.00 (C=N), 157.50 (C=O); MS: 899 (M+, 100); Anal. % Calcd. for C46H50N12O8: C, 62.33; H, 5.67; N, 18.17, Found: C, 62.29; H, 5.61; N, 18.05. 1-{4-[3,5-Di(4-acetylphenoxymethyl)benzyloxy]phenyl}-1-ethanone-N-aminocarbonyl semicarbazone (4c). Pale yellow powder (100% yield); m.p. 240°C (dec.); IR: ν 3417, 3250, 1681, 1579, 1508, 1470, 1226, 829 cm-1; 1H-NMR (DMSO-d6): 2.13 (s, 3H, CH3-C=N)), 5.15 (s, 2H, CH2O), 6.55 (s, 2H, NH2), 6.98, 7.76 (d, d, 2H, 2H, AA′BB′), 7.49 (s, central benzene ring CH), 9.25 (s, 1H, N-H); 13C-NMR (DMSO-d6): 13.38 (CH3-C=N), 69.15 (CH2O), 126.44 (central benzene ring CH), 137.76 (central benzene ring Cq), 114.54 and 127.50 (side chain benzene CH), 131.20, 158.73 (side chain benzene Cq), 144.11 (C=N), 157.90 (C=O); MS: 693.77 (M+, 100); Anal. % Calcd. for C36H39N9O6: C, 61.46; H, 5.61; N, 18.70, Found: C, 63.32; H, 5.51; N, 18.63. 1-{4-[4-(4-acetylphenoxymethyl)benzyloxy]phenyl}-1-ethanone-N-aminocarbonyl semicarbazone (4d). White powder (100% yield); m.p. 300°C (dec.); IR: ν 3417, 3212, 1681, 1604, 1502, 1418, 1239, 1014, 829 cm-1; 1H-NMR (DMSO-d6): 2.12 (s, 3H, CH3-C=N)), 5.20 (s, 2H, CH2O), 5.90 (s, 2H, NH2), 7.08, 7.89 (d, d, 2H, 2H, AA′BB′), 7.45 (d, central benzene ring CH), 9.21 (s, 1H, N-H); 13C-NMR (DMSO-d6): 13.30 (CH3-C=N), 69.30 (CH2O), 127.80 (central benzene ring CH), 136.41 (central benzene ring Cq), 114.50 and 128.00 (side chain benzene CH), 131.17, 159.70 (side chain benzene Cq), 143.00 (C=N), 158.70 (C=O); MS: 488.5 (M+, 100); Anal. % Calcd. for C26H28N6O4: C, 63.92; H, 5.78; N, 17.20, Found: C, 63.71; H, 5.72; N, 17.15. General procedure for the preparation of multiple hydrazones 5a-d [1]. A solution of di- or tri- or tetra- or hexaketone 3a-d (1 equivalent), a few drops of concentrated hydrochloric acid and 6, 9, 12 or 18 equivalents of ethyl hydrazinecarboxylate in dry chloroform (50 mL) was heated under reflux overnight with continuously removal of generated water. The solution was concentrated and the residue was washed with diethyl ether and chloroform. 1-{4-[2,3,4,5,6-Penta(4-acetylphenoxymethyl)benzyloxy]phenyl}-1-ethanone-N-ethoxycarbonyl hydrazone (5a). White solid powder (92% yield); m.p. 293°C (dec.); IR: ν 3231, 1719, 1610, 1502, 1233, 1047, 829 cm-1; 1H-NMR (DMSO-d6): 1.23 (t, 3H, CH3CH2), 2.12 (s, 3H, CH3-C=N)), 4.13 (q, 2H, CH3CH2), 5.29 (s, 2H, CH2O), 6.95, 7.58 (d, d, 2H, 2H, AA′BB′), 9.96 (s, 1H, N-H); 13C-NMR (DMSO-d6): 14.23 (CH3CH2), 15.01 (CH3-C=N), 60.95 (CH3CH2O), 64.36 (CH2O-ph), 138.09 (central benzene ring Cq), 114.86 and 127.94 (side chain benzene CH), 131.93, 159.41 (side chain benzene Cq), 149.25 (C=N), 154.76 (CO2); MS: 1484 (M+,100); Anal. % Calcd. for C78H90N12O18: C, 63.15; H, 6.11; N, 11.33, Found: C, 62.79; H, 6.27; N, 11.44. 1-{4-[2,4,5-Tri-(4-acetylphenoxymethyl)benzyloxy]phenyl}-1-ethanone-N-ethoxycarbonyl hydrazone (5b). White powder (87% yield), m.p. 211-213°C; IR: ν 3200, 3045, 1700, 1596, 1492, 1235, 1040, 828 cm-1; 1H-NMR (DMSO-d6): 1.24 (t, 3H, CH3CH2), 2.16 (s, 3H, CH3-C=N)), 4.16 (q, 2H, CH3CH2), 5.27 (s, 2H, CH2O), 7.03, 7.65 (d, d, 2H, 2H, AA′BB′), 7.71 (s, central benzene ring CH), 9.98 (s, 1H, N-H); 13C-NMR (DMSO-d6): 14.28 (CH3CH2), 15.19 (CH3-C=N), 60.98 (CH3CH2O), 67.40 (CH2O-ph), 129.37 (central benzene ring CH), 135.43 (central benzene ring Cq), 115.08 and 128.00 (side chain benzene CH), 131.76, 159.33 (side chain benzene Cq), 148.70 (C=N), 154.80 (CO2); MS: 1015 (M+, 100); Anal. % Calcd. for C54 H62N8O12: C, 63.89; H, 6.16; N, 11.04, Found: C, 63.58; H, 6.09; N, 10.97. 1-{4-[3,5-Di(4-acetylphenoxymethyl)benzyloxy]phenyl}-1-ethanone-N-ethoxycarbonyl hydrazone (5c). Pale yellow powder (85% yield), m.p. 202-204°C; IR: ν 3200, 3035, 1705, 1600, 1503, 1238, 1040, 830 cm-1; 1H-NMR (DMSO-d6): δ 1.24 (t, 3H, CH3CH2), 2.17 (s, 3H, CH3-C=N)), 4.15 (q, 2H, CH3CH2), 5.18 (s, 2H, CH2O), 7.03, 7.67 (d, d, 2H, 2H, AA′BB′), 7.52 (s, central benzene ring CH), 9.99 (s, 1H, N-H); 13C-NMR (DMSO-d6): δ 14.30 (CH3CH2), 15.20 (CH3-C=N), 60.90 (CH3CH2O), 69.60 (CH2O-ph), 126.90 (central benzene ring CH), 138.06 (central benzene ring Cq), 115.10 and 128.00 (side chain benzene CH), 131.60, 159.50 (side chain benzene Cq), 148.70 (C=N), 154.20 (CO2); MS: 781 (M+, 100); Anal. % Calcd. for C42H48N6O9: C, 64.60; H, 6.20; N, 10.76, Found: C, 64.48; H, 6.13; N, 10.50. 1-{4-[4-Mono(4-acetylphenoxymethyl)benzyloxy]phenyl}-1-ethanone-N-ethoxycarbonyl hydrazone (5d). White powder (91% yield); m.p. 259-260°C; IR: ν 3205, 3035, 1705, 1600, 1500, 1230, 822 cm-1; 1H-NMR (DMSO-d6): δ 1.22 (t, 3H, CH3CH2), 2.15 (s, 3H, CH3-C=N)), 4.16 (q, 2H, CH3CH2), 5.10 (s, 2H, CH2O), 6.99, 7.87 (d, d, 2H, 2H, AA′BB′), 7.43 (d, central benzene ring CH), 9.99 (s, 1H, N-H); 13C-NMR (DMSO-d6): δ 13.60 (CH3CH2), 14.70 (CH3-C=N), 60.80 (CH3CH2O), 67.80 (CH2O-Ph), 127.60 (central benzene ring CH), 136.30 (central benzene ring Cq), 114.50 and 127.60 (side chain benzene CH), 130.50, 160.10 (side chain benzene Cq), 148.81 (C=N), 154.30 (CO2); MS: 547 (M+, 100); Anal. % Calcd. for C30H34N4O6: C, 65.92; H, 6.27; N, 10.25, Found: C, 65.99; H, 6.18; N, 10.15. General procedure for preparation of multiple 1,2,3-selenadiazoles 1a-d Hydrazone 5a (0.47 mmol) or 5b (0.59 mmol) or 5c (0.9 mmol) or 5d (0.27 mmol) was dissolved in glacial acetic acid (30 mL) with vigorous stirring and gentle heating to 40-45°C. The solution was treated with selenium dioxide powder (8.46 mmol, 7.08 mmol, 8.1 mmol or 0.81 mmol, respectively) and the mixture was kept under gentle heating with vigorous stirring. After 2 min, the color of the mixture becomes red. Monitoring of the reaction by TLC showed that the reaction was complete in two days. The mixture was filtered and the filtrate was poured into ice water and extracted with chloroform (3 × 50 mL). The combined organic layers were washed with saturated sodium hydrogen carbonate solution and dried using magnesium sulphate. The solvent was removed under vacuum. The crude product was purified by chromatography using methanol or ethyl acetate as eluents, followed by recrystallization from chloroform/hexane. 4-(4-{2,3,4,5,6-Penta[4-(1,2,3-selenadiazole-4-yl)phenoxymethyl]benzyloxy}phenyl)-1,2,3-selenadiazole (1a). Yellow-orange solid (100%); m.p. 77-78°C; IR: ν 3096, 2975, 1739, 1611, 1527, 1469, 1220, 989, 841 cm-1; 1H-NMR (CDCl3): 5.27 (s, 2H, CH2O), 7.06, 7.17 (d, d, 2H, 2H, AA′BB′), 9.68 (s, 1H, CHSe); 13C-NMR (CDCl3): 63.62 (CH2O), 128.50 (central benzene ring Cq), 115.36 and 130.20 (side chain benzene CH), 119.20, 152.40 (side chain benzene Cq), 159.61 (C=N), 137.70 (CHSe); MS: 1500 (M+, 100); Anal. % Calcd. For C60H42N12O6Se6: C, 48.01; H, 2.82; N, 11.20; Se, 31.57, Found: C, 47.91; H, 2.74; N, 11.11. 4-(4-{2,4,5-Tri[4-(1,2,3-selenadiazole-4-yl)phenoxymethyl]benzyloxy}phenyl)-1,2,3-selenadiazole (1b). Yellow-orange solid (92% yield); m.p. 87-88°C; IR: ν 3096, 2930, 1725, 1611, 1527, 1469, 1245, 1040, 822 cm-1; 1H-NMR (CDCl3): 5.25 (s, 2H, CH2O), 7.12, 7.22 (d, d, 2H, 2H, AA′BB′), 7.70 (s, benzene central ring CH), 9.71 (s, 1H, CHSe); 13C-NMR (CDCl3): 67.70 (CH2O), 124.00 (central benzene ring CH), 129.80 (central benzene ring Cq), 115.89 and 130.12 (side chain benzene CH), 118.80, 152.60 (side chain benzene Cq), 159.92 (C=N), 136.40 (CHSe); MS: 1026 (M+, 100); Anal. % Calcd. For C42H30N8O4Se4: C, 49.13; H, 2.95; N, 10.91; Se, 30.77, Found: C, 49.10; H, 2.75; N, 10.71. 4-(4-{3,5-Di[4-(1,2,3-selenadiazole-4-yl)phenoxymethyl]benzyloxy} phenyl)-1,2,3-selenadiazole (1c). Yellow-orange solid (87% yield); m.p.100-102°C; IR: ν 3096, 2917, 1738, 1610, 1534, 1450, 1239, 1046, 835 cm-1; 1H-NMR (CDCl3): 5.15 (s, 2H, CH2O), 7.11, 7.20 (d, d, 2H, 2H, AA′BB′), 7.49 (s, central benzene ring CH), 9.72 (s, 1H, CHSe); 13C-NMR (CDCl3): 69.69 (CH2O), 126.00 (central benzene ring CH), 129.70 (central benzene ring Cq), 115.99 and 130.03 (side chain benzene CH), 118.80, 149.15 (side chain benzene Cq), 160.15 (C=N), 137.60 (CHSe); MS: 789 (M+, 100); Anal. % Calcd. For C33H24N6O3Se3: C, 50.20; H, 3.06; N, 10.64; Se, 30.01, Found: C, 50.10; H, 3.00; N, 10.59. 1,4-Bis[4-(1,2,3-selenadiazol-4-yl)phenoxymethyl]benzene (1d). Yellow-orange solid (60% yield); m.p. 160-162°C; IR: ν 3077, 2911, 1732, 1610, 1533, 1456, 1239, 1008, 822 cm-1; 1H-NMR (DMSO-d6): 5.15 (s, 2H, CH2O), 7.12, 7.21 (d, d, 2H, 2H, AA′BB′), 7.47 (d, benzene central ring CH), 9.72 (s, 1H, CHSe); 13C-NMR (DMSO-d6): 70.00 (CH2O), 127.89 (central benzene ring CH), 129.18 (central benzene ring Cq), 116.10 and 130.00 (side chain benzene CH), 121.00, 152.00 (side chain benzene Cq), 160.00 (C=N), 138.00 (CHSe); MS: 552 (M+, 100); Anal. % Calcd. For C24H18N4O2Se2: C, 52.18; H, 3.28; N, 10.14; Se, 28.60, Found: C, 52.03; H, 3.25; N, 10.20. Acknowledgements We are grateful to the Deanship of Scientific Research of the Jordan University for Science and Technology for financial support. Also, we thank Prof. H. Meier from Mainz University-Germany for helpful discussions. Sample availability: Contact the author. molecules-10-01126-t001_Table 1Table 1 Ring system R Cpd. Yield Cpd. Yield Cpd. Yield Cpd. Yield Br 2a ― 2b ― 2c ― 2d ― 3a 87 3b 90 3c 95 3d 89 4a 91 4b 81 4c 96 4d 98 5a 92 5b 87 5c 85 5d 91 1a 96 1b 92 1c 87 1d 60 ==== Refs References 1. Al-Smadi M. Meier H. Liebigs Ann. 1997 2357 2. Al-Smadi M. Hanold N. Meier H. J. Heterocycl. Chem. 1997 34 605 3. Reddy D.B. Babu N.C. Padmavathi V. Padmaja A. Tetrahedron 1997 53 17351 4. Bhaskar Reddy D. Somasekhar Reddy A. Padmavathi V. Synth. Commun. 2001 31 3429 5. Lalezari I. Shafiee A. Tetrahedron Lett. 1969 5105 6. Lalezari I. Shafiee A. J. Org. Chem. 1971 36 2836 7. Lalezari I. Shafiee A. Yalpani M. J. Org. Chem. 1973 38 338 8. Zhou Y. Heimgartner H. Helv. Chem. Acta 2000 83 539 9. Petrov M.L. Abramov M.A. Dehaen W. Toppet S. Tetrahedron Lett. 1999 40 3903 10. Detert H. Meier H. Liebigs Ann. 1997 1557 11. Zhou Y. Heimgartner H. Helv. Chem. Acta 2000 83 539 12. Nishiyama Y. Hada Y. Anjiki M. Hanita S. Sonoda N. Tetrahedron Lett. 1999 40 6293 13. Arsenyan P. Oberte K. Pudova O. Lukevics E. Chem. Heterocycl. Comp. 2002 38 1437 14. Burling F.T. Goldenstein B.M. J. Am. Chem.Soc. 1992 114 2313 15. Jalilian A.R. Sattari S. Bineshmarvasti M. Daneshtalab M. Shafiee A. Farmaco 2003 58 63 12595038 16. Lalezari I. Shafiee A. Khorrami J. Soltani A. J. Pharm. Sci. 1987 67 1336 17. Abramov M.A. Dehaen W. D’Hooge B. Petrov M.L. Smeets S. Toppet S. Voets M. Tetrahedron 2000 56 3933 18. D’Hooge B. Smeets S. Toppet S. Dehaeam S. J. Chem. Soc. Chem. Commun. 1997 1753 19. L’Abbe G. Haelterman B. Dehaen W. J. Chem. Soc. Perkin Trans. 1 1994 2203 20. Malek-Yazdi F. Yalpani M. Synthesis 1977 328	18007378	PMC6147698	NO-CC CODE	2021-01-05 20:52:04	yes	Molecules. 2005 Sep 30; 10(9):1126-1134
==== Front MoleculesMoleculesmoleculesMolecules : A Journal of Synthetic Chemistry and Natural Product Chemistry1420-3049MDPI 10.3390/60800647molecules-06-00647ArticleSynthesis, Structure and Antitumor Activity of Dibutyltin Oxide Complexes with 5-Fluorouracil Derivatives. Crystal Structure of [(5-Fluorouracil)-1-CH2CH2COOSn(n-Bu) 2]4O2 Zuo Dai-shu 1Jiang Tao 1Guan Hua-shi 1Wang Kui-qi 1Qi Xin 1Shi Zhan 21 Marine Drug and Food Institute, Ocean University of Qingdao, Qingdao, China 2660032 Key Laboratory of Inorganic Synthesis and Preparative Chemistry, Jilin University, Changchun, China, 130022 Author to whom correspondence should be addressed; Tel.: (+86) 0532-2032712; Fax: (+86) 0532-2033054; E-mail:[email protected] 7 2001 8 2001 6 8 647 654 19 3 2001 20 3 2001 02 5 2001 © 2001 by MDPI (http://www.mdpi.org).2001Reproduction is permitted for noncommercial purposes.Dibutyltin (IV) oxide complex reacts with the fluorouracil compounds 5-fluorouracil-1-propanonic or 5-fluorouracil-1-acetic acid (Fu) to give the complexes [(5-Fu)-1-(CH2)nCOOSn(n-Bu)2]4O2 (I, n=2; II, n=1) which were characterized by IR and 1H-NMR. The crystal structure of complex I shows that the molecular is a dimer, in which two [(5-Fu)-1-CH2CH2COOSn(n-Bu)2]2O units are linked by a bridging oxygen atom, and the tin atoms adopt distorted trigonal bipyramids via two carbons from a dibutyl moiety and three oxygen atoms from 5-Fu and bridging oxygen. These complexes have potential anti-tumour activity: in vitro tests showed that complexes I and II exhibit high cytotoxicity against OVCAR-3 and PC-14. Organotin complex5-Fusynthesiscrystal structureantitumor activity. ==== Body Introduction Many groups have reported the chemotherapeutic properties of tin compounds possessing anti-tumor activity [1,2,3,4,5]. Since then intense interest has developed in this field and a large number of organotin compounds have been synthesized and tested [6,7]. Among these compounds dibutyltin derivatives have displayed both higher activity and relatively low toxicity [8]. On the other hand, the cycle-specific schedule dependent antimetabolite 5-fluorouracil (5-Fu) has been in clinical use for 40 years and has evolved as an important agent in the treatment of a large spectrum of tumors, including all gastrointestinal cancers and breast cancer. While attempting to study organotin (IV) complexes as possible candidates for anti-tumor agents and the structure-activity relationships of these complexes, we successfully prepared two kinds of new 5-Fu dibutylorganotin (IV) derivatives and determined the X-ray crystal structure of one of them. At the same time, their anticancer activity was also tested. We report these results herein. Results and Discussion Synthesis The [(5-fluorouracil)-1-(CH2)nCOOSn(n-Bu)2]4O2 compounds I (n=2) and II (n=1) were synthesized by the reaction of (Bu)2SnO with (5-fluorouracil)-1-(CH2)nCOOH (n = 2,1) in the appropriate molar ratio of 1:1. A possible mechanism for the formation of compounds I and II is shown in Scheme 1 molecules-06-00647-scheme1_Scheme 1Scheme 1 The synthesis of [(5-Fu)-1-(CH2)2COOSn(n-Bu)2]4O2 The (5-fluorouracil)-1-(CH2)nCOOH (n = 2, 1) staring materials were obtained by the reaction of 5-fluorouracil with CH2=CHCN (n = 2) or ClCH2COOH (n = 1) in NaOH solution. 4Å molecular sieves were used as the catalyst for the dehydration reaction. If the reaction temperature were raised to 80˚C, the 2:1 molar ratio (5-Fu:Sn) complexes would be formed. The resulting complexes I and II are stable in the air and are difficult to dissolve in aromatic or ether solvents, but can be easily dissolved in methanol or H2O when heated. Molecular structure The IR and 1H-NMR spectroscopic spectra supported the fact that the complexes I and II contain the expected 5-Fu and butyl moieties. In order to confirm their molecular structure, the X-ray crystal structure of I was also determined. Its molecular structure and the unit cell are shown in Figure 1 and Figure 2, and selected interatomic bond distances (Å) and angles (°) are listed in Table 1. Figure 1 Molecular structure and atomic numbering system for [5-fluorouracil-1-(CH2)2COOSn(n-Bu)2]4O2 Figure 2 Unit cell of [5-fluorouracil-1-(CH2)2COOSn(n-Bu)2]4O2 molecules-06-00647-t001_Table 1Table 1 Selected Bond lengths [Å] and angles [°] for (I) Bond lengths Sn(1)-O(1A) 2.037(8) Sn(2)-C(20) 2.1432) Sn(1)-C(1) 2.083(17) Sn(2)-O(6) 2.151(9) Sn(1)-C(5) 2.086(15) Sn(2)-O(3A) 2.251(10) Sn(1)-O(1) 2.183(8) O(1)-Sn(1A) 2.037(8) Sn(1)-O(2) 2.269(8) O(2)-C(9) 1.258(14) Sn(2)-O(1) 2.043(7) O(3)-C(9) 1.261(14) Sn(2)-C(16) 2.119(19) O(3)-Sn(2A) 2.251(10) Bond angles O(1A) -Sn(1)-C(1) 106.6(5) C(5)-Sn(1)-O(2) 87.0(4) O(1A)-Sn(1)-C(5) 110.6(4) O(1)-Sn(1)-O(2) 165.6(3) C(1)-Sn(1)-C(5) 141.4(6) O(1)-Sn(2)-C(16) 109.0(5) O(1A)-Sn(1)-O(1) 76.4(3) O(1)-Sn(2)-C(20) 113.1(6) C(1)-Sn(1)-O(1) 100.0(5) C(16)-Sn(2)-C(20) 136.4(7) C(5)-Sn(1)-O(1) 98.2(4) O(1)-Sn(2)-O(6) 79.6(3) O(1A)-Sn(1)-O(2) 89.2(3) C(16)-Sn(2)-O(6) 102.5(6) C(1)-Sn(1)-O(2) 83.7(5) C(20)-Sn(2)-O(6) 95.8(6) O(1)-Sn(2)-O(3A) 91.9(4) Sn(1A)-O(1)-Sn(1) 103.6(3) C(16)-Sn(2)-O(3A) 84.6(6) Sn(2)-O(1)-Sn(1) 118.9(4) C(20)-Sn(2)-O(3A) 83.1(6) C(9)-O(2)-Sn(1) 138.2(8) O(6)-Sn(2)-O(3A) 170.4(3) C(9)-O(3)-Sn(2A) 135.4(9) Sn(1A)-O(1)-Sn(2) 137.4(4) C(24)-O(6)-Sn(2) 118.5(8) O(2)-C(9)-O(3) 123.1(12) This complex is a dimeric compound in which the [(5-Fu)-1-CH2CH2COOSn(n-Bu)2]2O units are linked by bridging oxygen atoms thus increasing coordinative saturation. The tin atom is pentacoordinated. The geometry about the four tin atoms are based on the trigonal bipyramids with the trigonal planes defined by the C (1), C(5), O(1A) atoms for Sn (1); C(16),C(20), O(1) for Sn(2); C(1A), C(5A), O(1) for Sn(1A) and C(16), C (20A), O(1A) for Sn(2A), while the O(1) and O(2), O(3A) and O(6), O(2A) and O(1A), O(6A) and O(3) are in the axial position for Sn(1), Sn(2), Sn(1A) and Sn(2A) respectively. The bonding of the bridging ligands is asymmetric with a Sn(1)-O(1A ) bond length of 2.037(8) Å and a Sn(1)-O(1) contact of 2.183 (8)Å, which forms a parallelogram. The complex also contains two five-member chelate rings, formed via carbonyl oxygen to tin coordination. The bond distances Sn(1)-O(2) (2.269(8)Å), Sn(2A)-O(3) (2.251 (10)Å) are longer than the bond distance of Sn(2)-O(6) or Sn(2A)-O(6A) ( 2.151(9)Å) , and the bond distances of C(9)-O(2) (1.258(14)Å), C(9)-O(3) (1.261(14)Å) are almost equal and both are between the length of C=O double bond (1.212Å) and C-O single bond (1.291Å) in the model complex shown in Figure 3 below [12]. This indicates that the coordination of two of the circular-linked 5-Fu ligand oxygen atoms to tin atoms is weaker than that of the other oxygen atom of the linearly-linked 5-Fu ligand. Figure 3 Biological activity The MTT method was used for a preliminary estimation of the in vitro tumor-inhibiting activity of complexes I and II. The data summarized in Table 2 shows that complexes I and II possess rather high cytotoxicity towards tumor cells of OVCAR-3 and PC-14 and the dibutyltin 5-fluorouracil-1-propanonic acid derivative is more active than the dibutyltin 5-fluorouracil-1-propanonic acid derivative. molecules-06-00647-t002_Table 2Table 2 Inhibitory effect of the complexes on tumor cells of OVCAR-3 and PC-14 (MTT) Tumor cell Complex Concentration of complex (μg/mL) Inhibitory Rate (%) OVCAR-3 I 1 - 10 72.99 100 97.8 II 1 - 10 20.51 100 95.30 PC-14 I 1 - 10 64.93 100 98.1 II 1 - 10 31.98 100 92.71 Experimental: General Methods IR spectra were recorded using KBr pellets on a Nicolet NEXUS 470 FT-IR. 1H-NMR spectra were recorded at 298K for CDCl3 solutions with TMS as the internal reference using a Unity-400MHz-NMR spectrometer. The elemental analyses were determined on a GmhH Varin EL analyzer. Tin was determined using complexometric titration with EDTA and lead nitrate as the volumetric solution. Toluene was dried over Na and distilled prior to use under N2. Dibutyltin oxide was synthesized following the literature method [9]. 5-Fluorouracil-1-acetic acid and 5-fluorouracil-1-propanoic acids were prepared according to the literature methods [10,11]. 5-Fluorouracil was a gift from Rudong Pharmaceutical Factory of Jiangsu. The tumor inhibiting effect of complex 1 and 2 was tested in vitro using the human ovarian carcinoma cell line OVCAR-3 and lung carcinoma cell line PC-14 by the MTT method. General preparation of the complexes of dibutyltin oxide and 5-fluorouracil -1-propanonic acids Both complexes were prepared in a similar fashion and therefore, only the synthesis of one of them, namely [(5-fluorouracil)-1-CH2CH2COOSn(n-Bu)2]4O2 (I) is described in detail herein. 5-Fluorouracil-1-propanonic acid (0.404g, 2mmol) was added to the solution of dibutyltin oxide (0.498g, 2mmol) in toluene (15mL) containing a few 4Å molecular sieves. The mixture was stirred under N2 for 8 hours at a temperature of 50˚C. After cooling down and filtration, the residue was extracted three times with methanol. Concentration of the resulting solution gave colorless needle-shaped crystals of [(5-fluorouracil)-1-CH2CH2COOSn (n-Bu)2]4O2 (I).Yield: 0.5613g (62%); m.p. 168-170˚C (from methanol); 1H-NMR: δ: 0.908 (6H, t, J1=5.6Hz, J2=4.8Hz, CH3), 1.243-1.370 (8H, m, CH2CH2), 1.642 (2H, m, Sn-CH2), 2.799 (2H, t, J1=6.0Hz, J2=5.6Hz, CH2CH2CO), 3.963 (2H, t, J1= 6.0Hz, J2=5.6Hz, N-CH2-CH2), 7.527 (1H, d, J=5.6Hz, =CH), 8.210 (b, NH); IR (cm-1): ν(N-H) 3412, ν(=C-H) 3168, ν(CH3) 3064, 2929, ν(CH2) 2959, 2868, ν(C=O) 1770 (-O-C=O), ν(C=O) 1693(-N-C=O), ν(C=C) 1576, ν(CH) 1339, ν(C-C) 1238, ν(Sn-O) 682, 543; Anal Calcd for C30H46F2N4O9Sn2: C 40.81, H 7.66, N 6.45, Sn 26.91; Found C 40.56, H 7.97, N 6.28, Sn 26.91. Complex II was similarly prepared. Yield: 63.7%; m.p 176-178°C (from methanol); 1H-NMR: δ: 0.881 (6H, t, J1=5.6Hz, J2=4.8Hz, CH3), 1.254-1.341 (8H, m, CH2CH2), 1.647 (2H, m, Sn-CH2), 4.448 (2H, s, N-CH2-CO), 7.521 (1H, s, =CH), 8.184 (b, NH); IR (cm-1): ν(N-H) 3420, ν(=C-H) 3187, ν(CH3) 3071, 2929, ν(CH2) 2956, 2866, ν(C=O) 1693(-O-C=O), ν(C=O) 1680(-N-C=O), ν(C=C) 1599, ν(CH) 1465, ν(CH) 1321, ν(C-C) 1241, ν(Sn-O) 685, 588. Anal Calcd for. C26H38F2N4O9Sn2: C 39.52, H 5.26, N 6.34, Sn 27.98; Found: C 39.28, H 5.18, N 6.54, Sn 27.73.. Crystal structure determination of I A single crystal of 0.34x0.20x0.04mm was placed in a Siemens SMART CCT four-circle diffractometer with MoKα (λ=0.71073Å) and a scan range 1.83≤θ≤23.28 at room temperature. Of the 9452 reflections collected, 5491 reflections with I>2σ(I) were considered to have been observed. The structure was solved by the heavy-atom method and refined by Full-matrix least-squares on F2 by the use of SHELXL Version 5.1 program. The final agreement factor was R=0.0693. Crystal data: C30H46F2N4O9Sn2, Fw=882.09, Triclinic, P1, a=11.423(3)Å, b=12.843(3)Å, c=14.770(4)Å, α=66.711(6)deg, β=79.319(6)deg, γ=78.172(7)deg, V=1935.0(8)Å3, Z=2, F(000)=888, Dc=1.514 mg/m3. Acknowledgements This project was supported by the Foundation for University Key Teachers of the Chinese Ministry of Education and the Foundation of the Key Laboratory of Inorganic Synthesis and Preparative Chemistry, Chinese Ministry of Education. Sample Availability: Available from the authors. ==== Refs References 1. Crown A. J. Drugs of the Future 1987 12 40 2. Biddle B.N. Grey J. S. Appl. Organomet. Chem. 1991 5 43 3. Keppler B.H. Metal Complexes in Cancer Chemotherapy VCH Publishers Weinheim 1993 4. (a) Guli G. Gennaro G. Pellerito L. Stocco G. C. Appl. Organomet. Chem. 1993 7 407 (b) Huber F. Vornefeld M. Preut H. Angerer E. Ruisi G. Appl. Organomet. Chem. 1992 6 597 5. Wang J. T. Progress in Natural Science 1998 8 180 6. Li Z. F. Fu F. X. Pan H. D. Xing Y. Lin Y. H. Acta Chimica Sinica 1999 57 820 7. Tian L. J. Zhou Z. Y. Zhao B. Yu W. T. Polyhedron 1998 17 1275 8. Pettina C. Pellei M. Marchetti F. Santini C. Miliani M. Polyhedron 1998 17 561 9. Hu C. Wang S. Zhao S. Chin. J. Med. Chem. 1994 4 32 (in Chinese) 10. Masao T. Bullet. Chem. Soc. Jap. 1975 48 3427 11. Zhou R. X. Fan C. L. Zhao R. L. Chem. J. Chinese Univ. 1986 7 508 12. Wang J. T. Zhang Y. W. Xu Y. M. Yang Z. W. Youji Huaxue (Chinese J. Org. Chem.) 1993 13 289	0	PMC6236383	NO-CC CODE	2021-01-05 21:43:01	yes	Molecules. 2001 Jul 31; 6(8):647-654
==== Front ACS Nano ACS Nano nn ancac3 ACS Nano 1936-0851 1936-086X American Chemical Society 10.1021/acsnano.8b05189 Article Breast Cancer Chemo-immunotherapy through Liposomal Delivery of an Immunogenic Cell Death Stimulus Plus Interference in the IDO-1 Pathway Lu Jianqin †‡ Liu Xiangsheng †‡ Liao Yu-Pei † Wang Xiang ‡ Ahmed Ayman † Jiang Wen † Ji Ying † Meng Huan †‡§ Nel Andre E. †‡§ †Division of NanoMedicine, Department of Medicine, David Geffen School of Medicine, ‡Center for Environmental Implications of Nanotechnology, California NanoSystems Institute, and §Jonsson Comprehensive Cancer Center, University of California, Los Angeles, Los Angeles, California 90095, United States * Phone: 310.825.0217. E-mail: [email protected]. * Phone: 310.825.6620. E-mail: [email protected]. 16 10 2018 27 11 2018 12 11 1104111061 09 07 2018 27 09 2018 Copyright © 2018 American Chemical Society 2018 American Chemical Society This is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes. Immunotherapy provides the best approach to reduce the high mortality of metastatic breast cancer (BC). We demonstrate a chemo-immunotherapy approach, which utilizes a liposomal carrier to simultaneously trigger immunogenic cell death (ICD) as well as interfere in the regionally overexpressed immunosuppressive effect of indoleamine 2,3-dioxygenase (IDO-1) at the BC tumor site. The liposome was constructed by self-assembly of a phospholipid-conjugated prodrug, indoximod (IND), which inhibits the IDO-1 pathway, followed by the remote loading of the ICD-inducing chemo drug, doxorubicin (DOX). Intravenous injection of the encapsulated two-drug combination dramatically improved the pharmacokinetics and tumor drug concentrations of DOX and IND in an orthotopic 4T1 tumor model in syngeneic mice. Delivery of a threshold ICD stimulus resulted in the uptake of dying BC cells by dendritic cells, tumor antigen presentation and the activation/recruitment of naïve T-cells. The subsequent activation of perforin- and IFN-γ releasing cytotoxic T-cells induced robust tumor cell killing at the primary as well as metastatic tumor sites. Immune phenotyping of the tumor tissues confirmed the recruitment of CD8+ cytotoxic T lymphocytes (CTLs), disappearance of Tregs, and an increase in CD8+/FOXP3+ T-cell ratios. Not only does the DOX/IND-Liposome provide a synergistic antitumor response that is superior to a DOX-only liposome, but it also demonstrated that the carrier could be effectively combined with PD-1 blocking antibodies to eradicate lung metastases. All considered, an innovative nano-enabled approach has been established to allow deliberate use of ICD to switch an immune deplete to an immune replete BC microenvironment, allowing further boosting of the response by coadministered IDO inhibitors or immune checkpoint blocking antibodies. immunogenic cell death indoleamine 2,3-dioxygenase immune checkpoint chemo-immunotherapy dual-delivery liposome doxorubicin breast cancer document-id-old-9nn8b05189 document-id-new-14nn-2018-05189g ccc-price ==== Body Athough the treatment of localized breast cancer (BC) is highly successful with a 5 year survival rate of ∼90%, metastatic breast cancer (MBC) is generally considered incurable with a high mortality rate, regardless of the use of radiation, chemotherapy, or estrogen blockers.1,2 In spite of the bleak picture for MBC, optimism has emerged with the advent of cancer immunotherapy, which utilizes the power of T-cell immunity to treat solid cancers, including BC. This is best exemplified by the use of immune checkpoint blocking antibodies, which have changed the treatment landscape for cancer, such as melanoma, renal cell carcinoma, and non-small cell lung cancer (NSCLC).3−5 However, BC is relatively resistant to treatment with checkpoint inhibitors,6 putatively because of its comparative immune deplete (“cold”) microenvironment, absence of tumor-infiltrating lymphocytes (TILs), and expression of poorly potent cytotoxic T lymphocytes (CTLs) for the initiation of tumor killing.6,7 These data are also in agreement with the observation that BC tumors tend to express a low burden of nonsynonymous DNA mutations, which serve as the tumor antigen neo-epitopes capable of inducing a robust T-cell response.8,9 This is also compatible with the notion that cancers with a high burden of nonsynonymous mutations are the most responsive to immune checkpoint inhibitors.10,11 In addition to its “cold” immune status, the strong immune suppressive micromilieu in the BC tumor site prevents effective T-cell priming.12,13 This includes a contribution from a number of immune suppressive mechanisms that are even more broadly oppressive than the checkpoint receptors being targeted by checkpoint blocking antibodies.6,7 In addition to the utility of immune checkpoint inhibitors to initiate immunotherapy, chemotherapy can positively impact the immune system, leading to increased density of TILs at the BC tumor site, which correlates with improved disease prognosis.14−16 In this regard, Denkert et al. demonstrated in a large BC study that the density of TILs predicts pathological complete responses (pCRs) to neoadjuvant chemotherapy, including during treatment with docetaxel, DOX, and cyclophosphamide.14 This is particularly evident in epidermal growth factor receptor 2-positive and triple negative (TNBC) disease.15 Moreover, neoadjuvant therapy with anthracycline drugs has demonstrated that an increase in the ratio of tumor-infiltrating CD8+ CTLs versus FOXP3+ regulatory T-cells correlates with the elimination of hyperploid BC cells in post-treatment biopsy specimens.17,18 Because the presence of activated CTLs is accompanied by IFN-γ production, which controls PD-L1 at the site of immune responsive cancers,19 it is noteworthy that PD-1 or PD-L1 receptor blocking antibodies could elicit significant objective response rates (∼20%) in TNBC or HER2–/ER+ breast cancer tumors expressing >1% PD-L1 on the tumor cell surface.20 In light of above observations, it is rational to ask whether the deliberate application of chemotherapeutic agents can reproducibly prime the immune response at the BC tumor site as a prelude to a practical approach for boosting immunotherapy to the immune checkpoint inhibitors. One possible approach is the use of chemo agents to induce immunogenic cell death (ICD) at the tumor site. ICD is a specialized form of tumor cell death that can be triggered by specific chemotherapeutic agents such as anthracyclines, taxanes, and oxaliplatin.21−23 ICD facilitates tumor antigen cross-presentation in dendritic cells as a result of calreticulin (CRT) expression on the dying tumor cell surface (Figure 1).21 CRT provides an “eat-me” signal for dendritic cell uptake via the CD91 receptor.24−27 Moreover, the delayed release of adjuvant stimuli, such as high mobility group box 1 protein (HMGB1; a TLR-4 ligand) and ATP (a danger signal that activates the NRLP3 inflammasome), provides additional stimuli for dendritic cell maturation and the ability to present tumor antigens to naïve T-cells.21,22,28−31 ICD provides a deliberate means of triggering TIL recruitment prior to response boosting by additional immune modulators, including antibodies that bind immune checkpoint receptors or metabolic immune surveillance pathways that prevent effective T-cell priming.32 An important example is the indoleamine 2,3-dioxygenase (IDO-1) pathway that is overexpressed at the BC tumor site.33 Figure 1 Schematic to explain BC immunotherapy by combined delivery of an immunogenic cell death stimulus plus an inhibitor of the IDO-1 pathway. Doxorubicin (DOX) delivery to the tumor site provides an effective stimulus for immunogenic cell death (ICD), which is characterized by calreticulin (CRT) expression (an “eat-me” signal for dendritic cell uptake) on the cancer cell surface. Subsequent release of adjuvant stimuli, HMGB-1 and ATP, by the dying cancer cells induce DC maturation and tumor antigen presentation to naïve T-cells. Recruitment of CD8+ cytotoxic T-lymphocytes (CTLs) triggers a full-fledged immune response, provided that the tumor infiltrating lymphocytes (TILs) can escape the immunosuppressive micromilieu at the BC tumor site. These immunosuppressive pathways include a contribution by FOXP-3+ regulatory T cells, autoregulatory effects of immune checkpoint receptors (e.g., PD-1) and the metabolic effects of the overexpressed IDO-1 immune surveillance pathway. The small molecule inhibitor, indoximod (IND), interferes in the IDO-1 pathway. We propose that simultaneous delivery of DOX and IND through the use of a nanocarrier can effectively combine the use of an ICD stimulus and interference in an immune surveillance pathway for the development of BC immunotherapy. The improved pharmacokinetics of drug delivery by the nanocarrier allows achievement of sufficiently high tumor drug levels to trigger an effective and sustained immune response for the reduction or elimination of the primary BC tumor and its metastases. How can ICD be used to invoke an orchestrated anti-BC immune response? Doxorubicin (DOX), an anthracycline agent, is a potent ICD inducer in addition to serving as a first-line chemotherapeutic drug for BC.26,34 It should be considered, however, that intravenous (IV) DOX administration is accompanied by off-target toxicity (e.g., of the heart and liver) and that the free drug has a relatively short circulatory half-life (t1/2), which limits tumor drug uptake.35−37 This could explain the poor potency or failure of free DOX to induce ICD at the BC tumor site in our preliminary animal studies (see later). To improve the pharmacokinetics (PK) for clinical use, encapsulated DOX delivery (e.g., the PEGylated liposomal DOX formulation, Doxil) has been successfully employed for the treatment of AIDS-related Kaposi’s sarcoma, recurrent ovarial carcinoma, MBC, and multiple myeloma.38−40 Moreover, our own preliminary data indicate that DOX encapsulation by an in-house liposome can effectively induce an ICD response at the tumor site in an orthotopic animal model (see later). We have also demonstrated in an orthotopic pancreas cancer (PC) model that it is possible to provide an effective ICD stimulus by using a mesoporous silica nanoparticle (MSNP) for delivery of oxaliplatin, which as a free drug was incapable of triggering an immunogenic response in vivo.22 All considered, these findings suggest that the use of a nanocarrier to improve the PK and tumor drug concentration of ICD-inducing chemotherapeutic agents could provide an effective means for initiating chemo-immunotherapy, which will be difficult to achieve on a reproducible basis by free drugs. Another potential advantage of using a nanocarrier is the ability to co-deliver synergistic drug combinations for improving treatment efficacy, as demonstrated in the PC tumor model, where contemporaneous delivery of 1-methyl-d-tryptophan (aka indoximod, IND) and oxaliplatin by the MSNP carrier triggered a synergistic immunotherapy response.22 Not only did the indoximod strongly synergize with oxaliplatin in calreticulin expression, but it also provided effective interference in the IDO-1 immune suppressive pathway, which is regionally overexpressed at the PC tumor site.22 The immunosuppressive effects of IDO-1 is due to its enzymatic conversion of tryptophan to kynurenine, resulting in tryptophan insufficiency, which interferes in the mTOR pathway or activation of the serine/threonine-protein kinase, GCN2 (general control nonderepressible), or kynurenine in excess, which activates the aryl hydrocarbon receptor (AhR) pathway.41,42 A possible basis for the synergy between ICD induction and interference in the IDO-1 pathway could be there closely linked paracrine relationship in the tumor microenvironment. Collectively, these effects interfere in activation of cytotoxic T cells, accumulation of Tregs and an overall immune suppressive outcome at the site of the regional tumor and draining lymph nodes. There is currently a strong interest in IDO-1 inhibitors for cancer treatment, including BC.43 Against this background, we set out to establish whether dual delivery of DOX and an indoximod (IND) prodrug by a liposome can initiate an anti-BC tumor immune response in an orthotopic tumor model. We constructed a phospholipid-conjugated IND prodrug that self-assembles into a lipid bilayer encapsulated nanovesicle or liposome. Following remote loading of DOX into the liposome through the use of a proton gradient, the innovative dual-delivery DOX/IND carrier was used to conduct PK, efficacy, and safety studies in a murine orthotopic model that resembles human triple negative breast cancer (TNBC). The ensuing innate and cognitive immune response dramatically reduced primary tumor volume, while also eliminating lung metastases. This treatment effect was further enhanced by treatment with an anti-PD-1 monoclonal antibody (mAb). Results Doxorubicin Is an Effective ICD-Inducing Chemotherapy Agent in Breast Cancer In addition to being considered as a first-line chemotherapeutic for breast cancer,34 the anthracycline drug, doxorubicin (DOX), is a potent inducer of ICD and therefore potentially useful to induce TILs in the BC tumor microenvironment.44 In order to provide proof-of-principle testing for the use of ICD in BC immunotherapy, consensus screening guidelines were used to compare the immunogenic effects of DOX with paclitaxel (PTX), cisplatin (CIS), and oxaliplatin (OX) in a 4T1 tumor model.45 Multiparameter cellular screening to assess cell surface expression of calreticulin (CRT) (cellular stress), ATP release (autophagy), and nuclear disintegration with HMGB1 release demonstrated that DOX, PTX, and OX are effective ICD inducers in 4T1 cells (Figure S1A–D). In contrast, CIS failed to induce the same ICD response parameters. In vivo confirmation of an ICD effect was provided by a vaccination approach in syngeneic Balb/c mice (Figure 2A). This requires subcutaneous injection of dying 4T1 cells exposed for 24 h to DOX (5 μM) or PTX (5 μM) in one flank of the animals on two occasions, 1 week apart. The animals were rechallenged by injection of live 4T1 cells in the contralateral flank 7 days later (Figure 2). This demonstrated that whereas vaccination with DOX- or PTX-treated cells could significantly suppress tumor growth at the challenged site, CIS (100 μM) had no effect (Figure 2 and Figure S1E). In addition to the growth inhibitory effect of the vaccination procedure, we confirmed that the effect is immune mediated by demonstrating perforin and granzyme B-mediated cytotoxic T-cell killing at the tumor site, with increased expression of CD8+ T-cells and an increase in CD8/Treg ratios (Figure S1F–H). Figure 2 Use of a vaccination approach to identify chemo agents that induce ICD in a BC model. Published consensus guidelines were used to identify effective ICD introducing chemotherapy agents by a combination of in vitro 4T1 screening, followed by use of the dying tumor cells for a tumor vaccination procedure in syngeneic Balb/c mice.45 Multiparameter in vitro screening showed that doxorubicin (DOX) and paclitaxel (PTX), but not cisplatin (CIS), induced surface expression of CRT on 4T1 cells in a dose-dependent fashion, as well as quantifiable HMGB1 and ATP release (Figure S1B–D). (A) Animal vaccination, using 2 rounds of subcutaneous (SC) injection of dying 4T1 cells 7 days apart, followed by SC injection of live cells on the contralateral side. Successful growth inhibition at the challenge site is suggestive of immune interference. (B) Spaghetti plots showing growth inhibition of the tumors in animals vaccinated by dying tumor cells treated with DOX and PTX, but not CIS or PBS (n = 6). Evidence for the involvement of the innate and cognitive immune systems in the vaccination experiment appears in Figure S1F–H. Synthesis of a Liposomal Carrier for Dual Delivery of DOX and IND In spite of the promising ex vivo ICD-inducing effects of DOX, our preliminary data demonstrate that IV administration of the free drug failed to induce CRT expression at the site of orthotopic 4T1 tumors (see Figure 9B). As it is known that the encapsulated DOX delivery in PEGylated liposomes improves the PK and treatment efficacy in malignancies such as Kaposi’s sarcoma, ovarian carcinoma, and MBC,38,40,46 we asked whether it is possible to improve ICD at the BC tumor site through the use of a DOX-only liposome, Dox-NP (identical composition as Doxil for preclinical research use), as well as a dual-delivery liposomal carrier for DOX plus the IDO-1 inhibitor, IND. Contemporaneous targeting of IDO-1 is appropriate in light of its overexpression at the BC tumor site (including 4T1 orthotopic tumors, as shown in Figure S2) and synergy with ICD in a PC model. The first step toward constructing a dual-delivery carrier was the covalent conjugation of IND (I-methyl-d-tryptophan or 1-d-MT) to 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (PL) to form an IND-PL prodrug, as schematically explained Figure 3A and Figure S3A.22 IND-PL self-assembles into a liposome (Figure 3A), which is constructed as outlined in Figure 3B.22 This requires mixing and suspension of IND-PL, cholesterol, and DSPE-PEG2K in an organic solvent, which is evaporated from the bottom of a round-bottom flask to form a uniform lipid film. Various molar ratios of IND-PL, cholesterol, and DSPE-PEG2K were tested to obtain optimal lipid bilayer stability and carrier size (Figure S4). Optimal results were obtained using a molar ratio of 75:20:5 for IND-PL, cholesterol, and DSPE-PEG2K, respectively. To accomplish remote DOX loading, the protonating agent, (NH4)2SO4, was introduced on top of the biofilm, followed by probe sonication and purification across a PD-10 column. The (NH4)2SO4-loaded IND-Liposomes were subsequently incubated in a DOX·HCl solution to allow amphiphilic DOX to be imported across the liposomal membrane (Figure 3B, bottom panel). Drug protonation leads to the formation of an intraliposomal (DOX-NH3)2SO4 precipitate, which is incapable of back-diffusion across the lipid bilayer. The combined synthesis process yielded a dual-delivery carrier with IND and DOX loading capacities (w/w) of 19.8 and 11.4%, respectively, as determined by UPLC-MS/MS. This is equivalent to an IND/DOX molar ratio of 4.3:1. Comprehensive liposome characterization demonstrated a carrier of 100 nm (DLS) size, a low polydispersity index, and a slight negative surface charge (Figure 3C). The liposome maintains its stability and size for up to one month in DI water, PBS, and 10% FBS-containing RPMI-1640 (Figure S4B). Noteworthy, ultrastructural viewing of the DOX/IND-Liposome by cryo electron microscopy showed a carrier that morphologically resembles Doxil47 as well as Dox-NP (Figure 3D), in addition to similarities in their drug loading capacities, size, and charge (Figure 3C). Both formulations had endotoxin levels of <0.1 EU/mL. Figure 3 Synthesis of the dual-delivery DOX/IND-Liposome. (A) Schematic to show that the carrier is synthesized by self-assembly of an IND prodrug to form a liposome, which is subsequently loaded with DOX. The synthesis commences by conjugating IND to a single chain phospholipid [1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (PL)] to derive the IND-PL prodrug, as previously described by us.22 The three-step synthesis process is schematically depicted in Figure S3. The prodrug is mixed with cholesterol and DSPE-PEG2K to form a lipid film for the construction of liposomes. (B) Schematic to outline the liposome synthesis steps. Briefly, the lipid film comprised of IND-PL, cholesterol, and DSPE-PEG2K at a molar ratio of 70:25:5, was hydrated in a (NH4)2SO4 solution, followed by sonication and removal of free (NH4)2SO4. DOX was remotely loaded by using a proton gradient, as shown in the bottom panel. (NH4)2SO4 dissociates into protons, NH3 and SO42–. DOX is a weak basic molecule that is capable of diffusing across the IND-PL lipid bilayer into the liposome, where it is converted to a (DOX-NH3)2SO4 precipitate, incapable of back diffusion across the lipid bilayer. (C) Side-by-side comparison of DOX/IND-Liposome and Dox-NP for drug loading capacity, size, polydispersity, charge, and endotoxin levels (n = 3). (D) CryoEM pictures to show the morphological similarity between Dox-NP and IND-Liposome, including the presence of the drug precipitate. Dual-Delivery Liposome Improves the PK of Drug Delivery and Tumor Drug Concentrations in a 4T1 BC Model The PK and drug biodistribution of the dual-delivery carrier was assessed in an orthotopic BC model. This model was established by injecting luciferase-transfected 4T1 cells into the right second mammary fat pad in Balb/c mice (Figure 4A).48 The animals develop rapidly growing primary breast tumors that can be detected by IVIS imaging within 2 weeks. This was followed by tumor metastasis to the lung within 30 days postimplantation.48 In order to determine a relevant and safe DOX dose for in vivo use, we first assessed the maximum tolerated dose (MTD) of free DOX, the DOX/IND-Liposome, and Dox-NP by a National Cancer Institute protocol, as described in the Methods section.49 The MTD in Balb/c mice was calculated as 8, 15, and 15 mg/kg for DOX, Dox-NP, and DOX/IND-Liposome, respectively. As Doxil is usually injected as an IV dose of 50 mg/m2 in patients,50 we used this as the basis to calculate an IV animal dose of 5 mg/kg of DOX in tumor-bearing mice, using dose conversion data.35−37 Figure 4 Pharmacokinetics (PK) of drug delivery by the DOX/IND-Liposome compared to Dox-NP in an orthotopic tumor model. (A) Syngeneic orthotopic model was established injecting luciferase-transfected 4T1 cells into the 2nd mammary fat pad of Balb/c mice (left). This is followed by the development of a primary tumor mass that can be viewed by IVIS imaging after 2 weeks. Animal sacrifice and collecting the tumors and organs confirmed the treatment effect on primary tumor mass as well as the presence of metastatic nodules in the lung. After 4 weeks, it was possible to visualize the large primary tumor mass and extensive lung metastases. (B) Drug dose calculations for the animal studies (first n = 2, then n = 6, refer to the Methods section): maximum tolerated dose (MTD) calculations were carried out for the DOX formulations shown, using a NCI protocol.49 (C) IVIS imaging of DOX fluorescence at the 4T1 orthotopic tumor site (n = 3). Three animals in each group received free DOX, Dox-NP, and the DOX/IND-Liposome at 5 mg/kg DOX IV.35−37 The mice were sacrificed after 24 h for IVIS imaging. DOX fluorescence intensity was quantified by Living Image software. (D,E) PK and tissue drug distribution in 4T1 orthotopic tumor-bearing mice (n = 6), receiving IV injection of free DOX, Dox-NP, and DOX/IND-Liposome at a DOX equivalent dose of 5 mg/kg. Panel D depicts the WinNonlin software calculation of the plasma concentration, expressed as the % of the injected DOX dose at the indicated time points (left panel). The corresponding tumor and tissue drug concentrations, expressed as % injected DOX dose/g tissue, appears in the right panel. The equivalent data for IND appears in panel E. Results are expressed as mean ± SD; *p < 0.01 (ANOVA). As DOX is a fluorescent drug substance, IVIS imaging was used in the first experiment to monitor the drug fluorescence intensity following a single IV dose of free drug, Dox-NP or the dual-delivery carrier at a DOX equivalent dose of 5 mg/kg (n = 3) (Figure 4C). Twenty-four hours postinjection, major organs were collected and DOX fluorescence was quantified by Living Image software (PerkinElmer, version 4.5), using an excitation filter of 500 nm and a DsRed emission filter. Compared to the weak fluorescence intensity of free DOX at the tumor site, there was an approximate ∼10-fold increase in fluorescence intensity in mice injected with the DOX/IND-Liposome or Dox-NP (Figure 4C). This was followed by a comprehensive PK study, in which orthotopic tumor-bearing mice (n = 6) were IV injected with free DOX, Dox-NP, and the DOX/IND-Liposome to deliver a DOX equivalent dose of 5 mg/kg. Blood was withdrawn at predetermined time points (0.083, 0.25, 0.5, 1, 2.5, 8, 24, and 48 h) and the plasma used for quantitation of the IND and DOX content by UPLC-MS/MS. Data calculation with WinNonlin software demonstrated a significant increase in the plasma t1/2 and intratumoral levels of both encapsulated DOX preparations compared to the free drug (Figure 4D). Whereas free DOX was rapidly eliminated from the circulation (t1/2 of <0.083 h) the circulatory t1/2 was increased to ∼3 h for Dox-NP and the DOX/IND-Liposome. Drug-dose calculation as a % of the total injected dose, demonstrated that up to ∼10 wt % (dose/g tissue) of the encapsulated DOX could be seen to distribute to the 4T1 tumor site by 48 h, as compared to ∼0.6% for free DOX (Figure 4D). There was also a significant improvement in the circulatory t1/2 of free IND as a result of liposomal encapsulation (Figure 4D). UPLC-MS/MS measurement of the IND content at the tumor site confirmed an increase from 0.6 to 9.6 wt % for free versus encapsulated drug delivery (Figure 4E). Effective Tumor Growth Reduction and Inhibition of Metastases by the DOX/IND-Liposome A comprehensive study was performed to compare the effect of free DOX with the effect of encapsulated DOX on tumor growth in the orthotopic 4T1 model. In addition, we also compared the result to treatment with the checkpoint blocking antibody, anti-PD-1, as well as combining anti-PD-1 with the dual-delivery liposome. The comprehensive array of treatment groups used in a single large experiment is shown in Table 1. For ease of data description, we will provide the data analysis as three groupings, which correspond to Figures 5–7. The first data set, outlined in Figure 5A–E compares 1, saline; 2, free DOX; 3, Dox-NP; 4, IND-Liposome (non-DOX-loaded, self-assembled nanovesicle); 5, DOX + IND-Liposome; and 7, DOX/IND-Liposome. The second data set, outlined in Figure 6A–F, compares 1, saline; 6, DOX + anti PD-1; 7, DOX/IND-Liposome; and 8, DOX/IND-Liposome + anti PD-1. The third data set, discussed in Figure 7A–D, compares 1, saline; 7, DOX/IND-Liposome, with or without the injection of an anti-CD8 monoclonal antibody, to determine the effect of CD8 depletion on the immune response. The dose equivalents of DOX and IND were 5 and 8.7 mg/kg based on an optimal constructed DOX/IND-Liposome (i.e., ratio 4, Figure S4), respectively, using a total of three injections 3 days apart (Figure 5A). Table 1 Treatment Groups for the Performance of the Efficacy Experimenta a We used nine groups of animals (each including nine mice) to perform a comparative efficacy study in the 4T1 model. For ease of description and to prevent data crowding, the data are displayed for three comparative groupings as outlined in Figures 5–7. Figure 5 addresses the synergy of DOX and IND-PL co-delivery in comparison to Dox-NP. Figure 6 shows the effect of combining the anti-PD-1 antibody with DOX or the dual-delivery liposome, and Figure 7 investigates the effect of anti-CD8 antibody on the immune response to the DOX/IND-Liposome. The experiment was repeated once, in which data were displayed to show animal survival. Figure 5 Treatment with the DOX/IND-Liposome impacts tumor growth and metastases in the orthotopic BC tumor model. (A) Outline of the experimental schedule (n = 9). The DOX/IND-Liposome was IV injected in animals in group 7 on days 8, 11, and 14 to deliver a DOX dose of 5 mg/kg and an IND dose of 8.7 mg/kg. Treatment was compared to IV injection of saline (#1), DOX (#2), Dox-NP (#3), (non-DOX-loaded) IND-PL-Liposome (#4), and DOX + IND-PL-Liposome (#5), using equivalent doses of DOX or IND. The inhibition of tumor growth by the DOX/IND-Liposome is significant compared to Dox-NP (p < 0.05) and other controls (*p < 0.01, ANOVA). (B,C) Representative tumor images and average tumor weights after animal sacrifice on day 22. (D) Representative IVIS imaging and quantification of bioluminescence intensity of lung metastases (p < 0.05; p < 0.01, ANOVA). (E) Kaplan–Meier analysis to show that the DOX/IND-Liposome dramatically prolongs animal survival (n = 9, p < 0.01, Log-rank Mantel-Cox test) in a separate experiment. Figure 6 Anti-PD-1 coadministration with the DOX/IND-Liposome augments growth inhibition and eradication of lung metastases. Green arrows represent the treatment time points including DOX formulations and/or anti PD-1. (A) IHC staining showing pronounced PD-1 expression in the 4T1 BC tissue. (B) Tumor growth was assessed as in Figure 5, demonstrating that the addition of anti-PD-1 (injected IP at 100 μg/mouse on days 8, 11, and 14) exerted additional growth inhibitory effects (n = 9, p < 0.05 ANOVA). (C,D) Representative tumor images and tumor weights for the treatment groups. (E) Representative IVIS images and quantitative data to show the complete disappearance of lung metastases in animals receiving coadministration of anti-PD-1. (F) Kaplan–Meier analysis to show prolonged animal survival by anti-PD-1 administration (p < 0.05, Log-rank Mantel-Cox test) in a separate experiment (n = 9). Figure 7 Anti-CD8 monoclonal antibody interferes in the antitumor efficacy of the DOX/IND-Liposome. In order to demonstrate the critical role of cytotoxic CD8+ T-lymphocytes in antitumor immunity, anti-CD8 monoclonal was IP injected in treatment group 9, 3 days prior to the first drug administration and repeated every 2–3 days until the termination of the study. (A) Comparative tumor growth inhibition as described in Figure 5 (n = 9). (B) IVIS imaging data align with the growth inhibitory effects. (C) Representative ex vivo IVIS imaging, with quantification of luciferase expression, to show interference of anti-CD8 on lung metastatic spread. (D) Kaplan–Meier analysis to show that CD8 depletion dramatically reduces animal survival. Results are expressed as mean ± SD (n = 9, ** p < 0.01, ANOVA). The results in the first comparative grouping demonstrated that the DOX/IND-Liposome was superior to free DOX and Dox-NP (p < 0.05) in shrinking primary tumor size (Figure 5A-C). Moreover, IND-PL enhanced the reduction in tumor size when co-delivered with DOX, but had no effect by itself or when combined with free DOX. Noteworthy, Dox-NP had a statistically significant effect (p < 0.01) compared to that with treatment groups, other than the dual-delivery liposome. Evidence that the reduction in tumor size by encapsulated DOX is due to an immunogenic effect with innate and cognate immune features, is discussed in Figures 8 and 9. In order to determine what effect treatment had on metastatic spread to the lung,48 mice were sacrificed on day 22, and the lungs were harvested for ex vivo IVIS imaging (Figure 5D). Quantitative expression of the imaging intensity demonstrated a highly significant reduction in metastatic spread in response to encapsulated co-delivery of DOX and IND compared to DOX only (Dox-NP). However, Dox-NP did exert a statistically significant effect compared to other treatment groups. A parallel survival study was performed, using nine animals in each group. Kaplan–Meier plots confirmed that the dual-delivery liposome resulted in a significant survival benefit, including in comparison to Dox-NP (Figure 5E). Figure 8 Immune phenotyping to demonstrate the effect of the DOX/IND-Liposome on initiating adaptive anti-BC immunity. Tumors were harvested from the different animal groups depicted in Table 1 to perform IHC staining and flow cytometry. (A) Multicolor flow cytometry analysis to show the impact on CD8/Treg ratios (n = 9). (B) IHC staining for CD8 expression in tumor tissue sections. (C) Flow cytometry analysis of T-cell IFN-γ+ expression in a CD45+CD3+CD8+IFN-γ+ gated cell population; granzyme B+ expression in a CD45+CD3+CD8+granzyme B+ gated cell population. (D) IHC staining for perforin expression. Additional IHC and flow data for the expression of FOXP-3, CC-3, IL12p70, and LC-3 are shown online (Figure S5C–E,G). The general treatment effect for the dual-delivery liposome (group 7) was to trigger effective cytotoxic killing at the tumor site, with significant boosting of the response by anti-PD-1 (group 8), and response decline during anti-CD8 administration (group 9). Figure 9 Immune phenotyping to demonstrate the effect of the DOX/IND-Liposome on innate anti-BC immunity. The grouping order in this figure is identical to that of shown in Figure 8A. (A) Multicolor flow cytometry analysis to assess CD91 (CRT binding receptor) and CD103 expression on DCs (n = 9). (B) IHC staining to show CRT expression at the primary tumor site. Additional IHC and flow data for the expression of CD91 and CD80/CD86 are shown online (Figure S5F,H). (C) Western blotting to assess phosphorylation (activation) of P-S6 kinase in tumor tissues obtained from animal groups treated with the IND-Liposome (n = 3). The staining intensity of P-S6 kinase in relation to the amount of total kinase protein was graphically expressed by ImageJ software. The right panel shows RT-PCR analysis of IL-6 m-RNA levels at the tumor site. The increased phosphorylation of P-S6 kinase and reduced expression of IL-6 message RNA reflects interference of IND in the local immunosuppressive effects of IDO-1 that is overexpressed at the BC tumor site (Figure S2). Antibodies that block the PD-1 and CTLA-4 receptors have been used with great success in soliciting of anticancer immunity in melanoma, NSCLC, and renal carcinoma.51,52 In contrast, the effect of immune checkpoint inhibitors in BC has been disappointing, possibly due to the immune deplete microenvironment in BC, where the absence of TILs as well as the failure to express PD-1 or its ligand may preclude an immune response to checkpoint blocking antibodies.12,13 As ICD may trigger an immune response that can be boosted by treatment with anti-PD-1, an antibody from the anti-PD clone, RMP1-14 (BioXcell), was administered intraperitoneally (IP) at 100 μg/mouse on day 8, 11, and 14. Immunohistochemistry (IHC) confirmed PD-1 expression at the 4T1 BC tumor site (Figure 6A). Assessment of tumor size showed a significant reduction of tumor volume in the animals receiving combined treatment with the DOX/IND-Liposome plus the anti-PD-1 antibody (Figure 6B–D). In addition, there was a total disappearance of lung metastases using the combination therapy (Figure 6E). Strikingly, the addition of the antibody was also responsible for significant further improvement in survival outcome, as demonstrated by Kaplan–Meier analysis (Figure 6F). DOX/IND-Liposome Induces Synergistic and Effective Innate and Adaptive Anti-BC Immune Responses in an Orthotopic Model In order to demonstrate the involvement of the immune response in the growth inhibitory effects of the dual-delivery liposome at the primary tumor site, we asked whether the depletion of CD8+ T-cells will affect treatment outcome. An anti-CD8 monoclonal antibody was IP administered 3 days prior to the first treatment with the liposome, and repeated every 2–3 days (group 9, Table 1). When compared to the response in group 7, which was treated with the DOX/IND-Liposome alone, we observed that the depletion of CD8+ T-cells (confirmed by IHC in Figure 8B) could dramatically interfere in the growth inhibitory effects of the liposome at the primary tumor site as well as the disappearance of metastases (Figure 7A–C). Anti-CD8 administration also interfered in survival outcome (Figure 7D). These findings are indicative of the generation of a CTL mediating the anti-BC immune response. Anti-CD8 administration also significantly reduced the immune response to Dox-NP as well as the DOX/IND-Liposome in Figure S6. In order to corroborate the above findings, extensive further immune phenotyping was performed on tumors harvested from the treatment groups in Table 1, using IHC staining and multiparameter flow cytometry (Figures 8 and 9). Flow cytometry showed that CD8/Treg ratios were markedly increased in DOX/IND-Liposome-treated mice compared to Dox-NP, which in turn, showed significant differences from other treatment groups (Figure 8A). The CD8/Treg ratio in group 7 was significantly enhanced by treatment with anti-PD-1, whereas the administration of anti-CD8 essentially restored the ratio to values seen in the saline treatment group (Figure 8A). These treatment effects were further corroborated by IHC staining for CD8, which clearly demonstrate the appearance of CTLs in response to DOX and IND co-delivery; the numbers were further boosted by the addition of anti-PD-1 (Figure 8B). A diagonally opposite trend was seen during IHC staining for FOXP3 expression, which showed disappearance of Tregs (Figure S5C). The effective induction of a cognate immune response was further corroborated by flow cytometry and IHC results looking at the local production or expression of IFN-γ, granzyme B, perforin, activated caspase 3 and IL12p70 (Figure 8C,D and Figure S5D,E). Apart from the impact on adaptive immunity, we also looked at innate parameters that reflect the induction of ICD (Figure 9A,B and Figure S5F,H). This was demonstrated by the dramatic increase in CRT expression as well as the levels of its counter receptor, CD91, on DCs in tumor tissue of animals treated by the dual-delivery liposome (Figure 9A,B). Moreover, the response was further enhanced or reduced by treatment with anti-PD-1 or anti-CD8 monoclonal antibodies, respectively, as described above. Similar responses were seen for biomarkers that reflect dendritic cell activation, including the integrin receptor, CD103 (Figure 9A), that is used by DCs to facilitate the CD8+ T-cell development and activation, as well as CD80 and CD86 (Figure S5H). We also obtained evidence for increased expression of microtubule-associated protein 1A/1B-like chain three (LC-3), which is involved in autophagy and responsible for ATP release during the ICD response (Figure S5G).31,59 All considered, these results demonstrate synergy between DOX and IND in generating a robust innate immune response at the tumor site. As it was not logistically possible to include additional treatment groups in the experiment in Table 1, it was necessary to perform an additional study to assess the comparative effect of anti-PD-1 on the response to Dox-NP (Figure S6). This experiment compared the effect of Dox-NP + anti-PD-1 to treatment with saline, anti-PD-1, Dox-NP, the DOX/IND-Liposome, and DOX/IND + anti-PD-1. We also compared the outcome to the use of anti-CD8 in the Dox-NP and the DOX/IND-Liposome groups (Figure S6). The data demonstrate that whereas both the DOX/IND-Liposome and Dox-NP groups benefited from anti-PD-1 administration, the outcome was significantly enhanced in the former treatment group after the first three 3 IV injections (day 17) (Figure S6A,B). The magnitude of this difference was further boosted by the addition of 2 further IV administrations (up to day 24). The use of flow cytometry to perform immunophenotyping of cells harvested from the tumor site confirmed that the increase in the CD8/Treg ratios was significantly higher during anti-PD-1 co-treatment in the DOX/IND-Liposome compared to the Dox-NP group (Figure S6D). There was no obvious weight loss or change in AST and ALT enzyme levels in response to any of the treatments (Figure S6C,E). In addition, anti-CD8 administration also significantly reduced the immune response to Dox-NP as well as the DOX/IND-Liposome in Figure S6. It was possible to discern the effective inhibitory effect of IND-PL on the IDO-1 metabolic pathway at the tumor site by conducting Western blotting to show the phosphorylation status of S6 kinase (Figure 9C, left panel). Activated P-S6 kinase plays a role in the mTOR pathway to reverse the immune suppressive effects of IDO-1 (Figure S2). This corroborates similar effects in 4T1 cells, in which IND-PL dramatically increased the intracellular retention of IND, including an exaggerated effect on S6 kinase phosphorylation (Figure S3B,C). We also observed in assessing IL-6 mRNA expression at the tumor site, that the message abundance was significantly decreased in animals receiving either one of the three IND-containing carriers (Figure 9C, right panel). IL-6 is involved in sustaining IDO-1 expression by an integrated IL-6/STAT3/AHR autocrine loop, which is disrupted by IND.22,42 A similar IND-PL effect was also seen in 4T1 cells, where the prodrug dose-dependently interfered in IL-6 release in the cellular supernatant (Figure S3C). Encapsulated DOX Delivery Improves Drug Safety One of the major advantages of the PEGylated liposomal DOX formulation has been the improvement of DOX safety. This was confirmed by assessing biomarkers of cardiac, liver, and kidney toxicity in animals that were treated in 4T1 orthotopic efficacy study. The results show that the co-delivery liposome is associated with normalization of the increased troponin I, creatine kinase, alanine aminotransferase (ALT), aspartate aminotransferase (AST), and creatinine levels in response to free DOX, similar to Dox-NP (Figure 10). Figure 10 Encapsulated DOX delivery prevents toxicity of the heart, liver, and kidney. Blood was withdrawn on day 22 during performance of the efficacy studies in Figures 5–7 to assess the DOX-related toxicity in the (A) heart (troponin I and creatine kinase), (B) liver (ALT, AST), and kidney (creatinine). Encapsulated DOX delivery by Dox-NP and the DOX/IND-Liposome reduced the free drug toxicity. Results are expressed as mean ± SD (n = 9, p < 0.05; p < 0.01, ANOVA). Discussion We demonstrate a chemo-immunotherapy approach for breast cancer, using a dual-drug delivery liposomal carrier that introduces an immunogenic cell death stimulus while, at the same time also interfering in the regionally overexpressed IDO-1 pathway that prevents effective T-cell priming. Through innovative design of an IND-PL prodrug that self-assembles into a liposome that can be remotely loaded with DOX, we were able to develop a dual-delivery carrier that could improve the pharmacokinetics of both drugs at the tumor site. Their combined action was to reduce the primary tumor growth as well as interfere in tumor metastases in a syngeneic mouse model (Figures 5–7). The antitumor immune effects were characterized by the expression of ICD biomarkers as well as enhanced dendritic cell and innate immune responses at the tumor site (Figure 9A,B and Figure S5H). Subsequent recruitment and activation of cytotoxic T-cells induced perforin and granzyme B-mediated tumor cell killing, as well as the local production of IFN-γ (Figure 8C,D). Immune phenotyping of the tumor tissues confirmed the recruitment of CD8+ CTLs, disappearance of Treg, and an increase in CD8+/FOXP3+ T-cell ratios (Figure 8A,B and Figure S5C). Not only did the DOX/IND-Liposome provide an improved PK and dual drug delivery effect compared to Dox-NP, but it also eliminated free DOX toxicity in the heart, liver, and kidney (similar to Dox-NP) (Figure 10). We also demonstrated that the dual-delivery liposome could be effectively combined with PD-1 blocking antibodies to eradicate tumor metastases (Figure 6E). This is in keeping with the notion that the generation of ICD in the BC tumor microenvironment introduces an exciting opportunity to improve the response rate to cancer immunotherapy. Problems with the dose and PK of free chemotherapy agents make this a challenging task toward a practical and reproducible approach. Equally important, we demonstrated that improved local delivery of IND constitutes an effective strategy for interfering in the regionally overexpressed IDO-1 pathway, which exerts immunosuppressive effects that are nonredundant with the effect of immune checkpoint inhibitors. All considered, this study demonstrates the value of a nanocarrier that improves the PK and action of a synergistic drug combination to trigger a robust anti-BC tumor immune response to the primary tumor as well as associated metastases. The use of chemotherapy to initiate immunotherapy is a logical choice in light of emerging clinical data indicating that neoadjuvant chemotherapy with DOX, docetaxel, cyclophosphamide and taxanes is capable of inducing pathological complete responses due to recruitment of TILs in BC patients.12,13,53 In addition, there are also indications that neoadjuvant chemotherapy may have a survival effect compared to adjuvant chemotherapy or surgery in patients with colon or urothelial cancer.54,55 It is interesting, therefore, that a number of chemotherapeutics, such as the anthracyclines (DOX, daunorubicin, and mitoxantrone), oxaliplatin, cyclophosphamide, and paclitaxel, can induce a programmed cell death response in tumor cells that is immunogenic in nature.22,28,29,56 This ICD response includes the translocation of CRT, which is normally expressed in the endoplasmic reticulum, to the cancer cell surface, where it provides an “eat-me” signal for dendritic cell uptake.26,27 ICD is also accompanied by the release of the alarmin, HMGB1, at a more advanced stage of cell death, with the ability to induce dendritic cell maturation and tumor antigen presentation through binding to TLR-4.57 Moreover, ICD also involves autophagy, which leads to the release of ATP.58,59 ATP serves as a danger signal that triggers a purinergic dendritic cell receptor which leads to assembly of the NRLP3 inflammasome and IL-1β production.60−63 Although the ICD concept dates back more than a decade, the deliberate use of chemo-immunotherapy has only been pursued in a limited number of animal studies, including in murine lung and colon cancer models.44,64 It is also possible to induce ICD through the use of physicochemical stimuli, such as ionizing radiation or photodynamic therapy.65 Here, we demonstrate the use of a liposomal carrier to induce ICD at an orthotopic BC tumor site, an outcome that is not accomplishable with free DOX (Figure 9B). We ascribe the tumor-associated ICD effect to increased DOX delivery to the tumor site as a result of the enhanced permeability and retention effect of the nanocarrier.39 Our animal PK data of Dox-NP (Figure 4) agrees with the highly significant improvement in the PK and tumor levels of DOX in clinical Doxil treatment studies.39,66−68 For instance, the clinical PK studies demonstrate an initial DOX distribution phase with a t1/2 of 1–3 h, followed by a second distribution phase with a t1/2 of 20–90 h.39,66−68 Moreover, the “area under the curve” for Doxil delivery in human studies is ∼300-fold bigger than the free drug, whereas the clearance and volume of distribution are reduced by factors of 250- and 60-fold, respectively.39,66−68 Although Dox-NP was effective in inducing CRT expression and initiating an effective BC tumor immune response in our animal studies, the efficacy of the response and survival outcome was significantly less than the impact of the DOX/IND-Liposome (Figures 5–9), even in the presence of anti-PD-1. As there was no difference between Dox-NP and the dual-delivery liposome in terms of the PK and intratumor concentrations of DOX, we ascribe the improved tumor growth and metastasis inhibition by the DOX/IND-Liposome to the action of co-delivered IND (Figures 5A,D). Moreover, the PK of IND-PL was also dramatically improved by encapsulated delivery (Figure 4E), with significant accentuation of CD8+ CTL recruitment (Figure 8B), disappearance of Tregs (Figure S5C), and increase in the CD8/Treg ratios compared to Dox-NP (Figure 8A). These findings are congruent with the demonstration that encapsulated IND-PL delivery effectively interfered IL-6 mRNA expression (Figure 9C, right) in parallel with phosphorylated S6 kinase activation (Figure 9C, left). These response outcomes are indicative of interference in the metabolic action of IDO-1, which is overexpressed at the BC tumor site (Figure S2). Cellular data further confirm that the increased uptake and retention of IND-PL exerts a stronger effect on P-S6 kinase and IL-6 production compared to the free IND (Figure S3B). A report was recently published describing the use of a polymeric carrier for the co-delivery of DOX and NLG919 (another IDO inhibitor).69 This study demonstrated a redox-responsive immunostimulatory polymeric prodrug micellar carrier for co-delivery of DOX and NLG919 with synergistic antitumor efficacy. However, there was no attempt to study the impact of the treatment on ICD, and this inhibitor (aka GDC-0919) also performed less favorably in two Phase 1 studies.70,71 In contrast, there are currently a total of 17 indoximod clinical studies, according to the NIH database (clinicaltrials.gov). Although it is clear that the dual-delivery carrier yields superior antitumor immune effects compared to Dox-NP, it is uncertain to what extent the improvement is due to the harmonized PK of the drugs, the regional delivery of IND to the site where IDO-1 is overexpressed or a synergistic mechanism of action (Figures 5–7). A case for synergy can be made insofar as the generation of INF-γ production by ICD-induced cytotoxic T-cells can induce additional IDO-1 expression and PD-1 ligand expression in a paracrine fashion. This will create a paradoxical increase in the metabolic and immune checkpoint receptor-mediated immune suppression at the same site in the tumor microenvironment where ICD is induced. Thus, inhibition of IDO-1 activity at the same site as ICD induction may be necessary for the effects of ICD to be propagated. Even though neoadjuvant chemotherapy is capable of improving TIL recruitment with a complete pathological response the BC tumor site, it is difficult to predict in advance which patients will respond. We propose that the improved delivery of the ICD stimulus by a nanocarrier provides a more predictable approach for the implementation of chemo-immunotherapy in terms of biodistribution, and dosimetry considerations. It is also important to consider that even though ICD-inducing drugs (such as DOX) may induce immunogenic effects at the tumor site, the free drug could exert immunosuppressive effects at systemic level to counter the local outcome. In addition to the use of the DOX/IND nanocarrier, it would also be possible to develop custom-design nanocarriers that deliver taxanes (e.g., paclitaxel, docetaxel) or cyclophosphamide to initiate ICD responses for cancer immunotherapy.45 It is also possible to include small molecule inhibitors of the IDO-1 pathway other than IND, including epacadostat.72−74 Aside from the ability of the DOX/IND-Liposome to serve as a standalone immunotherapy platform for BC, we demonstrate that the antitumor response of the nano-enabled platform can be enhanced by combining the liposome with an anti-PD-1 antibody (Figure 6). This finding could be of significance to a wide range of immune checkpoint inhibitors, which even under the best treatment circumstances (e.g., melanoma and NSCLC), yield response rates of 20–40% only.75−79 Although the exact explanation for the limited response rate is being researched, the current view is that responsiveness to immune checkpoint inhibitors is dependent on the presence of TILs that express immune checkpoint receptors.11,80−83 Moreover, there appears to be a requirement for a high burden of nonsynonymous DNA mutations at the cancer site to develop a hot immune microenvironment.10,11 These mutations give rise to potent neo-epitopes that are presented to T-cells by class I major histocompatibility complex on antigen-presenting dendritic cells.84−86 Although putatively this leads to robust activation of naïve T-cells and the recruitment of TILs,87,88 it is difficult to predict in a population of patients who the responders will be. We propose that the guesswork could be diminished by nanocarriers that introduce ICD stimuli in a predictable manner. Moreover, our study show that interference in the IDO-1 pathway is nonredundant with the effect of the PD-1 checkpoint receptor, allowing the DOX/IND-Liposome to be combined with anti-PD-1, which further boosts the immune response (Figure 6). We propose that the same treatment effect will also be accomplishable with additional immune checkpoint blocking antibodies, and that a combinatorial approach could also benefit other “cold” solid tumors. An interesting question regarding the use of ICD to improve immunotherapy is the identity of the tumor-associated antigens that are presented to the cognitive immune system. Theoretically, ICD should allow the presentation of mutational as well as nonmutational tumor antigens, which could expand the clonal diversity of the T-cell response. Antigen proofreading by the T-cell antigen receptors (TCR) could allow the selection of T cells that could mount an effective immune response to nonmutational antigens. Moreover, the expanded repertoire of responding T cells could improve T-cell memory, which is important for a durable immune response that controls disease resurgence and development of metastases.86,89−92 The robustness of the TCR activation response could also have a bearing on the severe immunological side effects that develop as a result of interference in the regulation of activated T cells by checkpoint receptors.93−96 One possibility is that ICD could lead to a lesser tendency toward an over-reactive immune response by involving less potent nonmutational antigens.97,98 This possibility will be investigated in future studies. In addition to the possibility of reducing immunological side effects, the encapsulated delivery of DOX by the dual-delivery liposome was equally effective to Dox-NP in protecting against cardiac, liver, and renal side effects (Figure 10). In studying a combination of therapies, it is difficult to include a comprehensive series of controls in every experiment due to the logistics and limitation of the number of animals that can be included in a single study. This was illustrated by the necessity to perform a separate experiment (from the data shown in Figures 5–7) to assess the combination of Dox-NP with anti-PD-1 (Figure S6). It was also not possible to independently assess the combination of Dox-NP with IND (either as a free drug or as a separate liposomal form). However, we did demonstrate previously that the combination of chemotherapy (free and encapsulated) with separately administered IND (free and encapsulated) did not achieve the same efficacy as combining the chemotherapy with IND-PL in a single nanocarrier (in pancreas cancer).22 Conclusion In summary, we developed a liposomal chemo-immunotherapy approach for use in BC. The liposome delivers DOX to provide an ICD stimulus as well as an IND prodrug to interfere in the metabolic immunosuppressive effects of the IDO-1 pathway in the tumor microenvironment. IV injection of the DOX/IND-Liposome favorably improved the PK and tumor uptake of both drugs at the tumor site of a syngeneic 4T1 orthotopic BC model. Compared to the DOX-only liposome, Dox-NP, the dual-delivery carrier significantly enhanced the anti-BC immune response at the primary as well as metastatic tumor sites. The response was further augmented by the addition of an anti-PD-1 monoclonal antibody, demonstrating that the potential use of ICD to generate a “hot” or immune replete BC tumor microenvironment to increase the number of responders in immunotherapy studies that use IDO inhibitors or immune checkpoint blocking antibodies. Although the concept of chemotherapy-induced ICD has been described in the literature for a number of years, this concept has not been deliberately pursued as a practical and reproducible immunotherapy principle that can be executed by a FDA-approved drug carrier. Our preclinical data clearly demonstrate the key benefit of dual delivery for an immunological perspective in comparison to free drugs or Dox-NP. This has obvious significance for possible clinical translation in human breast cancer where the use of liposomal carriers has already been approved, without the need to develop a new delivery platform from inception. Our demonstration that such a liposome can be made from a self-assembling, lipid-conjugated IND prodrug also provides significant innovation in how a synergistic drug combination can be accomplished. In summary, we report the innovative use of the triad of liposomal properties that provide: (1) effective co-packaging of an IND prodrug with DOX through self-assembly and drug import; (2) effective regional buildup of DOX and IND at the tumor site, suffice for concurrent ICD induction and immunological modulation; and (3) a combined in vivo effect, providing interference in primary tumor growth and elimination of tumor metastases. Methods Use of 4T1 Cells To Establish an Orthotopic Tumor Model in Syngeneic Balb/c Mice The 4T1 cell line was obtained from ATCC and was cultured in complete DMEM, containing 10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, and 2 mM l-glutamine at 37 °C in a CO2 incubator. 4T1 represents an aggressive phenotype that is representative of human triple-negative BC.99 In order to visualize tumor growth by bioluminescence imaging, 4T1 cells were stably transfected with a luciferase expressing lentivirus in the vector core facility at UCLA. A total of 1 × 106 cells in 30 μL DMEM/matrigel, 1/1, v/v, were injected into the right second mammary fat pad of female Balb/c mice (Charles Rivers), 6–8 weeks old (Figure 3). The orthotopic growing tumor, which mimics stage IV breast cancer in humans, leads to the development of lung metastases after 3–4 weeks.48 The mice were housed under pathogen-free conditions, and all animal experiments were approved by the UCLA Animal Research Committee. Confirmation of Immunogenic Cell Death (ICD) by Cellular Biomarkers A total of 1 × 105 4T1 cells were plated in 24-well plates overnight in complete DMEM medium. The culture medium was replenished with cisplatin (CIS), doxorubicin (DOX), paclitaxel (PTX), and oxaliplatin (OX) at the indicated concentrations for 24 h. One hundred microliters of supernatant from each well was obtained to measure HMGB-1 or ATP concentrations, using an HMGB-1 ELISA kit (IBL International GmbH) or ATPlite one-step luminescence assay kits (PerkinElmer) according to the manufacturers’ instructions. To determine CRT surface expression, 4T1 cells were trypsinized, washed in cold PBS three times, stained with a primary rabbit anti-CRT antibody (Ab2907, Abcam), followed by an Alexa Fluor 680-conjugated goat anti-rabbit IgG antibody for 30 min at 4 °C.22 The cells were suspended in 500 μL of staining buffer (BD Biosciences), to which 50 μg/mL propodium iodide was added before reading in a LSRII flow cytometer (BD Biosciences). Flow cytometry data were plotted as fold-change in mean fluorescence intensity compared to the PBS control. The experiment was repeated twice. To visualize the CRT translocation to the 4T1 cell surface, 1 × 104 4T1 cells were seeded in Lab-Tek 8-well chamber slides (Thermal Scientific) overnight and then treated with CIS (100 μM), DOX (5 μM), and PTX (5 μM) for 24 h. Cells were fixed in 4% PFA for 15 min, washed in cold PBS, and stained for 30 min with an Alexa Fluor 647-conjugated anti-CRT antibody (ab196159, 1/500, Abcam).22 The cell surface membrane was visualized by staining with 5 μg/mL Alexa Fluor 488-conjugated wheat germ agglutinin (W32466, 1/200, Thermo Fisher), followed by nuclear staining with Hoechst 33342 (H3570, 1/2000, Invitrogen) before visualization under a Leica SP8-SMD confocal microscope (63× objective lens). Assessment of Cellular Indoximod (IND) Content in 4T1 Cells A total of 1 × 105 cells were seeded in 24-well plates overnight in complete DMEM medium. The cells were treated with free IND or IND-Liposome to deliver a dose equivalent of 100 μg/mL IND for 4, 24, and 72 h. Cells were trypsinized, harvested, and dissolved in methanol overnight, before measuring the IND and IND-PL concentrations by UPLC-MS/MS, following our established protocol.22 Assessment of the IND Effect on IDO-1 Response Parameters at the Tumor Site The immunosuppressive effect of IDO-1 involves a number of downstream pathways, including inhibition of the mTOR pathway (Figure S2). In order to determine the impact of encapsulated IND delivery on the activation status of S6 kinase (a component in the mTOR pathway), Western blotting was used to assess total S6K protein as well as the level of kinase phosphorylation (P-S6K) in the tumor lysates.22,100 GAPDH was used as a comparative control. Tumors were cut into small pieces and homogenized in RIPA buffer, supplemented with a cocktail of proteinase and phosphatase inhibitors. After centrifugation of the lysates at 12000 rpm for 10 min, equal amounts of protein in the supernatants were loaded onto a 10–20% Tris-glycine gel (Novex gel, Invitrogen) and then transferred to a PDVF membrane. The membrane was blocked with 5% BSA/TBST, before incubation with primary and HRP-conjugated secondary antibodies that recognize S6K, P-S6K, and GAPDH. The blots were developed by soaking in ECL substrate (Thermo Scientific). The intensity of each protein band on the film was quantified by ImageJ software.22 To measure IL-6 mRNA expression by PCR, total RNA was extracted using TRIzol reagent (Invitrogen), treated with DNase I (Amplication grade, Invitrogen), and reverse transcribed using iScript cDNA synthese kit (Biorad).99 Quantitative RT-PCR (qPCR) was performed using iQ and SYBR Green detection kits (Bio-Rad, Hercules, CA). Primers were TCC ACG ATT TCC CAG AGA AC (forward); AGT TGC CTT CTT GGG ACT GA (reverse) (Invitrogen). PCR was conducted using a 3 min step at 95 °C, followed by 40 cycles at 94 °C for 15 s, 60 °C for 30 s, and 72 °C for 30 s. ICD Vaccination Screening in Balb/c Mice The procedure and experimental timeline are delineated in Figure 2. 4T1 cells were treated with PBS, 100 μM CIS, 5 μM DOX, and 5 μM PTX for 24 h. Cell suspensions were collected to confirm CRT surface expression by flow cytometry. Subsequently, 1 × 106 dying 4T1 cells in 0.1 mL of DMEM were administered on two occasions into the right flank of female Balb/c mice (n = 6), 1 week apart. The same mice received SC injection of healthy 1 × 106 4T1 cells in 0.1 mL of DMEM/Matrigel, 1/1, v/v in the left flank, 1 week later. Tumor size was closely monitored every 3–4 days by a digital caliper, using the formula: size = π/6 × length × width2. Mice were sacrificed on day 19, and the tumors were collected for flow cytometry and IHC analysis. The statistical difference between groups was calculated using two-way analysis of variance (SPSS software). Synthesis of the DOX/IND-Liposome The stepwise synthesis process is outlined in Figure 3B. The process begins with the synthesis of the IND prodrug, which requires IND conjugation to a phospholipid (PL), as described by us.22 This involves three steps: (i) protection of the amine group on IND by coupling to di-tert-butyl dicarbonate (Boc anhydride); (ii) conjugating the phospholipid–OH to the IND–COOH by an esterification reaction; (iii) removal of Boc to yield the IND-PL prodrug. Successful synthesis was verified by ESI-MS (Figure 3A). The liposome was constructed by preparing 50 mg of a lipid mixture in chloroform, containing 75% IND-PL, 20% cholesterol, and 5% DSPE-PEG2K (i.e., molar ratio of 15:4:1). An optimal ratio was determined by experimentation with different lipid mixtures (Figure S5). The chloroform solution was added to the bottom of a 50 mL round-bottom glass flask, followed by rotary evaporation of the solvent. This formed a thin lipid biofilm, which was further dried under a vacuum overnight. The film was subsequently hydrated in 2 mL of (NH4)2SO4 (123 mM) before probe sonication for 30 min, using a 20/15 s on/off cycle at a power output of 32.5 W. The free (NH4)2SO4 was removed over a PD-10 column (Sephadex G-25, GE Healthcare) using PBS elution. To obtain uniformly sized liposomes, the suspension was extruded 15 times through a mini-extruder (Avanti Polar Lipids), using a polycarbonate membrane (Avanti Polar Lipids) with 100 nm pore size at 80 °C. To achieve remote DOX loading, the (NH4)2SO4/IND-Liposome solution was incubated with 10 mg/mL DOX for 30 min at 65 °C. A further round of purification was carried out over a PD-10 column to obtain DOX/IND-Liposomes. The liposomes were comprehensively characterized for size, ζ-potential, loading capacity, morphology, and endotoxin level using DLS, UPLC-MS/MS, cryoEM, and the chromogenic LAL assay, respectively. DOX loading capacity was calculated using the reported35,36,102,103 formula shown below: An optimal batch was considered as liposomes with an average size ∼100 nm, slightly negative charge and stability for at least one month at 4 °C. The liposomes were stored at 4 °C in the dark until use. Calculation of Maximum Tolerated Dose (MTD) The MTDs for free DOX, the DOX/IND-Liposome, and Dox-NP were determined by a National Cancer Institute protocol.49 The protocol commences with two Balb/c mice receiving IV administration of a C1 dose equivalent of 2.5 mg/kg DOX. This is followed by incremental drug administration, using a dose escalation factor of 1.8 until the death of the animals in <24 h (aka the Cn dose). Subsequently, the Cn-1 dose was used with a 1.15 escalation factor (n = 2) to reach the MTD, which is characterized by the absence of morbidity or mortality. MTD was further corroborated in 6 healthy mice, which received IV injection at the calculated dose. These animals were closely monitored for 15 days, with the stipulation of <15% weight loss without morbidity and mortality. Assessment of the Pharmacokinetics (PK) and Biodistribution of Free and Encapsulated Drugs The experiment was carried out in 4T1 orthotopic tumor bearing mice. To assess the biodistribution of free DOX, Dox-NP, and DOX/IND-Liposome, these formulations were injected IV, at a dose equivalent of 5 mg/kg DOX (n = 3). Twenty-four hours post-administration, mice were sacrificed and ex vivo DOX fluorescence images were obtained for tumor tissue, heart, liver, spleen, lung, and kidneys, using an IVIS imager (excitation filter = 500 nm; emission filter = DsRed). Tissue autofluorescence was established at t = 0, before the drug injections. DOX fluorescence intensity was obtained by Living Image software (PerkinElmer, version 4.5). In the performance of the PK study, tumor-bearing mice (n = 6) received a single IV injection of free DOX, Dox-NP, and the DOX/IND-Liposome at a DOX dose equivalent of 5 mg/kg. At 0.08333, 0.25, 0.5, 1, 2.5, 8, 24, and 48 h post-IV injection, 50 μL of blood was drawn to obtain plasma for assessment of IND and DOX concentrations by UPLC-MS/MS analysis. In a separate study, tumor tissues, heart, liver, spleen, lung, and kidney were harvested 24 h post-IV administration and digested for 48 h in methanol to determine IND and DOX levels by UPLC-MS/MS.22,35,104 To quantify DOX, the following UPLC-MS/MS conditions were used: C18 Column (130 Å, 1.7 μm, 2.1 mm × 50 mm), connected to Waters LCT Premier with ACQUITY UPLC and Auto sampler; gradient elution sequence: (i) 0–4 min, 95% water +5% acetonitrile; (ii) 4–6.5 min, 5% water + 95% acetonitrile; and (iii) 6.5–10 min, 95% water + 5% acetonitrile. The flow rate was 0.4 L/min. The t1/2 of DOX formulations was calculated for display as a noncompartmental as well as a two-compartmental model, using WinNolin software.22,105,106 Therapeutic Efficacy Studies Using the DOX/IND-Liposome These experiments were carried out by injecting luciferase-expressing 4T1 cells in the mammary pad, as described above. The animals were randomly assigned to nine groups (nine animals each) at this stage when the tumor sizes approached 100–150 mm3 (∼day 8 days postimplantation). One group of animals (group 7) received IV injection with the DOX/IND-Liposome to deliver a dose of 5 mg/kg DOX and 8.7 mg/kg IND on days 8, 11, and 14 (Table 1). Other treatment groups included animals receiving saline or free DOX, Dox-NP, the IND-Liposome only, or free DOX + IND-Liposome at similar doses and administration frequency. We also tested whether intraperitoneal (IP) injection of an antimurine PD-1 monoclonal antibody (mAb clone: RMP1–14, BioXcell) on days 8, 11, and 14 will impact the treatment response to free DOX or the DOX/IND-Liposome. In order to demonstrate the role of cytotoxic T-cells in the antitumor response, we also assessed the effect of IP administration of 200 μg of an anti-CD8α mAb (clone 53–6.72, 200 μg/mouse, BioXcell) in animals receiving injection with the DOX/IND-Liposome. Treatment with the mAb commenced 3 days prior to the first IV injection of the DOX/IND-Liposome, and was repeated every 2–3 days until the end of the study. CD8 T-cell depletion was confirmed by immunohistochemistry (IHC). Tumor development was carefully monitored by a digital caliper on days 8, 11, 14, 17, and 22. Additionally, we visualized the tumor burden by IVIS imaging, following injection of 75 mg/kg D-Luciferin IP. We also assessed the tumor weight after tissue harvesting from euthanized animals. This included the collection of lung tissue for ex vivo imaging of metastatic spread, which was quantified by bioluminescence intensity in the region of interest, using Living Image software (PerkinElmer, version 4.5). The tumor tissues were also subdivided for performance of flow cytometry, IHC analysis, Western blotting and RT-PCR for IL-6 mRNA. Blood was withdrawn for the measurement of cardiac enzymes (troponin I and creatine kinase), liver enzymes (ALT and AST), and creatinine levels (renal function). In order to determine survival outcome, the animal efficacy study was repeated in a separate study, using the same treatment groups (n = 9) and procedures. The survival data were displayed as Kaplan–Meier plots. The criteria for animal sacrifice during Kaplan–Meier analysis included animal death or moribund status. Moribund status, as defined by our approved animal protocol, reflects lack of activity (unresponsive or unaware to appropriate stimuli), severe (>15%) body weight loss, lack of stool production, and severe dehydration. Immunohistochemistry In order to visualize the phenotypic changes in the immune system of the treated groups, IHC analysis of tumor slices was undertaken to visualize CD8, FOXP-3, CRT, CD91, IL12p70, activated caspase 3 (CC-3), LC-3, and IDO-1 expression, as previously described by us.22 Tumor chunks were fixed in 10% formalin, paraffin embedded, and sliced into 4 μm sections, which were mounted on glass slides in the UCLA Jonsson Comprehensive Cancer Center Translational Pathology Core Laboratory. The slides were deparaffinized and incubated in 3% methanol–hydrogen peroxide, prior to immersing in 1 mM sodium citrate (pH 6) or 10 mM EDTA (pH 8) at 95 °C in Decloaking NxGen Chambers (Biocare Medical, DC2012). Following rinsing in PBS containing 0.05% Tween-20 (PBST), the slides were overlaid with the different primary antibodies for 1 h, followed by addition of the corresponding HRP-conjugated secondary antibodies at room temperature for 30 min. For visualization of different immune cells, the slides were incubated with Vulcan Fast Red Chromogen Kit 2 (Biocare Medical, FR805) or DAB (3,3′-diaminobenzidine). After being rinsed in tap water, the slides were counterstained by Harris’ hematoxylin, dehydrated in ethanol, and mounted with media prior to scanning in an Aperio AT Turbo digital pathology scanner (Leica Biosystems). For PD-1 staining, slides were baked in 65 °C oven for 1 h and were then deparaffinized in xylene and rehydrated through graded ethanols to water. Heat-induced epitope retrieval was performed in a pressure cooker with high pH buffer (Leica Bond ER2 retrieval solution). Slides were cooled and washed with Leica Bond wash buffer and loaded onto Shandon Sequenza staining system. Leica Bond protein block was applied to slides for 5 min, and then primary anti-mouse PD-1 antibody was applied and incubated in refrigerator overnight. In the morning, slides were rinsed in Bond Wash buffer, and the remaining detection steps were performed on the Leica Bond III using bond refine detection reagents (anti-rabbit polymer, H2O2 quenching, DAB, and hematoxylin). Slides were then dehydrated through ethanols to xylene and coverslipped, and subsequently scanned by Aperio AT Turbo digital pathology scanner (Leica Biosystems). The slides were read by a veterinary pathologist. The following reagents were used. Primary antibodies: anti-CD8 (#14-0808, 1/100), and anti-FOXP3 (#13-5773, 1/200) were from eBioscience; anti-CRT (ab2907, 1/50), anti-LRP1(CD91) (ab92544, 1/50), antiperforin (ab16074, 1/100), and anti-PD-1 (ab214421, 1/1000) were from Abcam; anticleaved caspase 3 antibody (#9664, 1/200) was from Cell Signaling; anti-IFN-γ (NBP1-19761, 1/200) and anti-IL12p70 (NBP1-85564, 1/100) were from Novus Biologicals; anti-LC-3 (0231-100/LC3-5F10, 1/100) was from Nanotools; anti-IDO (#122402, 1/100) was from Biolegend. Secondary antibodies: Biomarkers were detected by a HRP-labeled polymeric anti-rabbit antibody (Dako, K4003), with the exception of CD91, which were visualized by a MACH2 rabbit AP-polymeric antibody (Biocare Medical, RALP525). Flow Cytometry Multiparameter staining for cell suspensions was performed as published previously.22 Briefly, tumor tissues collected during the vaccination and orthotopic efficacy studies were cut into small pieces, followed by digestion in collagenase type I (0.5 mg/mL, Worthington Biol Corporation) in a benchtop incubating shaker (MaxQ Digital 4450, Thermo Scientific) for 1 h at 37 °C. The digested tissues were meshed twice through a 70 μM cell strainer, and the cell pellets suspended in 5 mL of Ack lysis buffer (Gibco) at 4 °C for 5 min to lyse red blood cells. After centrifugation at 1500 rpm for 5 min, the single cell suspensions were washed twice with cold PBS twice and then resuspended in staining buffer (554656, BD Biosciences). To block nonspecific binding, cell suspensions were incubated with FcBlock (TruStain fcX anti-mouse CD16/32, clone 93, BioLegend) for 20 min. Multiparameter staining was performed by utilizing different combinations of fluorophore-conjugated antibodies for 40 min at 4 °C. Dead cells were excluded by 7-aminoactinomycin D (7AAD, Sigma) staining. Doublet cells were excluded based on their forward and side scatter characteristics. The following immune cell subpopulations were investigated, using multichannel gating: (i) CD8+ T cells (CD45+CD3+CD8+CD25+), (ii) Tregs (CD45+CD3+CD4+FOXP3+), (iii) IFN-γ+ T cells (CD45+CD3+CD8+IFN-γ+), (iv) granzyme B+ T cells (CD45+CD3+CD8+granzyme B+), (v) CD91+ DC-like cells (CD45+CD11b+CD11c+CD91+), (vi) CD80+/CD86+ DCs (CD45+CD11c+CD80+CD86+), and (vii) CD103+ DCs (CD45+CD11b+CD11c+CD103+). Anti-mouse antibodies sources are as follows: CD45-V450 (#560501, 1/100), CD45-APC-Cy7 (#557659, 1/100), CD4-Alexa Fluor 488 (#557667, 1/100), FOXP3-PE (#563101, 1/100), CD8α-PE (#561095, 1/100), CD11b-PE (553311, 1/100), and CD11c-V450 (560521, 1/100) were from BD Biosciences; CD103-Alexa Fluor 647 (#121410, 1/250) and IFN-γ-APC (505810, 1/100) were from BioLegend. LRP1 (CD91)-Alexa Fluor 647 (ab195568, 1/250) were from Abcam. CD3-APC-eFluor780 (#47-0032-82, 1/100), CD25-APC (#17-0251-82, 1/100) and granzyme B-eFluor 660 (50-8898-82, 1/100) were from eBiosciences. For intracellular staining of FOXP3, IFN-γ, and granzyme B, cells were fixed and permeabilized using a Staining Buffer Set (00-5523-00, eBioscience) followed by PBS washing prior to conducting flow cytometry in a LSRII (BD Biosciences). The data were plotted as a change in the normalized ratio in the experimental versus the control sample by FlowJo software (Tree Star). Statistical Analysis Differences among groups were estimated by the analysis of variance (ANOVA); Kaplan–Meier survival curves were compared using the Log-rank Mantel-Cox test (version 23, SPSS). Results were presented as mean ± standard deviation (SD), representing at least three independent experiments. Statistical significance was set at p < 0.05; **p < 0.01; #p < 0.001, as indicated in the figure legends. Supporting Information Available The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.8b05189.Additional figures, table, and results as described in the text (PDF) Supplementary Material nn8b05189_si_001.pdf The authors declare the following competing financial interest(s): Andre E. Nel and Huan Meng are co-founders and equity holders in Westwood Biosciences Inc. The remaining authors declare no conflict of interest. Acknowledgments Research reported in this publication was supported by the National Cancer Institute of the National Institutes of Health under Award Number, U01CA198846. J.L. is a recipient of the UCLA Tumor Immunology Training Grant (USHHS Ruth L. Kirschstein Institutional National Research Service Award No. T32 CA009120). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. We thank the Translational Pathology Core Laboratory (TPCL) at UCLA Jonsson Comprehensive Cancer Center for IHC staining, the Electron Imaging Center for Nanomachines the use of EM instruments at, the Molecular Instrumentation Center for NMRs and Mass Spectrometry, the CNSI Advanced Light Microscopy/Spectroscopy Shared Facility for confocal fluorescent microscopy. ==== Refs References Edwards B. K. ; Ward E. ; Kohler B. A. ; Eheman C. ; Zauber A. G. ; Anderson R. N. ; Jemal A. ; Schymura M. 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==== Front ACS OmegaACS OmegaaoacsodfACS Omega2470-1343American Chemical Society 3055598210.1021/acsomega.8b02090ArticleCisplatin-Loaded Graphene Oxide/Chitosan/Hydroxyapatite Composite as a Promising Tool for Osteosarcoma-Affected Bone Regeneration Sumathra Murugan †Sadasivuni Kishor Kumar ‡Kumar S. Suresh §Rajan Mariappan †† Biomaterials in Medicinal Chemistry Laboratory, Department of Natural Products Chemistry, School of Chemistry, Madurai Kamaraj University, Madurai625021, India‡ Centre for Advanced Materials, Qatar University, P.O. Box 2713, Doha, Qatar§ Department of Medical Microbiology and Parasitology, Universiti Putra Malaysia, Serdang 43400, Malaysia E-mail: [email protected]. Tel: +91 9488014084.01 11 2018 30 11 2018 3 11 14620 14633 18 08 2018 22 10 2018 Copyright © 2018 American Chemical Society2018American Chemical SocietyThis is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes. Presently, tissue engineering approaches have been focused toward finding new potential scaffolds with osteoconductivity on bone-disease-affected cells. This work focused on the cisplatin (CDDP)-loaded graphene oxide (GO)/hydroxyapatite (HAP)/chitosan (CS) composite for enhancing the growth of osteoblast cells and prevent the development of osteosarcoma cells. The prepared composites were characterized for the confirmation of composite formation using Fourier transform infrared spectroscopy, scanning electron microscopy, transmission electron microscopy, and X-ray diffraction techniques. A flowerlike morphology was observed for the GO/HAP/CS-3/CDDP composite. UV–vis spectroscopy was used to observe the controlled release of CDDP from the GO/HAP/CS-3/CDDP composite, and 67.34% of CDDP was released from the composite over a time period of 10 days. The GO/HAP/CS-3/CDDP nanocomposites showed higher viability in comparison with GO/HAP/CS-3 on MG63 osteoblast-like cells and higher cytotoxicity against cancer cells (A549). The synthesized composite was found to show enhanced proliferative, adhesive, and osteoinductive effects on the alkaline phosphatase activity of osteoblast-like cells. Our results suggested that the CDDP-loaded GO/HAP/CS-3 nanocomposite has an immense prospective as a bone tissue replacement in the bone-cancer-affected tissues. document-id-old-9ao8b02090document-id-new-14ao-2018-02090bccc-price ==== Body 1 Introduction Phosphates of calcium are of immense impact due to their astonishing biocompatibility and bioactivity making them useful in biomedicine fields analogous to orthopedics, drug delivery, and dentistry. Among the calcium phosphate compounds, hydroxyapatite (HAP) is the most valuable one because of its potential use in bone tissue engineering and also because it exhibits excellent osteoinductivity.1 The relevance of HAP in load-bearing implants is constrained because of the typical fragility with small fracture toughness.2 Generally, HAP can be combined with different varieties of natural polymers, synthetic polymers, and graphene family materials.3 The graphene family nanomaterials include several graphene derivatives, such as few-layered graphene, graphene oxide (GO), reduced graphene oxide, ultrathin graphite, and graphene nanosheets that were used in various biomedical applications.3 These all differ from each other in terms of surface properties, number of layers, and size. GO enhances or alters the required properties for a specific application. Nanosheets of GO is an attractive nanomaterial, which have two-dimensional property, mechanical potency, biocompatibility, quickly accumulated curiosity in the biomedical and pharmaceutical fields,4 potential release vehicles for drugs, biological macromolecules cellular coloring agents and implantable tissues.5,6 Current studies signify that the inclusion of GO could substantially provide mechanical strength to GO-related composites7,8 and also that GO might endorse the adhesion of osteoblasts and propagation of osteoblast activity.9,10 The concern of promoting apatite nucleation strength opens up a further advantage of GO in HAP-based composite preparation.11 Recently, chitosan (CS) has found diversity of applications such as in drug delivery, food packaging, forming a membrane for separation, environmental applications, and tissue engineering for admissible improvement of the bone graft materials.12 CS possesses a lot of superior biomedical properties such as anti-inflammatory and antimicrobial properties, biocompatibility, biodegradability, nonantigenicity, osteoconductivity, and nontoxicity.13,14 Moreover, for the CS molecule to be used as a structural sustainer in tissue regeneration, it should have sufficient surface area to accommodate live cells effectively; the CS structure allows appropriate transport of nutrients for cell growth. Its chemical structure possesses the ability to regenerate primary tissue cells.15,16 In tissue engineering, CS and HAP composites have the ability to promote a great proliferative activity in osteoblasts.17 A vastly permeable, three-dimensional structure is significant for tissue engineering materials for replicating the extracellular matrix (ECM) to set a proper microenvironment for cell attachment and propagation.18,19 Among these materials, GO, HAP, and CS are generally chosen because of the effectual functional features such as their bone-resembling properties, which can successfully encourage osteoblast enlargement, stimulate mineralization of osteoid, and suppress the osteosarcoma (OS) cells.20 In this study, we evaluate the impact of anticancer drug cisplatin (CDDP) loaded on CS-functionalized HAP on a GO platform composite on bone formation during bone repair in osteosarcoma (OS).21,22 OS is a greatly malignant mesenchymal cancer of bone in which the malignant cells create osteoid. Among all available chemotherapeutic agents, anthracycline and platinum-based drugs are used most commonly, and particularly CDDP is solitary of the mainly effectual anticancer agents for reducing the feasible cell quantity.23 This action could lead to the CDDP-induced cell death along with cell propagation reticence. 2 Results and Discussion 2.1 Fourier Transform Infrared (FTIR) Analysis Figure 1A,a shows the absorption bands at 473, 565, 602, 963, 1035, and 1098 cm–1, which are the distinguishing peaks of a PO43– group. The small peak at 473 cm–1 is ascribed to the ν2 bending vibration of the PO43– group. The triply degenerated ν4 bending vibrations appeared as the peaks at 565 and 602 cm–1. The band at 963 cm–1 corresponds to ν1, whereas the ν3 vibrations of PO43– ions showed the bands at 1033 and 1098 cm–1. Figure 1A,b CS polymer N–H and O–H stretching vibration peaks appeared at 1653 and 1635 cm–1, respectively. The spectrum of GO in Figure 1A,c clearly shows oxygen-possessing groups at 1054, 1223, 1395, 1622, and 1729 cm–1. These correspond to the C–O stretching vibration, C–OH stretching vibration, C–O–H deformation vibration, C–C stretching vibration, and CO stretching vibration of COOH groups.24 Meanwhile, the spectra of the GO/HAP composite in Figure 1A,d confirmed the presence of graphene oxide sheets by the emergence of clear absorption bands of methylene (CH2) groups nearly around 2854 and 2918 cm–1. The stretching band of pure HAP peaks shifted from 1035 to 1029 cm–1 (Figure 1A,d) due to the interaction of HAP with GO. This indicates the formation of strong hydrogen bonding between the HAP and GO sheets.25−28 In addition, the GO/HAP/CS composite, as revealed in Figure 1A,e–g, suggests that there is no observable variation in the three compositions following CS functionalization. However, a C=O absorption peak at 1654 cm–1 in CS shifted toward a lower region at 1621 cm–1 in the n-HA/CS/GO composite, which was due to the synergistic effects of hydrogen bonding between the CS and GO/HAP; also, the peak of −NH2 (1598 cm–1) did not appear, which may be due to the development of −NH3+. The peak of asymmetry stretching of −COO– was present at ∼1420 cm–1. These annotations stress upon the fact that there is a presence of electrostatic interaction between −COO– of GO and −NH3+ of CS, which resulted in the creation of GO/HAP/CS-1, GO/HAP/CS-2, and GO/HAP/CS-3 networks with the inclusion of HAP compound in Figure 1A,e–g. The FTIR spectrum of the CDDP composite exhibits only a peak in array of the corresponding CDDP characteristic peaks at 799, 1305, 1540, and 1651 cm–1 being visible in CDDP loaded in the GO/HAP/CS-3 composite was confirmed. The broadness of the peak is 3200–3300 cm–1 due to the intermolecular hydrogen bonding between the CDDP and GO/HAP/CS-3 composite. This is due to the electrostatic interaction between CDDP and the polymer matrix. In the spectrum of CS, there are two characteristic absorbance bands centered at 1653 and 1596 cm–1, which correspond to the C=O stretching vibration of −NHCO– and the N–H bending of −NH2, respectively. Compared with those of pure CS and GO, both peaks at 1596 cm–1 related to −NH2 absorbance vibration and at 1730 cm–1 belonging to the C=O stretch of the carboxylic group disappear in the spectra of CS/GO nanocomposites. Moreover, the band corresponding to the C=O characteristic stretching of the amide group shifts to a lower wavenumber, which could be ascribed to the synergistic effect of hydrogen bonding between CS and the oxygenated groups in GO and electrostatic interaction between polycationic CS and the negative charge on the surface of GO in the GO/HAP/CS composite. The transmittance percentage decreased with an increasing in the wt % of CS, as shown in Figure 1B,e–g. Figure 1 FTIR spectra of (A) (a) HAP, (b) CS, (c) GO, (d) GO/HAP, (e) GO/HAP/CS-1, (f) GO/HAP/CS-2, (g) GO/HAP/CS-3, and (h) GO/HAP/CS-3/CDDP composites. (B, C) Zoomed FTIR region with wavenumbers 1200–1800 and 3200–3800 cm–1. 2.2 X-ray Diffraction (XRD) The main peaks of HAP were formed at 26, 31.6, 32.92, 35.7, 40, 46.74, and 54.10°, which could be indexed to the (002), (211), (112), (300), (202), (222), and (213) lattice planes of the hexagonal HAP, respectively, as shown in Figure 2A,a. This demonstrates that diffraction is stronger than the standard diffraction pattern (JCPDS card no. 09-0432). The broad diffraction peaks at 2θ = 9.5 and 19.5° indicate the XRD spectrum of CS, as shown in Figure 2A,b. GO flakes showed a diffraction peak at 10.9° (Figure 2A,c). The XRD pattern of GO/HAP shows characteristic diffraction peaks at 2θ = 25.7, 28.79, 32.34, and 39.9° constant with the XRD pattern of pure HAP shown in Figure 2A,d. Typical peaks of CS around 2θ values of 11.3, 18.2, and 23° can be clearly seen from Figure 2A,e–g. The two primary peaks of CS and GO resembled the hydrated crystalline arrangement. The broadened peak at around 23° indicates the extension of the amorphous nature.30,31 In our case, together the hydrogen bonding and electrostatic interaction may have contributed toward a reasonably ordered array of CS chains along the rigid template offered by GO, Figure 2B,e–g.29 The two characteristic peaks of CDDP were observed at 2θ about 15 and 20°, as shown in Figure 2B,h. After incorporation of the CS matrix into GO/HAP, the XRD pattern of the GO/HAP/CS nanocomposite shows only the CS diffraction peaks, and the peak of GO disappears from the diffraction pattern of GO/HAP/CS (Figure 2A), which clearly demonstrates the formation of GO sheets in the composite CS polymer matrix and the disappearance of the regular and periodic structure of graphene oxide.30 It is noticed that incorporation of CS less than 30 wt % to GO/HAP slightly increases the intensity of the characteristic peaks of CS. However, the intensity of the characteristic peaks of CS obviously increases in the GO/HAP/CS composites, and characteristic peaks of CS at around (2θ) 8.4, 11.3, 18.2, and 23° can be clearly seen from Figure 2B. The first two peaks correspond to the hydrated crystalline structure, whereas the broadened peak at about 23° indicates the existence of an amorphous structure. Figure 2 (A) XRD spectra of (a) HAP, (b) CS, (c) GO, (d) GO/HAP, (e) GO/HAP/CS-1, (f) GO/HAP/CS-2, (g) GO/HAP/CS-3, and (h) GO/HAP/CS-3/CDDP composites. (B) Zoomed spectra of composites between 20 and 40°. 2.3 Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM) Morphological Analyses The surface morphologies of the GO/HAP, GO/HAP/CS, and CDDP-loaded GO/HAP/CS composites were investigated through SEM and TEM technologies and are presented in Figure 3. The images in Figure 3a,b indicate that HAP and GO had spherical and sheetlike morphologies, respectively. Figure 3c represents GO/HAP composites, and it shows the spherical particles of HAP on the GO surface. Figure 3d–i shows the morphology of the CS-functionalized GO/HAP composites after hydrothermal treatment with the increasing weight percent of CS. After the hydrothermal treatment, HAP from the GO/HAP/CS composite has grown, and the HAP fabricated by the hydrothermal method shows a typical flowerlike porous morphology. HAP spheres are composed of thin flakes with high aspect ratios of CS forming the flowerlike morphology (Figure 3d–i), which suggests significant difference in the synthesis of the composite biomaterial. The SEM image of the composite indicates unvarying porous morphology. The HAP spheres are composed of thin flakes with high aspect ratios of CS forming the flowerlike morphology, which suggests significant difference in the synthesis of the composite biomaterial. The c-axis-oriented hydroxyapatite surfaces are likely to promote preferentially oriented growth through a cyclic process of dissolution and re-precipitation, followed by homoepitaxial growth. The remarkable morphological and microstructural changes after dissolution suggest the capability of highly textured hydroxyapatite to act as a tissue engineering scaffold with an interconnecting porous network that may be beneficial for cellular activity. Figure 3j indicates the CDDP-loaded GO/HAP/CS-3 composites. Figure 3k,m represents the TEM images of the GO/HAP/CS-3 and CDDP-loaded GO/HAP/CS-3 composites. The TEM image of the GO/HAP/CS-3 flakes of flower structures is well correlated with the SEM images of GO/HAP/CS-3 and GO/HAP/CS-3/CDDP. The CDDP-loaded GO/HAP/CS-3 composite SADE spectrum is shown in the inset image of Figure 3l,n. It shows the amorphous nature of the overall composite. Figure 3 SEM images of (a) HAP, (b) GO, (c) GO/HAP, (d, g) GO/HAP/CS-1, (e, h) GO/HAP/CS-2, (f, i) GO/HAP/CS-3 and (j) GO/HAP/CS-3/CDDP composites. TEM images of (k) GO/HAP/CS-3 and (m) GO/HAP/CS-3/CDDP; SADE images of (l) GO/HAP/CS-3 and (n) GO/HAP/CS-3/CDDP. 2.4 Energy Dispersive X-ray Analysis (EDAX) Mapping Moreover, an elemental analysis mapping was carried out to recognize the distribution of elements in the composite material. Figure 4a,b reveals the SEM image and EDAX spectrum of the GO/HAP/CS-3/CDDP composite. From the EDAX mapping analysis, elemental distribution of the GO/HAP/CS-3/CDDP composite is presented in Figure 4c. Calcium, phosphorus, oxygen, carbon, and nitrogen were found and are shown in Figure 4d–h in different colors. This confirms that a part of the GO/HAP/CS-3/CDDP composite clearly shows the distribution of C, Ca, O, N, and P elements in the composite. It is thus clear that the GO/HAP/CS-3/CDDP composite has an immense prospective to be used in the progress of new bone formation and bone repair applications due to the presence of HAP, GO, CS, and CDDP. Figure 4 (a) SEM images of GO/HAP/CS-3/CDDP; (b) EDAX spectrum of GO/HAP/CS-3/CDDP; (c) elemental mapping of GO/HAP/CS-3/CDDP; and (d–h) elements present in GO/HAP/CS-3/CDDP: Ca, P, O, C, and N. 2.5 Barrett–Joyner–Halenda (BJH) and Brunauer–Emmett–Teller (BET) Analyses The pore volume, surface area, and pore diameter of the GO/HAP/CS-1, GO/HAP/CS-2, and GO/HAP/CS-3 composites were investigated through BET analysis, and BJH investigation revealed the mesoporous nature of the composite; the results are presented in Table 1. A distinctive adsorption/desorption graph of the mesosphere is shown in Figure 5. Figure 5 BET analysis: (A, C, E) adsorption of GO/HAP/CS-1, GO/HAP/CS-2, and GO/HAP/CS-3 and (B, D, F) desorption of GO/HAP/CS-1, GO/HAP/CS-2, and GO/HAP/CS-3. Table 1 Average Structural Parameters and Surface Properties of GO/HAP/CS-3 Nanocomposites composite ID surface area (m2/g) pore volume (cm3/g) pore diameter (nm) GO/HAP/CS-1 3.8 0.01854 4.489 GO/HAP/CS-2 5.552 0.01926 5.92 GO/HAP/CS-3 5.7887 0.0970 7.1798 2.6 CDDP Loading Capacity (LC) and In Vitro CDDP Release Analysis The aim of this study is self-curing of bone cancer through anticancer-drug-loaded composites. Thus, the investigation of the CDDP loading capacity and CDDP releasing properties of the GO/HAP/CS composite materials is very important in this regard. Figure 6a–c indicates UV–visible spectra of the CDDP loading capacity of GO/HAP/CS-1, GO/HAP/CS-2, and GO/HAP/CS-3 after 3 h. Initially, the CDDP absorption peak appearing at the intensity range is nearly zero, and then, for the composites vortexed for 3 h, the intensity increased to almost 0.89, 1.0, and 1.5, respectively. The loading capacities of GO/HAP/CS-1, GO/HAP/CS-2, and GO/HAP/CS-3 were around 44.7, 50.44, and 78% respectively. The in vitro UV–visible spectra of CDDP from GO/HAP/CS-3/CDDP were performed in phosphate buffer saline (PBS) medium at pH 7.4, and the corresponding discharge report is depicted in Figure 6d–f. The CDDP release was 84.84, 74.44, and 67.34% over a period of 10 days for the composites GO/HAP/CS-1/CDDP, GO/HAP/CS-2/CDDP, and GO/HAP/CS-3/CDDP. From the releasing profile, it could be understood that GO/HAP/CS-3/CDDP demonstrated the required quantity of drug release, which was observed with a constant releasing rate. This could be partially due to the flake pore geometry of the flowerlike morphology of the composite and the length of drug releases from the GO/HAP/CS-3/CDDP composites. The constant release rate further affirms that the composite can be a potential candidate for curing of cancer diseases as well as proved to be helpful in new bone formation.31Figure 6g represents the cumulative release of CDDP from the GO/HAP/CS-3/CDDP composites. Figure 6 UV–vis spectra of Loading Capacity of (a) GO/HAP/CS-1, (b) GO/HAP/CS-2; (c) GO/HAP/CS-3 and CDDP released from (d) GO/HAP/CS-1/CDDP, (e) GO/HAP/CS-2/CDDP, and (f) GO/HAP/CS-3/CDDP; and (g) cumulative release profile of CDDP released from GO/HAP/CS/CDDP composites. 2.7 Cell Viability and Cytotoxicity Figure S3 shows the MG63 osteoblast-like cells cultured with different concentrations (0.2, 0.4, 0.6, 0.8, and 1.0 μg/mL) of GO/HAP/CS-3 and GO/HAP/CS-3/CDDP for 24 h. The viability of MG63 osteoblast-like cells increased with the increasing sample concentration from 0.2 to 1.0 μg/mL. Figure 7 shows augmentation of MG63 osteoblast-like cells cultivated on the GO/HAP/CS-3 and GO/HAP/CS-3/CDDP composites. The 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay shows that the number of MG63 osteoblast-like cells can noticeably increase on HAP, GO/HAP, GO/HAP/CS-1, GO/HAP/CS, GO/HAP/CS-3, and GO/HAP/CS-3/CDDP in comparison to that in pure HAP as control. The culture time is extended from 1 to 7 and 14 days. The outcome shows that MG63 cells can proliferate well on the prepared composite. The viability activities of MG63 osteoblast-like cells cultivated on the as-prepared composite are observed by optical microscopy. As the main inorganic phase of the natural bone tissue, hydroxyapatite (HA) nanoparticles are chosen as ingredients of bone scaffolds, which have the potential to augment bone regeneration capability. Graphene oxide improves the biological properties of scaffold materials and promotes the osteoblast proliferation. Furthermore, the incorporation of GO into HAP (GO/HAP) was nontoxic to osteoblasts and augments propagation and osteogenic discrimination. In addition, one of the most promising polymeric materials seems to be chitosan, a biopolymer.32 An increase in the concentration of CS (10, 20, 30 wt %) in the GO/HAP composite results in the biocompatibility, osteoconductivity, and a lowest inflammatory response in MG63 osteoblast-like cells on day 1, 7, and 14. The composite exhibits an admirable cell viability performance because the MG63 osteoblast-like cells appear, which can be observed from optical microscopic images. When the culturing time of the MG63 cells is increased from 7 to 14 days, the culture is more obvious on the CDDP-loaded GO/HAP/CS-3 composite. The GO/HAP/CS-3/CDDP composite shows a major difference on day 14 compared with the GO/HAP/CS-3 composite, which is reported to encourage cell propagation and cell expansion. Figure 7 (A) Cell viability on HAP, GO/HAP, GO/HAP/CS-1, GO/HAP/CS-2, GO/HAP/CS-3, and GO/HAP/CS-3/CDDP analyzed by optical microscopy on 1, 7, and 14 days. (B) Quantification of cell viability of MG63 osteoblast-like cells measured by the MTT assay (n = 3, p < 0.005). Figure 8A shows the optical microscopy observation on A549 cells. The cytotoxicity analyses of HAP, GO/HAP/CS-3, and GO/HAP/CS-3/CDDP are revealed in Figure 8A,B. The considerable variations in the nature of toxicity of the GO/HAP/CS-3/CDDP composite appeared in A549 cells with an increase in the number of days of incubation, and it was attributed to the presence of CDDP in the composite (Figure 8A). Without CDDP loading, GO/HAP/CS-3 shows slightly toxic nature due to the presence of chitosan molecules in the composites.32 This consequence established that the GO/HAP/CS-3/CDDP composites abridged the growth of cancer cells (Figure 8B). The MTT assay showed that the cell viability exceeded 23% and the incubation of A549 cells with GO/HAP/CS-3/CDDP composites concealed cell augmentation after 21 days, as shown in Figure 8B. Hence, the GO/HAP/CS-3/CDDP composite diminishes osteosarcoma progression because one of the effectual anti-bone-cancer agents is cisplatin.33 HAP as a prospective material for CDDP confirmed unremitting discharge of the drug from HAP blocks.34 Chitosan has power over anticancer doings by itself and an eminent study is that chitosan diminishes the viability of osteosarcoma cells, except not the normal cells from which osteosarcoma cells begin osteoblasts.35 Chitosan was also able to condense the viability property on a prime bone cancer such as osteosarcoma. Thus, a chitosan can be of considerable use for bone cancer patients. Figure 8 (A) Cytotoxicity on HAP, GO/HAP/CS-3, and GO/HAP/CS-3/CDDP investigated by optical microscopy on 1, 7, 14, and 21 days. (B) Quantification of cell viability of A549 cells measured by the MTT assay (n = 3, p < 0.005). 2.8 Cell Adhesion For the investigation of effects of composites on sustaining cell growth, we seeded MG63 osteoblast-like cells on pure HAP, GO/HAP, GO/HAP/CS-1, GO/HAP/CS-2, GO/HAP/CS-3, and GO/HAP/CS-3/CDDP, which were examined by SEM for morphology after 1 day, 7 days, and 14 days. The SEM micrograph indicates that MG63 cells were spread over the composites. The ability of graphene oxide to improve the biological properties of composite materials and its ability to promote the adhesion of osteoblasts have been noticed in this study. After 7 days of culturing, there were plenty of MG63 osteoblast-like cells observed on the GO/HAP/CS-3/CDDP composite. Figure 9 indicates the micrographs of the adhesion on HAP, GO/HAP, GO/HAP/CS-1, GO/HAP/CS-2, GO/HAP/CS-3, and GO/HAP/CS-3/CDDP composites. Chitosan, a biopolymer, acts as a provider of better environment for MG63 osteoblasts cells. The GO/HAP/CS-3/CDDP composite has a suitable structure to mimic a temporary extracellular matrix (ECM), which can control cellular behaviors, promote MG63 cell adhesion, and provide appropriate microenvironments for MG63 osteoblast cells.36,37 The GO/HAP/CS-3/CDDP composite revealed an admirable cell adhesion performance because MG63 osteoblast-like cells show cytoplasmic extension and filopodia can be observed on the MG63 osteoblast-like cells. Figure 9 Morphology of cell adhesion of osteoblast-like cells (MG63) cultured in the presence of HAP, GO/HAP, GO/HAP/CS-1, GO/HAP/CS-2GO/HAP/CS-3, and GO/HAP/CS-3/CDDP on 1, 7, and 14 days. 2.9 Alkaline Phosphatase (ALP) Activity ALP is a significant osteogenic discrimination and biochemical pointer of osteoblasts. The GO/HAP/CS-3 and GO/HAP/CS-3/CDDP composites promote ALP production from osteoblast-like cells (Figure 10), which increases with the increasing culture time. For the GO/HAP/CS-3 composites alone, ALP concentration increased from 2.6 mM on day 1 to almost 4.9 mM on day 14. Indeed, with the GO/HAP/CS-3/CDDP composites, ALP activity was 4.2 mM at day 1 and 7.1 mM at day 14 (p < 0.005). The presence of CDDP in the GO/HAP/CS-3/CDDP composites significantly promoted the ALP activity as compared to that of GO/HAP/CS-3 alone.38 These data show that the ALP activity on osteoblast-like cells increases when CDDP is supplemented with GO/HAP/CS-3. Figure 10 ALP activity on GO/HAP/CS-3 and GO/HAP/CS-3/CDDP on 1, 7, and 14 days (n = 3, p < 0.005). 2.10 Gene Expression The analysis of gene expression on MG63 osteoblast-like cells using the osteogenic initiation culture was analyzed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) investigation, Figure 11a–c. The gene articulation like Runx2, ALP, and OCN on GO/HAP/CS-3 and GO/HAP/CS-3/CDDP was investigated. The mRNA transcript levels of ALP in GO/HAP/CS-3 and GO/HAP/CS-3/CDDP composites expanded fundamentally in contrast to those in GO/HAP/CS-3/CDDP congregation. A comparable prototype was seen in the osteogenic transformation factor Runx2 through amplification and partly covering to that on GO/HAP/CS-3 and GO/HAP/CS-3/CDDP. The OCN expression level of GO/HAP/CS-3 and GO/HAP/CS-3/CDDP get together was mainly up-managed with augment and superimpose than GO/HAP/CS-3 and GO/HAP/CS-3/CDDP congregation. It is well known that CDDP fulfills a major purpose in bone mineral homeostasis and that it might in addition perform as a bioactive protein that helps in augmentation of osteogenisis.39,40 Figure 11 Gene expression of (a) ALP, (b) Runx2, and (c) osteocalcin in MG63 osteoblast-like cells cultivated on GO/HAP/CS-3 and GO/HAP/CS-3/CDDP for days 1, 7, and 14 (n = 3, p < 0.005). 3 Conclusions Hydrothermal-assisted synthesis of GO/HAP/CS-3 was used to achieve a flowerlike morphology of HAP, GO, and CS, which exhibits a facile technique for scheming the morphological characteristics of composites. CDDP acts as a major chemotherapeutic remedy for the action of cancers. The CDDP-loaded composite GO/HAP/CS-3 exhibits an excellent cell viability behavior on MG63 osteoblast-like cells. The augmentation in cell viability of cells of MG63 osteoblasts enhances pertaining to the increase in the porosity, which serves a significant role in the medical trials. The GO/HAP/CS-3/CDDP composite exhibits a cytoplasmic extension, and filopodia can be observed on the MG63 osteoblast-like cells. The GO/HAP/CS-3/CDDP composite should allow artificially favorable biomaterial in the tissue engineering applications. 4 Materials and Methods 4.1 Materials Ammonia solution (NH4OH), calcium chloride dihydrate (CaCl2·2H2O), chitosan (molecular weight, 50–190 kDa), cisplatin (CDDP), diammonium hydrogen phosphate ((NH4)2HPO4), ethyl alcohol (C2H5OH), graphite, hydrogen peroxide (H2O2), potassium permanganate (KMnO4), phosphate buffer saline (PBS), phosphoric acid (H3PO4), and sulfuric acid (H2SO4) were brought from Sigma-Aldrich, Mumbai, India. Analytical-grade chemicals were used in all of the experiments with no any additional cleansing. Deionized water was used in all of the experiments. 4.2 Preparation of GO GO was synthesized using powder graphite following the modified Hummer’s method.35 The experimental procedures were slightly modified as follows: before preparation of GO, the graphite flakes were subjected to ultrasonication for the formation of graphene sheets. Then, KMnO4 (18.0 g, 6 wt equiv) was supplemented slowly to a 9:1 mixture of rigorous H2SO4/H3PO4 (360:40 mL) in six equal portions, producing a slight exothermic reaction that should not exceed the temperature 35–40 °C, and a graphene sheet (3.0 g, 1 equiv wt %) was formed. This reaction was passionate to 50 °C with continuous stirring for 12 h. The reaction temperature was reduced to room temperature (27 °C), and the reaction mixture was discharged onto ice (400 mL) containing 30% H2O2 (3 mL). 4.3 Preparation of GO/HAP Composite Distinctively, 3 mg of GO was dispersed in 5 mL of DD water with continuous stirring for 30 min. Then, 0.5 mM CaCl2·2H2O was added into each of the 5 mL GO suspension under stirring for fine mixing of each solution. Then, the 0.3 mM (NH4)2HPO4 aqueous solution was added slowly to the above mixture and stirred vigorously on a magnetic stirrer. The pH of the reaction mixture was maintained at pH 10.0 with the help of ammonia solution. Stirring was continued for about 30 min. The whole solution was ultrasonicated for 30 min, and the solution was slowly filtered using a vacuum-assisted Buchner funnel, followed by washing with 100 mL of water three times to obtain the HAP/GO composite. Finally, the resulting precipitate was dried at 50 °C. Throughout the synthesis, the oxygen-containing functional groups on GO surfaces act as receptor sites for Ca2P through electrostatic interactions; these anchored cations can in situ react with phosphate ions to obtain apatite nanoparticles. The distribution and the microstructures of HAP on graphene are mainly influenced by the amounts and types of oxygenated groups on the GO and the concentration of calcium and phosphorus in the reagents. Composites prepared in this method are expected to increase the interfacial bonding between GO and HAP. 4.4 Preparation of GO/HAP/CS Composites The quantity of CS has considerably influenced the potency of the synthesizing hydrogels. Considering the solubility boundary of CS in acetic acid solution, we used different weight percentages (10, 20, 30 wt %) of CS aqueous solutions to prepare the composites. Different weight percentages of the CS polymer solution were added into the GO/HAP composite, and then it was ultrasonicated for 30 min (5 s on, 3 s off), followed by pouring of the suspension into a sealed autoclave. It was heated to 180 °C for 2 h in a muffle furnace and, afterward, allowed to cool to room temperature (27 °C) to obtain a GO/HAP/CS-1, GO/HAP/CS-2, and GO/HAP/CS-3 composites with 10, 20, and 30 wt % CS, respectively. The schematic diagram of GO/HAP/CS-3/CDDP is shown in Figure S1. 4.5 Cisplatin (CDDP) Encapsulation/Entrapment on GO/HAP/CS-1, GO/HAP/CS-2, and GO/HAP/CS-3 Composites CDDP (5 mg) was dissolved in acetone (1 mg/1 mL in acetone, 5 mL) along with the addition of GO/HAP/CS-1, GO/HAP/CS-2, and GO/HAP/CS-3 with the help of stirring using a magnetic stirrer at 1000 rpm. The resultant mixture was centrifuged at 4000 rpm. Finally, the acquired GO/HAP/CS-1/CDDP, GO/HAP/CS-2/CDDP, GO/HAP/CS-3/CDDP powder was lyophilized using a lyophilizer (SSIPL-LYF/065/071216). 4.6 Physicochemical Characterizations 4.6.1 Fourier Transform Infrared (FTIR) Spectroscopy The HAP, GO/HAP, GO/HAP/CS composites and the CDDP-loaded GO/HAP/CS composites were tested by a Bruker Tensor 27 Series FTIR spectrometer, and 16 scans per sample were taken in the region of 400–4000 cm–1 with 2 cm–1 resolution. The pellets were made for the FTIR test by crushing 0.2 g of the sample powder together with 1 g of KBr and then pressing them into a transparent disc. 4.6.2 X-ray Diffraction The X-ray diffraction (XRD) characterization was done to analyze the phase composition and to precisely obtain the crystallinity of prepared HAP, GO/HAP, GO/HAP/CS composites, and CDDP-loaded GO/HAP/CS composites. This test was accomplished in a Bruker D8 Advance diffractometer with a monochromatic Cu Kα source operated at 40 kV and 30 mA. An acceleration voltage of 30 kV and a current of 15 mA were applied. The operating range of this test was over the 2θ range of 10–60° in step scan mode with a step size of 0.02° and a scan rate of 0.02°/min. 4.6.3 Scanning Electron Microscopy (SEM) The morphology, EDAX and elemental mapping, and morphologies of the GO/HAP, GO/HAP/CS, and CDDP-loaded GO/HAP/CS composites were examined by SEM (VEGA3 TESCAN) by operating it at a voltage of 10 kV. 4.6.4 Transmission Electron Microscopy The surface analyses of the synthesized GO/HAP and GO/HAP/CS composites and CDDP-loaded GO/HAP/CS composites were determined by high-resolution transmission electron microscopy (HR-TEM, TECNAI F30). For sample preparation for HR-TEM analysis, the synthesized nanoparticles and their composites were dispersed in ethanol by ultrasonication up to 15 min. Afterward, these were loaded on a carbon-coated copper mesh. 4.6.5 Barrett–Joyner–Halenda (BJH) and Brunauer–Emmett–Teller (BET) Analyses Nitrogen adsorption/desorption isotherms of GO/HAP/CS-1, GO/HAP/CS-2, and GO/HAP/CS-3 were assessed with a Tel Micro Tract analyzer (Bel cork, Japan) under a nonstop adsorption condition at a constant temperature (77 K). At the beginning of the analysis, GO/HAP/CS-1, GO/HAP/CS-2, and GO/HAP/CS-3 were degassed at 100 °C. BJH and BET analyses were used to find out the surface area, pore volume, and pore diameter.41 4.6.6 Loading Capacity (LC) on GO/HAP/CS-3 UV–visible spectroscopy was used to study the loading capacity of the composites formed. GO/HAP/CS-1/CDDP, GO/HAP/CS-2/CDDP, and GO/HAP/CS-3/CDDP composites (10 mg of each) were washed using 2 mL of acetone. Later, the solution was centrifuged. Afterward, complimentary CDDP having acetone was freeze-dried. Finally, hexane was used to extract the CDDP and a UV–vis spectrophotometer at 265 nm31 was used to measure the amount of CDDP loaded into GO/HAP/CS-3. 4.6.7 In Vitro Release Studies The in vitro discharge behavior of CDDP from CDDP-loaded GO/HAP/CS-1, GO/HAP/CS-2, GO/HAP/CS-3 composites was studied via a dialysis membrane technique using a PBS solution working at pH 7.4. Sample preparation included sealing of 50 mg of GO/HAP/CS-1/CDDP, GO/HAP/CS-2/CDDP, and GO/HAP/CS-3/CDDP composites into separate dialysis bags with the MWCO 12 000 Da. Then, 10 mL of the PBS solution containing CDDP-loaded composites was stirred at 100 rpm and at 37 °C. The supernatant solution was collected at different day interval by measured the concentration of CDDP solution λmax value of 265 nm in a UV-spectroscopy and replenishing among an identical quantity of new PBS medium.41,42 The following formula was used to calculate the % of drug release where AR is the absorbance of CDDP discharged from the composite and AC is the total amount of CDDP loaded in the composite. 4.6.8 Biodegradability The biodegradability of composites was analyzed for a time period of 28 days by putting them in PBS at pH 7.4 and ambient temperature 27 °C, while keeping the liquid-to-solid ratio at 0.5 mg/mL through stirring at 100 rpm. The buffer solution was freshly prepared every 3rd day at 1st, 7th, 14th, 21th, and 28th day; the days were the specimens were taken from them. The solution was then oven-dried at 60 °C for 24 h till the stable weight was achieved. The degradation proportion was calculated using the following formula where W0 is the early weight of the composite and Wt is the weight at later time t after treatment 4.6.9 Cell Viability on Osteoblast-like Cell (MG63) The National Centre for Cell Science (NCCS), Pune, India, was chosen for the procurement of the osteoblast-like cells MG63. The cells were maintained in a strict stringent condition in Dulbecco’s modified Eagle’s medium (DMEM) in a CO2 incubator at 37 °C (with a humidifier) along with low glucose concentration (1 g/L), fetal bovine serum (FBS-10%), and penicillin/streptomycin (1%). The trypsin/EDTA solution was used after every 3 days to harvest the cells. The effect of HAP, GO/HAP, GO/HAP/CS-1, GO/HAP/CS-2, GO/HAP/CS-3, and GO/HAP/CS-3/CDDP nanocomposites on MG63 was recorded with the help of the corresponding 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. A 24-well plate was used to seed the MG63 osteoblast-like cells at a density of 4 × 104 cells/well and co-cultivated with GO/HAP/CS-3 and GO/HAP/CS-3/CDDP composites with a concentration of 1 μg/mL. The MTT assay was used to assess the viability of MG63 osteoblast-like cells. After an incubation of 1, 7, and 14 days, the sample solutions were taken and the MTT solution (100 μL, 5 mg/mL) was added in 1 mL culture medium to each well plate and then again incubated for 4 h at 37 °C. Afterward, 1 mL of dimethyl sulfoxide was supplemented to it and the supernatant medium was collected separately, followed by centrifugation. The wavelength of 570 nm was used to record the ocular density of the superincumbent solution.43 4.6.10 Cytotoxicity Cell differentiation was studied on human lung cancer (A549) cells, and they were purchased from the National Center for Cell Science (NCCS), Pune, India. A549 cells were seeded in 24-well plates containing Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS and penicillin (100 U/mL)/streptomycin (100 U/mL) (Gibco, Grand Island, CA) and cultivated for 24 h. The cells were incubated at 37 °C (RT) in CO2 and were observed thorough MTT assay techniques. The composite was tested on cells incubated for different days, 1, 7, and 14. The OD values were recorded at a λmax value of 490 nm. The composite morphology was investigated by optical microscopy, and the following formula was used to calculate the cytotoxicity of the composite 4.6.11 MG63 Osteoblast-like Cell Adhesion on Composites Cell adhesion analysis was recorded using the cell viability protocol. Subsequent to day 1, day 7, and day 14 of incubation, cell-film assemblies were cleaned with PBS and fixed with 3% glutaraldehyde at 4 °C. These samples were dried in air. Dried samples were mounted on aluminum stubs with gold sputter-coating. The prepared samples were hence perspective under SEM at an extent voltage of 10 kV29 the investigations were continual three times. 4.6.12 Alkaline Phosphatase (ALP) Activity The expression of alkaline phosphatase was assessed with the composites on 24-well plates at a density of 4 × 104 cells. For each specimen of day 1, day 7, and day 14, the cells were thoroughly washed with PBS solution and lysated in Triton X-100 (0.1 vol %) using the typical freeze–defrost rotations. The calorimetric test was used to determine the ALP activity in the lysate with an ALP reagent consisting of a p-nitrophenyl phosphate substrate.40 A microplate reader quantifying at 405 nm was used to obtain the absorbance of p-nitrophenol. 4.6.13 Gene Expression Specific bone-related genes of transcript mRNA from MG63 osteoblast-like cells cultivated on the synthesized nanocomposite with a concentration of 10 μg/mL were tested by real-time qRT-PCR analysis. Full RNA was isolated, and indiscriminate hexamer-primed cDNA production was executed on them. A RevertAid first strand cDNA union pack was used. In a 40-cycle PCR using a Rotor-gene Q analyzer, the cDNA was used as a template base. To determine the real-time PCR, the Maxima SYBR green/ROX qPCR master mix was used. It was further followed by softening bend investigation to verify PCR specificity.39,40 In Rotor-Gene Q software (Corbett), limit cycle normal was used for calculation and all of the experiments were repeated twice. ΔCt analysis was used to calculate the relative gene expression. Each target gene’s comparative expression levels were normalized besides the general housekeeping gene’s Ct value. The reverse and forward primers of the handpicked genes are listed in Table 2. Table 2 Primary Sequences Used for PCR Amplification genes forward primer sequence (5′–3′) reverse primer sequence (5′–3′) gene bank no. size ALP CGGCCATCCTATATGGTAACGG CAGGAGGCCATACGCCATCACA NM_001287172 72 Runx2 CCAACTTCCTGTGCTCCGTG GTGAAACTCTTGCCTCGTCCG NM_001146038 151 osteocalcin GACCCTCTCTCTGCTCACTCT GACCTTACTGCCCTCCTGCTTG NM_007541.3 112 Supporting Information Available The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.8b02090.Schematic diagram, biodegradability, and in vitro cell viability (Figures S1–S3) (PDF) Supplementary Material ao8b02090_si_001.pdf M.R. acknowledges major financial support from the Department of Science and Technology, Science and Engineering Research Board (ref YSS/2015/001532; New Delhi, India) and also acknowledges the DST-PURSE program for the purchase of SEM and FTIR and UPE programs for the purchase of TEM. 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==== Front Cell Cycle Cell Cycle KCCY kccy20 Cell Cycle 1538-4101 1551-4005 Taylor & Francis 1542895 10.1080/15384101.2018.1542895 Research Paper Up-regulation of microRNA-497 inhibits the proliferation, migration and invasion but increases the apoptosis of multiple myeloma cells through the MAPK/ERK signaling pathway by targeting Raf-1 C.-Y. YE ET AL. CELL CYCLE Ye Cheng-Yu a Zheng Cui-Ping a Ying Wei-Wei b Weng Shan-Shan a a Department of Hematologic Oncology, Wenzhou Central Hospital, Dingli Clinical Medical School of Wenzhou Medical University, Wenzhou, P.R. China b Wenzhou Medical University, Wenzhou, P.R. China CONTACT Cui-Ping Zheng [email protected] 2018 11 12 2018 17 24 26662683 2 6 2018 14 9 2018 19 10 2018 © 2018 Informa UK Limited, trading as Taylor & Francis Group 2018 Informa UK Limited, trading as Taylor & Francis Group ABSTRACT Multiple myeloma (MM) is a cancer that occurs in plasma cells, which fall under the category of white blood cells that are in charge of antibody production. According to previous studies, microRNA-497 (miR-497) functions as a tumor suppressor in several types of cancer, including gastric cancer and colorectal cancer. Therefore, the present study aims to investigate the effects of miR-497 on cellular function of human MM cells through the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling pathway by targeting Raf-1. The differentially expressed genes and miRs in MM, and the relationship between the miR and gene were verified. It was found that Raf-1 was a target gene of miR-497. The data obtained from MM tissues showed increased Raf-1 level and decreased miR-497 level. MM cells were treated with mimic, inhibitor and siRNA in order to evaluate the role of miR-497, Raf-1 and MAPK/ERK in MM. The expression pattern of miR-497, Raf-1, ERK1/2, survivin, B-cell lymphoma-2 (Bcl-2) and BCL2-Associated X (Bax) as well as the extent of ERK1/2 phosphorylation were determined. Retored miR-497 and si-Raf-1 resulted in increases in the Bax expression and cell apoptosis and decreases in the expressions of Raf-1, MEK-2, survivin, Bcl-2, along with the extent of ERK1/2 phosphorylation. In addition, the biological function evaluations of MM cells revealed that miR-497 mimic or si-Raf-1 led to suppression in cell proliferation, invasion and migration. In conclusion, our results have demonstrated that miR-497 targets Raf-1 in order to inhibit the progression of MM by blocking the MAPK/ERK signaling pathway. Keywords MicroRNA-497 Raf-1 MAPK/ERK signaling pathway multiple myeloma proliferation migration invasion Wenzhou Municipal Science and Technology Bureau Projecty20150053 & y2016O117 the Platform Project of Zhejiang Provincial Health Department2016DTA010 This study was supported by the Platform Project of Zhejiang Provincial Health Department (No. 2016DTA010) and the Wenzhou Municipal Science and Technology Bureau Project (No. y20150053 and y2016O117). ==== Body 1. Introduction Multiple myeloma (MM) is a mature β-lymphoid cell malignancy of the bone marrow with a series of complex clinical presentations, including anemia, bone lesions, renal failure, and immune dysfunction [1–3]. It accounts for 1% of malignant diseases and 15% of hematological malignant tumors, as well as 20% of blood and bone marrow related cancer deaths [1,4]. In the early 1980s, MM inevitably resulted in a gradual decline in the life quality and even death within 2 years, while today, if properly diagnosed, there is a 50% chance of recovery with a mean survival of 5 years or even over 10 years in some cases [5]. The first three years after diagnosis is the optimal time for patients with MM to be cured, but MM remains one of the cancers with a high risk of mortality, especially in young patients [6]. The widely adopted therapies for MM are the bortezomib-based regimen and dexamethasone [7]. However, the efficacy is far from satisfactory. Hence, there is a need to thoroughly investigate the mechanism associated with the development of MM. A previous study has found that microRNAs (miRs) miR-15a and miR-16 regulate tumor proliferation in MM [8]. miRs have been proven to have essential effects on various biological processes, including cell proliferation and apoptosis, and have been considered as new therapeutic targets for cancers and cardiovascular diseases [9,10]. Due to the tumor suppressor role played by miRs, they have recently been reported to have a vital involvement in MM treatment and have been regarded as important regulators of the growth and survival of MM cells [11]. miR-497 functions as a potential biomarker and a tumor suppressor that is involved in tumor progression, proliferation and migration in various types of cancer and its expression has been found to be suppressed in several malignant tumors, such as human cervical cancer and non-small cell lung cancer [12–14]. miR-497 is predicted to promote apoptosis by regulating Bcl-2 expression and also to facilitate the inhibition of the invasion and migration of cancer cells [15,16]. Raf-1, a multifunctional protein, is a critical downstream target of several growth factors that encourages the proliferation and survival of various cancer cells [17]. A previous study has demonstrated that the expression of Raf-1 likely regulates the tumor metastatic potential and resistance to apoptosis [18]. Once activated, Raf-1 triggers the Raf-ERK signaling pathway, which, in turn, regulates the proliferation, migration, cell cycle entry, and apoptosis of cells [19]. Mitogen-activated protein kinases (MAPKs) are so-called evolutionarily well-conserved serine and threonine protein enzymes, which participate in signal transduction pathways linking cell surface receptors with major regulatory nuclear and intracellular targets [20]. In mammals, there are several MAPK enzymes that are responsible for cell proliferation, apoptosis, differentiation and survival [21]. Extracellular signal-regulated kinase (ERK) is embodied in the mammalian family of MAPKs [22]. In addition, the MAPK/ERK signaling pathway has been reported to be regulated by miRs [23]. These findings suggested that miR-497 could exert effects on cell proliferation, migration, invasion and apoptosis in human MM by targeting the Raf-1 gene through the MAPK/ERK signaling pathway and based on this hypothesis, we conducted the following experiments to provide further evidence on the effects of miR-497 in MM. 2. Methods Ethics statement All the patients involved had signed informed consent, and all experiments conducted below had been approved by the clinical trial ethics committee of Wenzhou Central Hospital, Dingli Clinical Medical School of Wenzhou Medical University. Study subjects A total of 152 MM patients hospitalized at the Department of Hematology of Wenzhou Central Hospital, Dingli Clinical Medical School of Wenzhou Medical University from March 2010 to September 2016 were selected for the current study, and their eligibility was determined using the MM diagnostic criteria adopted from the International Myeloma Working Group (IMWG) [24,25]. All cases had been pathologically confirmed MM, and the patients had no history of radiochemotherapy prior to the operation. Among the patients, there were 93 males and 59 females, between the ages of 24 to 85 years with a mean age of 59 years. According to the myeloma tumor node metastasis (TNM) staging criteria revised by the American Joint Committee on Cancer (AJCC) in 2010 [26], Thirty-nine cases were verified in stage II, and 113 cases were in stage III. Afterwards, MM tissue samples were collected from MM patients, while normal bone marrow tissue samples were collected from 3 healthy volunteers in order to be used as the control. All tissue samples were cut into small pieces, quickly placed into frozen tubes, stored in liquid nitrogen, and then preserved in a refrigerator at −80°C. Immunohistochemistry Tissue samples were rinsed 3 times with cold normal saline, embedded in optimal cutting temperature compound (OCT) and sliced into frozen sections, which were subjected to immunohistochemical staining. The sections were then incubated with rabbit polyclonal Raf-1 antibody (ab137435; Abcam, Cambridge, MA, USA) (1:1000) and were placed in a wet box at 4°C overnight. Next, the sections were incubated with biotin-labeled secondary antibody, namely, mouse anti-rabbit immunoglobulin G (IgG) antibody (ab6785; Abcam, Cambridge, MA, USA) (1:10,000) working fluid and were incubated at 37°C for 1 h, followed by the addition of diluted SP complex (horseradish peroxidase-labeled streptavidin; 1:100) (E03010; Beijing Hongyue Science and Technology Ltd., Beijing, China) and incubation at 37°C for 1 h. Thereafter, the diaminobenzidine (DAB) Kit (ab64238, Abcam, Cambridge, MA, USA) was added to the sections and color development was carried out by incubation for 10 min. The appearance of brown staining was considered a positive reaction [27]. Finally, five different fields were randomly selected from each section under an inverted microscope (XDS-800D; Shanghai Caikon Optical Instrument Co. Ltd., Shanghai, China) to obtain images. Positive cells were identified by the presence of brown-yellow granules in the cells. The Raf-1 protein staining rate was presented as the percentage of Raf-1 positive cells in a whole field of cells. The number of positive cells over 30% was considered to be positive (+) and the number of positive cells less than 30% was considered to be negative [28]. Hematoxylin-eosin (HE) staining Some of the MM tissues and normal bone marrow tissues were fixed in 3% neutral formaldehyde, made into paraffin sections, after which HE staining was performed. The sections were dewaxed routinely, hydrated with gradient alcohol, stained with hematoxylin for 2 min, rinsed with running water for 10 s, and underwent differentiation by 1% hydrochloric acid-ethanol for 10 s. After washing with distilled water for 1 min, sections were stained with eosin solution for 1 min, washed with distilled water again for 10 s, dehydrated by graded ethanol, cleared by xylene, and sealed with neutral balsam. Cell culture MM cell lines RPMI8226, U266, XG-6, XG-7, HEK293T and H929 were purchased from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. Firstly, all cells were cultured in RPMI 1640 culture medium (SP1355; Shanghai Shifeng Biological Technology Co., Ltd., Shanghai, China), 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 mg/mL streptomycin in a 5% CO2 incubator (DHP-9162; Shanghai Jiechen Laboratory Instrument Co., Ltd., Shanghai, China) with saturated humidity and a constant temperature of 37°C. The culture medium in U266, XG-6, XG-7 and H929 cells was changed every 1 ~ 2 d, and cell passaging was conducted when the cell confluence reached 80%~ 90%. Following the removal of the culture medium, the cells were rinsed with phosphate buffer saline (PBS) twice and were trypsinized in 0.25% trypsin for 2–5 min, resuspended in 5 mL of Dulbecco’s modified Eagle medium (DMEM) (190,040; Gibco, Gaithersburg, MD, USA) containing 10% FBS and sub-cultured. RPMI8226 cells were sub-cultured every two days, and, following a full mixture using a micropipette and the removal of 2/3 of the primary medium, 2/3 volume of fresh medium was added. Dual-luciferase reporter gene assay The biological prediction site microRNA.org was applied for the analysis of target genes of miR-497 and verification of whether Raf-1 was a target gene of miR-497. The full-length 3ʹuntranslated region (UTR) of the Raf-1 gene was cloned and amplified, and polymerase chain reaction (PCR) products were cloned downstream of the pmirGLO (Promega, Beijing, China) luciferase gene. Bioinformatics sites predicted the binding sites of miR-497 and its target genes, after which site-directed mutagenesis was performed. The pRL-TK vector (TaKaRa, Dalian, Liaoning, China) expressing Renilla luciferase was used to demonstrate the difference in the transfection efficiency from that of the internal reference to adjust the difference in the cell number. The miR-497 mimic (5ʹ-cagcagcacactgtggtttgt-3ʹ) and corresponding negative control (NC) (5ʹ-gtcgtcctctgtcaccagact-3ʹ) were co-transfected with luciferase reporter vectors into HEK293T cells. Dual luciferase activity detection was performed according to the methods provided by Promega. Reverse transcription-quantitative PCR (RT-qPCR) Cells of the P3 generation were subjected to miR-497 quantitative detection for cell line selection. The total RNA of 5 cell lines, RPMI8226, U266, XG-6, XG-7 and H929, was extracted using a Trizol Kit (15,596–018, Invitrogen Inc., Carlsbad, CA, USA). Thereafter, the ratio of the absorbance A260/A280 and RNA concentration were determined using an ultraviolet spectrophotometer (Nanodrop 2000; Thermo, Waltham, Massachusetts, USA), and the extracted RNA was stored at −80°C for further use. RNA was reversed transcribed into cDNA according to the manufacturer’s instructions of Applied Biosystems StepOneTM and StepOnePlusTM Real-Time PCR Systems (4,379,704; Applied Biosystems Inc, Carlsbad, CA, USA). The RT-qPCR RNA test kit was purchased from Ambion (Austin, Texas, USA). The reaction was performed by RT-qPCR (AM1005; Invitrogen Inc., Carlsbad, CA, USA). The reaction conditions for miR-497 were as follows: pre-denaturation at 95°C for 3 min, 35 cycles of denaturation at 95°C for 15 s, annealing at 60°C for 30 s, and extension at 72°C for 30 s. U6 served as the internal reference for the quantitative determination of miR-497 and glyceraldehyde phosphate dehydrogenase (GAPDH) as a reference for Raf-1, methyl ethyl ketone 2 (MEK-2), ERK1/2, survivin, B-cell lymphoma-2 (Bcl-2) and BCL2-Associated X (Bax). The primers were synthesized by Shanghai Boya Biotechnology Services Co., Ltd. (Shanghai, China). The aforementioned method was also applied to detect the miR-497 level and mRNA levels of Raf-1 and MAPK/ERK pathway- and apoptosis-related genes in tissues and transfected cells. The primers of miR-497, Raf-1, MEK-2, ERK1/2, survivin, Bcl-2, Bax, U6, and GAPDH are shown in Table 1. Each experiment was carried out 3 times. The dissolution curve was used for the evaluation of the reliability of the PCR results, taking the CT value (inflexions in a kinetic PCR amplification curve), and the relative expression of target genes, which was calculated by the 2−ΔΔCt method [29]. The formula was as follows: ΔΔCt = [Ct (target gene) – Ct (reference gene) experimental group – [Ct (target gene) – Ct (reference gene)] control group.10.1080/15384101.2018.1542895-T0001 Table 1. Primer sequences for RT-qPCR. Genes Sequences (5ʹ-3ʹ) miR-497 F: CAGCCCTGTCCAGTAGC R: GCCTGACTTTACTGTTGC Raf-1 F: CAGCCCTGTCCAGTA GC R: GCCTGACTTTACTGTTGC MEK-2 F: TGCTCACAAACCACACCTTCA R: ACACAACCAGCCGGCAAA ERK1/2 F: TGTTCCCAAATGCTGACTCCAA R: TCGGGTCGTAATACTGCTCCAGATA survivin F: CTGCCTGGCAGCCCTTT R: CCTCCAAGAAGGGCCAGTTC Bcl-2 F: GTTCGGTGGGGTCATGTGTGTGGAGAGCG R: TAGCTGATTCGACGTTTTGCCTGA Bax F: GAGGATGATTGCCGCCGTGGACA R: GGTGGGG GAGGAGGCTTGAGG U6 F: GCTTCGGCAGCACATATACTAAAAT R: CGCTTCACGAATTTGCGTGTCAT GAPDH F: GCCTTCCGTGTCCCCACTGC R: TGAGGGGGCCCTCGACG Note: RT-qPCR: reverse transcription quantitative polymerase chain reaction; miR: microRNA; MEK-2: methyl ethyl ketone 2; ERK: extracellular regulated protein kinases; Bcl-2: B-cell lymphoma-2; Bax: BCL2-Associated X; GAPDH: Glyceraldehyde 3-phosphate dehydrogenase; F: forward; R: reverse. Cell grouping and transfection RPMI8226 cells in the logarithmic growth phase were categorized into the blank (without any sequence transfection), NC (transfected with sequences of miR-497 NC), miR-497 mimic (transfected with sequences of miR-497 mimic), miR-497 inhibitor (transfected with sequences of miR-497 inhibitor), si-Raf-1 (transfected with sequences of si-Raf-1), and miR-497 inhibitor + si-Raf-1 groups (transfected with sequences of miR-497 inhibitor and si-Raf-1). The cells in the logarithmic growth phase were inoculated into a 6-well plate and were transfected in accordance with the instructions provided by the manufacturer’s for Lipofectamine 2000 (11,668,019; Invitrogen Inc., Carlsbad, CA, USA) when the cell density was increased to 30 ~ 50%. The transfection sequences of cells are shown in Table 2. Frozen-dried powder samples of the miR-497 mimic, si-Raf-1, miR-497 inhibitor, miR-497 inhibitor + si-Raf-1 and NC (YDSW-D18; Invitrogen Inc., Carlsbad, CA, USA) were centrifuged and dissolved in RNase-free water. A total of 250 µL of serum-free medium Opti-MEM (31,895–070, Gibco Company, Grand Island, NY, USA) was employed to dilute Lipofectamine 2000 (5 µL), and the solution was mixed slightly and cultured at room temperature for 5 min. The above two solutions were mixed and cultured at room temperature for 20 min and were added into the cell culture well. After cell culture was carried out for 6 ~ 8 h at 37°C in a 5% CO2 incubator, the medium was substituted by a complete medium. Following further incubation for 24 ~ 48 h, the RNA and protein collected were extracted for subsequent experiments.10.1080/15384101.2018.1542895-T0002 Table 2. Transfection sequences. Gene Sequence miR-497 mimic CAGCAGCACACUGUGGUUUGU miR-497 inhibitor ACAAACCACAGUGUGCUGCUG NC UUCUCCGAACGUGUCACGUTT Note: miR-497 mimic, micro RNA-497 mimic, miR-497 inhibitors, micro RNA-497 inhibitors; NC, negative control. Western blot analysis The cells taken from the frozen tissue were added to an appropriate amount of protein lysate containing 60% radio-immunoprecipitation assay (RIPA) cell lysis buffer, 39% sodium dodecyl sulfate (SDS) and 1% protease inhibitor and were collected into Eppendorf (EP) tubes and allowed to stand on ice for 30 min. Thereafter, the cells were centrifuged (36,684 × g) at 4°C for 30 min, the supernatants were collected, and the protein concentrations were measured using the bicinchoninic acid (BCA) method. Following quantification according to different concentrations, the protein (20 μg) in each group was separated by polyacrylamide gel electrophoresis (PAGE), transferred onto NC membranes by the wet transfer method, and sealed in 5% bovine serum albumin (BSA) at room temperature for 1 h. Diluted primary antibodies, rabbit anti-Raf-1 antibody (1:1000; ab173539), MEK-2 antibody (1:10,000; ab32517), ERK1/2 antibody (1:1000; ab17942), survivin antibody (1:5000; ab76424), Bax antibody (1:1000; ab32503), Bcl-2 antibody (1:1000; ab32124), and GAPDH antibody (1:2500; ab9485) (all of the above were purchased from Abcam Inc., Cambridge, MA, USA) were added. The following day, the membrane was incubated with the secondary antibody (ab7312; Abcam, Cambridge, MA, USA) at 4°C for 1 h. The imaging agent was added, and the Bio-Rad gel imaging system (MG8600; Beijing Thmorgan Biotech Ltd, Beijing, China) was used for development. Quantitative analysis was performed using IPP7.0 software (Media Cybernetics, Singapore, Republic of Singapore). The gray-value ratios of Raf-1, MEK-2, p-ERK1, ERK1/2, survivin, Bcl-2, Bax to GAPDH represented the respective content. This experiment was also applicable for cell experiments. 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay The RPMI8226 cell line was transfected at predetermined concentrations and was cultured in RPMI 1640 culture medium containing 10% FBS in a 5% CO2 incubator at 37°C for 48 h. The cells in the logarithmic growth phase in each group were selected for subsequent experiments. Cells were trypsinized at an ordinary temperature, and the cell suspension was transferred to a centrifuge tube. After being blown and sucked into a sterile micropipette, the cells were resuspended into a single-cell suspension. In addition, Trypan blue exclusion was conducted to count living cells. Cells in each group were then transferred into a 96-well plate and were plated according to the counting results, ensuring that living cells were added into the 96-well plate (5,000 cells/well), and then the plate was cultured in a 5% CO2 incubator at 37°C. Once the solution was substituted, PBS was added to the peripheral wells in the 96-well plate to prevent evaporation. Next, the 96-well plate was placed in a 5% CO2 incubator at 37°C for 24 h, 48 h, and 72 h. After the plate was removed, 20 μl of 5% MTT solution was added to each well, followed by incubation under dark conditions. The plate was gently shaken for the even mixture of the solution, after which the plate was cultured in a 5% CO2 incubator at 37°C for 4 h. Later, the plate was centrifuged at 453 × g for 5 min for the removal of the supernatant. Thereafter, the plate was added with 150 μL of dimethyl sulfoxide (DMSO) solution, followed by shaking on a table concentrator at 37°C for 30 min in the dark. Next, the plate was removed, and the optical density (OD) value at the wavelength of 490 nm was detected using an enzyme analyzer (SAF-680T, Multiskan, GO, Thermo, USA). The cell growth curve was plotted with transfection time on the X-axis and the OD value on the Y-axis. Scratch test Confluent RPMI-8226 cells on fibronectin (10 μg/mL)-coated (Sigma-Aldrich) 6-well plates [30] were incubated with serum-free RPMI 1640 following cell adhesion. When cells reached 90% – 100% confluency, a 10-μL pipette tip was employed to gently make vertical scratches at the bottom of the 6-well plate, with approximately 4 to 5 scratches with the same width made in each well. The cells were then rinsed with PBS 3 times with dripped cells eliminated and were placed in an incubator. At 0 h and 48 h following scratching, the distance of cell migration in the scratch area was observed under the inverted microscope, and several fields were randomly selected and photographed. IPP7.0 software (Media, Cybernetics, Singapore) was used for the analysis of the percentage of wound healing (cell surface in wound area/wound area). Three duplicated wells were established for each group, and each experiment was repeated 3 times. Transwell assay The Transwell chamber was placed into the 24-well plate, and the Matrigel-diluted solution (1:8) was used to cover the upper surface of the basement membrane of the Transwell chamber, which was dried at room temperature. Cells in the blank, NC, miR-497 mimic, miR-497 inhibitor, si-Raf-1, and miR-497 inhibitor + si-Raf-1 groups were centrifuged (1000 × g) for 3 min, rinsed with PBS twice and re-suspended with serum-free RPMI 1640 culture medium. The cell density was adjusted to 1 × 105 cells/mL, with 200 μL of the cell suspension added to the upper chamber, and 600 μL of serum-free RPMI 1640 culture medium added to the lower chamber. After conventional culture for 24 h, the Transwell chamber was removed and the cell suspension in the lower chamber was collected. The number of cells was then counted, and each group was established with 3 duplicated wells. The measurements were repeated 3 times in order to obtain the mean value. Flow cytometry After transfection for 48 h, the cells were removed and rinsed with cold PBS 3 times, centrifuged, and resuspended in PBS at a concentration of 1 × 105 cells/mL. Next, 1 mL of −20°C precooled 75% ethanol was added to fix the cells at 4°C for 1 h. Thereafter, centrifugation was carried out to remove the cold ethanol followed by washing twice with PBS to remove the supernatant, after which 100 μL of RNase A was added and the solution was incubated in a water bath at 37°C for 30 min under dark conditions. Later, 400 μL of propidium iodide (PI) (D0820, Sigma, San Francisco, California, USA) was added at 4°C for 30 min with the avoidance of light for staining. Flow cytometry (Gallios, Beckman Coulter Life Sciences, Brea, CA, USA) was performed to record red fluorescence at the excitation wavelength of 488 nm and to measure the cell cycle entry. Following a 48-h transfection, the cells were trypsinized with ethylene diamine tetraacetic acid (EDTA)-free trypsin, collected in a flow tube, and centrifuged, after which the supernatant was removed. The cells were then washed with cold PBS 3 times and were centrifuged in order to remove the supernatant. Using the manufacturer’s protocol of the Annexin-V-fluorescein isothiocyanate (FITC) cell apoptosis detection kit (4030ES20, Sigma, San Francisco, California, USA), an Annexin-V-FITC/PI dye mixture was prepared in the proportion of 1:2:50 for PI, HEPES and Annexin-V-FITC, respectively. Each 100 μL of the dye solution was used for the resuspension of 1 × 106 cells. After shaking and mixing, the cells were cultured at room temperature for 15 min, followed by the addition of 1 mL of HEPES buffer and the solution was evenly mixed. The 525- and 620-nm bandpass filter was excited at a 488-nm wavelength to determine FITC and PI fluorescence, respectively, along with cell apoptosis. Xenograft tumors in nude mice Eighteen male Kunming nude mice (aging 3 months old and weighing [20 ± 2] g) with clean grade were selected and purchased from the Animal Experimental Center of Southern Medical University. The RPMI8226 cell line was used for the preparation of the single-cell suspension, and PBS and Matrigel were mixed at a volume ratio of 1: 1. The cells were resuspended in the mixture, and the cell concentration was finally adjusted to 1 × 106 cells/200 μL. The 18 nude mice were then classified into the blank, NC, miR-497 mimic, miR-497 inhibitor, si-Raf-1, and miR-497 inhibitor + si-Raf-1 groups (n = 3). After the mice were anesthetized with ether, 1 × 106 cells/200 μL of RPMI8226 cells were subcutaneously inoculated into the right hind limb of nude mice in each group. The mice were raised in the same environment and observed every 7 d, with the length and width of the tumor recorded cautiously. The tumor volume was calculated using the formula: volume = length × width2/2. On the 35th day, the nude mice were sacrificed, and the tumors were dissected out. Three tumors were collected and weighed in each group. The animals enrolled in this study were fed in a pathogen-free environment ad libitum. All animal experiments were conducted in accordance with the animal care and use guidelines provided by the Ethics Committee of Wenzhou Medical University. This study also followed the applicable institutional governmental regulations concerning the ethical use of animals. Statistical analysis SPSS 18.0 statistical software (IBM Corp. Armonk, NY, USA) was used for data analysis. The measurement data were expressed using means ± standard deviation. The data of normal distribution were assessed by the D’Agostino & Pearson omnibus normality test, and the comparisons among multiple groups were conducted by one-way analysis of variance (ANOVA) using Prism 6.0 software (GraphPad Inc, La Jolla, CA, USA). Pairwise comparisons were performed using Turkey’s post hoc test. The comparisons of the data with skewed normal distribution were examined by Dunn’s multiple comparison post hoc test in Kruskal-Wallis test. A value of p < 0.05 was considered to be statistically significant. Bioinformatic analysis GenomicScape (http://www.genomicscape.com) was adopted for the analysis of differentially expressed genes and miRs in MM cells in comparison with the normal counterpart. All parameters were screened with p < 0.05 considered as the standard. 3. Results A higher positive expression rate of Raf-1 protein is found in MM tissues Raf-1 is one of the important signaling molecules during signal transduction related to tyrosine kinase, which is also a cross-linking point associated with multiple signaling pathways. Raf-1 regulates tumor progression by activating the down-stream signaling pathways such as the ERK signaling pathway [31]. As an oncogene, Raf-1 was rarely reported in MM, and its function in MM remains unclear. The positive expression rates of Raf-1 protein in MM tissues and normal bone marrow tissues were detected by immunohistochemistry (Figure 1(a)). Based on the results, the positive expression rate of Raf-1 protein was 36.18% in normal bone marrow tissues and 78.29% in MM tissues (Figure 1(b)). Compared with normal bone marrow tissues, there was a significant increase in positive expression rate of Raf-1 protein in MM tissues (p< 0.05). There was a high expression of Raf-1 in MM tissues, indicating that the Raf-1 signaling pathway was activated in MM.10.1080/15384101.2018.1542895-F0001 Figure 1. A higher positive expression rate of Raf-1 protein was found in MM tissues. a, Immunohistochemical staining of the positive Raf-1 protein expression in normal bone marrow tissues and MM tissues (× 200); b, positive expression rate of Raf-1 protein in normal bone marrow tissues (n = 3) and MM tissues (n = 152); , p < 0.05, vs. the normal bone marrow tissues; MM, multiple myeloma; PCs, plasma cells. The values refer to measurement data, which are expressed as mean ± standard deviation. Student t test was used for data analysis. The experiment was repeated 3 times. Identification of MM tissues and normal bone marrow tissues HE staining was applied for histopathological observation of MM tissues and normal bone marrow tissues in order to determine whether the collected samples were MM or bone marrow tissues. In the MM tissues, tumors presented with an invasive growth and invaded surrounding skeletal muscle tissues, and the tumor cells were also found to have typical morphological characteristics of malignant plasma cells, with an irregular nuclear pattern, a clear nucleolus, rich cytoplasm, and mitotic figures. There were no necrotic zones and infiltration of fibrous connective tissue observed. However, in the normal bone marrow tissues, the vessels were small and smooth, with a slightly increased diameter of individual vessels, a round lumen without distortion, and a regularly shaped nucleus (Figure 2).10.1080/15384101.2018.1542895-F0002 Figure 2. HE staining revealed that tumor cells in MM tissues presented with typical morphological characteristics of malignant plasma cells (× 400). HE, hematoxylin and eosin; MM, multiple myeloma; PCs, plasma cells. The arrow in the figures refers to malignant plasma cells and an irregular nucleus pattern. miR-497 was poorly expressed and Raf-1/ERK signaling pathway is activated in MM tissues RT-qPCR and western blot analysis were performed to detect the mRNA and protein levels of RAF-1, MEK-2, ERK1/2, and apoptosis-related factors (Bax, Bcl-2 and survivin) as well as the extent of ERK1/2 phosphorylation and verify the results of immunohistochemistry. As shown in Figure 3(a–c), compared with the normal bone marrow tissues, there were decreases in the levels of miR-497 and mRNA and protein levels of Bax in MM tissues, while mRNA and protein levels of Raf-1, MEK-2, Bcl-2 and surviving were elevated, along with the extent of ERK1/2 phosphorylation (all p< 0.05), and there was no significant difference observed in the ERK1/2 mRNA and protein levels (p> 0.05). The correlation analysis was conducted, and found that there was a negative correlation between miR-497 level and RAF-1 mRNA level, R value = −0.93 (Figure 3D). The above results suggested that there is a poor expression of miR-497, and a high expression of Raf-1, while the ERK signaling pathway was activated in MM tissues. In addition, the Raf-1/ERK signaling pathway activation is one of the key factors associated with MM progression.10.1080/15384101.2018.1542895-F0003 Figure 3. Lower miR-497 level and higher mRNA and protein levels of Raf-1, MEK-2 and apoptosis-related factors were found in MM tissues. a, miR-497 level and mRNA levels of Raf-1, MEK-2, ERK 1/2, Bcl-2, Bax, and survivin in tissues; b, Protein levels of Raf-1, MEK-2, ERK 1/2, Bcl-2, Bax, and survivin in tissues; c, protein bands of the protein levels of Raf-1, MEK-2, ERK 1/2, Bcl-2, Bax, survivin and GAPDH in tissues; d, correlation analysis of miR-497 expression and mRNA expression of RAF-1;, p < 0.05, vs. the normal bone marrow tissues; MM, multiple myeloma. The values refer to the measurement data that were expressed as mean ± standard deviation. Student t test was used for comparison between two groups (n = 152). The experiment was repeated 3 times. High miR-497 level and low Raf-1 mRNA level are associated with MM progression The above results showed that an increase in Raf-1 expression might be correlated with the activation of the ERK signaling pathway in MM. However, the key factor regulating Raf-1 is still unknown. We also found that miR-497 level was negatively correlated with Raf-1 mRNA level. Based on the bioinformatic analysis, miR-497 was predicted to be a candidate miR that regulates Raf-1. The potential role of miR-497 and Raf-1 in MM development were further analyzed by investigating the relationship between the miR-497 level and Raf-1 mRNA level and the pathological characteristics of MM patients (Table 3). The miR-497 level and Raf-1 mRNA level were unrelated to age or gender but related to the degree of anemia and renal function impairment, ISS staging and D-S staging. These findings indicated that the increase in the degree of anemia, the degree of renal function impairment, ISS staging and D-S staging resulted in a reduction of miR-497 level and an elevation in mRNA level of Raf-1 (p> 0.05). Hence, low miR-497 level but high Raf-1 level was correlated with higher degree of anemia and renal function impairment and ISS staging and D-S staging, which provided further proof on the hypothesis that miR-497 was negatively correlated with Raf-1 in MM progression.10.1080/15384101.2018.1542895-T0003 Table 3. The expression of miR-497 and Raf-1 and the pathological characteristics of patients. Pathological characteristics n Expression of miR-497 p value Expression of Raf-1 p value Age (years) 0.974 0.858 < 50 44 0.172 ± 0.021 2.223 ± 0.122 ≥ 50 108 0.170 ± 0.020 2.229 ± 0.120 Gender 0.999 0.960 Male 59 0.171 ± 0.019 2.227 ± 0.114 Female 93 0.171 ± 0.021 2.228 ± 0.124 Anemia < 0.0001 < 0.0001 Mild anemia 23 0.187 ± 0.016 2.124 ± 0.077 Moderate anemia 30 0.184 ± 0.006 2.132 ± 0.040 Severe anemia 99 0.163 ± 0.006 2.279 ± 0.040 Renal function impairment < 0.0001 < 0.0001 Renal inadequacy 27 0.193 ± 0.010 2.092 ± 0.036 Dropsical nephritis 33 0.182 ± 0.003 2.151 ± 0.029 No impairment 92 0.160 ± 0.003 2.294 ± 0.029 ISS staging < 0.0001 < 0.0001 Stage I 61 0.187 ± 0.012 2.123 ± 0.059 Stage II-III 91 0.160 ± 0.014 2.296 ± 0.079 D-S staging < 0.0001 < 0.0001 Stage I 35 0.191 ± 0.011 2.097 ± 0.041 Stage II 40 0.179 ± 0.005 2.171 ± 0.029 Stage III 77 0.157 ± 0.005 2.315 ± 0.029 Note: miR-497, microRNA-497. Raf-1 is confirmed as the target gene of miR-497, and the RPMI8226 cell line is used in subsequent experiments To verify whether miR-497 negatively regulates Raf-1, microRNA.org, a biology prediction site was employed and the results showed that miR-497 can target Raf-1 (Figure 4(a)). To confirm that Raf-1 was a target gene of miR-497, first, recombined luciferase reporter vectors pRaf-1-Wt and pRaf-1-Mut were constructed with Raf-1 mRNA 3ʹ-UTR inserted into the luciferase reporter vector. Additionally, the miR-497 mimic and NC were respectively co-transfected with recombined luciferase reporter vectors into HEK293T cells. The results of the dual luciferase reporter gene system showed that the luciferase activity of Raf-1wt-3'-UTR co-transfection was decreased by approximately 42% in the miR-497 mimic group compared with that in the NC group (p< 0.05) (Figure 4(b)). However, there was no significant difference in the luciferase activity of mutant Raf-1mut-3ʹ-UTR luciferase between the NC and miR-497 mimic groups (p> 0.05). Therefore, Raf-1 was confirmed to be a target gene of miR-497. Therefore, miR-497 can target Raf-1, in turn negatively regulating its mRNA level.10.1080/15384101.2018.1542895-F0004 Figure 4. Raf-1 was confirmed as the target gene of miR-497. a, microRNA.org predicted that Raf-1 is the target gene of miR-497; b, the result of the dual-luciferase reporter gene assay confirmed that Raf-1 was the target gene of miR-497 in RPMI8226 cells; the experiment was repeated 3 times, and the obtained mean value and standard deviation were presented as experiment results; Student’s t test was used for detection; * p < 0.05, vs. the control group; miR-497, microRNA-497; c, expression of miR-497 in 5 MM cell lines. The experiment was repeated 3 times, and the obtained mean value ± standard deviation was presented as experiment results. One-way analysis of variance (ANOVA) was used for analysis. * p < 0.05, vs. the RPMI8226 cells. RT-qPCR was used for cell line selection among the RPMI8226, U266, XG-6, XG-7 and H929 cell lines. As shown in Figure 4(c), the miR-497 level was decreased successively as RPMI8226 > H929 > U266 > XG-6 > XG-7. The highest level of miR-497 was detected in RPMI8226; thus, the RPMI8226 cell line was selected for subsequent experiments. miR-497 down-regulates Raf-1 to suppress the MAPK/ERK signaling pathway To investigate the underlying mechanism of miR-497, Raf-1 and the ERK signaling pathway in MM cells, the cells were treated with miR-497 mimic, inhibitor and siRNA targeting Raf-1 to interfere the expression of miR-497 and/or Raf-1. Following treatment, western blot analysis and RT-qPCR were performed to detect miR-497 level, mRNA and protein level of Raf-1 and the ERK signaling pathway-related genes, and the extent of ERK1/2 phosphorylation. As shown in Figure 5(a–c), there were no notable differences observed in the expression of ERK1/2 in all groups. There was no statistically significant difference in the miR-497 level and mRNA and protein levels of Bax, Raf-1, MEK-2, Bcl-2 and survivin, as well as in the extent of ERK1/2 phosphorylation between the blank and NC groups (p > 0.05). Compared with the blank and NC groups, the miR-497 mimic group presented with a significantly increased miR-497 level (p < 0.05), while the miR-497 inhibitor group has a markedly decreased miR-497 level (p< 0.05), and there were no significant differences in the si-Raf-1 group and miR-497 inhibitor + si-Raf-1 group (p > 0.05). Compared with the blank and NC groups, the miR-497 mimic group and si-Raf-1 group showed decreased mRNA and protein levels of Raf-1, MEK-2, Bcl-2 and survivin and increased Bax mRNA and protein levels (p< 0.05), while the miR-497 inhibitor group displayed decreased Bax expression but increased mRNA and protein levels of Raf-1, MEK-2, Bcl-2, ERK1/2, survivin and extent of ERK1/2 phosphorylation (p < 0.05), and there were no significant differences observed in the miR-497 inhibitor + si-Raf-1 group (p > 0.05). However, as compared to the blank and NC groups, the miR-497 mimic and si-Raf-1 groups showed decreased ERK1/2 mRNA level and extent of ERK1/2 phosphorylation, but there were insignificant changes in the level of ERK1/2 protein. Therefore, from the above findings, it can be concluded that the overexpression of miR-497 resulted in the inhibition of the Raf-1 expression so as to suppress the activation of the ERK signaling pathway.10.1080/15384101.2018.1542895-F0005 Figure 5. miR-497 resulted in a decrease in the expression of Raf-1 inhibiting the MAPK/ERK signaling pathway. a, miR-497 level and mRNA levels of Raf-1, MEK-2, ERK 1/2, Bcl-2, Bax, and survivin in cells; b, protein levels of Raf-1, MEK-2, ERK 1/2, Bcl-2, Bax, and survivin in cells; c, protein bands of the protein levels of Raf-1, MEK-2, ERK 1/2, Bcl-2, Bax, survivin and GAPDH in cells; the experiment was repeated 3 times, and the obtained mean value ± standard deviation were presented as experimental results. One-way analysis of variance (ANOVA) was used for analysis. , p < 0.05, vs. the blank group and NC group. NC, negative control; miR-497, microRNA-497. Overexpression of miR-497 or silencing of Raf-1 inhibits MM cell growth MTT assay was performed to detect MM cell viability to further examine the specific effect of miR-497 and Raf-1 on MM. The results from MTT showed that (Figure 6), compared with the MM cell viability at 0 h in each group, the MM cell viability at 24 h, 48 h and 72 h in each group was significantly different (all p < 0.05). Compared with the blank and NC groups, the MM cell viability was decreased in the miR-497 mimic and si-Raf-1 groups (p < 0.05) but increased in the miR-497 inhibitor group (p < 0.05), while the miR-497 inhibitor + si-Raf-1 group showed no significant difference (p > 0.05). These findings revealed that the overexpression of miR-497 or silencing of Raf-1 could lead to the inhibition of MM cell growth.10.1080/15384101.2018.1542895-F0006 Figure 6. miR-497 mimic or si-Raf-1 inhibited the MM cell viability, as detected by the MTT assay. The experiment was repeated 3 times, and the obtained mean value ± standard deviation was presented as experimental results. Two-way analysis of variance (ANOVA) was used for analysis. p < 0.05, vs. the blank and NC groups; #, p < 0.05, vs. 0 h; miR-497, microRNA-497; NC, negative control; MTT, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide. Overexpression of miR-497 or silencing of Raf-1 suppresses cell migration and invasion The scratch test and Transwell assay were performed to detect cell migration and invasion to show the effect of miR-497 and Raf-1 on MM cell migration and invasion. As shown in Figure 7(a,b), there were no significant differences among the blank, NC and miR-497 inhibitor + si-Raf-1 groups (all p > 0.05). Compared with the blank and NC groups, the cell migration ability was decreased in the miR-497 mimic and si-Raf-1 groups (p < 0.05) and increased in the miR-497 inhibitor group (p < 0.05).10.1080/15384101.2018.1542895-F0007 Figure 7. miR-497 mimic or si-Raf-1 suppressed the migration and invasion of MM cells. a, scratches on MM cells among the different transfection groups, as detected by the scratch test; b, wound healing rate of MM cells in each group; c, cell invasion ability in each group under a microscope, as assessed by Transwell assay (× 200); d, number of invading cells in each group. The experiment was repeated 3 times, and the obtained mean value ± standard deviation (SD) was presented as experimental results. One-way analysis of variance (ANOVA) was used for data comparison. , p < 0.05, vs. the blank group and NC group; NC, negative control; miR-497, microRNA-497. As shown in Figure 7(c–d), there were no significant differences among the blank, NC and miR-497 inhibitor + si-Raf-1 groups (all p > 0.05). Compared with the blank and NC groups, the cell invasion ability was decreased in the miR-497 mimic and si-Raf-1 groups (p < 0.05) and increased in the miR-497 inhibitor group (p < 0.05). These results implied that the enforcement of miR-497 or depletion in Raf-1 expression could inhibit cell migration and invasion. Overexpression of miR-497 or silencing of Raf-1 promotes cell apoptosis and arrests cells in the G1 phase Flow cytometry was applied to detect the cell cycle distribution and cell apoptosis in MM cells with overexpressed miR-497 or silenced Raf-1 expression. PI staining indicated (Figure 8(a–b)) that there were no significant differences among the blank, NC and miR-497 inhibitor + si-Raf-1 groups (all p > 0.05). Compared with the blank and NC groups, cell growth in the miR-497 mimic and si-Raf-1 groups was mainly arrested in G1 phase, increased in G1 phase, and decreased in S and G2/M phase (all p < 0.05).10.1080/15384101.2018.1542895-F0008 Figure 8. miR-497 mimic or si-Raf-1 promoted the apoptosis of MM cells, as detected by flow cytometry. a and b, cell cycle distribution in different transfection groups, G0/G1 were the early stage of DNA synthetic phase and G2 phase was the later phase stage of DNA synthetic phase; c and d, cell apoptosis in different transfection groups. The experiment was repeated 3 times, and the obtained mean value ± standard deviation was presented as experiment results. One-way analysis of variance (ANOVA) was used for data comparison. , p < 0.05, vs. the blank and NC group; NC, negative control; miR-497, microRNA-497. According to the result of Annexin V/PI double staining (Figure 8(c–d)), there were no significant differences among the blank, NC and miR-497 inhibitor + si-Raf-1 groups (all p > 0.05). In comparison to the blank and NC groups, the cell apoptosis rate decreased in the miR-497 inhibitor group (p < 0.05) and increased in the miR-497 mimic group and si-Raf-1 group (p < 0.05). Taken together, miR-497 arrests cells in G1 phase, and overexpression of miR-497 or si-Raf-1 promotes cell apoptosis. miR-497 up-regulation or Raf-1 knockdown contributes to a decrease in tumor size and growth Finally, in vivo experiments using xenograft tumors in nude mice were conducted to investigate the effect of miR-497 and Raf-1 on MM formation ability. Based on the findings, there were no significant differences in tumor growth in the blank and NC groups (p> 0.05). Compared with the blank group, nude mice in the miR-497 inhibitor group had increased subcutaneous tumors and the fastest tumor growth rate (p < 0.05). There were decreases in subcutaneous tumor size and tumor growth rate in nude mice in the miR-497 mimic and si-Raf-1 groups (p < 0.05), while the miR-497 inhibitor + si-Raf-1 group showed no significant difference (p > 0.05) (Figure 9(a–c)). Therefore, tumor size and growth was reduced in nude mice transfected with miR-497 mimic or si-Raf-1.10.1080/15384101.2018.1542895-F0009 Figure 9. Nude mice transfected with miR-497 mimic or si-Raf-1 showed a decreased tumor volume and weight. a, tumor growth curve of nude mice in each group examined by the experiment of xenograft tumors in nude mice; b, the tumor size in each group on the 35th day; c, comparison of the tumor weight in each group on the 35th day; the experiment was repeated 3 times, and the obtained mean value and standard deviation were presented as experimental results. One-way analysis of variance (ANOVA) was used for data comparison *, p < 0.05, vs. the blank and NC groups; NC, negative control; miR-497, microRNA-497. The differentially expressed genes and miRs in MM were analyzed on the bioinformatics platform GenomicScape. The obtained data displayed that there was a low expression of miR-497 but a high expression of RAF-1 in MM cells (Figure 11(a,b)), which is consistent with our experiments. Meanwhile, a co-expression network of RAF-1 and MM molecular subgroups (Wnt signaling pathway: ZNF family, CK1A, FZD family, BMP6 and inflammatory cytokine STAT3) was found on the basis of bioinformatics analysis (Figure 11(c)).10.1080/15384101.2018.1542895-F0010 Figure 10. miR-497 mediated the MAPK/ERK pathway by targeting Raf-1; miR-497 inhibited Raf-1 and then inhibited the activation of the MAPK/ERK pathway to promote the apoptosis of multiple myeloma cells. miR-497, microRNA-497; MAPK/ERK, mitogen-activated protein kinase/extracellular signal-regulated kinase. 10.1080/15384101.2018.1542895-F0011 Figure 11. miR-497 was lowly expressed but RAF-1 highly expressed in MM cells. a, RAF-1 was highly expressed in MM cells, as analyzed on the bioinformatics platform GenomicScape; b miR-497 was lowly expressed in MM cells, as analyzed on the bioinformatics platform GenomicScap; c, a co-expression network of RAF-1 and MM molecular subgroups (Wnt signaling, ZNF, CK1A, FZD, BMP6 and STAT3) on the basis of bioinformatic analysis. 4. Discussion MM is a disease related to a hematological disorder that appears at various stages of β-cells, and it accounts for 1% of all cancers, as well as 10% of hematological malignancies [32,33]. Based on recent studies, miRs have been identified as tumor-suppressor genes and play important roles in tumor pathogenesis [34,35]. In this study, we conducted a series of experiments in order to determine the effects of miR-497 on proliferation, migration, invasion and apoptosis in human MM cells by targeting Raf-1 through the MAPK/ERK signaling pathway. Finally, our experimental results indicated that the overexpression of miR-497 resulted in the inhibition in the proliferation, migration, and invasion of MM cells and promoted apoptosis through the reduction of Raf-1 expression. MM tissues presented with decreased levels of miR-497 and Bax and elevated levels of Raf-1, MEK-2, Bcl-2, survivin, along with an increase in the extent of ERK1/2 phosphorylation. Based on a global miR-profiling study conducted on miR expression in normal plasma cells and myeloma cells, 38 miRs were found to have been significantly down-regulated while 29 miRs were significantly up-regulated in myeloma cells in comparison with their normal counterpart [36]. A previous study has also identified miR-30, miR-106b and miR-16 as plasma cell differentiation stage specific miRNAs, which were aberrantly overexpressed in MM cells [37]. However, a number of studies revealed that the miR-497 level was reduced and the expression of Raf-1 was increased in MM tissues [18,38]. The high expression of Bcl-2 in MM cell lines was found in a former study [39]. Survivin is known to be expressed in most human cancer tissues instead of normal tissues [40]. In a study conducted by Tsubaki et al., increased levels of both ERK1/2 and survivin were found in MM [41]. The down-regulation of Bax was also observed in an MM cell line in a previous study [42]. Moreover, cells transfected with the miR-497 mimic and si-Raf-1 had lower expression levels of Raf-1, Bcl-2, survivin and there was also a decrease in the extent of ERK1/2 phosphorylation, while higher expression levels of Bax were observed. Survivin has been identified as a key factor in cell division and the inhibition of apoptosis in adult cancer tissues [40]. Bcl-2, ERK, Raf-1 and survivin are anti-apoptotic genes [43–45], while Bax is a pro-apoptotic gene [46,47]. There was a positive correlation between the levels of survivin, Bcl-2 and ERK with the anti-apoptotic action, while Bax was negatively associated with the levels of survivin and Bcl-2 [48]. The inhibition of Raf-1 expression could potentially result in the suppression of Bcl-2 expression [49]. Previous studies have also found that miRs negatively regulate gene expressions [50,51]. Additionally, the results from the dual fluorescence reporter assay confirmed that Raf-1 was a target gene of miR-497, which was consistent with a previous study suggesting that Raf-1 was a key activator of the ERK signaling pathway and could be negatively regulated by miR-497 [52,53]. Another study uncovered that the overexpression of miR-497 could decrease the level of Bcl-2 in human umbilical vein endothelial cells [54]. The down-regulation of Bcl-2 and up-regulation Bax could also occur as a result of miR-142 overexpression [55]. Therefore, the results implied that the overexpression of miR-497 results in the reduction of Raf-1 expression and anti-apoptotic genes, while it promotes the expression of pro-apoptotic genes via the MAPK/ERK signaling pathway. Finally, MM cells transfected with the miR-497 mimic and si-Raf-1 presented with decreased cell proliferation, migration and invasion and increased cell apoptosis, while the mice that had received miR-497 mimic or si-Raf-1 treatment had reduced subcutaneous tumor size and weight and tumor growth rate was also found to be inhibited. Zhang Y et al. and Han L et al. have also demonstrated that the overexpression of miR-497 resulted in decreased cell proliferation, migration and invasion [56,57]. Shen L et al. also found that the upregulation of miR-497 can lead to apoptotic enhancement [58]. miR-497 regulates the multidrug resistance of human cancer by targeting Bcl2 and inducing apoptosis and inhibiting proliferation via Bcl-2/Bax [54,59]. In addition, Raf-1 protein actively participates in cell growth and proliferation, and its inhibition is related to tumor growth arrest and cell apoptosis [60]. A study performed by Xu J et al. also showed that the up-regulation of miR-497 led to the inhibition of tumor growth [61]. The aforementioned findings suggested that the overexpression of miR-497 or Raf-1 silencing inhibits the proliferation, migration and invasion of MM cells, along with tumor growth. In summary, our data provided further evidence on previous observations in support of the hypothesis that the overexpression of miR-497 inhibits MM cell proliferation, migration and invasion by reducing the expression of Raf-1 through the MAPK/ERK signaling pathway (Figure 10), which can be considered as promising perspective for future clinical practice. However, due to the presence of a number of factors affecting miR-497 and the MAPK/ERK signaling pathway, additional studies are required in order to investigate investigating the direct relationship between miR-497 and the MAPK/ERK signaling pathway. Acknowledgments We thank the reviewers for critical comments. 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==== Front 88066056644Mod PatholMod. Pathol.Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc0893-39521530-02853025820910.1038/s41379-018-0123-6nihpa1500573ArticleCervical Adenosquamous Carcinoma: Detailed Analysis of Morphology, Immunohistochemical Profile, and Outcome in 59 cases Stolnicu Simona MD1Hoang Lien MD2Hanko-Bauer Orsolya MD3Barsan Iulia MD1Terinte Cristina MD4Pesci Anna MD5Aviel-Ronen Sarit MD6Kiyokawa Takako MD7Alvarado-Cabrero Isabel MD8Oliva Esther MD9Park Kay J. MD10Soslow Robert A. MD101 Department of Pathology, University of Medicine and Pharmacy of Targu Mures, Targu Mures, Romania2 Vancouver General Hospital, Vancouver, BC, Canada3 Department of Surgery, University of Medicine and Pharmacy of Targu Mures, Targu Mures, Romania4 Regional Institute of Oncology, Iasi, Romania5 Ospedale Sacro Cuore Don Calabria, Negrar, Italy6 Sheba Medical Center, Tel-Hashomer, Ramat Gan, Israel7 Jikei University School of Medicine, Tokyo, Japan8 Hospital de Oncología Mexico City, Mexico City, Mexico9 Massachusetts General Hospital, Boston, MA, USA10 Department of Pathology, Memorial Sloan Kettering Cancer Center, New York, NY, USACorresponding Author: Robert A. Soslow, MD, Department of Pathology, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York, NY 10065, USA, Fax: 929-321-5015, Phone: 212-639-5905, [email protected] 7 2018 26 9 2018 2 2019 26 3 2019 32 2 269 279 Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:http://www.nature.com/authors/editorial_policies/license.html#termsAlthough 2014 World Health Organization criteria require unequivocal glandular and squamous differentiation for a diagnosis of cervical adenosquamous carcinoma, in practice, adenosquamous carcinoma diagnoses are often made in tumors that lack unequivocal squamous and/or glandular differentiation. Considering the ambiguous etiologic, morphological, and clinical features and outcomes associated with adenosquamous carcinomas, we sought to redefine these tumors. We reviewed slides from 59 initially diagnosed adenosquamous carcinomas (including glassy cell carcinoma and related lesions) to confirm an adenosquamous carcinoma diagnosis only in the presence of unequivocal malignant glandular and squamous differentiation. Select cases underwent immunohistochemical profiling as well as human papillomavirus (HPV) testing by in situ hybridization. Of the 59 cases originally classified as adenosquamous carcinomas, 34 retained their adenosquamous carcinoma diagnosis, 9 were reclassified as pure invasive stratified mucin-producing carcinomas, 10 as invasive stratified mucin-producing carcinomas with other components (such as HPV-associated mucinous, usual-type, or adenosquamous carcinomas), and 4 as HPV-associated usual or mucinous adenocarcinomas with benign-appearing squamous metaplasia. Two glassy cell carcinomas were reclassified as poorly differentiated usual-type carcinomas based on morphology and immunophenotype. There were significant immunophenotypic differences between adenosquamous carcinomas and pure invasive stratified mucin-producing carcinomas with regard to HPV (p<0.0001), PAX8 (p=0.038; more in adenosquamous carcinoma), p40 (p<0.0001; more in adenosquamous carcinoma), p63 (p=0.0018; more in adenosquamous carcinoma) and MUC6 (p<0.0001; less in adenosquamous carcinoma), HNF1beta (p=0.0023), vimentin (p=0.0003), p53 (p=0.0004), and CK7 (p=0.0002) expression. Survival outcomes were similar between all groups. adenosquamous carcinomas should be diagnosed only in the presence of unequivocal malignant glandular and squamous differentiation. The two putative glassy cell carcinomas studied did not meet our criteria for adenosquamous carcinoma, and categorizing them as such should be reconsidered. cervical adenosquamous carcinomaglassy cell carcinomainvasive stratified mucin-producing carcinomasHPV ==== Body Introduction Invasive cervical adenosquamous carcinoma is a relatively uncommon histologic subtype of cervical malignant neoplasms classified by the World Health Organization Classification of Tumors of Female Reproductive Organs as a separate entity distinct from both squamous and glandular malignant tumors of the cervix (1). Adenosquamous carcinoma was first described as a “mixed carcinoma” by Glucksmann and Cherry in 1956, and later as “adenosquamous adenocarcinoma” by Greene in 1963 (2,3). The World Health Organization, since 2014, defines adenosquamous carcinoma as a malignant epithelial tumor composed of a mixture of invasive adenocarcinoma and squamous cell carcinoma (1). Historically, both glassy cell carcinoma (2,4,5) and “mucoepidermoid carcinoma” (6,7) have been considered malignancies lying within the spectrum of adenosquamous carcinoma. The 2014 World Health Organization classification system considers glassy cell carcinoma a subtype of adenosquamous carcinoma, but recommends the term “mucoepidermoid carcinoma” only for the extremely rare lesions identical to those occurring in the salivary glands, particularly those that contain squamous (epidermoid), intermediate, and mucin-producing cells (1). Tumors historically diagnosed as adenosquamous carcinoma in practice appear to represent a spectrum of lesions, some of which do not exhibit definitive malignant squamous and/or glandular differentiation. This degree of heterogeneity has led to variable reported estimates of adenosquamous carcinoma prevalence (2% to 50% of all invasive cervical carcinomas) (8,9). The principle rationale for classifying adenosquamous carcinomas separately from both squamous cell carcinomas and adenocarcinomas relates not only to morphology but also to reported differences in associated rates of human papillomavirus (HPV) (10–15) and clinical outcomes (9,11,16,17), with some of the older literature emphasizing the clinically aggressive nature of adenosquamous carcinoma. Given the enigmatic etiologic, morphological and clinical features of adenosquamous carcinomas, we sought to better define these tumors by examining a large series of cases originally diagnosed as adenosquamous carcinoma. Material and methods Case selection Slides from 462 endocervical adenocarcinomas and adenosquamous carcinomas (adenosquamous carcinomas and glassy cell carcinomas) were collected and reviewed by an international panel of pathologists from 8 institutions (USA: Memorial Sloan Kettering Cancer Center, New York, and Massachusetts General Hospital, Boston, MA; Romania: University of Medicine and Pharmacy of Targu Mures and Regional Institute of Oncology, Iasi; Japan: Jikei University School of Medicine, Tokyo; Mexico: Hospital de Oncología Mexico City, Mexico City; Israel: Sheba Medical Center, Tel-Hashomer, Ramat Gan; and Italy: Ospedale Sacro Cuore Don Calabria, Negrar). Only invasive tumors with at least 5-years’ follow-up were included. The following tumors were excluded: in situ carcinomas; squamous carcinomas; tumors with a neuroendocrine component; carcinosarcomas; any tumor demonstrating clinical, macroscopic, or microscopic features suggesting a lower uterine segment, uterine corpus, or adnexal primary; tumors represented by only biopsies and curettings; excisions lacking lymph node assessment; and specimens from patients treated with neoadjuvant chemotherapy and/or radiation therapy. Specimens from 70 loop electrosurgical excision procedures, 8 trachelectomies, 41 conizations, and 343 hysterectomies were collected. Fifty-three of the 462 total cases were excluded due to failure to meet entry criteria, missing blocks, or concern that the available slides were not representative of the lesion, leaving 409 cases for study. Institutional review board approval was obtained. Morphological assessment All microscopic subtypes of endocervical adenocarcinomas and adenosquamous carcinomas were included in the study. We required examination of all hematoxylin and eosin slides containing tumor (average of 12 slides per case). A consensus diagnosis was reached in every case, with at least 2, and as many as 4, study pathologists reviewing slides at a multi-head microscope. The 409 study cases were classified according to the new endocervical adenocarcinoma classification system (International Endocervical Adenocarcinoma Criteria and Classification system) (18). Cases were classified as adenosquamous carcinoma only when unequivocal invasive malignant glandular and squamous differentiation was present, each component representing at least 10% of the tumor. Cases were classified as pure glassy cell carcinoma when all tumor cells had sharp cytoplasmic margins, “ground glass” eosinophilic cytoplasm, and large round or ovoid nuclei with prominent nucleoli. The International Endocervical Adenocarcinoma Criteria and Classification system recognizes invasive stratified mucin-producing carcinomas as part of the spectrum of HPV-associated adenocarcinomas, while noting histologic similarity to lesions historically diagnosed as adenosquamous carcinoma (19). For the purposes of this study, pure invasive stratified mucin-producing carcinomas were classified separately from HPV-associated usual type, mucinous, or adenosquamous carcinomas with >10% but <90% invasive stratified mucin-producing carcinomas components (invasive stratified mucin-producing carcinomas with components). HPV-associated adenocarcinomas with a benign squamous component resembling squamous metaplasia were not considered adenosquamous carcinomas. Adenosquamous carcinomas were classified as high grade when either the glandular or squamous component was high grade. Otherwise, they were considered low grade. Solid architecture (>50%) or diffusely distributed high nuclear grade was considered “high grade” for the glandular component, while high nuclear to cytoplasmic ratios without keratinization was considered “high grade” for the squamous component. Clinical information on type of surgical treatment, tumor size, stage, follow-up, lymph node metastasis, distant metastasis, and recurrence and survival status was collected. Tissue microarray construction and immunohistochemical reactions Tissue microarrays were constructed using previously described methods (20,21). These included 25 putative adenosquamous carcinoma cases from New York, Boston, Mexico, Japan, and Romania for analysis of p16, p53, progesterone receptor, androgen receptor, Vimentin, HER2, HIK1083, MUC6, CAIX, SATB2, HNF-1beta, PAX8, CK7, CDX2, GATA3, p63, and p40 expression (Table 1). Each of the tumors from the New York, Mexican, and Romanian centers were represented by three 0.6-mm cores; those from Japan were represented by single 3-mm cores. Stains were scored by consensus among 2 study pathologists (RAS and SS). Disagreements were extremely rare (approximately 2–3%) and were adjudicated by re-reviewing the stated criteria for positivity, as described below. p16 was interpreted as positive if diffuse block-like staining was found in all cores, and negative if there was no or patchy staining. p53 was interpreted as positive if ≥75% of tumor cell nuclei were strongly positive or if no staining was present in the background of an intact internal control. progesterone receptor, androgen receptor, PAX8, CK7, and HNF-1beta were interpreted as positive if >25% (Score 3 or 4) of tumor cell nuclei or cytoplasm were stained. Scoring was as follows: Score 0: <5%; Score 1+: 5–10%; Score 2+: 11–25%; Score 3+: 26–75%; and Score 4+: >75%. Vimentin was interpreted as positive if ≥50% of tumor cells showed membranous/cytoplasmic staining. HER2 was scored using the College of American Pathologists guidelines for gastric carcinoma: 3+ membranous positive (22). HIK1083, MUC6, CAIX, SATB2, GATA3, p63, p40, and CDX2 were considered positive if any nuclear staining was noted in >5% of tumor cells. HIK1083 is currently not available in the United States. HPV detection HPV detection for high-risk HPV subtypes was performed on adenosquamous carcinomas in the tissue microarray that had sufficient tissue to score and had not been improperly fixed or stored (n=23). HPV in situ hybridization with a chromogen was performed using the Advanced Cell Diagnostics (Hayward, CA) RNAscope® system (catalogue no.312598). The RNAscope® Probe “HPV HR18” contains probes targeting E6 and E7 mRNA for the following high-risk subtypes: HPV16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, and 82. The methodology and interpretation were discussed in detail in a previous paper (18). Positive and negative control cases were used for optimal results. A full range of cytoplasmic and nuclear signals were encountered. Cases were interpreted to be HPV-positive if any brown signal appeared (nuclear, cytoplasmic) in the presence of a negative control. Statistical analysis GraphPad Prism 6 for Windows was used for statistical analyses. The log-rank (Mantel-Cox) test and Kaplan-Meier curves were used for survival analyses. p<0.05 was considered statistically significant. Results Composition of study group and pathologic findings Of the 409 cases, 57 were originally diagnosed as adenosquamous carcinoma and 2 as glassy cell carcinomas. After review for this study, 34 of 57 cases retained their diagnosis as pure adenosquamous carcinoma (Figures 1 and 2). The other cases were reclassified as pure invasive stratified mucin-producing carcinomas (n=9) (Figure 3) or invasive stratified mucin-producing carcinomas with components such as HPV-associated, usual-type adenocarcinoma (n=4) (Figure 4), adenosquamous carcinoma (n=3), or mucinous adenocarcinoma, not otherwise specified (n=3). There were also 4 mimickers of adenosquamous carcinoma—3 HPV-associated adenocarcinoma usual-type and 1 mucinous adenocarcinoma with benign-appearing squamous metaplasia (Figure 5). The 2 glassy cell carcinomas (Figure 6) were reinterpreted as poorly differentiated carcinoma, NOS after morphological evaluation (no overt squamous or glandular components) and after immunohistochemical results showed p63 and p40 negativity. None of the 34 patients with adenosquamous carcinoma was pregnant or had a recent history of pregnancy. Mean and median patient ages were 46 and 44 years, respectively (range, 24–68 years). International Federation of Gynecology and Obstetrics stage distribution was as follows: stage I, 65%; stage II, 32%; and stage III, 3%. Regional lymph nodes were assessed in all 34 adenosquamous carcinomas, with over 380 lymph nodes examined. Ten adenosquamous carcinomas had metastatic lymph node involvement (29%); one of the 10 had 5 involved lymph nodes. Pelvic metastasis occurred in 1 patient (to the ovary). Fifty percent of the adenosquamous carcinomas were high grade. Twenty-two (65%) of the 34 adenosquamous carcinomas had precursor lesions—high-grade squamous intraepithelial lesions, adenocarcinoma in situ, or stratified mucin-producing carcinomas. Lymph-vascular invasion was present in 26 adenosquamous carcinomas (76%). The immunohistochemical profiles of the adenosquamous carcinomas and pure invasive stratified mucin-producing carcinomas, the most common mimicker of adenosquamous carcinoma, are shown in Table 2. Adenosquamous carcinomas were block-like positive for p16 in 72% of cases and positive for HPV in 82.6% of cases. The pure invasive stratified mucin-producing carcinomas were positive for p16 in 62.5% of the cases, similar to the rate in adenosquamous carcinomas, while 100% of them were positive for HPV. Significant differences in immunophenotype between the adenosquamous carcinomas and pure invasive stratified mucin-producing carcinomas were observed for HPV (p<0.0001), PAX8 (p=0.038; more in adenosquamous carcinoma), p40 (p<0.0001; more in adenosquamous carcinoma), p63 (p=0.0018; more in adenosquamous carcinoma) and MUC6 (p<0.0001; less in adenosquamous carcinoma), HNF1beta (p=0.0023), vimentin (p=0.0003), p53 (p=0.0004), and CK7 (p=0.0002) expression (Table 2). Clinical findings and survival results Follow-up data were available for 26 of the patients with adenosquamous carcinoma. Mean and median times to follow-up were 87 months and 74 months, respectively (range, 6–189 months). Only 1 patient with adenosquamous carcinoma died of disease. She was 24 years old and had an International Federation of Gynecology and Obstetrics stage II, grade 3 adenosquamous carcinoma treated with radical hysterectomy, bilateral salpingo-oophorectomy and lymph node dissection followed by chemotherapy. She died 24 months after initial diagnosis with lung metastases. Two patients, both of whom presented with International Federation of Gynecology and Obstetrics stage II disease, are alive with disease. One had a histologically confirmed para-aortic nodal recurrence 14 months from diagnosis, and the other developed 2 pulmonary nodules, interpreted as metastases, 57 months after the initial diagnosis. Both patients were originally treated with radical hysterectomy and lymph node dissection followed by chemotherapy and radiation therapy. There was no difference in OS and DFS between pure bona fide adenosquamous carcinomas and HPV-associated adenocarcinomas (p=0.33 and p=0.83, respectively); originally diagnosed adenosquamous carcinomas and usual-type HPV-associated adenocarcinomas (p=0.8 and p=0.32, respectively); pure invasive stratified mucin-producing carcinomas and invasive stratified mucin-producing carcinomas with components (p=0.55 and p=0.75, respectively), and invasive stratified mucin-producing carcinomas (pure and with components) and HPV-associated mucinous carcinomas exclusive of invasive stratified mucinous carcinoma (p=0.89 and p=0.87, respectively) (Figures 7 and 8). Discussion Historically diagnosed adenosquamous carcinomas appear to be a heterogeneous group of tumors, which is in accordance with our series and the published literature (1,5–7,16,23–25). They have included cases of infiltrating tumors with distinct neoplastic squamous and glandular differentiation (i.e., pure adenosquamous carcinomas), HPV-associated adenocarcinomas with benign squamous differentiation, HPV-associated adenocarcinomas with invasive stratified mucin-producing carcinoma components, adenosquamous carcinomas with invasive stratified mucin-producing carcinoma components, pure invasive stratified mucin-producing carcinomas, and glassy cell carcinomas. In our study, only 34 of 59 cases originally diagnosed as pure adenosquamous carcinoma retained that diagnosis on review, while the 2 glassy cell carcinomas studied could not be supported as adenosquamous carcinoma variants on morphological or immunohistochemical evaluation. The remaining cases were pure invasive stratified mucin-producing carcinomas (n=9), invasive stratified mucin-producing carcinomas with components (in association with usual-type, HPV-associated mucinous, or adenosquamous carcinoma [n=10]) and benign squamous metaplasia in association with usual-type and HPV-associated mucinous endocervical adenocarcinomas (n=4). Tumor categorization used for this study overlaps significantly with that of the 2014 World Health Organization classification system. The only two notable exceptions relevant to this study are that 1) all mucinous carcinomas in this manuscript are HPV-associated carcinomas, unlike the “mucinous carcinomas” in the 2014 World Health Organization classification system, some of which are HPV-associated and others that are not, and 2) invasive stratified mucin-producing carcinomas are recognized as an entity in the International Endocervical Adenocarcinoma Criteria and Classification system but not in the 2014 World Health Organization classification system. The International Endocervical Adenocarcinoma Criteria and Classification system was specifically developed to enable practitioners to recognize HPV-associated and –unassociated variants of endocervical adenocarcinomas. In the original International Endocervical Adenocarcinoma Criteria and Classification paper, 95% of tumors demonstrating HPV-associated morphology were positive by high-risk HPV mRNA and 90% showed block-like p16 staining, indicating better performance characteristic for the in situ hybridization assay (18). In the current study, wherein only rare tumor types were studied, we report that 82.6% of adenosquamous carcinomas were HPV positive by in situ hybridization, compared to 72% with block-like p16 staining; 100% of invasive stratified mucin-producing carcinomas were HPV-positive, while only 62.5% showed block-like p16 staining. The reasons underlying these discrepancies and the existence of cases without detectable HPV are discussed in detail in a related manuscript (18). The use of an in situ hybridization probe targeting E6 and E7 mRNA of 18 different high-risk HPV types, instead of the more common DNA-based probes, underlies the superior performance of the former assay. Invasive stratified mucin-producing carcinoma is a newly recognized subtype of endocervical adenocarcinoma that differs from in situ stratified mucin-producing carcinomas (26). Invasive stratified mucin-producing carcinomas was first described by Lastra et al (19) in 2015 as an invasive adenocarcinoma containing nests of stratified columnar epithelium with round to ovoid hyperchromatic nuclei, intracytoplasmic mucin in the form of large mucin droplets, or more delicate and collapsing vacuoles that created spacing between adjacent nuclei and peripheral palisading. In the Lastra series, the amount of mucin among the cases (of which 7 were pure invasive stratified mucin-producing carcinomas and 1 was usual-type endocervical adenocarcinoma with an invasive stratified mucin-producing carcinoma component) varied from abundant to scarce. In a subsequent publication describing 3 additional cases of invasive stratified mucin-producing carcinomas, these HPV-related tumors were reported to show stratified mucinous epithelium that mimicked the appearance of immature squamous metaplasia, presumably in mucin-poor examples (27). This likely accounts for the historical misclassification of invasive stratified mucin-producing carcinomas as adenosquamous carcinomas. Invasive stratified mucin-producing carcinomas were recently reported to show some notable immunohistochemical differences from other HPV-associated endocervical adenocarcinoma subtypes, such as a higher prevalence of p40 and p63 expression and a lower prevalence of PAX8 expression, with possibly more frequent aberrant p53 staining (28). These data suggest that invasive stratified mucin-producing carcinomas diverge from other mucinous HPV-associated adenocarcinomas and could be categorized separately. This is the first work, to our knowledge, that addresses the clinical outcomes of invasive stratified mucin-producing carcinomas or the impact of an invasive stratified mucin-producing carcinoma component within an HPV-related adenocarcinoma or adenosquamous carcinoma. HPV-associated adenocarcinomas with benign squamous differentiation, HPV-associated adenocarcinomas with invasive stratified mucin-producing carcinoma components, and pure invasive stratified mucin-producing carcinomas should not be regarded as adenosquamous carcinomas, because they do not contain a malignant squamous component. There are, apparently, no differences in clinical outcomes between these categories; however, invasive stratified mucin-producing carcinomas are morphologically distinct and have a different immunophenotype from adenosquamous carcinomas. Most invasive stratified mucin-producing carcinomas and adenosquamous carcinomas are positive for p16 and HPV, but statistically significant differences were found in the expression of HPV, p40, p63, PAX8, HNF1beta, vimentin, p53, CK7, and MUC6. p40 and p63 are less often positive in invasive stratified mucin-producing carcinomas, and the positivity is patchy in the peripheral palisade of tumor cell nests, while MUC6 is more often positive in invasive stratified mucin-producing carcinomas, suggesting more glandular differentiation in invasive stratified mucin-producing carcinomas compared with adenosquamous carcinomas. Moreover, the scant p63 and p40 expression in pure invasive stratified mucin-producing carcinomas in the palisade around invasive nests and the relative lack of PAX8 suggest that these tumors may be of reserve cell origin, as has been suggested (26), compared with pure adenosquamous carcinomas. The older literature suggested that adenosquamous carcinoma is a relatively aggressive disease type, especially in advanced stages, occurring more frequently in pregnant and younger patients than either squamous carcinomas or endocervical adenocarcinomas (16,29,30). In our study, adenosquamous carcinomas were associated with clinical outcomes similar to those of HPV-associated adenocarcinomas, including mucinous endocervical adenocarcinomas, as reported previously (8,31,32), whether or not the invasive stratified mucin-producing carcinoma present was pure or with components. Tumor grade did not appear to be correlated with clinical outcomes. Ultrastructural studies that pointed to the presence of glandular and squamous differentiation (33,34) originally supported the idea that glassy cell carcinomas were a type of adenosquamous carcinoma. Although evidence of glandular differentiation is reportedly rather obvious using electron microscopy, only focal concentrations of tonofilaments have been construed as evidence of squamous differentiation. On examination of hematoxylin and eosin slides, however, these tumors display a uniform population of neoplastic cells, unlike adenosquamous carcinoma, although there are rare reports of glassy cell carcinomas with subtle intracytoplasmic mucin or squamous differentiation. glassy cell carcinoma has been thought of as an aggressive subtype of cervical carcinoma (35,36), although there are few studies describing this. In contrast, a recent publication reported the successful treatment of 5 glassy cell carcinoma patients with advanced-stage disease (37). Based on studying only 2 such cases, neither expressing markers associated with squamous differentiation, we conclude that glassy cell carcinomas are either not adenosquamous carcinomas or are so poorly differentiated that they can only be classified as such with difficulty. Further studies addressing this issue are therefore required for further elucidation of this entity. In summary, adenosquamous carcinomas can be diagnosed in the presence of unequivocal evidence of malignant glandular and squamous differentiation. Mimics such as invasive stratified mucin-producing carcinoma, invasive stratified mucin-producing carcinoma with components, and HPV-associated adenocarcinomas with benign-appearing squamous metaplasia should not be diagnosed as adenosquamous carcinomas based on distinguishing morphological features and some immunohistochemical differences, despite the fact that clinical outcomes appear similar. Since the two putative glassy cell carcinomas studied did not meet our criteria for adenosquamous carcinoma and lacked evidence of squamous differentiation with immunohistochemistry, one should reconsider whether these tumors should be categorized as adenosquamous carcinomas. Acknowledgments Funding: This study was funded in part through the NIH/NCI Memorial Sloan Kettering Cancer Center Support Grant P30 CA008748 (Dr. Soslow and Dr. Park). Disclosures/Conflicts of Interest: The authors have no conflicts of interest to disclose. Figure 1. Distribution of endocervical adenocarcinomas, adenosquamous carcinomas, and mimickers after microscopic evaluation. Figure 2. Adenosquamous carcinoma. A: Malignant glandular and squamous differentiation is present; B: Poorly differentiated squamous component; C: p63 expression; D: p40 expression; E: Positive high-risk human papillomavirus in situ hybridization; F: Block-like p16 expression Figure 3. Invasive stratified mucin-producing carcinoma. A: Invasive nests of cells with stratified mucinous cells surrounded by a palisade; B: p63 expression in the peripheral palisade; C: Block-like p16 expression Figure 4. Invasive stratified mucin-producing carcinoma combined with usual-type adenocarcinoma Figure 5. Usual-type adenocarcinoma with benign-appearing squamous differentiation Figure 6. Glassy cell carcinoma (A) with positive high-risk human papillomavirus in situ hybridization (B) Figure 7. Analysis of overall survival between cervical adenosquamous carcinomas, adenosquamous carcinomas with invasive stratified mucin-producing carcinomas components, invasive stratified mucin-producing carcinomas, and usual-type human papillomavirus HPV-associated adenocarcinomas Figure 8. Analysis of disease-free survival between adenosquamous carcinomas, adenosquamous carcinomas with invasive stratified mucin-producing carcinoma components, invasive stratified mucin-producing carcinomas, and usual-type human papillomavirus HPV-associated adenocarcinomas Table 1: Immunohistochemical Antibodies Antibody CLONE VENDOR Instrument (dilution) Vimentin V9 Roche Roche Discovery XT p53 D07 Roche Roche Benchmark Ultra p16 E6H4 Roche Roche Benchmark Ultra PAX8 Poly Protein Tech Roche Benchmark Ultra AR Poly Santa Cruz Biotechnology Roche Discovery XT PR 1E2 Roche Roche Discovery XT HER2 4B5 Roche Roche Discovery XT HIK1083 HIK1083 Kanto Manual (1/20) MUC 6 CLH5 Novocastra Manual (1/200) CA IX Poly Novus Roche Benchmark Ultra SATB2 EP281 Cell Marque Roche Benchmark Ultra HNF1beta CLO374 Sigma Leica Bond III CK7 OV-TV12/30 DAKO Roche Benchmark Ultra CDX2 CDX2–88 Biogenex Roche Benchmark Ultra p63 4A4 Roche Roche Benchmark Ultra p40 BC28 Biocare Roche Benchmark Ultra Table 2. The immunohistochemical profile of pure invasive stratified mucin-producing carcinomas in comparison with adenosquamous carcinomas Marker Pure invasive stratified mucin-producing carcinomas n (%) Adenosquamous carcinomas n (%) p HPV* 8/8 (100) 19/23 (82.6) < 0.0001 P16 5/8 (62.5) 18/25 (72) 0.1757 PAX8* 2/7 (28.5) 10/23 (43.4) 0.0382 p40* 2/7 (28.5) 15/23 (65.2) < 0.0001 p63* 3/8 (37.5) 15/25 (60) 0.0018 PR 2/8 (25) 6/25 (24) 1.0000 AR 0/8 (0) 0/25 (0) 1.0000 CAIX 4/7 (57.1) 16/23 (69.5) 0.4022 MUC6* 4/7 (57.1) 5/23 (21.7) < 0.0001 HIK1086 0/8 (0) 1/23 (4.3) 0.1212 HNF1beta* 1/7 (14.2) 7/23 (30.4) 0.0023 GATA3 1/7 (14.2) 2/23 (8.7) 0.2747 Vimentin* 1/8 (12.5) 0/25 (0) 0.0003 HER2 1/8 (12.5) 1/25 (4) 0.0652 SATB2 0/8 (0) 0/23 (0) 1.0000 CDX2 0/8 (0) 0/23 (0) 1.0000 p53* 2/7 (28.5) 2/25 (8) 0.0004 CK7* 8/8 (100) 20/23 (86.9) 0.0002 * statistically significant differences ==== Refs References 1. 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==== Front 04104626011NatureNatureNature0028-08361476-46873018590510.1038/s41586-018-0499-ynihpa1503236ArticleA Homing System Targets Therapeutic T-cells to Brain Cancer Samaha Heba 1234Pignata Antonella 234Fousek Kristen 2345Ren Jun 5Lam Fong 47Stossi Fabio 49Dubrulle Julien 49Salsman Vita S 234Krishnan Shanmugarajan 6Hong Sung-Ha 10Baker Matthew L 411Shree Ankita 234Gad Ahmed Z 12345Shum Thomas 245Fukumura Dai 6Byrd Tiara T. 2345Mukherjee Malini 3412Marrelli Sean P. 10Orange Jordan S 412Joseph Sujith K. 234Sorensen Poul H. 13Taylor Michael D 14Hegde Meenakshi 2341516Mamonkin Maksim 234517Jain Rakesh K 6El-Naggar Shahenda 1Ahmed Nabil 234571516171 Children’s Cancer Hospital Egypt −57357, Cairo, Egypt2 Center for Cell and Gene Therapy, Texas Children’s Hospital, Houston Methodist Hospital and Baylor College of Medicine, Houston, Texas, USA3 Texas Children’s Hospital, Houston, Texas, USA4 Baylor College of Medicine, Houston, USA5 Interdepartmental Program in Translational Biology and Molecular Medicine, Baylor College of Medicine, Houston, TX, USA6 Edwin L. Steele Laboratories for Tumor Biology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA7 Department of Pediatrics, Baylor College of Medicine, Houston, Texas, USA8 Center for Translational Research on Inflammatory Diseases at the Michael E DeBakey Veterans Affairs Medical Center, Houston, Texas, USA9 Integrated Microscopy Core, Advanced Technology Cores, Dan L Duncan Comprehensive Cancer Center, Baylor College of Medicine, Houston, Texas, USA10 Department of Neurology, McGovern Medical School at UT Health, Houston, Texas, USA11 National Center for Macromolecular Imaging, Baylor College of Medicine, Houston, Texas, USA12 Center for Human Immunobiology, Texas Children’s Hospital, Baylor College of Medicine, Houston, Texas, USA13 Department of Pathology & Laboratory Medicine, University of British Columbia, Vancouver, BC, Canada14 Developmental and Stem Cell Biology Program, The Arthur and Sonia Labatt Brain Tumour Research Centre, Division of Neurosurgery, Departments of Surgery, Laboratory Medicine and Pathobiology, and of Medical Biophysics, University of Toronto, Toronto, ON, Canada15 Houston Methodist Hospital, Houston, Texas, USA16 Texas Children’s Cancer and Hematology Centers, Texas Children’s Hospital, Baylor College of Medicine, Houston, Texas, USA17 Department of Pathology and Immunology, Baylor College of Medicine, Houston, Texas, USAAuthor Contributions: NA conceived the main study idea; and with HS conceived and implemented the study details. NA, KF, AP designed HS-molecules. HS, MDT, SM, PS, SE, MH, FS, JD, NA performed the CAM-studies. MB, the in-silico modeling. FL and HS designed and implemented microfluidics. MMa and AZG the molecular testing. HS, FS, JD, MM, JSO designed and performed the subcellular-imaging experiments. JR, HS, VSS, AS, TS, SM, SH, DF, SK, RKJ, NA implemented the animal microscopy and experiments. All authors gave their Corresponding Author: Nabil Ahmed. Baylor College of Medicine; 1102 Bates Street; Houston, TX 77030; Tel: +1 (832) 824 4611; Fax: +1 (832) 825 4732; [email protected] 9 2018 05 9 2018 9 2018 06 3 2019 561 7723 331 337 Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:http://www.nature.com/authors/editorial_policies/license.html#termsSummary Paragraph: Successful T-cell immunotherapy for brain cancer should adequately access tumor tissues, but strategies to achieve this have been elusive. We discovered that, in contrast to inflammatory brain diseases, such as multiple sclerosis, where endothelial-cells upregulate ICAM1 and VCAM1 to guide the extravasation of pro-inflammatory cells, cancer-endothelium downregulates these molecules to evade immune-recognition. In contrast, we found that cancer-endothelium upregulates ALCAM, which allowed us to overcome this immune-evasion mechanism by creating an ALCAM-restricted Homing System (HS). We re-engineered ALCAM’s natural ligand, CD6, in a manner that triggers initial anchorage of T-cells to ALCAM and conditionally mediates a secondary-wave of adhesion by sensitizing T-cells to low-level ICAM1 on the cancer-endothelium, thereby creating the adhesion forces necessary to capture T-cells from the bloodstream. Cytotoxic HS T-cells robustly infiltrated brain cancers after intravenous-injection and exhibited potent antitumor activity. We here describe a first-in-class molecule that targets the delivery of T-cells to brain cancer. ==== Body Main Text: The success of leukocyte trafficking from the bloodstream to the brain relies on well-concerted complementary waves of cell adhesion molecules (CAM) expressed on endothelial-cells (EC), the initial access point through the blood brain barrier (BBB) [1, 2]. This dynamic state becomes heightened in brain infiltrative-conditions, such as multiple sclerosis (MS), where preferential access is granted to disease-mediating immune-cells [3, 4]. Conversely, under the influence of cancer, homing of cytotoxic T-cells is often barricaded [5, 6]. Activated leukocyte cell adhesion molecule (ALCAM; CD166), a tissue-restricted CAM, plays a major role in triggering T-cell infiltration in inflammatory brain diseases [7, 8]. Indeed, antibodies blocking ALCAM or its T-cell cognate-ligand, CD6, decrease leukocyte access to the brain and are in clinical trial for MS, HIV-encephalitis and graft-versus-host disease [9–11]. After engaging ALCAM, successful transendothelial-migration (TEM) requires that T-cells sense a secondary-wave of more ubiquitous CAM on EC, predominantly mediated by ICAM1 and VCAM1, to reach the adhesion-threshold needed for T-cell capture from the bloodstream [12]. We found that, similar to MS, brain cancer-EC overexpress ALCAM but paradoxically downregulate ICAM1 and eliminate VCAM1, likely to abrogate the homing of antitumor T-cells. While ALCAM is widely expressed on cancer-cells and has been established as a mediator of tumor invasion and metastasis, its role in tumor-EC is yet to be defined [13]. We reasoned that lessons learnt from MS could perhaps give insight into how to overcome this cancer immune-evasion mechanism; specifically, how to enable therapeutic T-cells to infiltrate brain cancers. T-cell immunotherapy is an emerging field that has shown promise in clinical trials for cancer, infection, and more recently, autoimmune disease [14, 15]. Cell-engineering has extended the interest in this therapeutic modality; however, effective homing of therapeutic T-cells to the target site remains a major limiting factor, especially for brain tumors. Since cancer-EC express high levels of ALCAM, yet its cognate ligand, CD6, naturally-expressed on T-cells, fails to mediate adequate TEM, we hypothesized that optimizing ALCAM binding by rationally re-engineering CD6 will provide an entry point for T-cells through the otherwise restrictive tumor-endothelium. Cancer endothelium diverts T-cells from brain tumors We studied ALCAM expression in glioblastoma (GBM) and medulloblastoma (MB), the commonest brain cancers in adults and children, respectively, and detected intense ALCAM-immunoreactivity that co-localized with CD31, denoting its vascular expression (Fig. 1A–1C and Extended Data-[ED]-Fig. 1A). ALCAM was overexpressed on the surface of primary tumor-EC (pTEC; ED-Fig. 1B), isolated from GBM surgical-resections, in contrast to a panel of non-tumor EC in which ALCAM was only detected intracellularly (ED-Fig. 2A). Intriguingly, GBM-supernatant (supe) or TGFβ [16], which is highly-abundant in brain cancer [17], promoted EC-ALCAM expression, indicating that ALCAM is readily-inducible by tumor-derived factors (Fig. 1D and ED-Fig. 2B). During T-cell BBB-transmigration in MS, endothelial-ALCAM co-localizes in lipid rafts with T-cell CD6 [18, 19]. To investigate whether the observed ALCAM overexpression could enable T-cell transmigration through a cancer-BBB, we created an in vitro BBB-model by sandwiching a polycarbonate-membrane between pTEC and pericytes (Fig. 1E). Despite ALCAM overexpression (ED-Fig. 2C), cancer-BBB remained impermeable to T-cells both at baseline and after conditioning with GBM-supe or TGFβ (Fig. 1F). In contrast, IL-6 conditioning of a normal-BBB, where pTEC were substituted with normal brain-EC, rendered it highly permissive. The fact that cancer-BBB was resistant to the effect of pro-inflammatory and tumor stroma-secreted IL6, suggested that ALCAM overexpression alone is insufficient to enable transmigration of T-cells; indicating that perhaps the secondary-wave of adhesion, well-described MS, is lacking [12]. We therefore studied the dynamic expression of the principal mediators of the “secondary-wave” on cancer-EC. In contrast to normal brain-EC, we found that pTEC express lower levels of ICAM1 and no VCAM1 at baseline (Fig. 1G and ED-Fig. 2D-H). Culturing pTEC in the presence of GBM-supe, TGFβ or IL-6, further decreased ICAM1 and dramatically upregulated ALCAM. Expectedly, IL6 increased the expression of ICAM1, VCAM1 and ALCAM in normal brain-EC. Rather importantly, we observed this distinct pattern of adhesion molecule expression in the microvasculature of surgically-excised GBM (n=93) as well as MB (n=25) in contrast to normal brains (n=5; Fig. 1I, ED-Fig. 1A). Reduction of ICAM1 and elimination of VCAM1 under the influence of tumor-derived factors, suggested an inherent pTEC resistance to interacting with T-cells which explained the impermeability of the cancer-BBB seen in Fig. 1F. Engineering T-cells to traverse cancer endothelium ALCAM-expression was intensified in cancer-EC; we therefore reasoned that enhanced T-cell anchorage to ALCAM could compensate for the reduced expression of ICAM1. As the constitutively-expressed CD6 on T-cells was insufficient to promote effective TEM, we rationally re-engineered CD6, to create a system for guided T-cell homing to brain cancers. To extract CD6’s homing function, we computationally-mapped ALCAM’s binding-region to CD6’s extracellular domain-3 (D3), in agreement with previous reports (Fig 2A and ED-Fig. 3A-C) [9, 10]. The prototype HS-molecule included a D3 exodomain, an IgG1-hinge and transmembrane and CD6 signaling-domain (Fig. 2B and ED-Fig. 3D). We multimerized the exodomain (creating a trimer [3HS] and a pentamer [5HS]) to study the effect of enhancing HS’s crosslinking-avidity to ALCAM on T-cell behavior (Fig. 2C and ED-Fig. 3E) and created tailless HSΔ-molecules to study the role of HS-signaling (Fig. 2D and ED-Fig. 3F). We detected HS-molecules on the surface of human T-cells by probing D3 (ED-Fig. 3G-3H) and confirmed their ALCAM-specific binding (Fig. 2E, ED-Fig. 3G). The molecular interactions of T-cells with the endothelium at the transmigratory-cup are mediated by podosynaptic-CAMs engaging their cognate ligands to initiate TEM. [20] We detected D3/ALCAM-heterodimers in HS T-cell transmigratory-cups; these intensified with D3 multimerization (Fig. 2F; ED-Fig. 3I-J). [20] T-cell capture from the bloodstream, rolling along the vessel wall and firm adhesion are key steps prior to TEM [21]. To dissect the effect of HS on TEM-kinetics, we used microfluidics flow chambers lined by TGFβ-conditioned ALCAM+ endothelium, applying 2 dyne/cm2 shear force, akin to that found in tumor capillary-vessels (ED-Fig. 3K-3L and Supplementary Video 1). HS T-cells were captured more frequently, rolled slower, and stopped faster than NT (Fig 2G–2I and ED-Fig.3M-N). Multimerization of D3 increased their capture and arrest, while inclusion of a signaling-domain maximized this effect independent of the exodomain. Rolling-velocities were similar, likely because rolling is predominantly influenced by selectins; expressed similarly in all [22]. At their pre-extravasation status, HS T-cells were more resistant to mechanical-detachment (Fig. 2J) and could therefore resist fluctuations in blood-flow typical of tumor neo-vasculature. HS T-cells did not engage normal-(ALCAM-negative) EC. Next, we evaluated the ability of HS to enable T-cell transmigration through our BBB-model (Fig 2K). Resting-BBB allowed negligible T-cell migration, yet upon induction of ALCAM, migration of HS T-cells increased significantly. Multimerization of the exodomain further increased this migration (5HSΔ>3HSΔ>HSΔ) and a signaling-domain maximized migration across constructs. Blocking the D3/ALCAM-interaction using soluble ALCAM abrogated migration with all HS T-cells and washing it largely restored the pre-blocking pattern. Molecular interruption of ALCAM on pTEC using siRNA and on HUVEC using CRISPR-Cas9 muted their responsiveness to TGFβ (ED-Fig. 4A-F) and abrogated the transmigration of HS T-cells (yellow bars in Fig. 2O). ALCAM-ALCAM homotypic binding occurs but is decimated by its 50-fold stronger heterotypic ALCAM-CD6 interaction [1]. We found that elimination of T-cell ALCAM improved the transmigration of HSΔ (but not of HS) T-cells, indicating that the HS transmigration is largely mediated through heterotypic HS interaction with endothelial-ALCAM (ED-Fig. 4G-J) [1]. We concluded that enhanced ALCAM anchorage can trigger BBB-transmigration of HS T-cells. Multimerization of the HS-exodomain had an incremental advantage on TEM steps; yet signaling through the HS-endodomain maximized this effect, indicating the potential involvement of other mechanistic downstream events in enabling HS T-cell transmigration. HS harnesses the adhesive power of ICAM1/LFA-1 axis While the definite functions of CD6 remain obscure, its signaling is thought to be critical to T-cell motility and cell-cell contact. [11] CD6 is known to activate the downstream adapter SH2-domain containing Leukocyte Protein of 76kD (SLP-76), a shared pathway that results in activation of Lymphocyte Function-associated Antigen-1 (LFA-1). Upon activation, LFA-1 undergoes a conformational change exposing a ligand binding site for ICAM1. [23–25] We reasoned that if HS-signaling converges on SLP-76, ICAM1 could be brought into play, explaining the functional superiority of HS T-cells over HSΔ T-cells. We, sequentially interrogated individual mediators downstream of the HS-endodomain that could link to integrin-modulation. We found significant co-clustering of SLP-76 with eGFP-tagged HS-molecules (HS.eGFP) but not with eGFP-tagged HSΔ-molecules (HSΔ.eGFP) upon crosslinking ALCAM immobilized on a glass-surface (Fig 3A). SLP-76 binds to CD6 in a Zeta-chain-associated protein kinase-70 (Zap-70)–dependent manner, and we did find that, in contrast to HSΔ- and NT- T-cells, all HS T-cells showed significantly higher ZAP-70 phosphorylation after transmigration through the cancer-BBB-model.[26] HS T-cells also recruited cytosolic Talin-1, a high molecular-weigh cytoskeletal-protein known to unfold LFA-1 into a high-affinity conformation. [27, 28] Indeed, upon transmigration, Talin co-aggregated with and unfolded LFA-1 and engaged ICAM1 at transmigratory-cups formed by HS T-cells worming between and through ALCAM-expressing EC (Fig. 3B-C, ED-Fig 5A-C). These effects were absent with HSΔ T-cells. We functionally confirmed this important finding, since BIRT-377, an allosteric inhibitor of unfolded LFA-1[29], significantly reduced the migratory-capacity of HS T-cells to levels comparable to HSΔ T-cells, highlighting the contribution of the CD6-signaling endodomain (Fig. 3D). The effect of BIRT-377 on the migratory-capacity of HSΔ T-cells and NT was negligible. Thus, our data confirmed that CD6-signaling culminates in the unfolding of LFA-1 on T-cells, enabling HS T-cells to engage low-level ICAM1 and providing the deficient secondary-wave of adhesion to the cancer-EC (Fig. 3E). Cytoskeletal changes mediate adept transmigration Talin-1 is concentrated at regions of cell–cell contact, and is known to drive the extravasation mechanism of T-cells through cortical Actin polymerization and focal adhesions complex maturation mediated by Focal Adhesion Kinase (FAK) and Vinculin [30, 31]. We used Total Internal Reflection Fluorescence (TIRF) and found that upon landing of T-cells on ALCAM-coated glass surfaces, Actin was induced and co-localized with HS.eGFP- but not with HSΔ.eGFP- molecules on T-cells or with NT (Fig 4A and4B). Signaling from FAK promotes adhesion maturation of the migrating T-cells and mediates the rear retraction of the T-cell crawling on EC and their ultimate protrusion and extravasation [32, 33]. We found denser Actin and FAK after ALCAM interaction in HS T-cells compared to HSΔ T-cells and NT (Fig. 4C and4D). We also detected membrane-ruffling, formations of a motile cell-surface containing a meshwork of newly polymerized Actin, and an enrichment of Actin/FAK in lamellipodia and invadopodia using Structure Illuminating Microscopy (SIM), which offers a higher lateral resolution (Fig 4E). To quantify these findings in a technically-unbiased manner, we performed high-throughput Deconvolution Microscopy (DM) on T-cells from three donors at different levels of ALCAM (ED-Fig. 4F-G). HS T-cells had significantly higher Actin and FAK than NT. Additionally, Actin and FAK co-localized at the surface, enabling protrusion of the podosynaptic structure of the T-cells, which is needed for subsequent endothelial invasion. All HS T-cells showed higher podosynaptic lamellipodia per cell and focal adhesions per cell, and significantly larger area of spread, meaning that the cytoskeletal-rearrangement is well-tensioned to enable T-cell migration. Collectively, subcellular-microscopy demonstrated that HS-molecules anchor to the Actin-cytoskeleton and mature the FAK that T-cells need for transmigration. Targeted homing of HS T-cells to brain cancer ALCAM and its binding region on CD6 are highly conserved with 93–96% homology between human and mouse, enabling us to assess the ability of intravenous HS T-cells overcome the endothelial blockade and home to orthotopic U87-GBM in severe combined-immunodeficiency (SCID) mice (Fig. 5A). Flow-cytometry of tumor-infiltrating lymphocytes (TIL) demonstrated that all HS T-cells had superior specific homing capacity compared to NT, and that 5HS T-cells had the densest TIL infiltrate (Fig. 5B and ED-Fig. 6A). Next, we injected eGFP/firefly-luciferase (eGFP.FFLuc)-labeled T-cells intravenously in mice harboring U87-GBM (adult-GBM) and Daoy-MB (pediatric-MB) orthotopic grafts and quantified T-cell homing using bioluminescence-imaging (BLI). HS T-cells had a 1–2 log brighter signal than NT in U87-GBM (Fig 5C–5D) and Daoy-MB (Fig 5E–5F). Analysis of the spatial orientation of T-cells to the 3D-reconstituted tumor-vasculature in tumor explants showed significantly higher HS T-cell signals in the intra- and peri- vascular areas compared to NT (Fig. 5G–5H). Finally, we used a cranial window and video-rate multiphoton microscopy to examine the in vivo dynamics of HS T-cell homing to U87-GBM with single cell resolution (ED-Fig. 6B and Fig. 5I). Continuous videography of size-matched vasculature showed more rolling 5HS T-cells along tumor vessels and achieving firm-arrest compared to NT (Fig. 5J and5K and Supplementary Videos 2-5). Reconstitution of time-lapse images in 3D demonstrated more extravasating HS T-cells than NT at the tumor vascular bed (Fig. 5L). Importantly, we investigated if intravenous 5HS-equipped T-cells invaded normal tissues but found negligible TIL in the spleen, lungs and kidneys of U87-GBM-bearing mice, at levels no greater than NT (ED-Fig. 7A). We additionally observed no T-cell or alterations in the histo-morphology of normal brain tissues despite a heavy HS T-cell infiltrate in the tumor (ED-Fig. 7B). Anti-tumor activity of cytotoxic HS T-cells Next, we assessed the ability of the HS T-cells to deliver a therapeutic complex-biologic to brain cancers (Fig. 6A). We armed the winning design, 5HS T-cells, with chimeric antigen receptors (CAR) specific for human epidermal growth factor receptor 2 (HER2), a glioma antigen currently targeted by CAR T-cells in several clinical trials (ED-Fig. 8A) [34]. Prior to testing in animals, we confirmed that only HER2-CAR 5HS T-cells, but not 5HS T-cells or NT, efficiently killed U87-GBM cells in vitro (Fig. 6B). Importantly, HER2-CAR 5HS T-cells failed to lyse normal or tumorous human or murine EC (Fig. 6C) or any other ALCAM-expressing leukocytes (Fig. 6D). This indicated that HS T-cells have no cytolytic activity against ALCAM-expressing targets and that cytolysis is distinctly mediated by the CAR-molecules through engaging HER2. Similar to HER2-CAR T-cells, all HER2-CAR HS T-cells exhibited a predominantly effector-memory phenotype and a comparable exhaustion and proliferation profiles following transwell-migration (TWM; ED-Fig. 8B-D). To test the antitumor efficacy of HER2-CAR 5HS T-cells, we established eGFP.FFluc-labeled orthotopic U87-GBM tumors. Unlabeled T-cells were injected intravenously, and tumor growth was monitored. HER2-CAR 5HS T-cells induced regression of established tumors in all treated animals in contrast to HER2-CAR T-cells, which transiently slowed tumor-growth, and to NT (Fig. 6E and6F). HER2-CAR 5HS T-cells were significantly more abundant in GBM-explants compared to non-HS and NT (Fig. 6G and ED-Fig. 9A) but were absent in non-tumor areas of the brain (ED-Fig. 9B). Kaplan-Meier survival analysis showed mice treated with HER2-CAR 5HS T-cells had a median-survival exceeding 60 days, compared to 22 and 18 days for animals treated with HER2-CAR T-cells and NT, respectively (Fig. 6H). Discussion: The onset of the intricate process of TEM is mediated by waves of engagement of EC adhesion-molecules to their cognate ligands on T-cells, the signaling thereof mediates the adhesion-threshold necessary to pull T-cells from the bloodstream [35]. In MS, this threshold is reached by ALCAM crosslinking CD6, followed by a secondary-wave of interaction with other ubiquitous CAMs, predominantly ICAM1 [36]. Here, we show that similar to inflammatory-EC, cancer-EC overexpress ALCAM, but abrogate T-cell homing by downregulating ICAM1. Our results indicate that the interaction between endogenous-CD6 on T-cells with the ALCAM on cancer-EC alone is incapable of mediating T-cell transmigration. We thus created HS, a set of engineered ligands, to enhance the transmigration of T-cells through cancer-EC. We demonstrate how HS T-cells can harness the power of a preexisting pathway, ICAM1/LFA-1, akin to the secondary-wave seen in MS [37, 38]. This transforms the ineffective T-cell/cancer-EC interaction into a permissive inflammatory one. Subsequently, but very likely in concert, T-cells are guided by a chemokine gradient to their tumor target (Fig. 6I). We created HS to be an abstract ligand based on the minimal moiety on CD6 that could hetero-dimerize with ALCAM. In doing so, we extracted CD6’s homing function (mediated by D3) and equally importantly avoided carrying forward its other unwanted ones; these remain mostly elusive.[11] Indeed, when we cloned and expressed the full-length CD6 on human T-cells, their transmigration was improved but they were exceedingly activated at baseline, exhibited a rather exhausted phenotype, failing to expand (ED-Fig. 10). This confirms recent literature describing CD6’s D1 as a mediator for T-cells activation [39]. Importantly, it underscores the critical role of “rational-engineering” in the design of synthetic molecules to optimally mediate distinct functions, while simultaneously avoiding unwanted ones. Poor homing of CAR T-cells is a major obstacle to effective T-cell therapy. [40,41] We recently established a favorable safety-profile of intravenous HER2-CAR T-cells in GBM patients, yet only observed disease stabilization and partial responses [42]. As an alternate strategy, we and others have reverted to intra-tumoral administration to achieve the bioavailability needed to elicit a more uniform clinical-response (NCT02442297); an approach that is rather invasive and of limited applicability. At this stage, we ought to seek highly-specific refinements to cellular therapeutics, such as the HS-platform, if more durable tumor clinical responses are to be achieved. This first-in-class molecule enhances the ability of therapeutic T-cells to exert an antitumor activity while maintaining a favorable safety profile. Adaptations to the HS-platform as a modular delivery tool could be made to give access of complex therapeutics to diseased brain sites. Materials and Methods: Antibodies, recombinant proteins and chemicals. Antibodies: Anti-FAK, anti-ALCAM-PE, anti-ICAM1-PE, anti-VCAM1-PE, anti-VE cadherin, anti-von Willebrand (vWF) factor, anti-CHS1 were purchased from Abcam (Cambridge, MA, USA). OX124, mouse anti-human monoclonal antibody against HS, was a kind gift from Dr. Marion Brown, Oxford, UK. Biotin-labeled Anti-CD18 KIM127 was purchased from Exploratory Research Cell Tech Therapeutics Ltd. (Slough, UK). Anti-phospho-SLP-76; pTyr128, anti-VE-cadherin were purchased from Cell signaling Technologies (Danvers, MA, USA). Anti-HIF-1, anti-pZAP-70-FITC, anti-pZAP-70-PE, anti- LAG3, anti-TIM3, anti-PD-1, eFluor 670, anti-CD45-APC, anti-CHS-PerCP, anti-CCR7, anti-CD45RO were purchased from BD biosciences (Franklin Lakes, NJ, USA). Anti-Talin-1 and anti-Vinculin were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Goat anti-human Fc-PE was purchased from Millipore (Billerica, MA, USA). Alexa Fluor (AF) labelled secondary anti-mouse, anti-goat anti-rabbit antibodies, streptavidin AF 488 antibody; Texas Red Phalloidin and AF488 Phalloidin were purchased from Life Technologies (Carlsbad, CA, USA). Anti-Fab DyLight 488 was purchased from Jackson ImmunoResearch (Suffolk, UK) Recombinant proteins: Recombinant human TNFα, IFNγ, TGFβ, IL-6 were purchased from Genentech Inc. (San Francisco, CA, USA). Human IL-1α was purchased from Sigma- Aldrich, (St. Louis, MO, USA). IL-7 and IL-15 were purchased from Peprotec (Rocky Hill, NJ). ALCAM-Fc, HER2-Fc were purchased from R&D Systems (Minneapolis, MN, USA). Chemicals: BIRT377 was purchased from Tocris Cookson (Bristol, UK) Image analysis of primary glioblastoma and medulloblastoma samples and normal brain tissue. Tissue microarrays and individual tumor frozen sections were scanned on Leica microscope with 20x objective or a Cytation5 with a 10x objective. Images were acquired in the DAPI, ALCAM-FITC, CD31-TRITC and ICAM1Cy5 or VCAM-Cy5 channels, to create a 37×37×4 image dataset. Images (5 to 20 fields of view) were sorted and batch-processed using a custom-made Matlab® script. FITC, TRITC and Cy5 images were background-corrected by top-hat filtering, and signal noise was removed using the adaptive Wiener method. The positive signal in each channel (ALCAM-FITC or ICAM-cy5 or VCAM-cy5) was then segmented by channel-specific intensity thresholding and tissue masking. Regions of interest were gated based on CD31 fluorescence, and each pixel in identified regions was given a fluorescence intensity value. To confirm ALCAM as an endothelial marker, co-localization analysis between CD31-TRITC and ALCAM-FITC channels and also for control CD31-TRITC and ICAM-Cy5 was done by measuring the Manders Co-localization Coefficients (MCC [43]), using MATLAb® written algorithm. MCCs were then calculated between FITC signal (G) and TRITC or Cy5 signal (R) as: M1=∑Gi, colocal ∑Gi, where Gi, colocal = Gi if Ri>0 and Gi, colocal =0 if Ri = 0; and M2=∑Ri,colocal∑Ri,where Ri, colocal = Ri if Gi>0 and Ri, colocal =0 if Gi = 0; As a negative control, FITC images were rotated by 180° before calculating the MCCs. Cell culture. Primary brain tumor endothelial-cells (pTEC): Primary GBM surgical specimens and other primary tumor samples were obtained on a human protocol approved by the Institutional Review Board (IRB) of Baylor College of Medicine, The Houston Methodist Hospital and Texas Children’s and Toronto Children’s Hospitals, and were stained or processed to isolate patient derived primary tumor endothelial-cells (pTEC). Patients and healthy donors were consented prior to obtaining the samples unless these were from publicly available datasets. Briefly, the tissues were minced in DMEM (Gibco, Waltham, MA, USA) and digested with 1 mg/ml collagenase and 10 mg/ml DNAse (BD Biosciences, San Diego, CA) in DMEM for 30 minutes at 37 °C with continuous shaking at 180 rpm. The tissue suspension was filtered through mesh screen (200μm), washed with Dulbecco’s Phosphate-Buffered Saline (D-PBS), centrifuged at 1,000 x g for 10 min at 4°C and the pellet was re-suspended in DMEM containing 20% BSA. A second digestion was done for 2 hours on a shaker at 180 rpm using collagenase and dispase (1mg/mL) following which the digested layer was separated on 40% percoll gradient and centrifuged at 700 g for 10 minutes. The endothelial-cells were taken from the cloudy interphase, washed in D-PBS and grown in brain endothelial-cell medium (EBM; Lonza, Basel, Switzerland) supplemented with 20% fetal bovine serum (FBS), bFGF (20 μg/ml), heparin (100 μg/ml) and puromycin (500 μg/ml) on fibronectin (0.4 mg/ml) and collagen (0.1 mg/ml) pre-coated plates. After 7–10 days, cells were checked for endothelial-cells markers CD31, von Willebrand Factor and VE cadherin. They were sorted on pan endothelial marker CD31 at the Baylor College of Medicine Flow-cytometry Core. Viable Sorted CD31 positive propidium iodide (PI) negative population of isolated GBM endothelial-cells were regrown in EBM media in fibronectin/collagen coated flasks and used at passage 1–2 for the needed experiments. Other primary and established cell lines: Primary GBM cell lines were isolated from GBM surgical excision specimens as described [44]. Human Brain Microvascular Endothelial-cells (HBMEC), Human Brain Vascular Pericytes (HBVP) and CD1+ astrocytes were obtained from ScienCell Research Laboratories (Carlsbad, CA, USA). Human Lung Microvascular Endothelial-cells (HMVEC-L) were obtained from Lonza (Basel, Switzerland). Human Umbilical Cord Vascular Endothelial-cells (HUVEC), Human Embryonic Kidney-293A (HEK293) cells, U87-GBM cell line murine cerebral microvascular tumor endothelial-cells, bEnd.3, and the murine lymph node vascular endothelial-cells, 2-H11, were obtained from ATCC (Manassas, VA, USA). BALB/c mouse primary brain microvascular endothelial-cells and BALB/c Primary Mouse lung Microvascular Endothelial-cells (PMVEC) were obtained from Cell Biologics Inc. (Chicago, IL, USA). Astrocytes and brain endothelial-cells were cultured in EBM supplemented with 20% FBS and growth factors using supplement kit EGM-2 BulletKit containing VEGF, ECGS, Heparin, EGF, Hydrocortisone, l-Glutamine (2mM) and Puromycin (4 mg/ml). Human Embryonic Kidney-293A (HEK293) cells, U87- GBM cell lines and primary GBM cells lines processed in our lab were maintained in DMEM supplemented with 10 % FBS and 2mM GlutaMAX-I (Gibco). T-cells were maintained in T-cell media (250mL RPMI-1640, 200mL CLICKS with 10% FCS containing 2mMol/L GlutaMAX-I). All Cells were cultured in an incubator at 37 °C in a humid atmosphere of 5% CO2 and passaged at 70% confluency. All endothelial-cells were routinely analyzed for CD31 and vWF expression by flow-cytometry prior to use. Immunohistochemistry and immunofluorescence. Expression of ALCAM on primary GBM and MB tissue sections were detected by immunohistochemistry on formalin fixed paraffin embedded (FFPE) slides. Following de-paraffinization and rehydration, endogenous peroxidase activity was blocked with 30% H2O2 and antigen retrieval was performed using Dako antigen retrieval solution (BioGenex, Fremont, CA, USA) for 90 minutes at 90°C under pressure. Avidin, biotin (BioGenex) and Fc receptor (Innovex Biosciences, Richmond, CA, USA) blocking reagents were applied to the sections prior to a 4°C overnight incubation with rabbit anti-human anti-ALCAM (1:100). The sections were developed with HRP conjugated anti-rabbit antibody (1:1000; Abcam) using DAB as chromogen (BioGenex). All slides were counterstained in Harris hematoxylin, dehydrated and mounted. Images were acquired using an Olympus light microscope. Scoring of CD3 positive DAB signal was analyzed using IHC_Profiler® plugin in ImageJ®. Images were scored by a pathologist blinded to the conditions. Frozen tissue sections were also made from primary GBM surgical specimens at the Baylor College of Medicine pathology core and used for co-staining of CD31 and ALCAM. The slides were fixed with cold acetone/methanol for 15 minutes, antigen retrieval performed using 1× citrate buffer (Thermo Fisher Scientific), blocked with human goat serum for an hour and probed with mouse anti- human CHS1 (1:100) and rabbit anti- human ALCAM antibody (1:50) overnight. Slides were then incubated for an hour with secondary goat anti-rabbit Alexa-fluor 588 and goat anti-mouse Alexa Fluor 647 respectively, counterstained with DAPI and mounted. Tissue images were taken using a Zeiss confocal spinning disk microscope (Zeiss, Oberkochen, Germany) at 40x magnification. Flow-cytometry for surface expression of brain endothelial-cell adhesion molecules. For expression of cell adhesion molecules (CAM), 1×106 HBMEC cells and pTEC were harvested and stained using anti- human ALCAM-PE, anti- human ICAM1-FITC, anti-human VCAM1-FITC. Expression was analyzed by flow-cytometry at basal levels, after conditioning with IL-6 (100 ng/ml) TNFα (10, 100, 500 ng/ml), TGFβ (1 μg/ml), and GBM-supe for 6 hours. FlowJo data analysis software (FLOWJO, LLC, Ashland) was used for all flow cytometric analyses. ALCAM expression during basal and pathological conditions was measured on murine (2-H11, the BALB-C primary brain endothelium, bENd.3 and PMVECs) and human endothelial-cells (HBMEC, pTEC, HMVEC-L, HUVEC) by flow-cytometry. For inflammatory conditions, cells were cultured with TNFα, TGFβ, IL-6, IL-1, IFNγ at optimized concentration of 100 ng/ml for 6 hours. To simulate tumor environment, endothelial-cells were exposed 6 hours to fresh supernatants from GBM cell culture, collected 24 and 48 hours post addition. Conditioned and normal cells (1×106) were washed in PBS containing 2% FBS and 0.1% sodium azide (FACS buffer; Sigma Aldrich) and stained with ALCAM-PE for an hour in the dark along with matched isotype controls. Approximately 100,0000 events/tube were captured using a Gallios™ flow cytometer (Beckman Coulter Inc, Brea, CA) or BD Accuri™ C6 (Becton Dickinson, Franklin Lakes, NJ) and data analyzed by Kaluza software (Beckman Coulter Inc.) or FlowJo data analysis software (Flowjo LLC, Ashland, OR). Western Blotting. Conditioned and non-conditioned endothelial-cells (1×106) were lysed with RIPA lysis and extraction Buffer (ThermoFisher Scientific) per manufacturer recommendations and 10 μg each of protein extracts were separated by SDS-PAGE, blotted onto PVDF membranes (GE Healthcare, Buckinghamshire, U.K.) and probed with primary antibodies against ALCAM (1:1000) and B-actin (1:1000) at 4°C overnight. Following incubation with HRP-conjugated secondary anti-mouse antibody (1:25,000) and anti-rabbit (1:5.000) respectively, the blots were developed with ECL Prime Western blotting detection reagents (GE Healthcare, Chicago, IL, USA). Analysis was done using Image J (NIH) and ALCAM expression was normalized to beta-actin. HS Design, synthesis and production of HS T-cells. The minimal binding to ALCAM was mapped in silico to domain 3 (D3) of native CD6 and the adjacent stalk (ST). Using Clone Manager® (Sci-Ed Software, Cary, NC), the HS prototype molecule was designed a leader sequence followed by an exodomain; formed of domain 3 plus the stalk; followed by an IgG1 hinge, connected to CD6 transmembrane and a CD6 endodomain; formed of the full length CD6 signaling-domain (amino acids 30–400, NC_000011.10 (60971641..61020377). Subsequently, multimer of the prototype; a trimer (3HS) and pentamer (5HS); using multiples of the exodomain. Furthermore, to study the signaling-domain function, truncated versions; (HSΔ, 3HSΔ, 5HSΔ) were designed with a stop codon placed after 21 amino acids proximal to the transmembrane domain. Expression optimized DNA sequences with exodomain wobbled in multimers were synthesized by GeneArt® Inc. using oligonucleotides, cloned into the Gateway® entry vector pDONR™221. Then each HS construct transgene was cloned in the correct frame into an SFG retroviral vector and sequences were verified. For the in vivo bioluminescence tracking, all HS sequences were followed by a 2A sequence and a GFP and firefly luciferase fusion transgene. To produce retroviral supernatant, 293T-cells were co-transfected with SFG retroviral vector, Peq-Pam-e plasmid encoding the sequence for MoMLV gag-pol, and plasmid pME-VSVG containing the sequence for VSV-G, using GeneJuice transfection reagent (EMD Biosciences, San Diego, CA). Retroviral Supernatants were collected 48 and 72 hours later and cryopreserved. OKT3/CD28 activated T-cells were transduced with retroviral vectors as described [44]. Briefly, peripheral blood mononuclear cells (PBMCs) were isolated by Lymphoprep (Greiner Bio-One, Monroe, NC) Ficoll gradient centrifugation. 5×105 PBMC per well in a 24-well plate were activated with OKT3 (OrthoBiotech, Raritan, NJ) and CD28 monoclonal antibodies (BD Biosciences, Palo Alto, CA) at a final concentration of 1 μg/mL. For transduction, a non-tissue culture treated 24-well plate was pre-coated with a recombinant fibronectin fragment (Retronectin; Takara Bio USA, Madison, WI). Wells were washed with PBS and incubated twice for 30 minutes with the retrovirus supernatant. Subsequently, 2×105 T-cells per well were transduced with retrovirus in the presence of IL-7 at 10 ng/mL and IL-15 at 5 ng/mL. After 48–72 hours cells were removed and expanded in G-rex (Wilson Wolf, St Paul, MN) in T- cell media supplied with IL-7 at 10 ng/mL and IL-15 at 5 ng/mL for 10–15 days prior to use. Transduction efficiencies were assessed with flow-cytometry using mouse anti-human D3 monoclonal antibody followed by goat anti-mouse Alexa-fluor 488 conjugate, or human ALCAM-Fc followed by a APC-conjugated goat anti-human Fc. Secondary only controls were incorporated and all transductions percentage was normalized in comparison to native expression of CD6 on non-transduced (NT) control T-cells. HER2 CAR HS T-cells; used in the anti-tumor in vivo experiment; were generated by co-transduction with FRP5 HER2-specific scFv [34, 44] and the 5HS construct. Surface expression was detected with flow-cytometry using goat anti-mouse Fab fragment specific antibody conjugated with DyLight 488 and HS mAb followed by goat anti-mouse AF 488. Reverse transcriptase polymerase chain reaction (RNA-PCR). Using RNeasy extraction Kit (Qiagen), total RNA was extracted from 1×106 T-cells 8 days after electroporation with control Cas9 only or with gRNA. 4 mg of pre-treated RNA with an RNase-free DNase and was used for cDNA synthesis by using the SuperScript III First-Strand Synthesis System (LifeTechnologies). Aliquots of the RT product were used for regular RT-PCR amplification for ALCAM and GAPDH as positive control. The reaction was carried out in a total volume of 20μl containing 3μl reverse transcribed cDNA, 1 unit of Q5 high fidelity Taq polymerase (biolab), and 20μM of each primer. For ALCAM cDNA, the forward primer, 5′-GTCTGGGCAATAGTGACT CC-3′ and reverse primer 5′-AACCATTGCAAGTGGAAA CC-3 were used. For the control housekeeping gene, GAPDH, the forward primer 5’-TGCACCACCAACTGCTTAGC-3’ and reverse primer 5′- GGCATGGACTGTGGTCATGAG-3’ were used. The resulting gene amplicons were analyzed by agarose gel electrophoresis; ALCAM at 490bp and GAPDP at 660bp. Flow -cytometry. T-cell phenotype, exhaustion and proliferation: For phenotype analysis of the transmigrating T-cells, 2×105 T-cells, all HS T-cell groups and the control NT were stained with anti-human CCR7-PE-Cy7, and anti-human CD45RO-PE for 60 mins at room temperature. Similarly, for exhaustion analysis, 2×105 T-cells were collected from the bottom chamber of after transmigration and washed with PBS then incubated with LAG3, TIM3, PD-1 antibodies. Versacomp antibody capture beads (Beckman Coulter, Bria, CA) were stained with the same antibodies to allow accurate compensations. For proliferation analysis, 2×105 T-cells were collected before and after transmigration, cells were washed and labeled with eFluor 670 diluted to 10 μM and incubated for 25 minutes at 37 degree in a water bath in the dark before the assay, according to the manufacturer’s guidelines. Proliferation analysis and proliferation index comparison was done using FlowJo software. T-cell Signaling: For pZap70 and Talin-1 detection, 2.5×105 T-cells, all HS T-cells vs the NT control NT, were fixed and permeabilized using (4% PFA and 0.02% tween-20) then stained with anti-human pZap70-FITC and mouse monoclonal Talin-1 followed by anti-rabbit Alexa fluor 488. For LFA-1 open confirmation surface staining, 2.5×105 T-cells were collected before and after transmigration and stained for LFA-1 specific extended confirmation using biotin labeled KIM127 (recognizes the extended integrin β-chain) followed by a secondary streptavidin-FITC Ab. More than 100,000 events were acquired using Accuri C6 (Becton Dickinson, Franklin Lakes, NJ). FlowJo data analysis software (FLOWJO, LLC, Ashland) was used for all flow cytometric analyses. Confocal Microscopy. SLP-76: 50,000 GFP-tagged HS-cells and ΔHS-cells were collected; then seeded for 2 hours over a glass chamber slide (Thermo Fisher Scientific) pre-coated overnight with 1 μg ALCAM then fixed and permeabilized with Fixperm solution quenched with ammonium chloride, then incubated in blocking solution (PBS containing 0.01% Triton X-100 with 1% BSA). Finally, cells were immunolabeled with anti-phospho-SLP-76(pTyr128) followed by anti-mouse Alexa 647. Clustering images of SLP76 at the HS/ALCAM interface were captured using a Zeiss Axio-Observer Z1 confocal microscope equipped with a Yokogawa CSU10 spinning disc, a Zeiss 63x/1.43 NA objective, and a Hamamatsu Orca-AG camera. Single 0.1μm z-slices with SLP-76 puncta at the eGFP.G-expressing T-cell surface were detected and compared to ΔHS-cells. Talin-1/LFA-1/ICAM1 colocalization experiments: The endothelium/HS-T-cells interface was imaged using confocal microscopy. HS-cells and NT T-cells were seeded over HBMEC monolayer, pre-stimulated with TGFβ (0.1 μg/ml) for 4 hours to ensure ALCAM expression, and incubated for 2 hours at 37°C to allow conjugate interactions. Conjugates between HS-cells and HBMEC were then fixed and permeabilized with 4% paraformaldehyde and 0.02 % saponin (Tween), blocked with PBS containing 0.01% Triton X-100 with 1% BSA and stained for LFA-1 open conformation, ICAM1, and Talin-1. Primary antibodies were subsequently detected by anti-mouse AF 647, AF 488 secondary antibodies and anti-rabbit Alexa fluor pacific blue respectively. An extra blocking step was performed between the two anti-mouse human antibodies staining to eliminate background staining. HS-cells/HBMEC conjugates were imaged as 0.2 μm Z-steps to cover the entire volume of the podosynapse, determined individually for each conjugate using a Zeiss Axio-Observer Z1 confocal microscope equipped with a Yokogawa CSU10 spinning disc, a Zeiss 63x/1.43 NA objective, and a Hamamatsu Orca-AG camera. Images were analyzed with Volocity software (PerkinElmer, Waltham, MA). Cluster density of LFA-1, Talin-1, ICAM1 and Actin at the interface were calculated using the formula (volume×MFI) for an equal number of 1×1×1-μm voxels selected to cover the interface. Elisa of TGFβ and IL-6 in GBM supernatant. IL-6 and TGFβ were quantified using Elisa kits (ab46027) and (ab100647) were performed according to manufacture instruction (on 3 different samples of GBM supernatant collected 48 hours after culture in DMEM without serum. Cytotoxicity assay. Cytotoxicity assays were performed as previously described (20). Briefly, to test safety against ALCAM positive endotheium (U87-GBM and HBMEC) were used as targets and to test fratricide effect: (THP1, autologous NT T-cells, autologous PBMCs) were used. In all experiments: 1×106 targeT-cells labeled with 0.1 mCi (3.7MBq) 51Cr at effector to target (E:T) ratios of 40:1, 20:1, 10:1 and 5:1. T-cells incubated in complete medium alone or in 1% Triton X-100 were used to determine spontaneous and maximum 51Cr release, respectively. After 4 hours, cells were centrifuged; supernatants collected and radioactivity measured in a gamma counter (Cobra Quantum; PerkinElmer; Wellesley; MA). The mean percentage of specific lysis of triplicate wells was calculated according to the following formula: [test release − spontaneous release] / [maximal release − spontaneous release] × 100. Orthotopic GBM and MB xenogeneic SCID mouse model. Tumor Establishment: All animal experiments were conducted on a protocol approved by the Baylor College of Medicine Institutional Animal Care and Use Committee and all experiments complied with all relevant ethical regulations. Recipient ICR-SCID mice (C.B-Igh-1b/IcrTac-Prkdcscid) were purchased from Taconic (Hudson, NY). Male and female 9- to 11-week-old mice were anesthetized with isofluorane (Abbot Laboratories, England) followed by an intraperitoneal injection of 225–240 mg/kg xylazine solution and then maintained on isoflurane by inhalation throughout the procedure. After removing hair from the head region, the mice were immobilized in a Cunningham™ Mouse/Neonatal Rat Adaptor (Stoelting, Wood Dale, IL) stereotactic apparatus fitted into an E15600 Lab Standard Stereotaxic Instrument (Stoelting, Wood Dale, IL), and surgery area scrubbed with 1% povidone-iodine. A 10 mm skin incision was made along the midline. The tip of a 31G ½ inch needle mounted on a Hamilton syringe (Hamilton, Reno, NV) served as the reference point. A 1mm burr-hole was drilled into the skull. [34, 44] HS T-cell homing experiments: For the homing experiment, 5×104 unlabeled U87-GBM cells or DAOY-MB in 2.5 μL were injected orthotopically over 5 minutes. After 10 days of engraftment (based on previous experience xenograft will be vascularized [52, 53], 107 T-cells tagged with a GFP Firefly Luciferin fusion gene (eGFP.FFLuc) were injected intravenously through the tail vein. Groups of 15 mice received HS T- cells and 10 mice control NT. Homing T-cells to brain tumors were assessed by bioluminescence tracking in the brain of the animals using the IVIS® system (Perkin Elmer, Akron, OH) after intraperitoneal injection of 300 mg D-luciferin (Perkin Elmer). Mice under isofluorane anesthesia were imaged individually at highest sensitivity (level A) for 5 minutes each at hours 6, 24, 48 hours, and on days 3, 6 and 8 days post T-cell administration. Photon emission was quantified using the Living Image software (Perkin Elmer, Akron, OH). A pseudo-color image representing light intensity (blue: least intense, red: most intense) was generated and superimposed over the grayscale reference image. The region of signal in the brain were obtained and compared between all test animals (n=15 per group). In order to assess specific homing and T-cell infiltration to GBM xenografts, in a separate experiment, mice (n=5) were euthanized after 24 hours and the frontal right lobe containing the tumor were minced and tumor infiltrating lymphocytes (TIL) were enriched using Percoll gradient. Then cells were stained using CD45-PerCp, CHS-APC and mouse anti-human HS mAb followed by anti-mouse AF 647 and analyzed using flow-cytometry gating concentrically on CD45, CHS and HS positivity. The percentage of TILs were compared between various HS T-cell groups and NT. And to evaluate specificity of tumor area homing, random infiltration was evaluated after mincing the left lobe distal from the tumor explants, TILs were isolated and assessed following the same method described earlier. CAR HS T-cell efficacy experiments: 5×104 eGFP.FFLuc-labelled U87-GBM cells in 2.5 μL were injected orthotopically 5 minutes. 3 groups of mice were randomized to receive HER2-CAR HS T-cells (10 mice/group), HER2-CAR T-cells (10 mice/group), NT T-cells (5 mice/group) and tumor only group (3 mice/group) at day 6 and 11 days of engraftment through the tail vein. To evaluate the antitumor activity of T-cells, tumor sizes were monitored by BLI. Mice were imaged twice weekly for 1 minute using the IVIS® system (IVIS, Xenogen Corp., Alameda, CA) under isofluorane euthanasia and injecting IP 100μg D-luciferin (Xenogen, Alameda, CA). Images were acquired and quantified as described earlier. In order to assess the homing of HER2-CAR HS-cells, 3 mice of HER2-CAR HS T-cells, HER2-CAR T-cells only in a separate experiment were euthanized and lobes containing the tumor area were minced and TILs were assessed as described above. Mice were regularly examined for neurological deficits, weight loss, signs of stress, and a BLI signal >108, and euthanized according to pre-set criteria by the Baylor College of Medicine’s Center for Comparative Medicine’s guidelines. In none of the experiments were these criteria not fulfilled. Safety evaluation: The brain, heart, liver, spleen, lung, stomach, intestine, testicles and kidney were promptly collected after the mice of the homing experiments (n=5) were sacrificed and fixed in a 10% formalin solution. Then, the organs were embedded in paraffin, sectioned, and processed for H&E staining and pathologically assessed for histological abnormality or toxicity by a neuropathologist. IHC was carried out on 3 μm brain tumor tissues of different groups (n=3 per group) and probed using rabbit anti-human CD3 (Abcam). Statistical Analysis. Data were summarized using descriptive statistics. Comparisons between groups were carried out using one-way ANOVA or t-test. P-values were adjusted for multiple comparisons using the Tukey’s test and Dunnet’s test when appropriate. The Kaplan Meier method was used to estimate survival curves and the Log-Rank test was used to compare the curves. GraphPad Prism 7 software (La Jolla, CA) was used for statistical analysis. The sample-size for the animal experiments was calculated on the basis of the primary hypothesis and models derived from pilot studies. Animals were randomized between group and the operator was blinded to the agents tested. A P-value of less than 0.05 was considered significant Extended Data ED-Figure 1 │ Analysis of CAM expression in primary brain tumors. (A) High-throughput IFC analysis routine of the endothelial adhesion molecules; ICAM1, VCAM1, and ALCAM in primary 93 GBM, 25 MB and 5 normal brain. MATLAB segmentation and masking analysis algorithm of the co-immunofluorescence (co-IFC) of ICAM1 or VCAM (acquired on 647 channel), CD31 (acquired on 594 channel) and ALCAM (acquired on 488 channel), DAPI (acquired on blue/cyan channel). (B) Isolation and characterization of pTEC. Flow-cytometry sorting gating strategy of pTEC from freshly excised glioblastoma (GBM; n=5) based on CD31 positivity. GBM EC isolated also expressed the endothelial markers VE-cadherin, von Willebrand Factor (vWF) and ALCAM. Isotype shown in lighter grey and test shown in darker grey in individual histograms. n=5 surgical samples each interrogated at least twice. 100,000 or more events were acquired per condition. ED-Figure 2 │ ALCAM expression in a panel of human and murine endothelial-cells and their reactivity to inflammatory and cancerous conditioning. (A) Western Blot for ALCAM in a panel of human and murine EC lines: pTEC (primary Tumor EC from GBM surgical excision), HBMEC (Human Brain Microvascular Endothelial-cells), 1ry BMEC (Primary Brain Microvascular Endothelial-cells), 1ry PVEC (Primary Pulmonary Vein Endothelial-cells), HUVEC (Human Umbilical Cord Vascular EC), HMVEC-L (Human Microvascular EC of the Lung), bEnd.3 (murine brain tumor EC) and 2-H11 (murine SV40-transformed axillary lymph node vascular endothelium). The left panel shows basal ALCAM expression except in tumor EC (pTEC and 2-H11). Right panel shows the induction of ALCAM in all endothelial-cells after incubation with TNFα for 6 hours. (B) Expression of ALCAM at baseline and after 6 hours of conditioning in GBM-supe, TGFβ or IL6. Only tumorous-EC expressed ALCAM at baseline while normal-EC did not. C) IFC for ALCAM in 5×104 pTECs and HBMEC in the in vitro BBB-model at baseline and after culture in GBM-supe. Scale-bar=50μm. (D) Differential expression of key adhesion molecules at baseline and under the influence of cancer and inflammation in pTEC and HBMEC. Flow-cytometry dot plots detailing of baseline expression of ALCAM, VCAM1 and ICAM1 on 1×104 pTEC and HBMEC and conditioned expression after culture in GBM-supe, TGFβ or IL6. (E-H) Expression of adhesion molecules at baseline and under the influence of cancer and inflammation in pTEC (n=4) acquired from surgical resection samples (pTEC #1 is shown in Figure 1G). ED-Figure 3 │ In silico design of the prototype and derivative Homing System (HS) molecules, their forced-expression and detection on T-cells, and studies of their in vitro dynamic interaction with EC under shear-stress. (A) The potential interaction between ALCAM V1 (grey ribbon) and CD6 from computational docking. D1 of CD6 is colored blue, D2 is colored green and D3 is colored orange. (B) Details of the potential interaction interface between ALCAM V1 (grey ribbon) and CD6 D3 (orange ribbon) is shown. A rendering of the electrostatic surface of the ALCAM V1 (grey ribbon) with the D3 domain of CD6 (orange ribbon) in the same orientation. Potential interacting residues are highlighted in the models and (C) in a diagram generated from PDBe PISA and PDBSum. A small region of positively-charged residues in ALCAM V1 appears to interact with a negatively-charged patch of residues on CD6 D3. (D) Structure of the prototype HS-molecule. (E) HS-multimers 3HS and 5HS. (F) HS molecules with non-signaling endodomains, HSΔ, 3HSΔ and 5HSΔ. (G) Cartoon depicting the strategy used for surface detection of the HS-exodomain using a D3-specific antibody and specific binding of HS-exodomain to soluble ALCAM. (H) Flow-cytometry confirming HS surface expression (D3 mAB) on T-cells. (I) Cartoon depicting the design of the HS/ALCAM PLA experiment and (J) the digital rendition using ImageTool®. The ALCAM probe (–) binds to the D3 probe (+) to trigger the polymerase chain (PCR) reaction generating the red fluorescent signal that is quantified as total signal per region (TSR) in Fig. 2F. (K and L) Dynamic microfluidic studies showing (K) still image from the Supplementary Video 1 of Bioflux® channels with non-transduced control (NT) T-cells (top) vs. 1×106 HS T-cells interrogated under shear force over an ALCAM-expressing endothelium and (L) still image from MJtracker® demonstrating various T-cells under interrogation for various TEM dynamic measures, the standard grid used and the equation used for calculations (bottom). (M) Dynamic adhesion of T-cells to EC per field of view and (N) average dynamic rolling velocity against time; p<0.05, p<0.01, p <0.001. Two way ANOVA then Tukey for multiple-comparisons (compared to NT). ED-Figure 4 │ Functional effects of elimination of ALCAM on endothelial-cells and knock out of the human ALCAM gene using CRISPR-Cas9 technology and its effect on T-cell BBB migration. (A) Flow-cytometry of ALCAM expression on 1×106 wild-type pTEC at base-line, after TGFβ induction of ALCAM then after being transfected with 25nM ALCAM siRNA for 48 hours to knockdown (KD) ALCAM. Transmigration assay using pTECs to simulate a cancerous BBB showing percentage of migrant T-cells vs ALCAM-KD is shown in Figure 2K. (B) Highest 3 scoring guide RNA designs (sgRNA 44, sgRNA45 and sgRNA49) as seen on the SnapGene® software intended to disrupt ALCAM exons for the extracellular and transmembrane moiety. (C) CD5-KO and (D) CD19-KO were used as positive and negative experimental controls, respectively. (E) Flow-cytometry of ALCAM expression on wild-type human umbilical vein endothelial-cells (HUVEC) and HUVEC-ALCAM-KO using CRISPR/Cas9 (using the guide sgRNA 45) assessed at baseline and after TGFβ incubation. Isotype was used as control. (F) Transmigration assay showing percentage of 2×106 migrating T-cells on wild-type HUVEC before and after ALCAM induction compared to ALCAM-KO HUVEC. Both experiments were done at base line then after ALCAM induction was confirmed. Error bars are Mean±SD (n≥3 experiments; donor T-cells n=3), p<0.01, **p <0.001. Tukey Test (compared to wild type pTEC). (G) Real-time polymerase chain reaction (RT-PCR) analysis of representative of 1×106 ALCAM-KO HS T-cells in comparison to wild type normal T-cells. GAPDH is used as an internal control. (H) Flow-cytometry showing >90% knockout efficiency of the 3 sgRNA on 1×105 T-cells in comparison to wild-type normal T-cells; CRISPR-Cas9 only and isotype were used as experimental controls. (I) Sorted ALCAM negative KO T-cells were then successfully transduced with the 6 HS constructs. (J) Transmigration assay showing percentage of 2×105 migrating T-cells on a cancerous blood brain barrier (cBBB) model to compare wild type vs. ALCAM-KO T-cells in 4 conditions (ALCAM-, ALCAM+ conditioned with TGFβ, after blocking ALCAM, after washing the blocking away). Error bars in panels F and J are mean ±SD (n≥3 experiments; donors n=3), p<0.05, p <0.01. Tukey’s Test (compared to ALCAM+ T-cells). ED-Figure 5 │ Flow-cytometry quantification of nodes downstream of CD6 signaling endodomains and high-throughput analysis of super-resolution imaging using deconvolution microscopy (DM). Quantification of the flow cytometric data for (A) LFA-1 open configuration, (B) pZap70 and (C) Talin before (solid bars) and after (dotted bars) TWM of 1×105 T-cells. * p<0.001. (D) Characterization of migrant T cells cellular features using collective quantification of Actin MFI, focal adhesions at HS/ALCAM interface, area of spreading, and podosynapse formation by high throughput microscopy in 3 donors. n=200–800 cells. (E) Box plot summary representing single cell data distributions of all replicates between all three donors expressing HS vs. NT controls. ED-Figure 6 │ Assessment of TILs in GBM explants. (A) Flow-cytometry of 1×104 TILs; all HS T-cell designs vs. NT control gated on CD3+CD45+ then D3+ fractions in GBM explants 24 hours after iv infusion. Representative plots shown. n=5 animals per group. (B) Cranial window on a live mouse bearing U87-GBM tumor (black arrow in inset). ED-Figure 7 │ Analysis of T-cell infiltrates in vital organs and normal brain after infusion of HS T-cells. (A) CD3 immunohistochemistry (IHC) staining of normal vital tissues from animals receiving HS T-cells vs. NT control. n=3 mice/group. Scale bar=40μm (B) IHC showing HS T-cell infiltrate in micro-dissected GBM xenograft. Scoring of CD3 positive DAB signal was analyzed using IHC-Profiler® plugin in ImageJ®. Respective image analysis output and the score assigned using IHC-Profiler is also shown for each image. Total percentage of CD3+ DAB signal was more 66% in all mice brain with HS T-cells (score from 3 to 4) while percentage in control mice were less than 20% (score were 0–1). Scale bar=50μm. n=3 mice/group ED-Figure 8 │ Characterization of therapeutic T-cells after transmigration through an in vitro BBB-model. (A) Flow-cytometry assessing the HER2-CAR- and the HS-molecule expression in HS HER2 CAR T-cells. (B-D) 1×105 T-cells were collected from the bottom chamber after transmigration on ALCAM-expressing endothelium and analyzed for (B) CD45RO and CCR7 to assess their centrality, (C) expression of the exhaustion markers, PD-1 (black), TIM-3 (red) and LAG3 (orange); before transmigration is shown in grey, and (D) for their proliferative capacity before (red) and after (blue) transmigration, using efLuor 670. ED-Figure 9 │ Analysis of TIL isolated from tumor xenografts and normal brain for HER2-CAR HS T-cells. (A) Flow-cytometry of TIL isolated from orthotopic tumor xenografts 24 hours after intravenous-injection of HS T-cell products, HER2-CAR T-cells and NT control T-cells. Xenografts were micro-dissected and TIL were isolated and enriched on percoll/ficoll gradient. Cells were gated on D3+ subset inside a gate of D3+CD45+. A subset of HER2-CAR inside a gate of CD3+CD45+ 3+ to detect the HER2-CAR HS T-cell specifically. n=5 mice/group, representative data shown. (B) Flow-cytometry following the same gating strategy indicating the absence of HS T-cells in the contralateral lobe to the tumor xenograft; data representative of 3 mice. ED-Figure 10 │ Overexpression of whole-length native CD6 and its phenotypic and functional effects on T-cells. (A) Cartoon depicting the cloning strategy of native CD6 in an SFG retroviral backbone. (B) Flow-cytometry showing the transduction of 1×105 native CD6 relative to HS constructs on T-cells. (C) Flow-cytometry of the activation marker CD69 on day 8 transduction without additional stimulation, and (D) of the activation and exhaustion marker PD-1 stained with PD-1 PerCP on day 8 transduction at basal level without additional stimulation. (E) Expansion plot T-cells expressing the native CD6 relative to NT and various HS T-cells; cells were grown in Il-7/15 and collected at day 2 and day 12 post transduction. (F) Transmigration 2×105 T-cells through a cancerous BBB-model showing the percentage of migrant T-cells expressing native CD6 relative to various HS T-cells and the response of blocking ALCAM and its restitution. Error bars are mean ±SD (n≥3 experiments; donor T-cells n=3) *p <0.001 compared to migration of CD6 through ALCAM+ BBB. ANOVA with Tukey’s post-hoc analysis. Supplementary Material 1 2 Sup Vid 1 Sup Vid 2 Sup Vid 3 Sup Vid 4 Sup Vid 5 Acknowledgments: We thank Malcolm Brenner and Catherine Gillespie for the scientific-advice and linguistic-editing, respectively; Sylvie Roberge and Mark Duquette for their technical assistance. The D3-antibody was a generous gift from Professor Marion Brown, Oxford University, UK. This work was funded by SU2C-St./Baldrick’s Pediatric Dream Team Translational Research Grant (SU2C-AACR-DT1113; NA/PS/MDT). SU2C is a program of the Entertainment Industry Foundation administered by American Association for Cancer Research. Also funded by Alex’s Lemonade Stand, NIH-T32HL092332 (KF/TTB; Dr. Helen Heslop), R01AI067946 (JSO), T32GM08812, DK56338, CA125123, and 1S10OD020151–01, CPRIT (RP150578), the Dan L. Duncan CCC, P01-CA080124 (RKJ/DF); R35-CA197743, P50-CA165962 (RKJ). This content does not necessarily represent the official views of the funding agencies. Competing Interest Declaration: None identified that pertains to this work. Data Deposition and Availability: All relevant data are included in the manuscript linked as source data; more details are available from the corresponding author: Nabil Ahmed, MD; email: [email protected] on reasonable request. Figure 1 │ Adhesion-molecule expression and permeability of cancerous endothelium. (A) Representative confocal co-immunofluorescence (IFC) of ALCAM and CD31 in 93 GBM and 25 MB, performed twice with similar results. Nuclei DAPI-counterstained. Bar=100μm. (B) Pearson correlation of CD31:ALCAM pixel-mean fluorescence intensity (MFI). (C) Topographic co-localization of CD31:ALCAM over vascular segments (15 high-power fields [hpf] per tumor averaged; representative from n=3 with similar results). VTR, validation tandem-repeat. (D) ALCAM expression in human GBM pTEC (representative of n=5) and murine brain tumor endothelium (bEND.3) at baseline and after conditioning. (E) Cartoon depicting the BBB-model. HBVP, Human Brain Vascular Pericytes. (F) Transmigration of T-cells through BBB-model. Data represented as Mean±SD; Student’s t-test and One-way ANOVA with Tukey’s correction. * P<0.001; ns, not significant. All experiments done using human T-cells; validated for 3 donors in ≥3 independent experiments. (G) CAM expression in pTEC#1 (n=5 pTECs) and (H) HBMEC at baseline and after conditioning. (I) High-throughput CAM quantification in 5 normal brains, 93 GBM, and 25 MB, each examined twice. Each data-point is an average of MFI acquired from 15 confocal CD31(+)-gated vascular-patterned hpf and segmented by channel-specific intensity thresholding per tumor. Data represented in G-I as Mean±SD; ANOVA with Tukey’s correction; P<0.01, P<0.001. Figure 2 │ Rational-engineering of the Homing System (HS). (A) Cartoon outlining the ALCAM binding-region on CD6, (B) prototype HS-molecule, (C) multimerized exodomains and (D) tailless HSΔ-molecules lacking signaling-domains. (E) Specific binding to soluble ALCAM; assessed independently 10x with similar results. (F) PLA identifying D3/ALCAM heterodimers in T-cell/endothelium-conjugates. 83–103 cell-conjugates analyzed per condition; repeated 3x independently with similar results. TSR=Total Signals-per-Region. (G-J) TEM kinetics of 1×106 cells/condition in microfluidic channels under 1–3dyne/cm2 shear over ALCAM+ pTEC. (K) Transmigration of 2×105 T-cells per well through pTEC cBBB and the effect of soluble ALCAM blockade and washing. Yellow bars show the effect of ALCAM-siRNA knockdown on the permissivity of EC. Three experiments independently performed in triplicates with similar results. All data represented as Mean±SD. P-values represented as P<0.05, P<0.01, P<0.001. One-way ANOVA then Dunnett’s test for multiple-comparisons (compared to NT). Figure 3 │ Signaling events downstream of HS-molecules. (A) Confocal-IFC images after 5×104 T-cells land on an ALCAM-coated glass-surface, showing micro-clusters of SLP-76 (red) and eGFP-tagged HS and HSΔ T-cells (green). Scale-bar=50μm. (B-C) Intracellular flow-cytometry for (B) pZAP70, Talin-1 and surface staining for unfolded LFA-1 using KIM127, monoclonal-antibodies that bind exclusively to the extended β2-chain (CD18), before and after transmigration of 2×105 T-cells through an ALCAM+ cBBB-model. (D) Confocal-images of the transmigratory-cup at the HS T-cell/ EC interface co-stained for Talin-1 (blue), ICAM1 (green) and unfolded LFA-1 (red). Data represented as Mean ±SD, 4 independent experiments with similar results. P=0.774. Man-Whitney test; ns, not significant. (E) Cartoon depicting the HS-signaling events culminating in unfolding of LFA-1. Figure 4 │ Cytoskeletal changes mediated by HS-signaling. (A) Representative TIRF-micrographs of HS.eGFP T-cells upon landing on an ALCAM-coated glass surface, correlated with Actin in (B), Pearson coefficient=0.8. Scale-bar=10μm. Comparison of (C) Actin- and (D) FAK- MFI among HS- and HSΔ- T-cells. (E) Representative SIM-images depicting HS T-cell membrane ruffles. (F) The MATLAB® script used to analyze DM data of podosynaptic protrusions and their spread in 2×106 HS T-cells. (G) Characterization of migrating T-cells through collective quantification of Actin-MFI, focal adhesions, area of spreading, and podosynapse formation by high-throughput DM at HS/ALCAM interface in a representative donor (n=200–800 cells/condition). All assessments independently repeated 3 times with similar results. Data represented as Mean ±SD, P<0.001. Tukey’s test used in panels C and D; Student’s t-test used in panel G. Figure 5 │ Homing of HS T-cells to brain tumors. (A) eGFP-labeled T-cells were injected intravenously in orthotopic tumor bearing mice. (B) Flow-cytometry analyzing TILs in GBM-explants (n=5 mice/group). (C) BLI of T-cells after intravenous-injection in GBM (quantified in D) and MB (E, quantified in F). Data represented as Mean±SD (n=5 mice/group) P=0.001, *P<0.0001. ANOVA with Tukey’s correction. (G) Iso-surface 3D-rendering of tumor-explant confocal-images showing eGFP.HS T-cells relative to ALCAM+ vessels (red). Cryo-sections imaged at 40x, 50μm z-stacks, bar =50μm. (H) Quantification of GFP+ T-cells in and around the ALCAM+ (red) signal indicating perivascular and intravascular locations, respectively. Error bars mean±SD (n=4 explants) p=0.0015, **p <0.0001. 2-tailed t-test. Dynamics of T-cell homing: (I) Snapshot image taken at 15 seconds of Supplemental Video-M2 showing rolling (arrow), adherent (dashed-arrow) 5HS T-cells inside the blood vessels, and a 5HS T-cell extravasating (arrow-head) into a U87-GBM tumor. Green, T-cells; Red, TAMRA-dextran (blood vessels). Scale-bar 100μm. (J and K) Quantification of rolling or adherent T-cells in U87-GBM vasculature. n=3 mice/group. Data represent mean ±SEM. p< 0.05, 2-tailed t-test. P= 0.0219 and 0.0033. (L) Time-lapse 3D-reconstructed images showing extravasation of T-cells. Green, T-cells; Red, TAMRA-dextran (blood vessels). Scale bar 100μm Figure 6 │ Anti-tumor activity of cytotoxic HS T-cells. (A) Cartoon depicting experiment. (B) 51Cr-cytotoxicity assessing the cytolytic activity of HS T-cells at indicated E:T ratio against 5×103 targets; (B) HER2+ U87-GBM, (C) human- and murine-ECs, and (D) ALCAM-expressing leukocytes. THP-1, human monocytic cells. PMBC, peripheral blood mononuclear cells. 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==== Front Cell Cycle Cell Cycle KCCY kccy20 Cell Cycle 1538-4101 1551-4005 Taylor & Francis 30810438 1580494 10.1080/15384101.2019.1580494 Research Paper Overexpression of microRNA-202-3p protects against myocardial ischemia-reperfusion injury through activation of TGF-β1/Smads signaling pathway by targeting TRPM6 H.-Y. WU ET AL. CELL CYCLE Wu Hui-Ying a Wu Jian-Li b Ni Zhan-Ling a a Department of Cardiovascular Medicine, Fuwai Central China Cardiovascular Hospital, Zhengzhou, P.R. China b Medical School, Huanghe S & T University, Zhengzhou, P.R. China CONTACT Hui-Ying Wu [email protected] 2019 27 2 2019 18 5 621637 19 7 2018 27 1 2019 31 1 2019 © 2019 Informa UK Limited, trading as Taylor & Francis Group 2019 Informa UK Limited, trading as Taylor & Francis Group ABSTRACT MicroRNAs (miRNAs) have been found to act as key regulators in the pathogenesis of myocardial ischemic-reperfusion (I/R) injury. In this study, we explore the role and mechanism of microRNA-202-3p (miR-202-3p) in regulating cardiomyocyte apoptosis, in respective of the TGF-β1/Smads signaling pathway by targeting the transient receptor potential cation channel, subfamily M, member 6 (TRPM6). The targeting relationship between miR-202-3p and TRPM6 was verified by a dual-luciferase reporter gene assay. Sprague-Dawley rat models of myocardial I/R injury were initially established and treated with different mimics, inhibitors and siRNAs to test the effects of miR-202-3p and TRPM6 on myocardial I/R injury. The levels of inflammatory factors; IL-1β, IL-6, TNF-α as well as the degree of myocardial fibrosis and cardiomyocyte apoptosis were determined in rats transfected with different plasmids. TRPM6 was found to be the target of miR-202-3p. Up-regulated miR-202-3p or knockdown of TRPM-6 alleviated oxidative stress and inflammatory response, reduced ventricular mass, altered cardiac hemodynamics, suppressed myocardial infarction, attenuated cell apoptosis, and inhibited myocardial fibrosis. MiR-202-3p overexpression activates the TGF-β1/Smads signaling pathway by negatively regulating TRPM6 expression. Taken together, these findings suggest that miR-202-3p offers protection against ventricular remodeling after myocardial I/R injury via activation of the TGF-β1/Smads signaling pathway. KEYWORDS MicroRNA-202-3p TRPM6 TGF-β1/Smads signaling pathway myocardial ischemia/reperfusion injury ventricular remodeling None ==== Body Introduction Ischemia-reperfusion (I/R) injury usually arises in patients who present with an acute ST-segment elevation myocardial infarction. The most effective therapeutic treatment for reducing acute I/R injury and mitigating the myocardial infarction (MI) size is effective and timely myocardial reperfusion [1]. However, uncontrolled reperfusion may lead to additional damage, which may contribute to myocardial dysfunction and even death [2]. Current knowledge generally supports that cellular processes such as cell apoptosis, free radical injury and inflammation play a vital role in myocardial I/R injury [3]. I/R injury can produce impaired cardiac function as a consequence of ventricular remodeling [4]. Recently, microRNAs (miRNAs) have been reported to participate in the regulating cellular and molecular mechanisms such as cardiac injury and dysfunction induced by I/R injury, as well as ventricular remodeling after I/R injury [5]. miRNAs are involved in many cardiac pathological events such as cardiac remodeling, arrhythmias, hypertrophy, and heart failure, and have been reported to play a role in the pathogenesis towards I/R injury by altering key signaling elements, thereby making them potential therapeutic targets [6–8]. Dysregulation of specific miRNAs in both mice and humans were contributors to myocardial infarction [9]. Increasing evidences using animal models have demonstrated that cardiac-specific miRNAs including miR-1, miR-21, miR-133a, and miR-320 are involved in cardiovascular diseases, myocardial cell apoptosis, heart development and including MI [10–12]. The role of miR-22 has been recently found to effectively attenuate I/R injury by regulating related gene expression to inhibit apoptosis [12]. The loss of ventricular remodeling during re-perfused ST-segment-elevation myocardial infarction is of critical importance in reversing I/R injury which provides a significant therapeutic target for dysregulating ventricular remodeling [13]. Transient receptor potential (TRP) channels are actively expressed in every type of cardiac tissue, including vascular smooth muscle cells, endothelial cells, cardiomyocytes and fibroblasts [14]. The transient receptor potential cation channel, subfamily M, member 6 (TRPM6) is a member of the TRP channel gene family [15]. It has been reported that TRPM6 gene is highly expressed in the atrial tissues of patients with atrial fibrosis [16]. TRPM6 also been found to play an important role in the regulation of extracellular divalent cations in cardiomyocytes [17]. In a former study, TRPM7, also a subordination of TPR melastatin subfamily, was found to be up-regulated in cardiac fibroblasts of human auricular fibrillation (AF) and plays a pivotal role in activating the fibrogenic effect of TGF-β1. This suggests that TRPM7 may serve as a therapeutic target for treating patients suffering from cardiac fibrosis [18]. Transforming growth factor (TGF) and Smad3 proteins are found to be linked with fibrosis via activation of transforming growth factor-beta (TGF-β) cellular activities including differentiation, inflammation, apoptosis, and proliferation [19]. TGF-β1, normally used as myocardial fibrosis marker, was reported to be closely linked with the TRP family that regulates the cardiovascular system [16]. It has been reported that TGF-β1 may play a critical role in cardiovascular diseases by a process which allows the loss of its protective properties [20]. For instance, TGF-β/Smad pathway was implicated in the modulation of hyperglycemia-induced cardiac remodeling [21]. Therefore, we aimed to investigate the role and mechanism of miR-202–3p in ventricular remodeling after I/R injury by regulating the expression of TRPM6 and activating the TGF-β1/Smads signaling pathway. Materials and methods Ethics statement The study was approved by the institutional ethics committee at Fuwai Central China Cardiovascular Hospital. All painful procedures performed in animal subjects were performed with anesthesia. Best efforts were made to minimize suffering in animal subjects. Animal treatment A total of 105 specific pathogen-free (SPF) male Sprague-Dawley (SD) rats weighing 200 ± 20 g were purchased from the Laboratory Animal Center of Third Military Medical University (Chongqing, China) and housed in quiet and clean cages. The cage environment was set at 23 ± 1°C with a humidity of 55–70%, and illuminated by a fluorescent lamp with natural light, alternating between 12 h of light, and 12 h without light. Rats were adaptively raised with free access to water and food for one week. All rats fasted for 12 h before the experiment. Model establishment and control group set up SD rats were randomly assigned into the myocardial I/R injury (90 rats) and control groups (15 rats). The rats in the myocardial I/R injury group were anesthetized with an intraperitoneal injection of 1000 mg/kg 0.5% urethane, followed by the removal of hair from the neck. Rats were then intubated and connected to a ventilator followed by a tracheotomy procedure. The procedure was performed on rats in the supine position (with a respiratory frequency of 60 times/minute, respiration rate of 1.5: 1 and tidal volume of 5 mL/100 g). A micro needle electrode connected to an electrocardiograph was then inserted into the epidermal layer of the limbs of the rats to record the electrocardiogram (ECG). Ventricular remodeling in rat models with myocardial I/R injury was established after 30 min of ischemia and 120 min of reperfusion [22]. Rats in the control group were subjected to a thoracotomy and received no ligation of the left anterior descending artery (LAD). All rats were treated with an intramuscular injection of 10000 U/kg penicillin every day, lasting for 7 days. Plasmid construction and grouping TRPM6 siRNA was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). MiR-202-3p mimic, miR-202-3p inhibitor and negative control (NC) were purchased from Shanghai Rainbow Chemistry Co., Ltd. (Shanghai, China). Adenovirus type 5 was used as the vector for all of the recombinant adenovirus (Ad) mentioned above. The sequences of recombination of siRNA-TRPM6, miR-202-3p mimic, miR-202-3p inhibitor (an anti-miR of micR-202-3p), miR-202-3p inhibitor + siRNA-TRPM6 and NC were used along with the Ad vector. During I/R injury model establishment, Ad was perfused with 5 × 109 pfu/gm by 0.5 mL/min at the reperfusion phase for 35–45 min using a micro-pump with a perfusion pressure of 5.33 kPa. The SD rats were evenly grouped into the sham, I/R injury (without any sequence), NC (SD rats transfected with NC), siRNA-TRPM6 (transfected with TRPM6 siRNA Ad), miR-202-3p mimic (SD rats transfected with miR-202-3p mimic Ad), miR-202-3p inhibitor (SD rats transfected with miR-202-3p inhibitor Ad), and miR-202-3p inhibitor + siRNA-TRPM6 (SD rats transfected with miR-202-3p inhibitor + siRNA-TRPM6 Ad) groups, with 15 rats in each group. Determination of peroxidase and antioxidant enzyme activities After 4 weeks, the rats in each group were anesthetized by intraperitoneal injection of 0.5% urethane at 1000 mg/kg. 5 mL of blood was collected from the abdominal aorta and centrifuged at 4°C at 1610 × g for 10 min. The serum was then collected and frozen for preservation and later use. The rat serum sample was collected for determinating the serum contents of superoxide dismutase (SOD), catalase (CAT), malondialdehyde (MDA) and glutathione peroxidase (GSH-Px) in accordance with the instructions provided by the CAT, SOD, GSH-Px and MDA kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, Jiangsu, China) instructions. Addition of sample: the blank (added with 100 μL of sample dilution), standard (added with 100 μL of standard products) and sample (added with sample to be tested) wells were set up, respectively, and degassed. The samples were added to the bottom of the microplate wells and gently shaken before incubating at 37°C for 2 h. After aspirating and removing the solution, the plate was dried and mixed with 100 μL of biotin-labelled antibody at 37°C for 60 min. After the removal of the solution, the plate was dried, washed 3 times (1–2 min for each) and reacted with 100 μL of horseradish peroxidase (HRP)-labelled avidin at 37°C for 60 min. The solution was removed and the plate was dried, washed 5 times (1–2 min for each) and incubated with 90 μL of substrate solution at 37°C, in the dark. Subsequently, 50 μL of stop solution was added orderly to terminate the reaction. Then, the activities were determined within 15 min. All the experiments were repeated three times. Enzyme-linked immunosorbent assay (ELISA) ELISA kits were used to determine the levels of inflammatory factors in serum samples. The procedures were carried out according to the following protocol. A microplate was obtained followed by the addition of the standard sample and specimen dilutions. In total, 100 μL of each sample were added to each well or standard sample with varying concentrations. The reaction wells were sealed with adhesive tape, and incubated at 36°C for 90 min, and washed afterwards Biotinylated antibody dilution was prepared 20 min prior to the experiment. In total, 100 μL of biotinylated antibody dilution was then added into the blank well, and the standard and sample wells were added with biotinylated antibody solution. The reaction wells were sealed with a new sealing adhesive tape and incubated at 36°C for 60 min. An enzyme conjugate solution was prepared and allowed to stand at room temperature (22°C–25°C) in the dark. The plate was washed for 5 times, and blank wells were added with enzyme conjugate dilution. The standard and sample wells were added with 100 μL of enzyme binding solution. Subsequently, the reaction wells were sealed with a new sealing tape and incubated at 36°C for 30 min in the dark. The levels of interleukin 1 beta (IL-1β), interleukin 6 (IL-6) and tumor necrosis factor-α (TNF-α) were determined according to instructions provided by the ELISA kit: IL-1β (E0900197), IL-6 (E0900198), TNF-α ELISA (E0900199). The above-mentioned ELISA kits were purchased from R&D Systems (Minneapolis, MN, USA). All the experiments were repeated three times. Measurement of ventricular weight After drawing blood, the hearts were extracted from 5 rats using scalpel and scissors, followed by the removal of atrial tissue, fat and great vessels. The blood in the ventricles of the heart were washed with normal saline and left to dried. The left and right ventricles were cut off along the interventricular septum, and the interventricular septum was cut into the left ventricle. Next, the left ventricular absolute weight (LVAW) and right ventricular absolute weight (RVAW) were measured using an electronic balance (Shenyang Longteng Co., Ltd., Shenyang, Liaoning, China). The left ventricular relative weight (LVRW) and right ventricular relative weight (RVRW) were calculated according to the formula: LVRW = LVAW/BW, RVRW = RVAW/BW (BW referred to the weight of rats). Measurement of ventricular hemodynamic parameters Five rats were randomly selected from each group. A cannula was then inserted into the left ventricles via the right common carotid artery to establish a connection with the pressure transducer. The RM-6000 type eight channel physiological recorder (Nihon Kohden Co., Ltd., Shanghai, China) was used to record the left ventricular systolic pressure (LVSP), left ventricular end diastolic pressure (LVEDP), systolic arterial pressure (SBP) and diastolic blood pressure (DBP). Measurement of myocardial infarct size (MIS) After arterial blood sample collection, 5 rats were randomly selected from each group to have their LADs ligated. Then, 2–3 mL of 1% Evans blue was injected into the rats along the internal jugular vein. When a blue color appeared on the rat mouth and limb skin, the heart was quickly extracted and the residual blood inside the ventricle was cleaned up followed by removal of the left and right atrium and the right ventricle. The left ventricle was stored in a plastic wrap in a refrigerator at −20°C. The left ventricle was vertically cut into 4–5 pieces (with a thickness of approximately 1 mm). The blue part of a slice represents the non-ischemic area while the rest of the heart is designated as the area at risk (AAR). The slices were immersed in 1% tripheny tetrazolium chloride (QiyiBio Biological Technology Co., Ltd., Shanghai, China) phosphate buffer for 10–15 min in dark after being photographed. The gray area represents the infarction zone (IZ) of the myocardium, and the slices of the myocardium were fixed in 10% neutral formalin overnight, and re-photographed. Adobe Photoshop software was applied to calculate the percentage of the AAR and the IZ in the total cross-sectional area: MIS = the percentage of IZ/the percentage of AAR. Masson staining The left ventricle was obtained from the remaining 5 rats, fixed with 4% formaldehyde and embedded in paraffin in order to prepare slices with a thickness of approximately 4 μm. One slice was selected from 5 pieces, and 3 pieces were selected from each section and used for Masson staining after dewaxing. Slices were observed under an optical light microscope (BM2000, Shanghai Wanheng Precision Instruments, Co., Ltd., Shanghai, China). The red area represents the myocardial cells, while the bluish green part represents the collagen fibers. Five visual fields were selected randomly for photograph under a microscope in each slice. Image-Pro-Plus 5.0 image processing system was used to calculate the proportion of the positive area of collagen fibers in an observed area. Immunohistochemistry The cardiac tissues of 5 rats after measurement of hemodynamic parameters were first obtained. The tissues were routinely fixed, dehydrated, embedded and sliced into 4 μm sections. The sections were added with peroxidase and 1% H2O2 and then immersed in antigen repair solution at 97°C, and cooled down at room temperature for 15 min. Next, goat serum blocking solution (Beijing Solarbio, Co., Ltd., Beijing, China) was added and the excess liquid was discarded after 20 min. The antibodies TRPM6 (ab47017, 1: 1000 dilution, Abcam, Inc., Cambridge, MA, USA) or cleaved-poly-adenosine diphosphate-ribose polymerase (PARP, ab32064, Abcam, Inc., Cambridge, MA, USA) were added to the slides and left to incubate overnight at 4°C. On the following day, the biotinylated secondary antibodies (Beijing Zhongshan Golden Bridge Co., Ltd., Beijing, China) were added for incubation at 37°C for 40 min. Diaminobenzidine (DAB) substrate kit (Beijing Zhongshan Golden Bridge Co., Ltd., Beijing, China) was used for color development and the multi-functional true color cell image analysis management system (Media Cybernetics, Inc., San Diego, CA, USA) was used for image analysis. Four sections were selected from each specimen, and three random visual fields were randomly selected. The number of positive cells was identified and counted under a microscope. The average value of positive cells in the three fields was used to calculate the positive expression rate of the TRPM6 protein. Dual-luciferase reporter gene assay According to the prediction on the http://www.targetscan.org/vert72/website, TRPM6 is the target of miR-202-3p. The fragment of a synthetic TRPM6 3ʹUTR gene was inserted into pMIR-reporter (Promega, Madison, WI, USA) using endonuclease sites SpeI and Hind III. The complementary sequence mutation (MUT) sites of seed sequences were designed according to the wild type (WT) of TRPM6. The target fragments were inserted into the pMIR-reporter plasmid using restriction enzyme digest and T4 DNA Ligase. HEK-293T cells (Shanghai BeinuoBio Biological Technology Co., Ltd., Shanghai, China) were transfected with miR-202-3p and luciferase reporter plasmids WT and MUT, respectively. After transfection for 48 h, the supernatant was discarded and then cells were washed with phosphate buffered saline (PBS). Cells were then lysed for 5–10 min at room temperature with diluted cell lysis, and then triturated. The cell fragments were removed by centrifugation at 1610 × g for 5 min. A total of 50 μL luciferase was added into each group, and transferred to the detection plate. Luciferase activity was confirmed by the dual-channel fluorescence of luciferase assay kit. RNA isolation and quantitation The mRNA levels of genes in rat myocardium collected from each group were determined by RNA isolation and quantitation. Trizol total RNA extraction kit (Aidlab Biotechnologies Co., Ltd., Beijing, China) was used to extract the total RNA. RNA sample (5 μL) was diluted 20 times with RNA-enzyme-free pure water. The ratio of optical density (OD) 260 nm/OD 280 nm was measured by an ultraviolet spectrophotometer, and the RNA purity was determined to ensure that the OD value was 1.8 to 2.1. The synthesis of cDNA and antisense-mediated reverse transcription of miRNA were determined using a PCR amplifier (Thermo Fisher Scientific, Waltham, MA, USA). RNA isolation and quantitation was carried out by quantitative polymerase chain reaction (q-PCR) (ABI7500, Thermo Fisher Scientific, Waltham, MA, USA). Primers used are displayed in the Table 1. U6 was used as the internal control. 2−ΔCt implicated the multiple proportions of genes between the experimental and the control group. ΔCt = Ct target gene – Ct β-actin/U6 [23]. Ct refers to the number of amplification cycles when the real-time fluorescence intensity reached the set threshold whereby the amplification procedure was in the logarithmic growth phase. The measurement was repeated 3 times [24–26].10.1080/15384101.2019.1580494-T0001 Table 1. The primer sequences for reverse transcription quantitative polymerase chain reaction. Genes Primer sequences miR-202-3p F: 3ʹ-AGAAGCATTCGCGTCGGTTC-5’ T: 5ʹ-GAAAGCTCGTCCACGTCAGAC-3’ TRPM6 F: 3ʹ-GCAATGGCTTGGGATAGAAT-5’ T: 5ʹ-CAGTGTGCTTTCCGAAGACTC-3’ TGF-β1 F: 3ʹ-GCTACTGCCGCTTCTGC-5’ T: 5ʹ-GCCACTCAGGCGTATCAG-3’ Smad2 F: 3ʹ-AAGCCATCACCACTCAGAATTG-5’ T: 5ʹ-CACTGATCTACCGTATTTGCTGT-3’ Smad7 F: 3ʹ-CCAACTGCAGACTGTCCAGA-5’ T: 5ʹ-CAGGCTCCAGAAGAAGTTGG-3’ U6 F: 3ʹ-ACACGACGGCTTCGCTC-5’ T: 5ʹ-AACGCTTCACGAATTTGCGT-3’ Bcl-2 F: 3ʹ-TGGGATGCCTTTGTGGAACTA-5’ T: 5ʹ-GCTGATTTGACCATTTGCCTAA-3’ Bax F: 3ʹ-GAGCGAGTGTCTCCGGCGAAT-5’ T: 5ʹ-GCCACAAAGATGGTCACTGTCTG-3’ β-actin F: 3ʹ-CTTCGACATCGGTGCGAGC-5’ T: 5ʹ-GTCACGCACGATTTCCCTCT-3’ miR-202-3p, microRNA-202-3p; TRPM6, transient receptor potential cation channel, subfamily M, member 6; TGF-β1, transforming growth factor, beta 1; Smad2, SMAD family, number 2; Smad7, SMAD family, number 7; Bcl-2, B cell leukemia/lymphoma 2; Bax, BCL2-associated X protein. Western blot analysis The total protein was extracted from rat myocardium. The concentration of sample protein was determined using a bicinchoninic acid (BCA) kit (Thermo Fisher Scientific, Waltham, MA, USA). The total protein (30 μg) was separated by polyacrylamide gel electrophoresis and then transferred onto a polyvinylidene fluoride (PVDF) membrane. After being sealed with 5% skimmed milk powder at room temperature for 1 h, the membrane was incubated with primary antibodies for TRPM6 (Rb719-221108-WS, 1: 2000, Osenses, Adelaide, Australia), TGF-β1 (ab15715, 1: 1000), Smad2 (ab33875, 1: 1500), p-Smad2 (ab188334, 1: 1500), Smad7 (ab216428, 1: 550), Bcl-2 (ab196495, 1: 1500), Bax (ab32503, 1: 2000), and β-actin antibody (ab8227, 1: 2500) at 4°C overnight. The primary antibodies except for TRPM6 were purchased from Abcam Inc. (Cambridge, MA, USA). After rinsing three times with PBS containing 0.05% Tween20 (PBST) (5 min each time), the membrane was incubated with HRP-labeled IgG secondary antibodies (1: 1000, Wuhan Boster Biological Technology Co., Ltd., Wuhan, Hubei, China) at room temperature for 1 h. Next, the electrogenerated chemiluminescence (ECL) reagent was prepared according to the instructions of SuperSignal® West Dura Extended Duration Substrate to visualize the results using X-ray film. Finally, Image Pro Plus 6 software (Media Cybernetics, Inc., Bethesda, MD, USA) was used to analyze the gray values with β-actin as the internal control. The measurement was repeated 3 times Terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling (TUNEL) assay Cardiac tissues were fixed in 4% paraformaldehyde and embedded in paraffin to prepare the slice. The slices were incubated with 100 μL equilibrium solution and reacted with 100 μL of TUNEL solution (Boehringer Mannheim Co., Ltd., Indianapolis, IN, USA) for 1 h. Then the slices were sealed in neutral gum for 15 min after enzyme labeling. A DAB substrate kit was used for color development in the dark. The slices were then counter-stained with hematoxylin and mounted. Five random visual fields with high magnification (400 ×) were randomly selected under a light optical microscope and used to count the number of apoptotic myocardial cells and the total number of live myocardial cells. Myocardial cell apoptosis rate was calculated by = apoptotic myocardial cell number/total number of myocardial cells × 100%. The measurement was repeated 3 times. Statistical analysis Statistical analyses were conducted by using SPSS 20.0 (IBM, Armonk, N.Y., USA). Measurement data were expressed as a mean ± standard deviation. Differences of data between two groups were compared by a t test. Multiple groups were compared by one-way analysis of variance followed by a Tukey’s post hoc test. Values of p < 0.05 were considered statistically significant. Results TRPM6 and miR-202-3p are associated with I/R injury As early as 1989, Shinohara K et al. found that TRPM6 gene was highly expressed in the atrial tissues of patients with atrial fibrosis [16]. Subsequently, a study has shown that the TRPM6 gene plays an important role in the regulation of extracellular divalent cations in cardiac myocytes [17]. In addition, the expression of TRPM family, including TRPM6 gene, was significantly increased in heart tissues after I/R [27]. Furthermore, it is found that there is a close relationship between the TGF signaling pathway and I/R [28,29]. Among the TRPM family, a small number of studies suggested that TRPM6 gene can play a regulatory role through the TGF signaling pathway [16,30]. However, the mechanism of TRPM6 gene in myocardial I/R remains unclear. To understand the upstream regulation mechanism of TRPM6 gene, the TargetScan database (http://www.targetscan.org/vert71/) and the microDB database (http://mirdb.org/microDB/index.html) were used to predict the regulatory miRs of TRPM6 gene. A website http://bioinformatics.psb.ugent.be/webtools/Venn/was employed to construct the Venn diagram of the intersection of prediction results of the two databases (Figure 1(a)). The Venn diagram showed that there were 11 miRs in the intersection, among which rno-miR-202-3p has the highest prediction score. Therefore, miR-202-3p was selected for further study. The expression of TRPM6 and miR-202-3p in the control and myocardial I/R injury groups were analyzed by qPCR (Figure 1(b)). The results showed that myocardial I/R rats were featured by highly expressed TRPM6 and poorly expressed miR-202-3p (all p < 0. 05).10.1080/15384101.2019.1580494-F0001 Figure 1. Upregulated TRPM6 and downregulated miR-202-3p are related to the progression of myocardial I/R. (a) the regulatory miRs prediction of TRPM6 gene. The left blue circle represents the top 15 miRs in the TargetScan database, and the right red circle represents the top 15 miRs in the microRNADB database. The overlapping section indicates the intersection of the two databases. (b) expression of TRPM6 and miR-202-3p in normal and I/R injury rats, as determined by qPCR; , p < 0.05 vs. the sham group; differences between two groups were compared by independent t test followed by a Tukey’s post hoc test; TRPM6, transient receptor potential cation channel; miR-202-3p, microRNA-202-3p. Rat models of I/R injury are established successfully Initially, we used ECG to verify the success of myocardial I/R modeling. According to the comparisons of ECG between the control and myocardial I/R injury groups (Figure 2), we verified the successful establishment of rat models with I/R. The ECG band of the control group appeared relatively stable; along with an increase in the width of QRS peak after 2–5 min. A fusion was found between ST segment and T wave, it shows a bow-shaped one-way curve in the myocardial I/R injury group, which indicated that the ligation position was right. These findings provide evidence that the myocardial I/R rat models were successfully established.10.1080/15384101.2019.1580494-F0002 Figure 2. Rat models of myocardial I/R are successfully established. ECG, electrocardiogram; QRS, a name for the combination of three of the graphical deflections seen on a typical electrocardiogram (ECG). Up-regulated miR-202-3p or knockdown TRPM-6 alleviates oxidative stress and attenuates inflammatory response In order to investigate the effects of miR-202-3p and TRPM6 on oxidative stress and inflammatory response after myocardial I/R, we evaluated the levels of antioxidant enzyme markers SOD, CAT, and GSH, oxidative stress marker MDA and inflammatory factors IL-1β, IL-6 as well as TNF-α. At the same time, we also measured the serum Troponin T content to reflect the degree of myocardial injury. SOD, CAT, GSH-Px levels were significantly decreased (p < 0.05) while MDA was significantly increased (p < 0.05) in other groups compared with the sham group. Compared with the I/R injury group, SOD, CAT, GSH-Px levels in the miR-202-3p mimic and siRNA-TRPM6 groups were significantly elevated (p < 0.05), whereas MDA levels was substantially decreased (p < 0.05). Next, SOD, CAT, GSH-Px levels significantly decreased in the miR-202-3p inhibitor group in addition to increased MDA level (p < 0.05). The levels of SOD, CAT, MDA, GSH-Px did not significantly differ between the NC and miR-202-3p inhibitor + siRNA-TRPM6 groups (p > 0.05) (Table 2). Levels of inflammatory factors included IL-1β, IL-6, TNF-α and Troponin T in the NC, I/R injury, miR-202-3p mimic, miR-202-3p inhibitor, siRNA-TRPM6 and miR-202-3p inhibitor + siRNA-TRPM6 groups were significantly up-regulated (p < 0.05) compared with those in the sham group. Meanwhile, as opposed to the I/R injury group, IL-1β, IL-6, TNF-α and Troponin T levels in the miR-202-3p mimic and siRNA-TRPM6 groups were down-regulated (p < 0.05), but substantially increased in the miR-202-3p inhibitor group (p < 0.05). Moreover, the IL-1β, IL-6, TNF-α and Troponin T levels in the NC and miR-202-3p inhibitor + siRNA-TRPM6 groups did not differ significantly (p > 0.05) (Table 3). These results suggest that miR-202-3p overexpression and TRPM6 knockdown can lead to a decreased level of peroxides, a reduced release of inflammatory factors, as well as an increase in levels of antioxidant enzymes by inhibiting TPRM6. These findings suggest that miR-202-3p can help to improve the oxidative stress and inflammation induced by I/R injury and reduce myocardial injury.10.1080/15384101.2019.1580494-T0002 Table 2. MiR-202-3p overexpression and TRPM6 knockdown lead to reduced levels of peroxides and enhanced level of antioxidant enzymes. Groups SOD/U•mg−1 CAT/U•mg−1 GSH-Px/U•mg−1 MDA/nmol•mg−1 sham 795.05 ± 79.26 85.61 ± 9.02 214.71 ± 18.45 1.74 ± 1.34 I/R injury 530.45 ± 46.86 32.73 ± 3.28* 127.11 ± 13.75* 29.71 ± 1.36* NC 531.54 ± 46.77* 31.83 ± 3.05* 127.55 ± 14.73* 29.52 ± 1.56* miR-202-3p mimic 661.32 ± 57.56# 53.11 ± 7.18# 170.51 ± 18.58# 15.86 ± 1.02# siRNA-TRPM6 671.31 ± 56.89# 53.16 ± 6.02# 171.34 ± 19.57# 15.45 ± 1.21# miR-202-3p inhibitor 366.62 ± 36.83# 12.04 ± 1.03# 82.11 ± 10.26# 43.88 ± 3.93# miR-202-3p inhibitor + siRNA-TRPM6 531.43 ± 46.61* 32.96 ± 3.26* 126.88 ± 12.15* 29.48 ± 1.87* , p < 0.05 vs. the sham group; #, p < 0.05 vs. the I/R injury group; n = 5; multiple groups were compared by one-way analysis of variance followed by a Tukey’s post hoc test; NC, negative control; miR-202-3p, microRNA-202-3p; TRPM6, transient receptor potential cation channel, subfamily M, member 6; SOD, superoxide dismutase; CAT, catalase; GSH-Px, glutathione peroxidase; MDA, malondialdehyde. 10.1080/15384101.2019.1580494-T0003 Table 3. Up-regulated miR-202-3p and knockdown TRPM-6 result in declines in inflammatory response and Troponin. Groups IL-1β/pg•mL−1 IL-6/pg•mL−1 TNF-α/pg•mL−1 Troponin T/pg•mL−1 Sham 59.05 ± 7.38 16.28 ± 2.83 10.73 ± 2.73 8.96 ± 2.79 I/R injury 129.36 ± 15.94 56.55 ± 6.21* 51.51 ± 6.78* 49.39 ± 3.39* NC 128.79 ± 14.88* 57.56 ± 6.47* 50.91 ± 6.95* 48.47 ± 3.34* miR-202-3p mimic 89.83 ± 9.94# 35.34 ± 4.41# 29.57 ± 4.12# 26.76 ± 3.21# siRNA-TRPM6 88.43 ± 9.14# 35.14 ± 3.98# 30.24 ± 3.12# 27.33 ± 3.08# miR-202-3p inhibitor 184.45 ± 23.05# 83.34 ± 13.14# 78.78 ± 8.91# 76.22 ± 4.12# miR-202-3p inhibitor + siRNA- TRPM6 129.13 ± 16.14* 56.34 ± 8.66* 51.76 ± 6.32* 50.29 ± 3.29* , p < 0.05 vs. the sham group; #, p < 0.05 vs. the I/R injury group; n = 5; multiple groups were compared by one-way analysis of variance followed by a Tukey’s post hoc test; NC, negative control; miR-202-3p, microRNA-202-3p; TRPM6, transient receptor potential cation channel, subfamily M, member 6; IL-1β, interleukin 1 beta; IL-6, interleukin 6; TNF-α, tumor necrosis factor-α. Up-regulated miR-202-3p or knockdown TRPM-6 inhibits ventricular remodeling and promotes functional recovery To further investigate the effects of miR-202-3p and TRPM6 on ventricular remodeling, we measured the ventricular weight of rat hearts. The weight of the left and right ventricles is illustrated in Figure 3(a). In contrast to the sham group, LVRW and RVRW in the NC, I/R injury, miR-202-3p mimic, miR-202-3p inhibitor, siRNA-TRPM6 and miR-202-3p inhibitor + siRNA-TRPM6 groups significantly increased (p < 0.05). When compared with the I/R injury group, the LVRW and RVRW were remarkably lower in the miR-202-3p mimic and siRNA-TRPM6 groups (p < 0.05) but higher in the miR-202-3p inhibitor group (p < 0.05). LVRW and RVRW did not differ significantly between the miR-202-3p inhibitor + siRNA-TRPM6 and NC groups (p > 0.05). These results suggest that miR-202-3p can inhibit ventricular remodeling after myocardial I/R injury by inhibiting TRPM6. In order to detect the function of ventricular remodeling, we further examined the hemodynamic parameters. As shown in Figure 3(b), the NC, I/R injury, miR-202-3p mimic, miR-202-3p inhibitor, siRNA-TRPM6 and miR-202-3p inhibitor + siRNA-TRPM6 groups presented a higher level of LVEDP (p < 0.05) and a lower level of SBP, DBP and LVSP than those in the sham group (p < 0.05). Compared to the I/R injury group, LVEDP significantly decreased in the miR-202-3p mimic and siRNA-TRPM6 groups (p < 0.05), whereas, SBP, DBP and LVSP increased (p < 0.05). LVEDP in the miR-202-3p inhibitor group increased markedly (p < 0.05), while SBP, DBP, and LVSP decreased observably (p < 0.05). Moreover, LVEDP, SBP, DBP and LVSP did not differ significantly in the miR-202-3p inhibitor + siRNA-TRPM6 and NC groups (p > 0.05). These results imply that miR-202-3p is capable of enhancing the functional recovery after ventricular remodeling by inhibiting TRPM6.10.1080/15384101.2019.1580494-F0003 Figure 3. Ventricular remodeling is inhibited and functional recovery is promoted by up-regulated miR-202-3p or knockdown TRPM-6 after 4 weeks. (a) the relative weight of the left and right ventricles in rats treated with miR-202-3p mimic, siRNA-TRPM6, miR-202-3p inhibitor or miR-202-3p inhibitor + siRNA-TRPM6 after 4 weeks; (b) the differences of ventricular hemodynamic parameters LVEDP, SBP, DBP and LVSP in rats treated with miR-202-3p mimic, siRNA-TRPM6, miR-202-3p inhibitor or miR-202-3p inhibitor + siRNA-TRPM6 after 4 weeks; n = 5; multiple groups were compared by one-way analysis of variance followed by a Tukey’s post hoc test; , p < 0.05 vs. the sham group; #, p < 0.05 vs. the I/R injury group; LVRW, left ventricular relative weight; RVRW, right ventricular relative weight; NC, negative control; siRNA, small interfering RNA; TRPM6, transient receptor potential cation channel, subfamily M, member 6; miR-202-3p, microRNA-202-3p; LVEDP, left ventricular end diastolic pressure; LVSP, left ventricular systolic pressure; SBP, systolic arterial pressure; DBP, diastolic blood pressure. Up-regulated miR-202-3p or knockdown TRPM-6 in rats reduces MIS and myocardial fibrosis We measured the MIS and myocardial fibrosis after myocardial I/R. As seen in Figure 4(a,b), the MIS in the NC, I/R injury, miR-202-3p mimic, miR-202-3p inhibitor, siRNA-TRPM6 and miR-202-3p inhibitor + siRNA-TRPM6 groups increased significantly compared with the sham group (p < 0.05). When compared with the I/R injury group, the MIS was reduced prominently in the miR-202-3p mimic and siRNA-TRPM6 groups (p < 0.05); whereas, the miR-202-3p inhibitor group exhibited a larger MIS (p < 0.05). Additionally, the MIS did not differ significantly between the miR-202-3p inhibitor + siRNA-TRPM6 and NC groups (p > 0.05). Following Masson staining, the degree of myocardial fibrosis in each group is shown in Figure 4(c,d), the blue part represents myocardial fibrosis, and the red part represents the normal myocardial cells. Compared with the sham group, myocardial fibrosis in the NC, I/R injury, miR-202-3p mimic, miR-202-3p inhibitor, siRNA-TRPM6 and miR-202-3p inhibitor + siRNA-TRPM6 groups increased significantly (p < 0.05). In comparison to the I/R injury group, the myocardial fibrosis in the miR-202-3p mimic and siRNA-TRPM6 groups was significantly decreased (p < 0.05); myocardial fibrosis in the miR-202-3p inhibitor group was significantly increased (p < 0.05); myocardial fibrosis presented no significant difference between the miR-202-3p inhibitor + siRNA-TRPM6 and NC groups (p > 0.05). The above findings revealed that miR-202-3p can inhibit myocardial fibrosis and myocardial infarction by decreasing TRPM6.10.1080/15384101.2019.1580494-F0004 Figure 4. miR-202-3p inhibits MIS and myocardial fibrosis. (a) photographs of MIS in rats treated with miR-202-3p mimic, siRNA-TRPM6, miR-202-3p inhibitor or miR-202-3p inhibitor + siRNA-TRPM6 after 4 weeks (the white area was the infarction area); (b) comparisons of the MIS in rats treated with miR-202-3p mimic, siRNA-TRPM6, miR-202-3p inhibitor or miR-202-3p inhibitor + siRNA-TRPM6 after 4 weeks; (c) myocardial Masson staining of rats treated with miR-202-3p mimic, siRNA-TRPM6, miR-202-3p inhibitor or miR-202-3p inhibitor + siRNA-TRPM6 after 4 weeks; (d) comparison of the degree of myocardial fibrosis in rats treated with miR-202-3p mimic, siRNA-TRPM6, miR-202-3p inhibitor or miR-202-3p inhibitor + siRNA-TRPM6 after 4 weeks; n = 5; multiple groups were compared by one-way analysis of variance followed by a Tukey’s post hoc test; , p < 0.05 vs. the sham group; #, p < 0.05 vs. the I/R injury group; NC, negative control; siRNA, small interfering RNA; TRPM6, transient receptor potential cation channel, subfamily M, member 6; miR-202-3p, microRNA-202-3p; MIS, myocardial infarct size. MiR-202-3p suppresses positive expression of TRPM6 To verify whether TRPM6 is involved in ventricular remodeling after myocardial I/R, we examined the expression of TRPM6 in myocardial tissue. Under the light microscope, the positive staining of TRPM6 appears tan or brown, and the positive areas were mainly localized in the cardiac interstitial region (Figure 5(a)). The positive expression of TRPM6 in the NC, I/R injury, miR-202-3p mimic, miR-202-3p inhibitor, siRNA-TRPM6 and miR-202-3p inhibitor + siRNA-TRPM6 groups was expressed at a high level compared with the sham group. In contrast to the I/R injury group, TRPM6 was found to be expressed at a lower levels in the miR-202-3p mimic and siRNA-TRPM6 groups (p < 0.05); meanwhile, the miR-202-3p inhibitor group exhibited higher levels of TRPM6 (p < 0.05). There were no significant differences in TRPM6 expression in the miR-202-3p inhibitor + siRNA-TRPM6 and NC groups compared to the I/R injury group (p > 0.05) (Figure 5(b)). In conclusion, miR-202-3p inhibits the expression of TRPM6 induced by myocardial I/R in rats.10.1080/15384101.2019.1580494-F0005 Figure 5. miR-202-3p leads to decreased TRPM6 expression. (a) the immunohistochemical staining of TRPM6 in myocardial tissues treated with miR-202-3p mimic, siRNA-TRPM6, miR-202-3p inhibitor or miR-202-3p inhibitor + siRNA-TRPM6 after 4 weeks (× 400); (b) the positive expression rate of TRPM6 in myocardial tissues treated with miR-202-3p mimic, siRNA-TRPM6, miR-202-3p inhibitor or miR-202-3p inhibitor + siRNA-TRPM6 after 4 weeks; n = 5; multiple groups were compared by one-way analysis of variance followed by a Tukey’s post hoc test; , p < 0.05 vs. the sham group; #, p < 0.05 vs. the I/R injury group. NC, negative control; siRNA, small interfering RNA; TRPM6, transient receptor potential cation channel, subfamily M, member 6; miR-202-3p, microRNA-202-3p. TRPM6 is confirmed to be a target gene of miR-202-3p With the aim of determining whether miR-202-3p works by targeting TRPM6 gene, we analyzed the targeting relationship between TRPM6 and miR-202-3p. The http://www.targetscan.org/vert72/website showed that TRPM6 was a target of miR-202-3p (Figure 6(a)). The dual-luciferase reporter gene assay demonstrated that luciferase activity of Wt-miR-202-3p/TRPM6 in the miR-202-3p mimic group was down-regulated substantially (p < 0.05). However, we found no significant changes in luciferase activity of Mut-miR-202-3p/TRPM6 (p > 0.05) in the NC group (Figure 6(b)). These findings provide evidence that miR-202-3p binds to TRPM6.10.1080/15384101.2019.1580494-F0006 Figure 6. TRPM6 is verified to be the target gene of miR-202-3p. (a) the binding site between miR-202-3p and TRPM6 3ʹ-UTR; (b) luciferase activity of the TRPM6-Wt and TRPM6-Mut after transfection; n = 5; differences between two groups were compared by independent t test; , p < 0.05 vs. the NC group; TRPM6, transient receptor potential cation channel, subfamily M, member 6; miR-202-3p, microRNA-202-3p; siRNA, small interfering RNA. MiR-202-3p negatively regulates TRPM6 gene and the TGF-β1/Smads signaling pathway activates It has been reported that the TGFβ signaling pathway is involved in regulating myocardial injury. In order to investigate whether TGFβ is involved in this process, we measured the expression of TGF-β1, Smad2, and Smad7 as well as Smad2 phosphorylation. We also measured the expression of Bcl-2 and Bax to investigate whether miR-202 and TRPM6 affects myocardial apoptosis after myocardial ischemia (Figure 7(a–c)). MiR-202-3p expression and the mRNA and protein levels of Bcl-2 and Smad7 decreased significantly, whereas the mRNA and protein levels of TRPM6, Bax, TGF-β1 and Smad2 as well as Smad2 phosphorylation increased in the NC, I/R injury, miR-202-3p mimic, miR-202-3p inhibitor, siRNA-TRPM6 and miR-202-3p inhibitor + siRNA-TRPM6 groups when compared with the sham group (p < 0.05). Compared to the I/R injury group, mRNA and protein levels of TRPM6, Bax, TGF-β1 and Smad2 and Smad2 phosphorylation reduced significantly in the miR-202-3p mimic and siRNA-TRPM6 group, accompanied with a higher mRNA level of Bcl-2 and Smad7 (p < 0.05). Additionally, miR-202-3p expression in the miR-202-3p mimic group increased markedly (p < 0.05) and did not differ significantly in the siRNA-TRPM6 group (p > 0.05) while mRNA and protein levels of TRPM6, Bax, TGF-β1 and Smad2 along with Smad2 phosphorylation increased significantly but the expression of miR-202-3p and mRNA and protein levels of Bcl-2 and Smad7 decreased evidently in the miR-202-3p inhibitor group (p < 0.05); the expression of miR-202-3p decreased significantly (p < 0.05), and there was no signficant difference of mRNA and protein levels of TRPM6, TGF-β1, Smad2, Smad7, Bcl-2 and Bax as well as the extent of Smad2 phosphorylation (p < 0.05) in the miR-202-3p inhibitor + siRNA-TRPM6 group; no significant difference was found of miR-202-3p expression and mRNA and protein levels of TRPM6, TGF-β1, Smad2, Smad7, Bcl-2 and Bax as well as the extent of Smad2 phosphorylation in the NC group (p > 0.05). These results suggest that miR-202-3p expression and inhibition of TRPM6 reduces myocardial cell apoptosis by suppressing the TGF-β1/Smads signaling pathway.10.1080/15384101.2019.1580494-F0007 Figure 7. miR-202-3p decreases TRPM6 expression and inhibits the activation of TGF-β1/Smads signaling pathway after 4 weeks. (a) the miR-202-3p expression and mRNA levels of TRPM6, TGF-β1, Bcl-2, Bax, Smad7 and Smad2 in response to the treatment of miR-202-3p mimic, siRNA-TRPM6, miR-202-3p inhibitor or miR-202-3p inhibitor + siRNA-TRPM6; (b) the protein levels of TRPM6, TGF-β1, Bcl-2, Bax, Smad7 and Smad2 and the extent of Smad2 phosphorylation in response to the treatment of miR-202-3p mimic, siRNA-TRPM6, miR-202-3p inhibitor or miR-202-3p inhibitor + siRNA-TRPM6; (c) the gray values of TRPM6, TGF-β1, Bcl-2, Bax, Smad7, Smad2 and β-actin protein bands in response to the treatment of miR-202-3p mimic, siRNA-TRPM6, miR-202-3p inhibitor or miR-202-3p inhibitor + siRNA-TRPM6; n = 5; multiple groups were compared by one-way analysis of variance followed by a Tukey’s post hoc test; , p < 0.05 vs. the sham group; #, p < 0.05 vs. the I/R injury group; TGF-β1, transforming growth factor, beta 1; Bcl-2, Panel B cell leukemia/lymphoma 2; Bax, BCL2-associated X protein; Smad, SMAD family; NC, negative control; mRNA, messenger RNAs; miR, microRNA; siRNA, small interfering RNA; TRPM6,transient receptor potential cation channel, subfamily M, member 6; miR-202-3p, microRNA-202-3p. Up-regulated miR-202-3p or knockdown TRPM-6 suppresses cell apoptosis in myocardium The effect of miR-202-3p on cell apoptosis was further verified by TUNEL (Figure 8(a,b)) and cleaved-PARP staining [31] (Figure 8(c,d)). Apoptotic cells in myocardial tissue were stained and appeared with brown whereas normal cells were blue. A heavier stain with brown granules implied that there were much more apoptotic cells. Compared with the sham group, cell apoptosis of myocardial tissue in the NC, I/R injury, miR-202-3p mimic, miR-202-3p inhibitor, siRNA-TRPM6 and miR-202-3p inhibitor + siRNA-TRPM6 groups increased significantly (p < 0.05). As opposed to the I/R injury group, cell apoptosis in the miR-202-3p mimic and siRNA-TRPM6 groups was significantly lowered (p < 0.05); the miR-202-3p inhibitor group presented a higher level of cell apoptosis (p < 0.05); no significant changes of cell apoptosis were found in the miR-202-3p inhibitor + siRNA-TRPM6 and NC groups (p > 0.05). The results of cleaved-PARP staining were similar to those above, suggesting that miR-202-3p inhibits myocardial cell apoptosis in myocardium.10.1080/15384101.2019.1580494-F0008 Figure 8. miR-202-3p represses myocardial cell apoptosis. (a) photographs of TUNEL staining (× 400); (b) apoptotic cell rate of each group in response to the treatment of miR-202-3p mimic, siRNA-TRPM6, miR-202-3p inhibitor or miR-202-3p inhibitor + siRNA-TRPM6; (c) the immunohistochemical staining of cleaved-PARP in response to the treatment of miR-202-3p mimic, siRNA-TRPM6, miR-202-3p inhibitor or miR-202-3p inhibitor + siRNA-TRPM6 (× 400); (d) the proportion of cleaved-PARP positive cells in response to the treatment of miR-202-3p mimic, siRNA-TRPM6, miR-202-3p inhibitor or miR-202-3p inhibitor + siRNA-TRPM6; n = 5; multiple groups were compared by one-way analysis of variance followed by a Tukey’s post hoc test; *, p < 0.05 vs. the sham group; #, p < 0.05 vs. the I/R injury group; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end labeling; NC, negative control; siRNA, small interfering RNA; TRPM6, transient receptor potential cation channel, subfamily M, member 6; miR-202-3p, microRNA-202-3p. Discussion Myocardial I/R injury is a pathological process that results in DNA, plasma membrane and and protein damage, contributing to severe myocardial injury and inflicting pain and damage on I/R injury victims [32]. Robinson et al. have revealed that miRNAs are being studied more in depth due to their uses as potential markers for acute myocardial infarction (AMI), as some specific miRNAs may be associated with AMI and I/R injury [33]. Investigations on functional roles of miRNAs in I/R injury and ventricular remodeling may allow us to understand more as to how miRNAs affect cellular behaviors, such as cell apoptosis, fibrosis or infarction. We hypothesized that miR-202-3p plays a role during I/R injury or ventricular remodeling. Our results demonstrated that miR-202-3p could activate the TGF-β1/Smads signaling pathway by inhibiting TRPM6, thus regulating myocardial cell activity and cardiac fibroblasts. One of the most important finding was that TRPM6 is highly expressed after myocardial I/R injury. Based on the target prediction program and the luciferase activity determination, we found that TRPM6 is a putative target gene of and negatively regulated by miR-202-3p. It has been reported that calcium channels (TRPM) play important roles in myocardial ischemia and ischemia-reperfusion; additionally, TRPM7 and TRPM2 have been verified to be involved in delayed neuronal death after ischemia [34]. Furthermore, the necessary function of TPRM6 in I/R injury on its protein expression was close to its initial level after 48 h of reperfusion [35]. Magenta et al. found that in addition to common conditions such as aging and diabetes, cardiovascular diseases are also prominently associated with up-regulated miR-200 expression, as well as those in the brain and I/R injury [36]. To investigate the mechanism of miR-202-3p in ventricular remodel after myocardial I/R injury, we established a myocardial I/R injury model in SD rats that were transfected with miR-202-3p mimic, miR-202-3p inhibitor and siRNA-TRPM6. The results showed that miR-202-3p overexpression activates the TGF-β1/Smads signaling pathway by negatively regulating TRPM6 expression. TGF-β1 plays a role mainly via the Smad-dependent and Smad-independent pathways, including ERK, MAPK, Rac, PI3K, and GTPases [37]. According to an aforementioned study, TGF-β could induce the expression of miR-202-3p in U2OS cell lines [38]. In the ischemic heart, SMAD-3 is directly regulated by GSK-3β by modulating the canonical TGF-β1 signaling pathway, and negatively regulates fibrotic remodeling [39], which indicates that TGF-β1/Smads signaling pathway is involved in ventricular remodeling after myocardial I/R injury. Interestingly, TGF-β1 has been found to enhance expression of TRPM7 via TGF-β/Smad signaling in hepatic stellate cells and elimination of TRPM7 inhibited phosphorylation of Smad2 and Smad3 [40]. Several previous studies have shown that miR-202 mediates the proliferation and apoptosis of gastric cancer, multiple myeloma and osteosarcoma cells by targeting different genes and thus regulates the tumorigenesis and development [41–43]. Our results suggest that miR-202-3p can directly participate in the process of ventricular remodeling after I/R by targeting TRPM6. TRPM6 may also be regulated by other miRs. Similarly, miR-202-3p may be involved in ventricular remodeling by targeting other genes. More mechanisms and signaling pathways need to be further explored in the future. miR-202-3p also had the potential to alleviate myocardial fibrosis and inflammation as well as inhibit cardiac fibroblast apoptosis. The potential mechanisms might be associated with; down-regulated expression of pro-inflammatory cytokines (IL-1β, IL-6, TNF-α), MDA, and up-regulated expression of SOD, CAT, GSH-Px. All of these mechanisms may lead to the subsequent activation of the TGF-β1/Smads signaling pathway to help ameliorate myocardial ischemia and accelerate the cardiac repair process. If reperfusion of oxygen is initiated in the area of infarction, an intense inflammatory reaction will occur, leading to cellular injury. Although it may lead to cardiac cell injury, reperfusion could also enhance cardiac repair and this effect may be partially connected with the inflammatory response [44]. TGF-β1 has been considered as a powerful mediator in the pathogenesis of myocardial I/R injury, which is able to initiate inflammatory responses and activate myofibroblasts [37,45]. A previous study has demonstrated that TGF-β1 induces cell apoptosis by mediating Bcl-2 and Bax expression [46]. Another study showed that TGF-β1 inhibits cardiac fibroblast apoptosis induced by simulated I/R through the non-canonical (ERK1/2 and Akt) and canonical (Smad3) signaling pathways [47]. These further highlight the multi-functional role of the TGF-β1 cytokine that was also in line with our results. The measurements of ventricular weight, ventricular hemodynamic parameters and MIS in this study support the results of our in vitro experiment. To sum up, we demonstrated that the overexpression of miR-202-3p alleviates oxidative stress and inflammatory response, reduces ventricular mass, alters cardiac hemodynamics, suppresses myocardial infarction and cell apoptosis, and inhibits myocardial fibrosis by activating the TGF-β1/Smads signaling pathway through inhibiting TRPM6 expression. These findings highlight the potential therapeutic value of miR-202-3p in the treatment of ventricular remodeling after I/R injury. 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==== Front Cell Cycle Cell Cycle KCCY kccy20 Cell Cycle 1538-4101 1551-4005 Taylor & Francis 30990350 1608128 10.1080/15384101.2019.1608128 Research Paper NF-κB signaling pathway inhibition suppresses hippocampal neuronal apoptosis and cognitive impairment via RCAN1 in neonatal rats with hypoxic-ischemic brain damage H. FANG ET AL. CELL CYCLE Fang Hua ab* Li Hua-Feng c* Yang Miao ab Liao Ren d Wang Ru-Rong d Wang Quan-Yun d Zheng Peng-Cheng e Zhang Fang-Xiang ab Zhang Jian-Ping ab a Department of Anesthesiology, Guizhou Provincial People‘s Hospital, Guiyang, P. R. China b Department of Anesthesiology, Guizhou University People’s Hospital, , Guiyang, P. R. China c Department of Anesthesiology, West China Second University Hospital, Sichuan University, Chengdu, P. R. China d Department of Anesthesiology, West China Hospital, Sichuan University, Chengdu, P. R. China e Guizhou University Research Center for Analysis of Drugs and Metabolites, Guizhou University, Chengdu, P. R. China CONTACT Jian-Ping Zhang [email protected] * These authors contributed equally to this work. 2019 3 5 2019 18 9 10011018 5 1 2019 19 3 2019 25 3 2019 © 2019 Informa UK Limited, trading as Taylor & Francis Group 2019 Informa UK Limited, trading as Taylor & Francis Group ABSTRACT NF-κB is a core transcription factor, the activation of which can lead to hypoxic-ischemic brain damage (HIBD), while RCAN1 plays a protective role in HIBD. However, the relationship between NF-κB and RCAN1 in HIBD remains unclear. This study aimed to explore the mechanism of NF-κB signaling pathway in hippocampal neuron apoptosis and cognitive impairment of neonatal rats with HIBD in relation to RCAN1. Initially, microarray analysis was used to determine the differentially expressed genes related to HIBD. After the establishment of HIBD rat models, gain- or loss-of-function assay was performed to explore the functional role of NF-κB signaling pathway in HIBD. Then, the learning and memory ability of rats was evaluated. Expression of RCAN1, NF-κB signaling pathway-related genes and glial fibrillary acidic protein (GFAP), S-100β and acetylcholine (Ach) level, and acetylcholinesterase (AchE) activity were determined with neuron apoptosis detected to further explore the function of NF-κB signaling pathway. RCAN1 could influence the development of HIBD. In the HIBD model, the expression of RCAN1 and NF-κB-related genes increased, and NF-κB p65 showed a significant nuclear shift. By activation of NF-κB or overexpression of RCAN1, the number of neuronal apoptosis, S-100β protein level, and AchE level increased significantly, Ach activity decreased significantly, and GFAP positive cells increased. In addition, after the activation of NF-κB or overexpression of RCAN1, the learning and memory ability of HIBD rats was inhibited. All the results show that activation of NF-κB signaling pathway promotes RCAN1 expression, thus increasing neuronal apoptosis and aggravating cognitive impairment in HIBD rats. KEYWORDS RCAN1 NF-κB signaling pathway hypoxic-ischemic brain damage neuronal apoptosis cognitive impairment the Foundation of Science and Technology Department of Guizhou ProvinceQiankehe SY zi [2012] 001 the Foundation of Science and Technology Department of Guizhou ProvinceQiankehe SY zi [2012] 3090 National Key Technology R&D Program2014BAI05B05 Foundation of Science and Technology Department of Sichuan ProvinceChuanrenshebanfa (2017) 919-26 This study was supported by the Foundation of Science and Technology Department of Guizhou Province (Qiankehe SY zi [2012] 001), the Foundation of Science and Technology Department of Guizhou Province (Qiankehe SY zi [2012] 3090), the National Key Technology R&D Program (2014BAI05B05), the Foundation of Science and Technology Department of Sichuan Province (Chuanrenshebanfa (2017) 919-26). ==== Body Introduction Hypoxic–ischemic brain damage (HIBD) is a disease which can induce inﬂammatory lung injury [1]. It is also one of the major causes of death and long-term neurological impairment in infants and children [2]. Hypoxic-ischemic encephalopathy induced by HIBD is a severe brain disorder for children but without effective treatment [3]. It has been proved that the pathological mechanisms of HIBD are involved in factors like oxidative stress, inﬂammation and nerve cell apoptosis [4,5]. It requires extensive resources and wealth for the treatment and care of the sequelae of HIBD, and there is frequently little improvement in the overall ability of children with the disease even with the best care [6]. Therefore, it is necessary to elucidate the molecular mechanism underlying HIBD to explore a treatment regimen for HIBD. Nuclear factor κB (NF-κB) is involved in many types of tumors in which it plays a confounding role [7]. Moreover, as a core transcription factor of many signaling pathways, NF-κB also plays an important role in cell survival and apoptotic cell-death via regulating the relative expression of selected genes [8]. A previous study has proved that when NF-κB signaling pathway is inhibited, the apoptosis of damaged nerve cells can be reduced resulting in an improvement of learning-memory function in HIBD rats [9]. Moreover, the regulator of calcineurin 1 (RCAN1) has been proved to be regulated by NF-κB [10]. RCAN1 can regulate the activity of calcineurin phosphatase and suppress inflammation [11]. Recently, evidence suggests that RCAN1 is highly expressed around the infarct area after experimental stroke and are associated with brain ischemia/reperfusion injury [12]. Overexpression of RCAN1 has been proved to induce caspase-9 and caspase-3 and subsequently promotes neuronal apoptosis in primary neurons in Alzheimer disease [13]. Based on these findings, it is of great importance to study the exact roles of NF-κB signaling pathway and RCAN1 in the treatment of HIBD. Thus, in this study, we aim to explore the hypothesis that RCAN1 down-regulation by the inhibition of the NF-κB signaling pathway can suppress the apoptosis of hippocampal neurons and improve cognitive impairment of rats with HIBD. Materials and methods Ethics statement This study was approved by the ethics committee of Guizhou Provincial People’s Hospital and was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals. Best efforts were made to minimize the sufferings of animals. Microarray analysis The HIBD microarrays, GSE2161 and GSE11686, were retrieved from the National Center for Biotechnology information (NCBI). The data of the microarrays were downloaded from the Gene Expression Omnibus (GEO) database (http://www.ncbi.nlm.nih.gov/geo). GSE2161 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE2161) included four HIBD samples and four normal samples with annotation probe [Mouse430_2] Affymetrix Mouse Genome 430 2.0 Array (GPL1261). GSE11686 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc = GSE11686) included 6 HIBD samples and 10 normal samples with annotation probe [HG-U133A] Affymetrix Human Genome U133A Array (GPL96). The “limma” package (http://www.bioconductor.org/packages/release/bioc/html/limma.html) in the R Language Programming based on Bioconductor was used to select important differentially expressed genes through Empirical Bayes [14]. At last, the differentially expressed genes were annotated using the “annotate” package (http://www.bioconductor.org/packages/release/bioc/html/annotate.html). Differentially expressed mRNAs were judged by the threshold of p< 0.05 and \|Log2FoldChange\| >1.5. HIBD model establishment in rats Sixty healthy Sprague-Dawley (SD) rats (no limitation with gender; aged 7 days; weighing 12–20 g), provided by Experimental Animal Center of Guizhou University, were selected and randomly divided into two groups: the sham group (n = 10) and the HIBD group (n = 50). Artery ligation improved by Nakajima et al. [15] was conducted to establish hypoxia-ischemia animal models at room temperature of 20°C ± 5°C after all animals were weighed preoperatively. The neonatal rats were anesthetized with ether and placed in a supine position with limbs fixed on a small operation board. After the skin was sterilized with 75% alcohol, an incision was made in the midline of the neck, and the left common carotid artery was aseptically isolated and ligated twice with a 7–0 sterile surgical silk. The wound was sutured and the skin was disinfected again. The animals were allowed to recover in an incubator at 32.5°C for 2 h. The surgical rat was then placed in a 2 L airtight jar, and the bottom layer was covered with sodium lime (to absorb CO2 and moisture). The jar was submerged in a 37°C water bath to maintain a constant thermal environment. A mixture of 8% oxygen and 92% nitrogen were introduced at a rate of 1 to 2 L/min, and the internal oxygen concentration was measured with an oxygen meter to maintain the oxygen concentration in the hypoxic chamber at about 8% (7% ~ 9%) with continuous hypoxia kept for 2 h. After successful establishment of HIBD model, the rats were placed in air at room temperature for about 1 ~ 2 h, and finally put back into the dams for breastfeeding. The entire procedure was completed within 40 min and the anesthetic time should not be too long. Intraoperative procedures should be refined to minimize bleeding and to avoid stimulating the surrounding tissue. After surgery, the bloodstains of neonatal rats should be wiped off to prevent the dams from refusing to breastfeed because of strange smells. Sham-operated rats underwent the same operative procedures except that the exposed carotid artery was not ligated. In addition, after the skin was sutured, no hypoxic treatment was performed, and the rats were returned to the dams at the same time as the model group. Heart perfusion and sectioning The state of consciousness, general behavior, and physical activity of the rats were observed before the experiment, after the anesthesia, after the surgery, during the hypoxia, and at the end of the hypoxia. At the 7th d after establishment of HIBD model, three rats in the sham group or the HIBD group were randomly selected for anesthesia. The rats were fixed on a surgical plate to open the chest cavity. A 20 mL sterile medical syringe attached to a puncture needle was carefully inserted to the left ventricle of the neonatal rat, and the right atrial appendage of the rat was cut, which was injected with sterile saline continuously until the effluent fluid became clear and then injected with about 10 to 20 mL of 4% paraformaldehyde. After the limbs and liver of rats became white and hardened, the brain was extracted, observed with naked eyes and externally fixed in paraformaldehyde. The section was prepared vibrantly. The structure, neuronal morphology and the arrangement of the rat cerebral cortex and hippocampus were observed by the Nissl staining under a normal optical microscope. Nissl staining The paraffin-embedded sections were routinely dewaxed and immersed in a 1% toluidine blue solution for 10 min at 37°C. The excess staining solution was washed away with distilled water. The sections were color-saturated by 80–95% gradient alcohol to an appropriate rate and observed under a microscope. After the cytoplasmic staining could be clearly compared, the sections were dehydrated with anhydrous alcohol, cleared with transparent xylene, and sealed with neutral balsam. Then the morphological changes of neurons in the hippocampus were observed under the microscope. Grouping Besides the sham group (n = 10) and the HIBD group (n = 10), the remaining successfully modeled rats were randomly divided into the following groups: Phorbol-12-myristate-13-acetate (PMA) group: PMA (ab120297, Abcam Inc., Cambridge, MA, USA) was dissolved in dimethyl sulfoxide (DMSO) with the final concentration 100 mM and intraperitoneally injected into the HIBD rats (n = 10); BAY-11–7805 group: The NF-κB inhibitor BAY-11–7805 (ab141574, Abcam Inc., Cambridge, MA, USA) was dissolved in DMSO with the final concentration 10 μM and injected intraperitoneally into the HIBD rats (n = 10); si-RCAN1 group: RCAN1 AAV siRNA (iAAV05614009, ABM Inc., Richmond, BC, Canada) packaged viruses were injected into the left hippocampus of successfully modeled HIBD rats by stereo injection (n = 10); PMA + si-RCAN1 group: PMA and RCAN1 AAV siRNA packaged viruses were injected stereoscopically into the successfully modeled HIBD rats (n = 10). Test of learning and memory ability The Morris water maze (MWM) test and swimming training were performed when rats were 1-month-old to evaluate the spatial learning and memory of rats. Before the day of training, the platform was removed, and a fixed point was selected on the wall of the pool. The rats swam into the water in the direction of facing the pool wall, and swimming trajectory were recorded. The purpose was to allow the rats to adapt to the environment, to perform preliminary tests on the swimming ability of the rats and to eliminate those who have obvious abnormalities. The navigation test was adopted to test the ability of rats to learn and memorize the water maze. The rats were continuously trained. Each day, the rats were placed into the water from different fixed points facing the pool wall. In each experiment, the time that the rat sought and boarded the platform (escape latency) was recorded as the learning and memory performance. If the rat failed to find the platform within the set time, the computer would stop tracking and record the time. Rats failing to find the platform would be brought to the platform by the experimenter. The training interval (after a training session, the rats were allowed to rest on the platform for a while) was very important for rats that were highly stressed in water. It can prevent rats from jumping back into the water and guarantee the success of the experiment. After the day’s training, the rats would be washed, dried and placed next to the heater to prevent them from hypothermia. Then, the average escape latency for each group of rats was calculated. And the spatial probe trail was used to detect rats‘ exact memory of the spatial location of the platform after the rats learn to search for the platform, namely the memory retention ability. After the navigation test (the 5th d of MWM test), the platform was withdrawn, and then a fixed quadrant was selected randomly as the entry and the rat was placed here to swim into water in the direction of facing the pool wall. The swimming trajectory within 120 s and the times of crossing the original platform position was recorded. At the end of the spatial probe trail, after rats rested for 1 d (the 6th d of MWM test), a water maze test was performed again on all rats. The method was the same as the spatial probe trail to exam the ability of long-term memory retention. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) The total RNA was extracted by the Trizol one-step method, and the microplate reader (DNM-9606, Beijing Perlong New Technology Co., Ltd., Beijing, China) was used to determine the purity and concentration of the sample RNA. The RNA was reversely transcribed into cDNA Using the PrimeScriptTM RT-PCR Kit (Perfect Real-Time) Kit (RR047A, Takara Bio Inc., Otsu, Shiga, Japan), which was used as a template. The expression of RCAN1 was determined using β-actin as an internal control. The reverse transcription reaction conditions were: at 37°C for 60 min, and at 85°C for 5 min. The relative expression of mRNA was measured by using the SYBR Green Real-time fluorescence quantitative PCR kit (1725270, Bio-Rad, Hercules, CA, USA) on the Bio-Rad 9700 instrument (10021337, Bio-Rad, Hercules, CA, USA), and the experiment was repeated 3 times. The RT-qPCR system was 25 µL, including 21 μL of 1 × SYBR premix Ex Taq mix (Takara Bio Inc., Otsu, Shiga, Japan), 2 μL of cDNA and 1 μL of (10 nM) forward and reverse primers, respectively. The reaction conditions were as follows: pre-denaturation at 95°C for 3 min, 45 cycles of denaturation at 95°C for 15 s, annealing at 60°C for 20 s and extension at 72°C for 30 s. the mRNA level in each group was calculated using the 2−ΔΔCt method [16]. The used primers are shown in Table 1.10.1080/15384101.2019.1608128-T0001 Table 1. Primer sequences for RT-qPCR. Gene Sequences (5ʹ – 3ʹ) RCAN1 F: GACTGGAGCTTCATCGACTGC R: CCCAGGAACTCGGTCTTGT β-actin F: AGCTGAGAGGGAAATCGTGC R: ACCAGACAGCACTGTGTT Note: RT-qPCR: reverse transcription-quantitative polymerase chain reaction; RCAN1: regulator of calcineurin 1; F: forward; R: reverse. Western blot analysis The total protein was extracted from the tissue homogenate. The lysate was collected after centrifugation at 4°C and 45 × g for 15 min. The protein concentration was determined by the Bradford method. Then, 30 μg of the sample protein was obtained and mixed well with immobilized pH gradient (IPG) loading buffer and underwent electrophoresis with 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel for 2 h. The protein on the gel was transferred onto a polyvinylidene fluoride (PVDF) membrane. After being blocked with 5% skimmed milk for 2 h at room temperature, the membrane was washed 3 times with tris-buffered saline with tween (TBST) buffer, and incubated with rabbit anti-rat NF-κB p65 polyclonal antibody (ab16502, 1: 500), NF-κB phosphorylated (p)-p65 polyclonal antibody (ab86299, 1: 2000), RCAN1 monoclonal antibody (ab185931, 1: 500) and β-actin monoclonal antibody (ab8227, 1: 2000) overnight at 4°C. β-actin was used as an internal reference. Then, the membrane was washed three times with TBST buffer and then incubated with goat anti-rabbit secondary antibody (ab6721, 1: 2000) at room temperature for 2 h. Subsequently, the membrane was washed 3 times with TBST buffer. The primary and secondary antibodies were all obtained from Abcam Inc. (Cambridge, UK). The color was developed by Electro-Chemi-Luminescence (ECL) method. After tableting and computer scanning, the gray value of the target bands was measured by the image analysis software Image J (National Institutes of Health, Bethesda, Maryland, USA). The relative expression of the target proteins was expressed by the ratio of the gray value of the target protein bands to that of the reference protein band. Statistical mapping was performed and the experiment was repeated 3 times. Terminal deoxynucleotidyl transferase (TdT)-mediated dUTPbiotin nick end labeling (TUNEL) staining The apoptotic cells in hippocampus tissues of rats in each group were detected by TUNEL staining and counted. The hippocampus tissues were dehydrated by gradient alcohol, cleared by xylene, immersed in wax, embedded in paraffin, and cut into sections. The continuous coronal sections were obtained by the paraffin machine from the chiasma opticum to the posterior horn of the lateral ventricle and loaded to the slides treated by polylysine. The sections were dewaxed with xylene for 10 min, immersed in 100–75% gradient alcohol for 1 min, respectively, and soaked in distilled water for 1 min. The sections were washed twice with 0.01 M phosphate buffer saline (PBS) for 5 min each time. Proteinase K was used for detachment at 37°C for 20 min. The sections were washed twice with 0.01 M PBS for 5 min each time, soaked in 0.3% H2O2-methanol for 20 min at room temperature and washed twice with 0.01 M PBS with 5 min each time. The terminal deoxyribonuclease reaction solution was prepared when the cells were in equilibrium. The compositions in each well were 45 μL balance buffer, 5 μL nucleotide mixture and 1 μL terminal deoxyribonuclease. After the equilibrium solution was sucked out, each well was incubated with 50 μL of terminal deoxyribonuclease reaction solution at 37°C for 1 h. After 20 × SSC was diluted with water to 2 × SSC, each well was added with 50 μL of 2 × SSC to terminate the reaction, allowed to stand for 15 min at the room temperature under the environment void of light. The sections were washed with PBS 3 times, incubated with 1 μL/mL DAPI for 15 min at the room temperature under the environment void of light, and washed with PBS 3 times. The sections were added with anti-fade solution. Under the fluorescence microscope, the cells were observed under the excitation luminescence at 488 nm and 405 nm wavelength and photographed. About 8–10 visual fields were randomly selected and at least 100–200 cells in each visual field were counted. The number of apoptosis cells was counted, and the apoptosis rate was analyzed. The experiment was repeated three times. Enzyme-linked immunosorbent assay (ELISA) The soluble protein-100β (S-100β) content in the brain tissues was determined by the rat S-100β protein ELISA kit (R&D Systems, Minneapolis, MN, USA). The tissue homogenate (10%, 1000 μL) was centrifuged by a high-speed cryogenic centrifuge at 2863 × g at 4°C for 10 min, and the supernatant was obtained. The quantitative standard substance was added with 2 mL of distilled water to prepare a 20 ng/mL standard solution. Eight standard tubes were set-up. The first standard tube was added with 900 μL of the sample dilution, and the second to the seventh tubes were added with 500 μL sample dilution. Standard solution (100 μL, 20 ng/mL) was added to the first tube and mixed, and 500 μL of the mixture was aspirated with a pipette and transferred into the second tube. Dilutions were repeated until 500 μL of the mixture was removed from the 7th tube. The 8th tube was a blank control. The 10 × sample dilution was diluted with distilled water at the ratio of 1: 10, and the 20 × concentrated washing buffer were diluted with double distilled water at the ratio of 1: 20. Each tube was added with 100 μL of standard or test sample. After being mixed thoroughly, the plate was incubated at 37°C for 120 min. The subsequent operations were carried out following the kit instructions. A standard curve was plotted with standards of 2000, 1000, 500, 250, 62.5, 31.2 and 0 pg/mL as the abscissa and optical density (OD) as the ordinate. According to the OD value of the sample on the graph, the corresponding S-100β content was calculated. Colorimetry Colorimetry was applied to determine the levels of acetylcholine (Ach) and acetylcholinesterase (AchE) activity in the rat hippocampus. The tissue homogenate (10%) was centrifuged at 2863 × g for 10 min at 4°C and the supernatant was obtained. According to the instructions of the kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, Jiangsu, China), the sample tubes, standard tubes, blank tubes and control tubes were prepared. The mixture in the tube was centrifuged were mixed and centrifuged at 2863 × g for 5 min at 4°C to obtain the supernatant. The OD value of Ach was measured with the spectrophotometer, and λ was set to 550 nm. The supernatant was placed in a slit cuvette and zeroed with distilled water, and the OD value of each tube was measured. The Ach level of the sample was calculated. The OD value of AchE was measured with the same procedure except for λ was set to 412 nm. Immunofluorescence staining and laser confocal scanning The samples were sectioned and put in a 60°C incubator for 20 min. The sections were dewaxed, added into 200 mL of ethylenediaminetetraacetic acid (EDTA) solution, steamed in a high-pressure cook for 10 min, and immersed in 3% H2O2-methanol for 10 min. Afterwards, the sections were incubated with rabbit anti-rat glial fibrillary acidic protein (GFAP, 1: 400, Beijing Bioss Biotechnology Co., Ltd., Beijing, China) and rabbit anti-rat NF-κB p65 polyclonal antibody (ab16502, 1: 500, Abcam Inc., Cambridge, UK) at 4°C overnight. After washing with PBS, the sections were added with CY-3-conjugated goat anti-rabbit Immunoglobulin G (IgG) and incubated in a wet box at 37°C for 30 min under the environment void of light. After PBS washing under the environment void of light, 4’,6-diamidino-2-phenylindole (DAPI) solution was added to counterstain the nucleus for 30 s under the environment void of light at 37°C. And after PBS washing under the environment void of light and distilled water washing, the sections were sealed with 50% buffer glycerol. Except for sealing, after each step was completed, the samples were washed with PBS three times, 10 min each time. Sealing, antibody incubation, and rinsing steps were all performed on a shaker. Finally, the brain sections were spread on the slides. The hippocampal CA1 region was observed, and the number of GFAP immune-positive cells in three adjacent fields per section was counted, and the results were averaged from five fields. After the calculation of the number of GFAP positive cells, the nuclear shift of NF-κB p65 was observed using the NIS-Elements Viewer 4.0 software under a laser confocal microscope, and the emission wavelengths of CY-3 and DAPI were 565 nm and 460 nm, respectively. Statistical analysis The statistical analyses were conducted by the SPSS17.0 statistical software (SPSS Inc., Chicago, IL, USA). The experimental data were expressed as mean ± standard deviation. The escape latency among multiple groups was analyzed by repeated measures analysis of variance (ANOVA). The times of crossing platforms and the long-term memory latency in the spatial probe trial were analyzed by one-way ANOVA. Pairwise comparison within group was analyzed by means of Least Significant Difference (LSD) test. Data between two groups were compared by independent sample t test, with Bonferroni method for correction analysis. p < 0.05 was considered statistical significance. Results RCAN1 is related to the development of HIBD GSE2161 and GSE11686 microarrays were downloaded from the GEO database. From the GSE2161 microarray, 2094 differentially expressed genes were screened out, among which 985 differentially expressed genes were upregulated and 1109 were downregulated. From the GSE11686 microarray, 241 differentially expressed genes were screened out with 106 genes were upregulated and 135 were downregulated. Previous studies suggest that RCAN1-encoded protein interacts with calcineurin A, inhibits phosphatase-dependent signaling pathway, and influences the central nervous system development, such as HIBD [17,18]. RCAN1 has been reported to serve as an oncogene in many cancers [19,20]. However, the specific effect of RCAN1 on HIBD is elusive. With RCAN1 as a candidate gene, we aimed to explore the relevance of RCAN1 to drug in HIBD and its clinical significance. Among the differentially expressed genes, RCAN1 has a high rank. Through the expression heatmaps of the first 50 differentially expressed genes from GSE2161 microarray (Figure 1(a)) and GSE11686 microarray (Figure 1(b)), we found that RCAN1 existed in the first 50 differentially expressed genes of both GSE2161 and GSE11686 microarrays and that RCAN1 was highly expressed in these two microarrays, especially in GSE2161 microarray. Moreover, the intersection of the first 50 differentially expressed genes of the microarrays were obtained, suggesting that only RCAN1 existed in the intersection (Figure 1(c)). The NF-κB signaling pathway could cure neuroglioma by regulating RCAN1 gene [18,21]. Thus, we hypothesized that the NF-κB signaling pathway could influence HIBD by regulating RCAN1 gene.10.1080/15384101.2019.1608128-F0001 Figure 1. RCAN1 is involved in HIBD. (a) and (b), the expression heatmaps of the first 50 differentially expressed genes of GSE2161 and GSE11686 microarrays, in which the abscissa refers to sample number, the ordinate refers to gene name, the upper dendrogram represents clustering of sample type, and the right color histogram represents gene expression. Each circle refers to the expression of a gene in a sample. The left dendrogram shows the clustering of gene expression; (c), RCAN1 is determined as the only gene existed in the first 50 differentially expressed genes of both GSE2161 and GSE11686; NF-κB: nuclear factor-kappa B; HIBD: hypoxic-ischemic brain damage; RCAN1: regulator of calcineurin 1. Successful establishment of HIBD model The Nissl-stained tissue sections were observed under the microscope to detect the pathological changes in the sham group and the HIBD group. The study found that there was no obvious neuronal damage on both sides of the section in the sham group. The morphology and structure of the cells were clear and complete, the nucleolus was clear, and the Nissl bodies without vacuoles were evenly distributed around the nucleus in order. It was demonstrated that in the HIBD group obvious pathological changes on the left cerebral hemisphere modeling side were observed, showing a large number of nuclear pyknosis, nuclear debris. Nissl body shape blurred or disappeared with vacuolar formation, and Nissl body was disorderly arranged forming a net. Compared with the sham group, the morphology of pyramidal cells in the HIBD group was changed significantly with disordered cell arrangement, significantly reduced volume, concentrated cytoplasm, slightly stained Nissl bodies, and deformed or disappeared nucleus. There were apparent cell death and cell loss with normal pyramidal cells distributing among the dead cells (Figure 2). Therefore, the HIBD model was successfully established.10.1080/15384101.2019.1608128-F0002 Figure 2. The HIBD model is established which was identified by Nissl staining (400 ×). HIBD, hypoxic-ischemic brain damage. RCAN1 is highly expressed and the Nf-κB signaling pathway is activated in HIBD rats The mRNA and protein levels of RCAN1 and the NF-κB signaling pathway-related factors in hippocampal tissues were determined by RT-qPCR and Western blot analysis. The results showed that compared with the sham group, the HIBD group had significantly increased mRNA and protein levels of RCAN1 and the protein level of NF-κB p65 (all p< 0.05) (Figure 3), suggesting that RCAN1 and NF-κB p65 were highly expressed in the HIBD rats.10.1080/15384101.2019.1608128-F0003 Figure 3. Higher expression of RCAN1 and NF-κB p65 are identified in HIBD models. (a), the mRNA levels of RCAN1 in hippocampal tissues detected by RT-qPCR; (b), the gray value of RCAN1, NF-κB p65 and NF-κB p-p65 protein bands; (c), the protein levels of RCAN1, NF-κB p65 and NF-κB p-p65 in hippocampal tissues; , p< 0.05 vs. the sham group; NF-κB, nuclear factor κB; RCAN1: regulator of calcineurin 1; HIBD: hypoxic-ischemic brain damage; p-p65: phosphorylated p65. Activation of Nf-κB signaling pathway upregulates RCAN1 expression RT-qPCR and Western blot analysis were used to detect the expression of RCAN1 with activation of NF-κB signaling pathway. Compared with the sham group, the mRNA and protein levels of RCAN1 in the HIBD group, the PMA group, and the PMA + si-RCAN1 group were significantly increased (all p < 0.05), whereas in the BAY-11-7805 and si-RCAN1 groups, the expression of RCAN1 was not statistically different (all p > 0.05). Compared with the HIBD group, the mRNA and protein levels of RCAN1 in the PMA group were significantly increased (p < 0.05), whereas in the BAY-11–7805 group, si-RCAN1, and PMA + si-RCAN1 groups, the mRNA and protein levels of RCAN1 were significantly decreased (all p < 0.05) (Figure 4). The results demonstrated that the activation of NF-κB signaling pathway up-regulated the expression of RCAN1.10.1080/15384101.2019.1608128-F0004 Figure 4. Activation of NF-κB signaling leads to upregulation of RCAN1. (a), the mRNA level of RCAN1 in response to the treatment of PMA, BAY-11–7805, si-RCAN1, PMA + si-RCAN1; (b), the gray value of RCAN1 protein band in response to the treatment of PMA, BAY-11–7805, si-RCAN1, PMA + si-RCAN1; (c), the protein level of RCAN1 in response to the treatment of PMA, BAY-11–7805, si-RCAN1, PMA + si-RCAN1; , p < 0.05 vs. the sham group; #, p < 0.05 vs. the HIBD group; NF-κB: nuclear factor κB; RCAN1: regulator of calcineurin 1; HIBD: hypoxic-ischemic brain damage. Silencing RCAN1 has no significant effect on the NF-κB signaling pathway The subcellular localization of NF-κB p65 was detected by immunofluorescence confocal laser scanning (Figure 5(a)). Blue was a DAPI marker indicating the nuclear region; red was a CY-3 marker indicating NF-κB p65; two fluorescent confocal overlapped into pink, indicating the translocation of NF-κB p65 into the nucleus. The observation of hippocampal tissues in the sham group revealed that a small amount of NF-κB p65 always presented in the cytoplasm, with no apparent nuclear translocation, and was mainly located in the cytoplasm. Compared with the sham group, in the HIBD, PMA, si-RCAN1, and PMA + si-RCAN1 groups, significant amount of NF-κB p65 was observed moving from cytoplasm to the nucleus, and the protein levels also increased significantly (all p < 0.05), while there was no significant difference in the BAY-11-7805 group (p > 0.05). Compared with the HIBD group, the PMA group, and the PMA + si-RCAN1 group appeared a large amount of NF-κB p65 moving from cytoplasm to the nucleus, and the relative protein expression was also significantly increased (all p < 0.05). However, in the si-RCAN1 group, NF-κB p65 had no significant sign of nuclear shift, and there was no statistical difference in the number of positive cells and the relative protein expression (p > 0.05). In the BAY-11-7805 group, NF-κB p65 showed no significant nuclear shift (p > 0.05), but the relative protein expression and the number of positive cells decreased (p < 0.05) (Figure 5(b–d)). It was suggested that the activation of NF-κB signaling pathway up-regulated the expression of NF-κB p65, but RCAN1 silencing had no significant effect on the NF-κB signaling pathway.10.1080/15384101.2019.1608128-F0005 Figure 5. Inhibition of the NF-κB signaling pathway down-regulated NF-κB p65 while silencing RCAN1 had no significant effect on the NF-κB signaling pathway. (a), Immunofluorescence copolymerization for detection of NF-κB p65 expression in hippocampus (400 ×); (b), the number of positive cells of NF-κB p65; (c), the gray value of NF-κB p65 and NF-κB p-p65 protein band in response to the treatment of PMA, BAY-11-7805, si-RCAN1, PMA + si-RCAN1; (d), the relative expression of NF-κB p65 and NF-κB p-p65 protein; , p < 0.05 vs. the sham group; #, p < 0.05 vs. the HIBD group; NF-κB: nuclear factor κB; RCAN1: regulator of calcineurin 1; HIBD: hypoxic-ischemic brain damage. Activation of the NF-κB signaling pathway increases GFAP expression in glial cells of the hippocampus The number of GFAP-positive cells with activation of the NF-κB signaling pathway was measured by immunohistochemical staining. After immunohistochemical staining of rat hippocampal tissue, it was found that GFAP-positive cells were stained with green. Compared with the sham group, the number of positive cells in the HIBD, PMA, si-RCAN1, and PMA + si-RCAN1 groups increased significantly (all p < 0.05), whereas in the BAY-11-7805 group, GFAP positive cells showed no statistical difference between the two groups (p > 0.05). Compared with the HIBD group, the number of positive cells in the BAY-11–7805 group and the si-RCAN1 group decreased significantly, while it increased significantly in the PMA group (Figure 6). All these results showed that with the activation of the NF-κB signaling pathway, the GFAP expression in glial cells of the hippocampus was elevated.10.1080/15384101.2019.1608128-F0006 Figure 6. Activation of NF-κB signaling pathway upregulates GFAP in glial cells of the hippocampus. (a), Positive cell immunofluorescence analysis of GFAP expression in hippocampus of rats in each group (200 ×); (b), GFAP positive cells in hippocampus in each group; , p < 0.05 vs. the sham group; #, p < 0.05 vs. the HIBD group; NF-κB: nuclear factor κB; GFAP: glial fibrillary acidic protein. Inhibition of NF-κB and RCAN1 silencing reduces the neuronal apoptosis The changes of neuronal apoptosis with inhibition of NF-κB and RCAN1 silencing were observed by TUNEL staining. Under a light microscope, there was no obvious apoptotic cell was observed in the hippocampal tissues of the sham group. Compared with the sham group, the HIBD, PMA, si-RCAN1 and PMA + si-RCAN1 groups showed different degrees of apoptosis. As shown in Figure 7, visible bright red fluorescence increased, and the number of apoptosis was also significantly increased (p < 0.05). Compared with the HIBD group, the apoptosis of the PMA group was significantly increased (p < 0.05), while the number of apoptosis cells in the BAY-11-7805 group and the si-RCAN1 group was significantly decreased (p < 0.05), and there was no statistical difference in the PMA + si-RCAN1 group (p > 0.05). All these results suggested that inhibition of NF-κB and silencing of RCAN1 reduced the neuronal apoptosis.10.1080/15384101.2019.1608128-F0007 Figure 7. RCAN1 gene silencing or inhibition of the NF-κB signaling pathway inhibits the neuronal apoptosis in rats with HIBD. (a), TUNEL staining reflects the apoptotic neurons in the hippocampus (200 ×); (b), the apoptosis rate of neurons in the hippocampus; , p < 0.05 vs. the sham group; #, p < 0.05 vs. the HIBD group; NF-κB: nuclear factor κB; RCAN1: regulator of calcineurin 1; HIBD: hypoxic-ischemic brain damage. Inhibition of NF-κB and RCAN1 silencing decreases the levels of S-100β and AchE but increases Ach activity ELISA and colorimetry were applied to determine the levels of S-100β, Ach and AchE in the rat hippocampus. Compared with the sham group, S-100β protein content and AchE activity were significantly increased but the Ach activity was significantly decreased in the HIBD group, the PMA group, the si-RCAN1 group, and the PMA + si-RCAN1 group (all p < 0.05), and there was no statistical difference in BAY-11-7805 group (all p > 0.05). Compared with HIBD group, S-100β protein content and AchE activity significantly decreased in the BAY-11-7805 and si-RCAN1 groups (all p < 0.05) and increased in the PMA group, while the Ach activity increased significantly in the BAY-11–7805 and si-RCAN1 groups, but significantly decreased in the PMA group (all p < 0.05), and there was no statistical difference in the PMA + si-RCAN1 group (all p > 0.05) (Figure 8). Based on these results, it was found that inhibition of NF-κB and RCAN1 silencing decreased the levels of S-100β and AchE but increased the Ach activity.10.1080/15384101.2019.1608128-F0008 Figure 8. Activated NF-κB signaling pathway elevates the levels of S-100β and AchE but reduces Ach activity. (a), the relative expression level of S-100β; (b), the relative expression level of Ach; (c), the relative expression level of AchE; , p < 0.05 vs. the sham group; #, p < 0.05 vs. the HIBD group; NF-κB: nuclear factor κB; RCAN1: regulator of calcineurin 1; HIBD: hypoxic-ischemic brain damage; S-100β: soluble protein-100β; AchE: acetyl cholinesterase; Ach: acetylcholine. The NF-κB signaling pathway correlates to long-term learning ability of rats The MWM test was used to test the learning and memory ability of rats in each group (Figure 9). In each group, on the first day of swimming training, it was found that all rats had normal swimming ability. In the spatial probe trail, the escape latency of neonatal rats in each group changed with time, and the trend decreased most obviously in the sham group, and the memory remained good on the 6th d. From the 2nd d, compared with the sham group, the differences in the HIBD, PMA, si-RCAN1, and PMA + si-RCAN1 groups gradually increased with time. Compared with the HIBD group, PMA, si-RCAN1 and BAY-11-7805 groups differed greatly, and the difference was most obvious at the 6th d (p < 0.05). It was suggested that hypoxia-ischemia impairs the spatial learning acquisition ability and recent memory ability of rats, which was related to the NF-κB signaling pathway. On the 6th d of training, rats in the sham group still quickly found a hidden platform with a short swimming trajectory and a clear goal, while the swimming trajectories in the HIBD, PMA, si-RCAN1, and PMA + si-RCAN1 groups were disorganized. Compared with the HIBD group, there were significant differences in the PMA, si-RCAN1, and BAY-11-7805 groups, suggesting that the rats lost their ability to locate and maintain memory after HIBD, and their long-term memory deficits were associated with the NF-κB signaling pathway. On the 5th day of the MWM test, rats in the sham group crossed the platform significantly more than those in the HIBD, PMA, si-RCAN1 and PMA + si-RCAN1 groups. Compared with the HIBD group, there were significant differences in the PMA, si-RCAN1 and BAY-11-7805 groups (p < 0.05). It was demonstrated that the sham-operated rats had better spatial memory retention and spatial localization accuracy than the HIBD rats, which was associated with the NF-κB signaling pathway.10.1080/15384101.2019.1608128-F0009 Figure 9. The NF-κB signaling pathway inhibition and RCAN1 silencing partially restore the long-term learning ability of rats induced by HIBD. (a), the escape latency changes in different groups of rats at the 1st d, 2nd d, 3rd d, 4th d, 5th d and 6th d; (b), the time of crossing the platform in each group; *, p < 0.05 vs. the sham group; #, p < 0.05 vs. the HIBD group; NF-κB: nuclear factor κB; RCAN1: regulator of calcineurin 1; HIBD: hypoxic-ischemic brain damage. Discussion HIBD is one of the main causes of infant mortality and neurologic damage including cognitive impairment and mental retardation [22]. More and more evidence shows that the increase of apoptosis plays an important role in HIBD, which has led to an in-depth study of the mechanism of targeted therapy for HIBD [23]. Therefore, how to inhibit cell apoptosis in patients with HIBD is of great importance. Previous findings have shown that when NF-κB is activated in different situations, it can inhibit or promote cell apoptosis [24]. Besides, it has also been proved that RCAN1 is involved in neuronal apoptosis and the abnormal expression of RCAN1 can induce neurological disease [25]. Thus, aiming to determine the role of NF-κB signaling pathway and RCAN1 in apoptosis of hippocampal neurons and cognitive impairment, this study found that RCAN1 down-regulated by inhibition of NF-κB signaling pathway suppressed the apoptosis of hippocampal neurons and improves the cognitive impairment in rats with HIBD. Initially, our results found that RCAN1 was overexpressed and that the NF-κB signaling pathway was activated in HIBD rats. Furthermore, we also observed downregulation of RCAN1 after treatment of the NF-κB signaling pathway inhibitor. A previous study shows that in HIBD, NF-κB was activated [8]. Besides, it has also been proved that in rats with hypoxic-ischemic encephalopathy, the level of NF-κB p65 was significantly higher [26]. Moreover, a study reveals the overexpression of RCAN1-4 in brain ischemia/reperfusion (I/R) injury [12]. In line with our findings, a previous study has shown that the NF-κB signaling pathway can up-regulate RCAN1 [10]. More specifically, it has been proved that overexpression of RCAN1 can induce neuronal apoptosis, but when the NF-κB signaling pathway is inhibited, the expression of RCAN1 is down-regulated [27]. Furthermore, this study showed that the inhibition of NF-κB signaling pathway or silencing of RCAN1 reduced the apoptosis of hippocampal neurons in HIBD rats, which was indicated by the decrease of S-100β and AchE expression and increase of Ach activity. A previous study shows that S-100β protein was closely associated with HIBD, therefore the level of S-100β is of predictive values in patients with HIBD [28]. Another study points out that apoptosis may be associated with increased level of AchE [29]. Ach is a major neurotransmitter which is closely related to the inflammatory response [30]. And it has been found that Ach has the ability to suppress apoptosis induced by Fas [31]. As a transcription factor, NF-κB can control the expression of many genes which play a dominating role in cell programmed death and cell survival [32]. Consistent with our study, a previous research verifies that in neurons with hippocampal damage, the NF-κB signaling pathway is activated and the expression of NF-κB is increased [33]. Furthermore, another study confirms that the inhibition of the NF-κB signaling pathway leads to suppressed apoptosis of hippocampal neurons and promoted memory recovery [34]. A previous study reports that RCAN1 can regulate calcineurin signaling which is able to control apoptosis [35]. Additionally, another finding shows that when RCAN1 expression is decreased, neuronal apoptosis can be inhibited [36]. The present study also proved that the cognitive impairment of rats with HIBD was attenuated via the inhibition of NF-κB signaling pathway or RCAN1 silencing. Consistent with our study, a previous research finds that lycopene can ameliorate cognitive impairment through inactivating NF-κB signaling pathway [37]. Another study proves that naringenin can be used in pre-treatment to improve cognitive impairment via inhibiting inﬂammation regulated by NF-κB [38]. Furthermore, it has also been proved that when RCAN1 isoform 4 is overexpressed, cognitive behavioral impairment can be aggravated [39]. These findings confirm our result that inhibition of NF-κB signaling pathway can improve the cognitive impairment of rats with HIBD. Conclusion Based on the previous researches, the present study has confirmed that the inhibition of NF-κB signaling pathway reduce the RCAN1 expression in HIBD rats, which decreases the hippocampal neuronal apoptosis and improve the cognitive impairment in rats with HIBD (Figure 10). These findings identify the NF-κB signaling pathway and RCAN1 as potential therapeutic targets for the treatment of HIBD. Given that all the experiments in the present study were conducted in rats, we will try to perform human clinical trials in future studies.10.1080/15384101.2019.1608128-F0010 Figure 10. Molecular mechanism underlying the NF-κB signaling pathway regulated HIBD progression. The NF-κB signaling pathway is activated in HIBD rats. Activation of the NF-κB signaling pathway promotes RCAN1 expression, which increased the neuronal apoptosis and aggravated the cognitive impairment in HIBD rats. Future perspectives Different genes and non-coding RNAs were also reported to be differentially expressed in several eye-related neurodegenerative disorders [40–42]. Therefore, in order to develop the idea that they might be used as molecular targets for future clinical trials, the possibility to realize a transcriptomic experiment should be conducted to analyze which genes and non-coding RNAs could be implicated in biochemical pathway involving NF-κB and RCAN1. For instance, glyoxalase I (GLO1), one of the oxidative stress-related enzymes, was involved both in oxidative stress and apoptotic pathway mediated by NF-κB [43], that was also found to be associated with other pathologies caused by neuronal death, such as retinitis pigmentosa [44]. Any new discoveries of genes or non-coding RNA or mechanisms in HIBD, may ultimately improve knowledge of pathologies and benefit therapeutically from neurodegenerative diseases. Another point was that the cause of RCAN1 over-expression in HIBD rats should be evaluated, and RCAN1 promoter should be analyzed to evaluate if the presence of variants could alter binding of transcription factors. Therefore, the dual luciferase reporter gene assay, which was already applied to other neurodegenerative pathologies, such as Stargardt disease [45], should be conducted to validate the promoter integrity in the future study. Acknowledgments We would like to thank our researchers for their hard work and reviewers for their valuable advice. Disclosure statement No potential conflict of interest was reported by the authors. ==== Refs References [1] Arruza L , Pazos MR , Mohammed N , et al Hypoxic-ischemic brain damage induces distant inflammatory lung injury in newborn piglets. Pediatr Res. 2016;79 (3 ):401–408. .PMID: 25950454 25950454 [2] Sekhon MS , Ainslie PN , Griesdale DE. 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==== Front ACS Cent SciACS Cent SciocacsciiACS Central Science2374-79432374-7951American Chemical Society 3126376710.1021/acscentsci.9b00224Research ArticleTargeted Protein Internalization and Degradation by ENDosome TArgeting Chimeras (ENDTACs) Nalawansha Dhanusha A. †Paiva Stacey-Lynn †Rafizadeh Diane N. ‡Pettersson Mariell †Qin Liena †Crews Craig M. †‡§† Department of Molecular, Cellular and Developmental Biology, Yale University, 219 Prospect Street, New Haven, Connecticut 06511, United States‡ Department of Chemistry, Yale University, New Haven, Connecticut 06511, United States§ Department of Pharmacology, Yale University, New Haven, Connecticut 06511, United States E-mail: [email protected] 05 2019 26 06 2019 5 6 1079 1084 05 03 2019 Copyright © 2019 American Chemical Society2019American Chemical SocietyThis is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes. Targeted protein degradation has generated excitement in chemical biology and drug discovery throughout academia and industry. By hijacking the machinery responsible for protein degradation via the ubiquitin proteasome system (UPS), various cellular targets have been selectively degraded. However, since the tools used, often termed PROteolysis TArgeting Chimeras (PROTACs), hijack the intracellular quality control machinery, this technology can only access targets within the cell. Extracellular targets such as growth factors, cytokines, and chemokines bind to cell surface receptors, often initiating aberrant signaling in multiple diseases such as cancer and inflammation. However, efforts to develop small molecule inhibitors for these extracellular target proteins have been challenging. Herein, we developed a proof-of-concept approach to evaluate if extracellular proteins can be internalized and degraded via the receptor-mediated endolysosomal pathway. Using a heterodimeric molecule, termed “ENDosome TArgeting Chimera” (ENDTAC), internalization and degradation of an extracellular recombinant eGFP-HT7 fusion protein was achieved by hijacking the decoy GPCR receptor, CXCR7. This proof-of-concept study suggests that using ENDTACs to co-opt the endosomal–lysosomal degradation pathway, in contrast to PROTACs using the UPS, may provide an avenue for degrading extracellular targets such as cytokines. Overall, the technology described herein provides a novel expansion to the field of targeted protein degradation. Chimeric molecules, “ENDTACs”, induce targeted protein internalization and degradation by hijacking the receptor-mediated endocytosis pathway. document-id-old-9oc9b00224document-id-new-14oc-2019-00224sccc-price ==== Body Introduction Traditional drug development efforts are focused mainly on small molecules that target druggable protein classes such as enzymes and receptors.1 The majority of drugs operate as inhibitors of protein function; however, because this mode of action utilizes a target occupancy paradigm requiring high drug concentrations to sustain the biological response, it also can lead to undesirable off-target effects. As an alternative, PROteolysis TArgeting Chimeras (PROTACs) hold great promise as a therapeutic modality since they require only a transient interaction with the target protein to promote its degradation.2−4 However, despite promoting the degradation of various proteins, PROTACs are limited to target engagement within the intracellular space for ubiquitination.5−9 PROTACs are therefore unable to act on secreted proteins, such as cytokines and chemokines, which exert their biological activity from the extracellular space.10,11 These proteins bind to cell surface receptors and can initiate the aberrant signaling implicated in multiple diseases (Figure 1A). While monoclonal antibodies can target secreted proteins or their cognate receptors to block signaling, efforts to develop small molecule inhibitors for secreted proteins have so far been less successful.12,13 Given the functional importance of secreted proteins in diseases and the current limitations on inhibiting their activities, alternative technologies to efficiently target them are needed. Figure 1 ENDTAC technology. (A) In the absence of the ENDTAC, extracellular target protein (e.g., cytokine) binds to its cognate receptor (e.g., cytokine receptor) and activates downstream signaling leading to a cellular response (e.g., cell proliferation, apoptosis, and/or inflammation). (B) Upon ENDTAC addition, the target extracellular protein of interest (POI) is endocytosed via a decoy GPCR, CXCR7, and subsequently degraded by the lysosome. EE = early endosome, LE = late endosome. (C) An ENDosome TArgeting Chimera (ENDTAC) is a heterodimeric molecule consisting of an agonist ligand that binds to a cell surface receptor (e.g., CXCR7) coupled to a ligand that recruits the extracellular POI (e.g., cytokine). Receptor–ligand-mediated delivery systems have gained significant attention in the past few years.14,15 Several studies have reported that cell surface receptors such as transferrin receptor, asialoglycoprotein (ASGPr), and folate receptor can be used to selectively deliver a wide range of therapeutic agents into cancer cells via receptor-mediated endocytosis.16−18 In addition, a recent study has shown that Cas9 could be selectively delivered into cells by harnessing the receptor–ligand interactions followed by endosomal escape.19 Here we report a new approach to potentially target extracellular proteins for receptor-facilitated lysosomal degradation using chimeric molecules termed ENDosome TArgeting Chimeras (ENDTACs) (Figure 1B). An ENDTAC is a heterodimeric molecule consisting of a small molecule (agonist) that binds to a plasma membrane-localized receptor of interest (e.g., a GPCR) while the other end, connected via a linker, of the ENDTAC binds and recruits the extracellular protein of interest (POI) (e.g., a cytokine) (Figure 1C). Upon binding to the GPCR, the tethered extracellular protein can undergo receptor-mediated endocytosis and subsequently degradation by the lysosome (Figure 1B). Results and Discussion To validate the feasibility of the ENDTAC approach, we performed a proof-of-concept study using the decoy receptor, CXCR7 (ACKR3), as the cell surface receptor and an engineered HA-eGFP-HaloTag7 (eGFP-HT7) fusion protein as the extracellular target protein. The GPCR CXCR7 is constitutively endocytosed at a low level to degrade its cognate chemokines via transport to the lysosome.20 A previous report has shown that two potent small molecule agonists VUF11207 and VUF11403 induce CXCR7 internalization (Figure S1).21 Since both these 3,4-dimethoxy and 3,4,5-trimethoxy styrene amides (VUF11403 and VUF11207, respectively) exhibit similar agonist activity in various pairwise comparisons,21 we incorporated the HT7-recruiting chloroalkane at their 5-position (Figure 2A). Using a facile synthetic method (Scheme S1, in the Supporting Information), linkers ranging from a diethylene glycol to pentaethylene glycol were incorporated in both series (R1: H or OMe) to afford ENDTACs-1–8 (Figure 2A). Agonist activity for ENDTACs-1–8 was evaluated by the Tango GPCR assay22,23 (Table 1). Interestingly, the dimethoxy-containing ENDTAC series (ENDTACs-1–4; R1, OMe; R2, H) displayed only slightly reduced potency compared to the parent warhead VUF11207 (Figure 2B; Table 1; left EC50 column), while the monomethoxy-containing ENDTACs (ENDTACs-5–8; R1, H; R2, H) exhibited greater than 10- to 40-fold reduction in activity compared to the respective warhead, VUF11403 (Figure S1 and Table 1). Accordingly, we focused on the dimethoxy-containing ENDTAC series and measured the ability of the prereacted ENDTAC:eGFP-HT7 complex to activate the CXCR7 receptor. Purified eGFP-HT7 was used as the reporter POI (Figures S2 and S3) to evaluate ENDTACs-1–4. The reaction between purified eGFP-HT7 and ENDTACs was confirmed by LC-MS analysis (Figures S4 and S5 and Table S1). Figure 2 Characterization of ENDTACs. (A) Core structure of ENDTACs. (B) Characterization of agonist activity of ENDTACs-1–4 using the Tango assay (n = 4). (C) Activity of ENDTAC-1 and ENDTAC-neg after prereacting with eGFP-HT7 measured by Tango assay (n = 2). (D) Pulse chase Tango assay for warhead VUF11207, ENDTAC-1, and precomplexed ENDTAC-1:eGFP-HT7 (4 μM) (n = 2). Curves were fitted using GraphPad Prism 5. All data represent mean ± SEM. Table 1 EC50 Values of VUF11207 and CXCR7-Recruiting ENDTACs As Determined in the Tango Assaya entry compound ID n EC50 (nM) compound alone EC50 (nM) compound + eGFP – HT7 EC50 (nM) compound + Nanoluc – HT7 1 VUF11207 N/Ab 68 ± 1.2 2 ENDTAC-1 2 113 ± 1.3 169 ± 1.5 146 ± 1.3 3 ENDTAC-2 3 111 ± 1.2 251 ± 1.5 4 ENDTAC-3 4 145 ± 1.1 373 ± 2.0 5 ENDTAC-4 5 158 ± 1.2 287 ± 3.0 6 VUF11403 N/Ab 76 ± 1.3 7 ENDTAC-5 2 1360 ± 3.8 ND 8 ENDTAC-6 3 2453 ± 1.1 ND 9 ENDTAC-7 4 4705 ± 5.2 ND 10 ENDTAC-8 5 1815 ± 1.4 ND 11 ENDTAC-neg 2 ND ND a ENDTACs (1–4) contain OMe at the R1 position and H at the R2 position whereas ENDTACs (5–8) contain H at both R1 and R2 positions. These ENDTACs are generated by adding a chloroalkane to VUF11207 or VUF11403 via varying linker lengths. The ENDTAC-neg structure is slightly different than ENDTACs-1–4 where R1 is H, and R2 is OMe. VUF11207 and VUF11403 are known small molecule agonists of CXCR7. eGFP-HT7 is HA-eGFP-Halotag7 protein, and Nanoluc-HT7 is a HA-nanoluciferase-Halotag7 fusion protein. ND: not determined. All data represent mean ± SEM. b N/A: not applicable. Among these four ENDTACs, ENDTAC-1 retained a similar potency compared to VUF11207 after reacting with eGFP-HT7 (Table 1). On the basis of this initial activity profile, we selected ENDTAC-1 for use in subsequent experiments. To ensure that eGFP-HT7 internalization is mediated via CXCR7 activity, we synthesized a negative control molecule, ENDTAC-neg, using a similar synthetic approach (Figure 2A; Scheme S2). The para-methoxy group on the styrene ring (R1, H; and R2, OMe) of ENDTAC-neg was previously identified to abrogate CXCR7 agonist activity.21 Therefore, ENDTAC-neg was evaluated for activity in the absence or presence of eGFP-HT7. According to Tango assay data, prereacted ENDTAC-neg:eGFP-HT7 did not show any CXCR7 activation compared to ENDTAC-1:eGFP-HT7, supporting the use of ENDTAC-neg as a negative control. (Figure 2C and Figure S6). Furthermore, we also analyzed the saturation kinetics of the prereacted ENDTAC-1:eGFP-HT7 complex using the Tango assay. The warhead VUF11207 and ENDTAC-1 show fast saturation kinetics, whereas ENDTAC-1:eGFP-HT7 reaches saturation within 4 h (Figure 2D). Given the similar agonistic activities of the warhead and corresponding ENDTAC, as well as favorable binding kinetics, we next proceeded to study ENDTAC-induced internalization of eGFP-HT7 protein. CXCR7-expressing MCF7 cells were treated for 4 h with either ENDTAC-1 or ENDTAC-neg conjugated to purified eGFP-HT7 (10 μM), and internalization was monitored by confocal microscopy. Internalized eGFP-HT7 was visualized as GFP-positive puncta (green), and we observed a greater uptake of eGFP-HT7 in the presence of 10 μM ENDTAC-1 after 4 h (Figure 3A). The quantitation of GFP-positive cells suggests ENDTAC-1 induced internalization of eGFP-HT7, as compared to ENDTAC-neg (Figure 3B). We also analyzed eGFP-HT7 internalization by immunostaining with HA antibody and observed an overlay between the GFP and HA puncta in the presence of ENDTAC-1 (Figure S7). eGFP-HT7 uptake was confirmed as being ENDTAC-dependent in CXCR7-expressing MCF7s and MIA PaCa-2 cells, where greater uptake is observed in the presence of 500 nM ENDTAC-1, compared to ENDTAC-neg, via immunoblotting (Figure 3C). To corroborate this finding, we used purified Nanoluc-HT7 (Figures S2 and S3) as the extracellular POI and evaluated Nanoluc-HT7 uptake by measuring luciferase activity in MCF7 and HTLA cells (Figure 3D and Figure S8). As was observed with the ENDTAC-1:eGFP-HT7 adduct, ENDTAC-1 retained CXCR7 agonistic activity after reacting with Nanoluc-HT7 (Table 1). We first incubated prereacted Nanoluc-HT7 + ENDTAC-1 (1 μM) with MCF7 cells for 2 h and then changed to ENDTAC-free media and assayed for Nanoluc-HT7 uptake over 24 h. Consistent with previous data, we observed a 2.5-fold uptake of Nanoluc-HT7 in the presence of ENDTAC-1 compared to ENDTAC-neg in the first 2–6 h (Figure 3D, Figure S8). In both MCF7 and HTLA cells, there is an observed reduction in Nanoluc-HT7 activity at 24 h, suggesting that internalized protein has been degraded in the lysosome. Figure 3 Internalization of eGFP-HT7 in the presence of ENDTAC-1. (A) Confocal microscopy analysis of internalized eGFP-HT7 (green puncta) with 10 μM ENDTAC-1. Nuclei are stained with Hoechst stain (blue). Scale bar: 5 μm. (B) eGFP-positive cells were quantified and presented as a percentage. Quantified data represent mean ± SEM, n = 3. p < 0.05. (C) Cellular uptake of eGFP-HT7 in MCF7 and MIA PaCa-2 cells was analyzed by immunoblotting after incubating for 4 h with ENDTAC-neg:eGFP-HT7 or ENDTAC-1:eGFP-HT7 (500 nM). (D) Cellular uptake of Nanoluc-HT7 in MCF7 cells was analyzed by evaluating luciferase activity. The relative luminescence units of ENDTAC-1:Nanoluc-HT7 were normalized to ENDTAC-neg:Nanoluc-HT7 and presented as the fold uptake using GraphPad Prism 5 (n = 6). Data represent mean ± SEM; p < 0.05; ns, not significant. (E) Cellular uptake of eGFP-HT7 in MCF7 cells was analyzed by immunoblotting after incubating for 4 h with DMSO:eGFP-HT7, ENDTAC-neg:eGFP-HT7, or ENDTAC-1:eGFP-HT7 (250 nM) in the absence or presence of excess warhead, VUF11207 (25 μM). Given the possibility that bulk endocytosis could lead to nonspecific uptake of proteins, we performed a ligand competition assay with excess CXCR7 agonist VUF11207 to probe that the ENDTAC-1-mediated HT7 uptake is CXCR7-dependent. We incubated MCF7 cells with DMSO:eGFP-HT7, ENDTAC-neg:eGFP-HT7, and ENDTAC-1:eGFP-HT7 in the absence or presence of excess warhead (VUF11207). The ENDTAC-1-mediated HT7 uptake was completely inhibited in the presence of the excess warhead (Figure 3E), suggesting that the ternary complex formation between eGFP-HT7, ENDTAC-1, and CXCR7 is required to facilitate selective internalization of eGFP-HT7. We next sought to determine whether the level of CXCR7 expression could play a key role in efficiency of the ENDTAC system. To probe the hypothesis, we first compared the uptake of eGFP-HT7 in CXCR7-overexpressing 293T cells. Upon incubation of ENDTAC-1:eGFP-HT7 with 293T cells, we observed an ENDTAC-1-dependent uptake of eGFP-HT7 compared to nontransfected cells (Figure S9A), suggesting that increased expression of CXCR7 enhances the ENDTAC-1-mediated internalization of HT7. To further support this result, we compared the ENDTAC-1-dependent cellular uptake in MDA-MB-231 cells (CXCR7-negative) and MCF7 cells (CXCR7-positive). Compared to MDA-MB-231 cells, we observed an increased eGFP-HT7 uptake in ENDTAC-1 treated MCF7 cells (Figure S9B), suggesting that the level of CXCR7 surface expression is a key parameter that dictates ENDTAC efficiency. Upon receptor-mediated endocytosis, we propose that the eGFP-HT7 protein traffics sequentially to the early endosome and the late endosome and subsequently is degraded in the lysosome. To evaluate this hypothesis, we treated MCF7 and MIA PaCa-2 cells with ENDTAC-1:eGFP-HT7 or ENDTAC-neg:eGFP-HT7 for 4 h, washed the cells, replaced the medium with fresh medium, and cultured the cells for another 3 h, to monitor the fate of internalized eGFP-HT7. We observed eGFP-HT7 uptake by the cells after a continuous 4 h ENDTAC-1:eGFP-HT7 incubation, followed by the disappearance of eGFP-HT7 after a 3 h chase (Figure 4A,B). Therefore, it is possible that internalized eGFP-HT7 protein traffics to the lysosome and is therein degraded, given the mechanism of CXCR7 internalization following agonist treatment.20 To evaluate if lysosomal-mediated eGFP-HT7 degradation occurs, we incubated cells with ENDTAC-neg:eGFP-HT7 or ENDTAC-1:eGFP-HT7 for 4 h, washed the cells, and cultured them in the absence or presence of the lysosome inhibitor, bafilomycin A1 (bafA1). Interestingly, eGFP-HT7 is degraded only in the absence of bafA1, suggesting that eGFP-HT7 degradation indeed occurs via the lysosomal pathway (Figure 4C,D). Figure 4 Cellular uptake and lysosomal degradation of eGFP-HT7 in the presence of ENDTAC-1. (A) Immunoblot analysis of uptake and fate of internalized eGFP-HT7 after 4 h of incubation followed by a 3 h chase in ENDTAC-neg/ENDTAC-1:eGFP-HT7-free medium in MCF7 (n = 4) and (B) MIA PaCa-2 cells (n = 3). (C) MCF7 or (D) MIA PaCa-2 cells were incubated with ENDTAC-neg/ENDTAC-1:eGFP-HT7 (500 nM) for 4 h, released to ENDTAC-free medium, and chased for an additional 3 h in the absence or presence of bafilomycin A1 (bafA1) (100 nM) (n = 3). To further confirm endolysosomal accumulation of eGFP-HT7 in the presence of ENDTAC, we examined the uptake and degradation of eGFP-HT7 in MCF7 cells by confocal microscopy. MCF7 cells were treated with either Alexa Fluor 488-conjugated transferrin (TFN488), a positive control for endocytosis,24 or the ENDTAC-1/ENDTAC-neg:eGFP-HT7 conjugate (10 μM) for 4 h, fixed, and analyzed by confocal microscopy. Interestingly, as similarly observed with TFN488 treatment, eGFP-HT7-positive cells were observed in the presence of ENDTAC-1:eGFP-HT7 (Figure 5A–C). These eGFP-HT7 foci colocalize with the early endosome marker (EEA1) suggesting that internalized eGFP-HT7 traffics to the endosome (Figure 5, panel C). Furthermore, we also observed that eGFP-HT7-positive foci partially colocalize with the lysosome marker (LysoTracker), suggesting that ENDTAC-1 promotes receptor-mediated uptake and lysosomal degradation of eGFP-HT7 protein (Figure 5D,E). Figure 5 Endolysosomal trafficking of eGFP-HT7 in the presence of ENDTAC-1. Confocal microscopy analysis of (A) AF488-conjugated transferrin (TFN488) was used as a positive control for endocytosis. (B–E) Trafficking of internalized eGFP-HT7 (10 μM) to the endolysosome compartment after 4 h. (B, C) EEA1, early endosome marker, and (D, E) LysoTracker, lysosome marker. Red, EEA1 and LysoTracker; green, eGFP-HT7; yellow, merged images; blue, Hoechst stain for nuclei. Scale bar: 5 μm. While PROTACs are widely used in the field to target many intracellular proteins,9 a key limitation is their inability to target extracellular proteins. Here we show for the first time that chimeric molecules, which we designate as “ENDTACs”, are capable of recruiting an extracellular protein (eGFP-HT7), for internalization and lysosomal degradation by hijacking a GPCR-mediated endocytosis pathway. Although we present a proof-of-concept approach using a covalent HaloTag-based system, we anticipate that the ENDTAC technology could be further optimized and modified for a noncovalent setting. In summary, the ENDTAC technology represents a new chemical biology tool to study extracellular proteins and has the potential for depleting disease-causing extracellular proteins in the future. Supporting Information Available The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acscentsci.9b00224.Experimental procedures and synthesis and characterization of ENDTACs (PDF) Supplementary Material oc9b00224_si_001.pdf The authors declare the following competing financial interest(s): C.M.C. is a consultant to and shareholder in Arvinas, which partially supports research in his lab. Acknowledgments This work was supported by the NIH (R35 CA197589). M.P. gratefully acknowledges the Swedish Research Council (Vetenskapsrådet) for international postdoctoral funding (2016-00294). HTLA (a HEK293 cell line stably expressing a tTA-dependent luciferase reporter and a β-arrestin2-TEV fusion gene) cells were generously provided by Gilad Barnea (Brown University, RI). We thank the Alanna Schepartz lab for their technical help and John Hines for reviewing the manuscript. ==== Refs References Hopkins A. L. ; Groom C. R. The druggable genome . Nat. Rev. Drug Discovery 2002 , 1 (9 ), 727 –730 . 10.1038/nrd892 .12209152 Sakamoto K. M. ; Kim K. B. ; Kumagai A. ; Mercurio F. ; Crews C. M. ; Deshaies R. J. Protacs: chimeric molecules that target proteins to the Skp1-Cullin-F box complex for ubiquitination and degradation . Proc. Natl. Acad. Sci. U. S. A. 2001 , 98 (15 ), 8554 –8559 . 10.1073/pnas.141230798 .11438690 Sakamoto K. M. ; Kim K. B. ; Verma R. ; Ransick A. ; Stein B. ; Crews C. M. ; Deshaies R. J. Development of Protacs to target cancer-promoting proteins for ubiquitination and degradation . Mol. Cell. 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==== Front 95020158791Nat MedNat. Med.Nature medicine1078-89561546-170X3116081410.1038/s41591-019-0459-6nihpa1527245ArticleCCR5-Δ32 is deleterious in the homozygous state in humans Wei Xinzhu 1Nielsen Rasmus 121 Department of Integrative Biology and Statistics, University of California, Berkeley, Berkeley, CA, 94720, USA2 GeoGenetics Centre, University of Copenhagen, 1350 Copenhagen, DenmarkAuthor contributions X.W. and R.N. designed the study and wrote the manuscript; X.W. analyzed the data. Correspondence to: [email protected]; [email protected] 5 2019 03 6 2019 6 2019 03 12 2019 25 6 909 910 Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:http://www.nature.com/authors/editorial_policies/license.html#termsWe use the genotyping and death register information of 409,693 British individuals to investigate fitness effects of the CCR5-Δ32 mutation. We estimate that individuals homozygous for the Δ32 allele have a 21% increase in all-cause mortality rate. A deleterious effect of the Δ32/Δ32 mutation is also independently supported by a significant deviation from Hardy-Weinberg equilibrium due to a deficiency of Δ32/Δ32 individuals at the time of recruitment. ==== Body In the fall of 2018, a scientist from the Southern University of Science and Technology in Shenzhen, Jiankui He, announced the birth of two CRISPR edited human babies1. While no presentation of the experiment has appeared in the scientific literature, online information2 describes an introduction of mutations in the CCR5 gene aimed at mimicking the effect of the CCR5-Δ32 mutation, which provides protection against HIV in Europeans3. Although the mutations were not identical to CCR5-Δ322, and the consequences of these mutations are unknown, the stated purpose was nonetheless HIV prevention. The CRISPR experiment raises a number of obvious ethical issues. In addition, it is not clear if the Δ32 mutation is beneficial. A mutation can be advantageous or disadvantageous depending on environmental conditions4 and developmental stages5. In fact, even though Δ32 provides protection against HIV, and possibly other pathogens such as smallpox6 and flavivirus7, and facilitates recovery after stroke8, it also appears to reduce protection against certain other infectious diseases such as influenza9. Direct fitness effects of individual segregating mutations are expected to be small, and are therefore very hard to measure directly. However, due to the recent availability of large databases with genomic data, direct studies of fitness effects of individual mutations have now become feasible10. We might expect that the Δ32 mutation is deleterious in the homozygous state based on previous reports, in smaller data sets, showing that individuals with the Δ32/Δ32 genotype have increased mortality when infected by influenza9 and are four times more likely to develop certain infectious diseases11. We here investigate this hypothesis using the genotyping and death register information of 409,693 individuals of British ancestry in the UK Biobank12. Δ32 has a frequency of 0.1159 in the British population and the UK Biobank contains approx. 5500 homozygous individuals, providing an opportunity to compare the longevity of these individuals to that of Δ32/+ and +/+ individuals. We calculate the survival rate (1 - death rate) per year for each of the three Δ32 genotypes from age 41 till age 78 (see Materials and methods), which is the entire range allowed by the data available (Fig. 1a). Due to the small sample size at age 77 and 78, we primarily report the survival probability before age 76 (see Materials and methods). The death rate at age 70–74 years in the UK Biobank volunteers is 46–56% lower than that in the general UK population of the same age13, likely due to an ascertainment bias known as the “healthy-volunteer effect”14. Nonetheless, the relative death rates among different genotypes can still be compared to provide information about the fitness effects of specific mutations. The uncorrected survival probabilities to age 76 of individuals enrolled in the study is 0.8351 for Δ32/Δ32, 0.8654 for Δ32/+, and 0.8638 for +/+ (Fig. 1a), which implies that Δ32/Δ32 has an approx. 21% higher aggregated death rate before age 76 compared to the other genotypes. The average age of enrollment is 56.5 so this largely reflects differences in mortality in individuals above age 56.5. We can partially correct for the death registration delay and biased ascertainment, provided the general population’s death rate per year. After correction, the Δ32/Δ32 individuals are about 20% less likely to reach age 76 (see Materials and Methods). To test the significance of the nominally lower survival rate of Δ32/Δ32, we first perform a log-rank test comparing the death rate of Δ32/Δ32 individuals to that of the other two genotypes (Z-score = 2.37, one-tailed P = 0.0089). We also bootstrap the sample 1000 times and find that Δ32/Δ32 individuals have significantly higher death rate than the other two genotypes, while Δ32/+ and +/+ individuals have similar death rate (Supplementary Table 1). The increase in mortality of Δ32/Δ32 individuals is the highest at age 74, where it is 26.4% higher than the mortality of +/+ individuals (95% bootstrap confidence interval [3.0%,49.5%]). Similarly, a Cox-model15 for left truncated and right censored data also suggests that Δ32/Δ32 individuals have an average of 21.4% elevated death rate across all ages (95% confidence interval 3.4% and 42.6%, one-tailed P = 0.0089). The 5th principal component is associated with Irish ancestry12 and is also associated with a difference in mortality (two-sided P = 2.5×10−16) in the Cox-model. However, when correcting for this effect using PCA loadings as covariates, the increase in mortality of Δ32 is maintained (see Supplemental information). We note that despite the nominally large detected effect on survivorship, the P-value is only moderately small, due to the low frequency of Δ32/Δ32 individuals and the generally low mortality in the cohort. The accuracy of the estimates will likely improve in future years as the mortality rate of the cohort increases. Selection against homozygous individuals will lead to deviations from Hardy-Weinberg Equilibrium (HWE), which can be measured by the inbreeding coefficient (F). Deviations from HWE at the time of enrollment, which is the time at which samples are obtained for genotyping, provides an assessment of differential fitness of Δ32 genotypes that is independent from the previous analyses using death registry information obtained after enrollment. We test for deviations from HWE consistent with a deleterious effect of Δ32 in homozygous individuals by calculating the allele-specific inbreeding coefficient FΔ32/Δ32. However, there might be deviations from HWE in the data for multiple other reasons, including inbreeding and population structure. Therefore, we compare FΔ32/Δ32 (see Materials and Methods) with the locus specific value of F for other variants in the data with minor allele frequencies similar (plus/minus 0.0025) to that of Δ32. Only 20/5932 variants have a smaller F than FΔ32/Δ32 (Fig. 1b; empirical one-tailed P = 0.0034). In addition, the deviation from HWE for each age group also correlates with the deviation predicted by the survival probability (Spearman’s ρ = 0.67, P = 1.4 × 10−4; see Supplementary information and Extended Data Figure 1). These two independent analyses are largely consistent with each other and both indicate a substantial increase in mortality associated with the Δ32/Δ32 genotype. Our results show that being homozygous for the Δ32 mutation is associated with reduced life expectancy in a modern cohort, despite the protective effect of the mutation against HIV3. This finding echoes the previous reports that the Δ32 reduces resistance against influenza9 and other infectious diseases11. We did not observe any difference in mortality between Δ32/+ and +/+ individuals (Supplementary Table 1), despite the fact that Δ32/+ also provides protection against HIV3. It could reflect the “healthy volunteer effect” in the UK Biobank cohort13 if individuals affected by HIV, or suffering from mortality due to HIV infection, are less likely to be recruited. In that case, our estimates of death rates reflect individuals that have reduced exposure to HIV, and the conclusion regarding increased mortality of Δ32/Δ32 is then with reference to such individuals. If so, it would also imply that in the presence of HIV, Δ32 is overdominant, i.e. that individuals heterozygous for the mutation have the highest fitness. In the absence of HIV or other infectious agents for which the mutation provides protection, the mutation will be under negative directional selection. But because only about 0.16% of the current British population is infected by HIV16, the benefit from this protection is likely too small to have a detectable influence on survival probability in our study. It is unclear exactly which factors are most important for the fitness effects of the Δ32 mutation. There are many phenotypic associations significant at 5% significance level after correction for multiple testing in the UK Biobank (see Supplementary information for the phenotypes), and the mutation is likely highly pleiotropic. Out of the 5932 SNPs with matching allele frequencies, only 76 have more phenotypic associations than Δ32 in terms of the UK Biobank phenotypes (empirical one-tail P = 0.0128, see Supplementary information). It is perhaps not unexpected that homozygosity for a deletion in a functional gene is associated with reduced fitness. It underscores the notion that introduction of new or derived mutations in humans using CRISPR technology, or other methods for genetic engineering, comes with considerable risk even if the mutations provide a perceived advantage. In this case, the cost of resistance to HIV may be increased susceptibility to other, and perhaps more common, diseases. Materials and Methods The study population This study uses the UK Biobank data under application number 33672 and basket ids 10997 and 2000429. It is regulated under ethical regulations of UC Berkeley and the data is accessed under the Material Transfer Agreement between the UK Biobank and UC Berkeley. In the UK Biobank, 409,693 volunteers have self-reported British ancestry confirmed by principle component analysis12, which constitutes roughly 0.62% of the entire British population. Our main analysis are performed on the British ancestry volunteers, unless otherwise stated. There are 75,970 volunteers in the UK Biobank labeled as non-British ancestry, which are used to investigate the effect of Δ32 in other populations than the British. The UK Biobank volunteers were recruited during 2006–2010 and 2.9% of the volunteers (13,831) have a recorded age at death (all cause). Marker selection and validation SNP rs62625034 (coordinate 3:46414975 in GRCh37) is a directly genotyped SNP which is used to identify Δ32 (rs333) based on the following validations: First, the Affymetrix probe used for this SNP is ‘CCATACAGTCAGTATCAATTCTGGAAGAATTTCCA[G/T]ACATTAAAGATAGTCATCTTGGGGCTGGTCCTGCC’ based on annotation files ‘Axiom_UKBiLEVE.na34.annot.csv’ and ‘Axiom_UKB_WCSG.na34.annot.csv’. The targeted region of this probe fully includes the 32 bp deletion in rs333, given rs333 (Δ32) has coordinate 3: 46414947–46414978 in GRCh37. Second, rs62625034 is not called as a SNP in the 1000 Genome database, and a recent study on variants in CCR5 gene17 also confirmed that it could only be detected in one of the Denisovian samples. However, the detected allele frequency by the probe of rs62625034 in the UK Biobank is 0.1159 among the British ancestry genomes, which does not resemble the frequency of rs62625034, but closely resembles the frequency of rs333 (0.1237) in the European and the British population (CEU and GBR) in the 1000 Genomes data. Third, SNP rs113010081, a directly genotyped SNP in the UK Biobank data, is in strong linkage disequilibrium (LD) with rs333 in the 1000 Genomes data, with a r2 of 0.93 combining CEU and GBR in 1000 Genomes data (https://ldlink.nci.nih.gov/?var1=rs333&var2=rs113010081&pop=CEU%2BGBR&tab=ldpair). We calculate the Pearson correlation between rs113010081 and the probe of rs62625034 using the UK Biboank British ancestry genotypes, and obtain r2 = 0.94, which again resembles the correct LD between rs113010081 and rs333. In addition, there is no other SNP that is in as strong LD with rs113010081 in the targeted region of this probe (https://ldlink.nci.nih.gov/?var=rs113010081&pop=CEU%2BGBR&r2_d=r2&tab=ldproxy). Lastly, we also estimate the survival probability for rs113010081, and the results are similar to that obtained for rs62625034 (not shown). Estimation of survival probability The UK Biobank death records are updated quarterly with the NHS Information Centre for participants from England and Wales and by NHS Central Register, Scotland for participants from Scotland. However, the death records are not made available immediately to researchers. The latest date of death among all registered deaths in the downloaded data is 2016–02–16, and we use this date to approximate the time of last death entry, and assume that after that date we have no mortality/viability information of the volunteers. We use five entries from the UK Biobank data, the age at recruitment, the date of recruitment, the year of birth, month of birth, and the age at death, to calculate the number of individuals (Ni) who are ascertained from age i to age i + 1, and the occurrence of death observed from these Ni individuals during the interval of age i to age i + 1 is Oi. Using this information, we calculate the ascertained age for each individual. We ignore the partially ascertained age to avoid biases from censoring. For example, an individual recruited at age 45.2, and reaching age 52.3 on 2016–02–16, who does not have a reported death in our data, is treated as being observed from age 46 to age 52, thus this volunteer contributes to N46, N47, N48, N49, N50, N51. As another example, a person who is recruited at age 65.7, and could have reached age 72.6 by 2016–02–16, but has a reported death at age 69.7 will contribute to N66, N67, N68, N69, and this volunteer will also contribute to O69. This volunteer does not contribute to N70, because death has already occurred before age 70. The death rate per year is then calculated as hi = Oi/Ni, and the probability of surviving to age i + 1 is Si = ∏n=1n=ihn. The UK Biobank data allows estimation of death rates from h41 to h77, but because N77 is smaller than 800, we have to assume that h76 = h77 and combined these two ages in our estimation. We estimate hi separately for the three different Δ32 genotypes. We mainly report the survival probability before age 76, where there is sufficient data to obtain accurate estimates, but the estimated survival probabilities to age 77 and 78 are also shown in Fig. 1. Because the exact birth dates of the volunteers are considered sensitive, we do not have access to them. The age at recruitment in the UK Biobank is rounded down to nearest integer age, and we approximate the exact age using the date of recruitment, the year of birth, and month of birth, assuming everyone is born on the 15th of their birth month. In rare cases, when the date of recruitment is very close to a person’s birthday, the approximated age could be smaller than the age at recruitment provided by the UK Biobank and in these rare cases we instead round up the estimated age. After applying this rounding scheme, if there are no errors in the data, under no scenario should the estimated age be smaller than the integer age at recruitment. However, there are 17 individuals whose estimated age is smaller than the age at recruitment, and we exclude these individuals in the death rate calculation. Among them, 15 are British ancestry. Although the UK Biobank routinely imports death records from the national databases, the “healthy volunteer effect”13 can still lead to a substantial underestimation of the death rate per year hi compared to the general population. The delay of the death records may be affected by many factors including time of recruitment, age of death, cause of death, and various socio-economic factors18. However, if we assume that these biases are independent of the Δ32 genotype, we can then estimate the death rate correction factor Ci for each age i and estimate the death rate per year and the survival probability for the three different Δ32 genotypes in the general population. To do this, we download the national life tables in the UK (“nltuk1517reg.xls”) from the Office of National Statistics (https://www.ons.gov.uk) which contain the death rate per year for the entire British population each year from 1980 to 2017, estimated for males and females separately. We average the death rate per year from 2006 to 2016 to represent the death rate Hi of the general population. We then use hi/Hi to estimate Ci. We then calculate a corrected death rate for each Δ32 genotype. For example, the corrected death rate for +/+ is hi,+/+/Ci. We use the corrected death rates to estimate the corrected survival probability (SC). The inferred survival probability after correction (SC) to age 76 are 0.7565, 0.7589 and 0.7111 for genotypes +/+, Δ32/+, and Δ32/Δ32, respectively. With this crude correction, the probability of death before age 76 in the general population is (1 - SC,Δ32/Δ32)/(1 - SC,Δ32/+) - 1, about 20% higher for Δ32/Δ32 individuals compared to heterozygous individuals. We note that while the calculations of death rates could be done more accurately, for example by using exact birthday (which we did not have access to), the significant difference in death rates between genotypes is unlikely to be explained by this effect. However, our survival analyses may underestimate the beneficial effects of Δ32 in some age groups due to ascertainment biases caused by the “healthy volunteer effect”13. Estimation of F FΔ32/Δ32 is estimated from the equation PΔ32/Δ32 = (1 + FΔ32/Δ32)PΔ32PΔ32, where PΔ32 and PΔ32/Δ32 are the observed frequencies of Δ32 and Δ32/Δ32, respectively. When FΔ32/Δ32 is significantly smaller than 0, it implies that the observed fraction of Δ32/Δ32 individuals is lower than expected under HWE, consistent with increased mortality of Δ32/Δ32 individuals. The F of other SNPs are similarly estimated. Statistical analysis One-tail P-values from log-rank test are used in Fig. 1a and Supplementary Table 1. In Fig. 1b, empirical one-tail P-values are used from the F of 5932 SNPs. 95% confidence intervals from bootstrap are shown as error bars in Extended Data Figure 1a, and are used in Supplementary Table 1. Spearman’s correlation is used in Extended Data Figure 1. In addition, the details of the statistical tests are given at places they are mentioned. Life sciences reporting summary Further information on experimental design is available in the Nature Research Reporting Summary linked to this article. Data, code, and research notebook availability The genotype and death registry information are available with the permission of the UK Biobank. Analytical results and scripts are accessible through (https://github.com/AprilWei001/CCR5-delta32). In addition, a detailed experimental notebook covering the entire development of this project is available at depository (https://xinzhuaprilwei.weebly.com/download/ccr5-delta32). Extended Data Extended Data Figure 1. The deviation from HWE with age. a, The observed deviation using age at recruitment estimated. Each dot represents one age group. The grey error bars show the 95% confidence intervals estimated from bootstrap the genotypes of individuals recruited at each age 1000 times. The sample size used for each error bar ranges from 15191 to 100117 with a mean of 65479. b, The predicted deviation from HWE using the corrected survival probability. A total of 395704 samples are used. The observed and predicted values are coefficient ρ = 0.67, P = 1.4 × 10−4). Supplementary Material Reporting summary pdf Supplementary Information Acknowledgements The authors thank D. Feehan, M. Slatkin, P. Wilton for discussions about death rate estimation, and R. Durbin, C. Freeman, G. McVean for discussions about UK Biobank marker. This work is supported by NIH grant R01GM116044 to R.N. Supplementary information Supplementary information including supplementary materials and methods, one figure, and one table. Competing interests The authors declare no competing interests. Figure 1. Δ32 is deleterious at homozygous state. a, Survival probabilities of Δ32 genotypes. The observed survival probabilities of the three genotypes (+/+, Δ32/+ and Δ32/Δ32) are shown in red, blue, and black, respectively. The x-axis shows the age and the y-axis shows the survival probability. The one-tail P-values from the log-rank test till age 76 is shown on the panel. The number of samples whose genotype at Δ32 and age information are both available is 395704. b, The histogram of inbreeding coefficients, F, from 5932 SNPs whose allele frequencies closely resemble that of Δ32. The black arrow points to the observed F of Δ32 (FΔ32/Δ32 = −0.19), calculated for the Δ32/Δ32 individuals. The sample size used in estimating F for each of the 5932 SNPs varies from 7896 to 409607 with a mean of 405428, and the sample size for Δ32 is 395714. ==== Refs References 1. Normile D Shock greets claim of CRISPR-edited babies (2018 ). DOI: 10.1126/science.362.6418.978 2. Cyranoski D First CRISPR babies: six questions that remain (2018 ). DOI: 10.1038/d41586-018-07607-3 3. Samson M Resistance to HIV-1 infection in Caucasian individuals bearing mutant alleles of the CCR-5 chemokine receptor gene . Nature 382 , 722 (1996 ).8751444 4. Wei X & Zhang J The genomic architecture of interactions between natural genetic polymorphisms and environments in yeast growth . Genetics 205 , 925 –937 (2017 ).27903611 5. Pavlicev M & Wagner GP A model of developmental evolution: selection, pleiotropy and compensation . Trends in Ecology & Evolution 27 , 316 –322 (2012 ).22385978 6. Galvani AP & Slatkin M Evaluating plague and smallpox as historical selective pressures for the CCR5-δ32 HIV-resistance allele . Proceedings of the National Academy of Sciences 100 , 15276 –15279 (2003 ). 7. Cahill ME , Conley S , DeWan AT & Montgomery RR Identification of genetic variants associated with dengue or West Nile virus disease: a systematic review and meta-analysis . BMC infectious diseases 18 , 282 (2018 ).29929468 8. Joy MT CCR5 is a therapeutic target for recovery after stroke and traumatic brain injury . Cell 176 , 1143 –1157 (2019 ).30794775 9. Falcon A CCR5 deficiency predisposes to fatal outcome in influenza virus infection . Journal of General Virology 96 , 2074 –2078 (2015 ).25918237 10. Mostafavi H Identifying genetic variants that affect viability in large cohorts . PLoS biology 15 , e2002458 (2017 ).28873088 11. Lim JK & Murphy PM Chemokine control of West Nile virus infection . Experimental cell research 317 , 569 –574 (2011 ).21376172 12. Bycroft C The UK Biobank resource with deep phenotyping and genomic data . Nature 562 , 203 (2018 ).30305743 13. Fry A Comparison of sociodemographic and health-related characteristics of UK Biobank participants with those of the general population . American journal of epidemiology 186 , 1026 –1034 (2017 ).28641372 14. Delgado-Rodriguez M & Llorca J Bias. Journal of Epidemiology & Community Health 58 , 635 –641 (2004 ). 15. Cox DR Analysis of survival data (Routledge , 2018 ). 16. Nash S , Desai S , Croxford S Progress towards ending the HIV epidemic in the United Kingdom: 2018 report . London : Public Health England (2018 ). 17. Hoover KC Intragenus (homo) variation in a chemokine receptor gene (CCR5) . PloS one 13 , e0204989 (2018 ).30278065 18. Patel V Impact of registration delays on mortality statistics: 2016 (2016 ).	31160814	PMC6613792	NO-CC CODE	2021-01-06 02:45:44	yes	Nat Med. 2019 Jun 3; 25(6):909-910
==== Front ACS OmegaACS OmegaaoacsodfACS Omega2470-1343American Chemical Society 10.1021/acsomega.8b00488ArticleRegulating the Microstructure of Intumescent Flame-Retardant Linear Low-Density Polyethylene/Nylon Six Blends for Simultaneously Improving the Flame Retardancy, Mechanical Properties, and Water Resistance Zhao Pan Lu Chang Gao Xi-ping Yao Da-Hu Cao Cheng-Lin Luo Yu-Jing Chemical Engineering & Pharmaceutics School, Henan University of Science and Technology, 263, Kaiyuan Avenue, Luoyang 471023, China E-mail: [email protected] (C.L.).27 06 2018 30 06 2018 3 6 6962 6970 15 03 2018 14 06 2018 Copyright © 2018 American Chemical Society2018American Chemical SocietyThis is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes. A compatibilizer was melt-blended with intumescent flame-retardant linear low-density polyethylene/nylon six blends (LLDPE/PA6/IFR) by different methods, and the effect of microstructure on the flame retardancy, mechanical properties, and water resistance was investigated. Melt-blending compatibilizers with LLDPE/PA6/IFR above the polyamide-6 (PA6) melt temperature formed the microstructure with IFR dispersion in the LLDPE matrix and good interphase adhesion between the PA6 phase and the matrix. Compared with the blends with the lack of compatibilizers, although good interphase adhesion improved the mechanical properties and water resistance, IFR dispersion in the LLDPE matrix reduced the flame retardancy sharply. To obtain the microstructure with IFR dispersion in the PA6 phase and strong interface adhesion of the PA6 phase with a matrix, a novel method in which a compatibilizer was melt-blended with LLDPE/PA6/IFR between the melt temperatures of LLDPE and PA6 was employed. The results showed that the flame retardancy, mechanical properties, and water resistance were improved simultaneously. document-id-old-9ao8b00488document-id-new-14ao-2018-00488tccc-price ==== Body 1 Introduction Intumescent flame retardants are now being used more and more in polymer flame retardants because they are more environmentally friendly than traditional halogen-containing flame retardants. The characteristic of intumescent flame retardants is that the combustion chamber can form a honeycomb expansion carbon layer, which reduces the mass transfer and heat transfer between the gas phase and the solidification phase.1−3 Acid source, carbon source, and blowing agent are the three elements of chemically intumescent flame retardants.4−6 In general, ammonium polyphosphate (APP as the acid source and blowing agent), pentaerythritol (PER as the charring agent) and melamine (as the blowing agent) form a traditional IFR system together. However, IFR has poor water resistance and compatibility with many polymer matrices, resulting in the decrease of flame retardancy and the damage to the mechanical properties of polymer composites. These drawbacks will restrict their wide industrial applications. Microencapsulation of IFR is regarded as an efficient method to get rid of these drawbacks mentioned above.7−11 The microencapsulated IFR particles have a core–shell structure, which allows the isolation of encapsulated substances from the surrounding and thus improves their compatibility with the polymer matrix and the water resistance. In general, the encapsulating shell, such as polyurethane (PU), melamine–formaldehyde, melamine, silicon resin, or urea–melamine–formaldehyde, was prepared by the in situ polymerization method.12−17 Physics encapsulation technology was also employed to prepare microencapsulated IFR particles. Wang18,19 provided a novel method of preparing microencapsulated IFR by the extrusion of melamine phosphate and PER, together with a polypropylene (PP) carrier. The results showed that the IFR encapsulated by the PP carrier improved flame retardancy and water resistance. Zeng20 used PU as a carrier resin to encapsulate melamine pyrophosphate/PER through melt blending, and encapsulated IFR was adopted to flame-retard PP. The results showed that encapsulated IFR endowed PP with better flame retardancy, water resistance, and mechanical properties. Compared with the in situ polymerization method, physics encapsulation technology has the advantages of a simple process and less environmental problems. Polymer blending has gained considerable interest as a suitable way to tailor the properties of polymeric materials without investing in new chemistry.21 However, the flame retardancy of polymer blends is more complex than that of pure polymers because of the complicated microstructure caused by the dispersion of IFR and their synergists in the multiphase.22−24 Lu25 reported that APP had a tendency to disperse in the polyamide-6 (PA6) phase of polystyrene (PS)/PA6 blends. The blends with cocontinuous phase structures have better flame retardancy than that of the blends with the sea-island structure. For the blends of PS/PA6 and acrylonitrile–butadiene–styrene/PA6 with sea-island morphology, Lu26,27 found that blends with organic montmorillonite and APP, respectively, distributed in the phase interface and PA6 phase have better flame retardancy than that of the blends with clay and APP dispersed in the PA6 phase. Jin28 employed the compatibilizer to adjust the microstructure for improving the mechanical properties and flame retardancy of PP/ethylene-octene copolymer/IFR. Along with the increase of compatibilizer content, the blends exhibited different microstructures. The addition of compatibilizers in a proper range caused the simultaneous improvement on mechanical properties and flame retardancy. IFR for flame-retardant polymer blends also encounters the challenges of poor water resistance and damage on mechanical properties. Although the microstructure of polymer blends contributes to the augment of the flame retardancy to a great extent, improving IFR water resistance and decreasing its damage on mechanical properties with resorting to regulating the microstructure have not been studied systematically. In this paper, we attempted to regulate the microstructure of polymer blends for the purpose of overcoming these disadvantages. The microstructure in which IFR localizes in the dispersed phase can be regarded as the microencapsulated IFR in which dispersed phase as the shell encapsulates IFR to improve water resistance. Compatibilizers can be employed to improve the compatibility. Moreover, char-forming polymers chosen as dispersed phase to encapsulate IFR particles could endow the blends with better flame retardancy. The flammability of linear low-density polyethylene (LLDPE)/PA6 blends has limited its applications in packaging and automotive fields. In the LLDPE/PA6 blends, higher affinity of IFR for PA6 than for LLDPE should exhibit the dispersion of IFR in the PA6 phase. Furthermore, it was reported that PA6 acted as the charring agent of APP to enhance the charring performance, resulting in better flame retardancy.29−31 Therefore, the use of IFR as flame retardants was investigated for their potential in improving the fire-retardant behavior of LLDPE/PA6. The IFR system used consists of APP and PER. Meanwhile, maleic anhydride grafted with LLDPE (LLDPE-g-MAH) was chosen as a compatibilizer for enhancing the compatibility of LLDPE/PA6 blends. The compatibility of LLDPE-g-MAH may prohibit the dispersion of IFR in the PA6 phase, while LLDPE, PA6, and IFR are melt-blended with LLDPE-g-MAH simultaneously. In order to prepare the blends with IFR dispersion in the PA6 phase, a two-step processing method was employed: PA6, LLDPE, and IFR were melted above the melting temperatures of LLDPE and PA6 to first prepare blends of LLDPE/PA6/IFR, and then LLDPE-g-MAH was melt-blended with LLDPE/PA6/IFR between the melt temperatures of LLDPE and PA6. The effects of the microstructure on the flame retardancy, mechanical properties, and water resistance were investigated. 2 Results and Discussion 2.1 Dispersion of APP and PER in LLDPE/PA6 Blends and Their Microstructure In order to investigate the spontaneous dispersion of APP or PER in the blends of LLDPE/PA6, PA6/LLDPE/APP or PA6/LLDPE/PER prepared by method one was investigated by Fourier transform infrared (FTIR) spectra, respectively. Figure 1 shows the FTIR spectra of the remaining parts of the PA6/LLDPE/IFR or PA6/LLDPE/PER extracted by formic acid or formic acid and ethyl alcohol, respectively. The −CH2– asymmetric stretching (2919 cm–1), symmetric stretching (2849.5 cm–1), bending vibrations (1469 cm–1), and the wagging vibration of C–H (719.4 cm–1) were observed, indicating that the remaining parts consisted of PE. In these blends, PA6 formed a continuous phase because of high PA6 content. The absence of PA6 in the remaining parts indicated that continuous PA6 phase was completely extracted by formic acid. The absence of APP or PER in the remaining parts showed that APP or PER was also completely extracted by formic acid or ethyl alcohol, respectively. If APP or PER was localized in the LLDPE phase of blends, they could not be extracted by formic acid or ethyl alcohol, with the result that APP or PER could be detected in the remaining parts. Therefore, the results that APP or PER was not detected in the remaining parts indicated that APP or PER is localized in the PA6 phase. In the blends tested by FTIR, APP or PER should disperse spontaneously in the polymer with high affinity, due to which the processing temperature was higher than the melting temperature of LLDPE and PA6. Therefore, the localization of APP or PER in the PA6 phase indicated that the affinity of APP or PER is higher for PA6 than for LLDPE because of similar polarity between PA6 and IFR. Figure 1 FTIR spectra of the (a) remaining part of LLDPE/PA6/APP extracted by formic acid and (b) remaining part of LLDPE/PA6/PER extracted by formic acid and ethyl alcohol. Scanning electron microscopy–energy-dispersive spectroscopy (SEM–EDS) was employed to characterize the dispersion of IFR in the blends and the microstructure of the blends, as shown in Figure 2. SEM results showed that APP and PER were irregular particles. The spherical particles with a large dimension were observed in the matrix of C-0. The results of FTIR showed that IFR is spontaneously distributed in the PA6 phase. Therefore, IFR particles were coated by the PA6 phase to form the spherical particles. The EDS results showed that the P content was 1.83 wt %. Generally, the detected depth for the EDS measurement is about 1000 nm, so small amounts of elemental P dispersed in the PA6 phase were detected. Some cavities were seen on the surface of C-0, and there were clear interfaces between PA6 particles and LLDPE, indicating poor compatibility between PA6 and LLDPE, which is due to the different polarity between PA6 and LLDPE, exhibiting a weak interfacial adhesion. The results showed that C-0 formed sea-island morphology in which IFR localized in the dispersed PA6 phase and PA6 phase and the LLDPE matrix exhibited poor interface adhesion. Figure 2 SEM–EDS of IFR and blends prepared by different methods. For blends of C2-M1 and C8-M1, irregular cavities with lengths of about some 10 μm and spherical particles with a dimension of about 3–5 μm were observed. The exfoliation of IFR particles from the LLDPE matrix formed larger irregular cavities, while the PA6 phase formed the small spherical particles in the LLDPE matrix. The results indicated that PA6 and IFR particles were both dispersed in the LLDPE matrix. The EDS results of C2-M1 and C8-M1 also indicated that IFR was dispersed in the LLDPE matrix rather than in the PA6 phase, due to which the percentages of elemental P and O in C2-M1 and C8-M1, respectively, were higher than that of C-0. The cavities formed by the exfoliation of IFR from the LLDPE matrix and clear interfaces between IFR particles and the LLDPE matrix indicated poor interfacial adhesion. Meanwhile, strong interfacial adhesion between PA6 particles and the LLDPE matrix was observed because of vague interface. The results indicated that the compatibilizer improved the interfacial adhesion between the PA6 phase and the LLDPE matrix. The different polarity between IFR and LLDPE caused a weak interfacial adhesion. The SEM–EDS results showed that LLDPE/PA6/IFR/LLDPE-g-MAH blends prepared by method one formed the sea-island morphology such that both IFR and PA6 localized in the LLDPE matrix and LLDPE-g-MAH improved the interfacial adhesion of the PA6 phase and the LLDPE matrix. Interfacial tensions between the filler and each polymer determine the filler distribution in the filler-filled polymer blends.32,33 In the uncompatibilized blends, IFR dispersion in the PA6 phase rather than in the LLDPE phase indicated that the interfacial tension between IFR and PA6 was lower than that of IFR and LLDPE. When LLDPE-g-MAH was melt-blended with LLDPE/PA6/IFR, LLDPE-g-MAH was reacted with PA6 to form a copolymer. The copolymer should migrate to the interface of PA6 and LLDPE to reduce the interfacial tension and phase size, indicating that the interfacial tension between LLDPE and PA6 was lower than that of IFR and PA6. As a result, IFR particles were found to localize in the LLDPE matrix rather than in the PA6 phase. The blends of C2-M2 and C8-M2 were prepared via a two-step process. At the first step, IFR should disperse in the PA6 phase because of similar polarity between PA6 and IFR. When LLDPE-g-MAH was melt-blended with LLDPE/PA6/IFR at the second step, LLDPE-g-MAH should not change the dispersion of IFR, due to which the PA6 phase remained solid, exhibiting IFR dispersion in the PA6 phase. The typical empty holes and the exfoliation phenomenon that appeared in C-0, C2-M1, and C8-M1 almost vanished, which suggested good interfacial adhesion. The results indicated that LLDPE-g-MAH improved the compatibility between PA6 and LLDPE. The PA6 phase remained solid when LLDPE-g-MAH was melt-blended with LLDPE/PA6/IFR. Therefore, the improvement of interfacial adhesion and compatibility between PA6 particles and LLDPE should be attributed to the reaction between the maleic anhydride group in the LLDPE-g-MAH melt and the amidogen on the surface of solid PA6 particles. The EDS results showed that the percentages of elemental P and O in C2-M2 and C8-M2, respectively, were lower than that of C-0, indicating that LLDPE-g-MAH encapsulated the PA6 phase, demonstrating that IFR dispersed in the PA6 phase was hard to be detected by EDS. As the content of LLDPE-g-MAH increased from 2 to 8 wt %, the coating thickness of LLDPE-g-MAH on the surface was also increased, causing the percentages of P and O to decrease from 14.88 and 1.03 to 14.12 and 0.6%, respectively. Therefore, the sea-island morphology in which IFR localized in the dispersed PA6 phase and LLDPE-g-MAH improved the compatibility of the PA6 phase and the LLDPE matrix was formed in LLDPE/PA6/IFR/LLDPE-g-MAH blends prepared by method 2. In order to confirm the reaction between LLDPE-g-MAH and PA6 in the blends prepared by method 2, the blends of LLDPE/PA6 (85/15) or LLDPE/PA6/LLDPE-g-MAH (80/15/5) were extracted by dimethylbenzene to remove LLDPE and LLDPE-g-MAH and the remaining parts were tested by FTIR, as shown in Figure 3. The blend of LLDPE/PA6 was melt-blended above the melting temperature of LLDPE and PA6. LLDPE/PA6/LLDPE-g-MAH blend was prepared by method 2. The typical infrared spectra of PA6 were shown, and no characteristic peaks of LLDPE were found in the remaining part of LLDPE/PA6, indicating that whole LLDPE was dissolved by dimethylbenzene and the remaining part consisted of PA6. For the remaining part of LLDPE/PA6/LLDPE-g-MAH prepared by method 2, the peaks at 2934 and 2968 cm–1 shown in LLDPE/PA6 were replaced by 2918 and 2850 cm–1, respectively. The peaks at 2934, 2968 cm–1 in the remaining part of LLDPE/PA6 were the −CH2– asymmetric stretching and symmetric stretching of PA6, respectively. From Figure 1, it was observed that the peaks at 2918 and 2850 cm–1 were the −CH2– asymmetric stretching and symmetric stretching of LLDPE. The results indicated that the remaining part of LLDPE/PA6/LLDPE-g-MAH contained LLDPE or LLDPE-g-MAH. LLDPE or unreacted LLDPE-g-MAH can be extracted by dimethylbenzene, but LLDPE-g-MAH reacted with PA6 should not dissolve in dimethylbenzene. Therefore, it can be concluded that the maleic anhydride group in the LLDPE-g-MAH melt can react with the amidogen on the surface of solid PA6 particles in the blends prepared by method 2. Figure 3 FTIR spectra of the remaining part of LLDPE/PA6 (a) or LLDPE/PA6/LLDPE-g-MAH (b) extracted by dimethylbenzene. 2.2 Flame Retardancy The flame retardancy of LLDPE/PA6/IFR and LLDPE/PA6/IFR/LLDPE-g-MAH prepared by different methods was investigated by limiting oxygen index (LOI) and UL-94 test, as shown in Table 1. For C-0, the LOI value was 29.7, and the samples achieved a V-2 rating in UL94 testing because of the melt dripping during combustion. Processing methods showed a remarkable influence on the flame retardancy. Blends of LLDPE/PA6/IFR/LLDPE-g-MAH prepared by method 1 exhibited poor flame retardancy, and the LLDPE-g-MAH content showed weak influence on the flame retardancy. The LOI values were only 24–24.7% and had no vertical rating in the UL-94 test. However, the flame retardancy of the blends prepared by method 2 was significantly improved, compared with the blends prepared by method 1. LOI values of C2-M2 and C5-M2 were both 28.6 and the samples passed the UL-94 V-2 rating, indicating that their flame retardancy was close to C-0. The best flame retardancy was exhibited in C8-M2. The LOI value reached 30.0%. The samples passed the UL-94 V-0 rating, indicating that the dripping properties exhibited in C-0, C2-M2, and C5-M2 were restrained in C8-M2. Table 1 Flammability Characteristics of Blends sample code LOI (%) UL-94 rating C-0 29.7 V-2 C2-M1 24.0 no rating C5-M1 24.0 no rating C8-M1 24.7 no rating C2-M2 28.6 V-2 C5-M2 28.6 V-2 C8-M2 30.0 V-0 The results showed that blends with IFR dispersion in the PA6 phase (C-0, C2-M2, C5-M2, and C8-M2) exhibited better flame retardancy than the blends with IFR dispersion in the LLDPE phase (C2-M1, C5-M1, and C8-M1). It was reported that PA6 can act as the charring agent of APP to enhance the charring performance and flame retardancy.29−31 Therefore, IFR dispersion in the PA6 phase was more beneficial for the reaction between APP and PA6, exhibiting better flame retardancy, compared with the blends with IFR dispersion in the LLDPE phase. C8-M2 samples had no melt dripping during combustion. The reason for this may be the case that LLDPE-g-MAH increases the viscosity of blends and enhances the antidripping property. Melt flow index (MFI) can relate to viscosity indirectly, which indicates the dripping properties of flame-retardant LLDPE/PA6 blends. MFI experiments were used to test the flow rate of flame-retardant LLDPE/PA6 blends at different LLDPE-g-MAH contents, as shown in Table 2. The highest MFI value observed in C-0 indicated the lowest melt viscosity. Obviously, the melt dripping of C-0 should be attributed to the low melt viscosity. MFI values of the LLDPE/PA6/IFR/LLDPE-g-MAH blends prepared by method 1 were lower than that of C-0. Moreover, the increase of LLDPE-g-MAH contents caused a sharp reduction of MFI values. The results indicated that LLDPE-g-MAH increased the melt viscosity. The copolymer formed by the reaction of LLDPE-g-MAH and PA6 can entangle with PA6 and LLDPE molecular chains at the interface to increase the flow resistance of melt, resulting in high melt viscosity. The increase of LLDPE-g-MAH contents in the blends produced more copolymers, resulting in higher melt viscosity. For the LLDPE/PA6/IFR/LLDPE-g-MAH blends prepared by method 2, the MFI values were also lower than that of C-0 and the increase of LLDPE-g-MAH contents caused the reduction of MFI values. The MFI value of C8-M2 was about one-third of C-0, indicating a sharp increase of melt viscosity, resulting in the enhancement of antidripping property. The increase of melt viscosity can also be attributed to the copolymer formed by the reaction of LLDPE-g-MAH and PA6. Table 2 Effect of LLDPE-g-MAH Contents on the MFI of the Blends Prepared by Different Methods sample code C-0 C2-M1 C5-M1 C8-M1 C2-M2 C5-M2 C8-M2 MFI (g/10 min) 105 71 52 25 67 43 34 2.3 Cone Calorimeter Analysis The cone calorimeter was also employed to evaluate the flame retardancy. Samples of C-0, C8-M1, and C8-M2 were chosen for testing by cone calorimetry. The heat release rate (HRR), total heat released (THR), and mass loss curves recorded during cone calorimeter tests are presented in Figure 4. The related data are presented in Table 3. Figure 4 Relationship between (a) HRR, (b) THR, and (c) mass loss and time of blends prepared by different methods. Table 3 Cone Calorimeter Test Data of Blends sample code PHHR (kW/m2) THR (kJ/m2) char residues (wt %) MAHRE (kW/m2 s) FPI (m2 s/kW) FGI (kW/m2 s) C-0 223 99 32 153 0.35 0.44 C8-M1 296 113 25 209 0.23 1.04 C8-M2 246 88 29 139 0.31 0.56 The peak HRR (PHRR) value of C8-M1 was higher than that of C-0 and C8-M2, indicating that the flame retardancy of C8-M1 was poorer than that of C-0 and C8-M2. The results showed that IFR dispersion in the PA6 phase enhanced flame retardancy in comparison with IFR dispersion in the LLDPE phase. The HRR curves of C-0 and C8-M2 displayed two peaks: the first peak was assigned to the formation of expandable char layers and the second peak was assigned to the further decomposition of a carbonaceous residue.34 The first PHHR and the HRR values for the initial 250 s of C8-M2 were lower than that of C-0, indicating that the char layer of C8-M2 formed in the initial period can provide better protection against the combustion of the matrix than that of C-0. Combustion experiments performed in the UL94 or LOI test were similar to the scenario of cone calorimetry at the ignition stage.35−37 Therefore, the results that the LOI and the vertical combustion level of C8-M2 were better than that of C-0 can be due to reduced combustion intensity in the initial period. The second PHHR value of C8-M2 was higher than that of C-0, indicating that the stability of the carbonaceous residue in C8-M2 was poorer than that of C-0. The THR of C-0 and C8-M2 was lower than that of C8-M1, and the mass loss curve showed that the char residue of C-0 and C8-M2 was higher than that of C-0, indicating that the dispersion of IFR in the PA6 phase was more favorable to the formation of a carbonaceous residue, resulting in the decrease of THR. Fire performance index (FPI) is defined as the proportion of TTI and PHRR; fire growth index (FGI) is defined as the proportion of PHRR and time to PHRR; MARHE is the maximum of the average rate of heat emission. The values of MARHE, FPI, and FGI can evaluate the fire hazard. It can be seen from Table 1 that the highest MAHRE and FGI values or the lowest FPI was observed in C8-M1, suggesting that C8-M1 possessed the strongest fire hazard in comparison with C-0 and C8-M2. 2.4 Characterizations of Residue Char Figure 5 shows the residue of the blends at the end of the cone calorimeter test. Broken char residue was observed in C8-M1. C-0 and C8-M2 formed a coherent and dense char residue with high intumescentia. The formation of highly intumescent, coherent, and dense char layer could provide a better protective shield; thus, the heat and mass transfer between the gas and condensed phase could be slowed, and the underlying materials are protected from further burning. For this reason, C-0 and C8-M2 exhibited much lower PHRR values than C8-M1. Figure 5 Photos of the aspect of the crust of blends after the cone calorimeter test. Char residues of LLDPE/PA6/IFR and LLDPE/PA6/IFR/LLDPE-g-MAH prepared by different methods were examined by SEM, as shown in Figure 6. The char residue of C2-M1 and C8-M1 was completely a loose structure; many voids can be observed. A continuous and compact char residue was formed in C-0, C2-M2, and C8-M2. Compared with the loose structure displayed in C2-M1 and C8-M1, a continuous and compact char residue can accumulate flammable gas and improve the blocking ability of heat and gas, resulting in the improvement of flame retardancy. Figure 6 SEM of intumescent char residues for blends prepared by different methods. The different morphologies of char residues should be attributed to the different dispersion of IFR in polymer blends. Compared with IFR dispersion in the LLDPE phase, IFR dispersion in the PA6 phase was beneficial for the reaction between APP and PA6, forming a high char residue, resulting in the formation of a continuous and compact char residue. However, IFR dispersion in the LLDPE phase was against the charring performance, forming loose char residues. 2.5 Water Resistance of Flame-Retardant LLDPE/PA6 Table 5 shows the mass loss percentages, LOI, and the UL-94 test results of flame-retardant LLDPE/PA6 after water immersion. The highest mass loss percentage was exhibited in C-0. Mass loss percentages of LLDPE/PA6/LLDPE-g-MAH/IFR blends prepared by method 1 were higher than that of blends prepared by method 2. The results indicated that LLDPE/PA6/LLDPE-g-MAH/IFR prepared by method 2 exhibited the best water resistance. The LOI and UL-94 test results were in accordance with the mass loss results. After water immersion, the LOI value of C-0 reduced from 29.7 to 24%, and the samples failed to pass the UL-94 test, indicating remarkable reduction in flame retardancy because of poor water resistance. Blends of C2-M1, C5-M1, and C8-M1 had poor flame retardancy before water immersion. Therefore, little change in flame retardancy was observed after water immersion. Good water resistance was exhibited in C2-M2, C5-M2, and C8-M2. After water immersion, the LOI values were reduced slightly, and UL-94 rating grades were unchanged. The different water resistance exhibited in Table 4 should be attributed to different microstructures of the blends. The sample of C-0 had the microstructure in which IFR dispersed in the dispersed PA6 phase and the interfacial adhesion between the PA6 phase and the LLDPE matrix was poor. Poor interfacial adhesion contained a number of microgaps, through which the water entered to dissolve IFR, resulting in poor water resistance. Blends of LLDPE/PA6/LLDPE-g-MAH/IFR prepared by method 1 possessed the microstructure in which IFR and PA6 phase both dispersed in the LLDPE matrix and the interfacial adhesion between the PA6 phase and the LLDPE matrix or IFR particles and the LLDPE matrix was strong or poor, respectively. Although the compatibilization of LLDPE-g-MAH on LLDPE/PA6 decreased the microgaps, the dispersion of IFR in the LLDPE matrix and the poor interfacial adhesion between IFR particles and the LLDPE matrix were both against the improvement of water resistance. In blends of LLDPE/PA6/LLDPE-g-MAH/IFR prepared by method 2, the microstructure in which IFR dispersed in the dispersed PA6 phase and LLDPE-g-MAH improved the interfacial adhesion between the PA6 phase and the LLDPE matrix was formed. Good interfacial adhesion reduced the microgaps, indicating that the water was hard to diffuse into the matrix. The PA6 phase can protect the IFR component from attack by water. Accordingly, good water resistance was observed in these blends. Table 4 Mass Loss, UL-94, and LOI Results of Blends after Water Treatment at 70 °C for 168 h sample code mass loss percentage (%) LOI (%) UL-94 rating C-0 3.2 ± 0.1 24 failed C2-M1 1.7 ± 0.2 24 failed C5-M1 1.9 ± 0.1 24.7 failed C8-M1 1.6 ± 0.1 24 failed C2-M2 0.9 ± 0.1 26.3 V-2 C5-M2 0.9 ± 0.1 27.1 V-2 C8-M2 0.7 ± 0.1 28.6 V-0 2.6 Mechanical Properties The mechanical properties of flame-retardant LLDPE/PA6 are compiled in Table 5. The tensile strength, elongation at break, and impact strength of C-0 were 7.1 MPa, 9.4%, and 3.0 kJ/m2, respectively. Poor compatibility between PA6 and LLDPE should be responsible for the deteriorated mechanical properties. For the blends prepared by method 1, compatibilizer LLDPE-g-MAH improved the tensile strength and elongation at break. Introduction of LLDPE-g-MAH to C-0 through method 1 also improved the impact strength except for C2-M1. The mechanical properties of the blends prepared by method 2 were higher than that of C-0 and the blends prepared by method 1. This phenomenon should be attributed to different morphologies. For the blends prepared by method 1, PA6 phase and IFR were both dispersed in continuous LLDPE phase. The compatibilizer elevated the interphase adhesion of PA6 and LLDPE phases and decreased PA6 phase size. Therefore, the tensile strength and elongation at break were increased from 7.1 MPa and 9.4% for C-0 to 9.0 MPa and 23.4% for C5-M1, respectively. For the blends prepared by method 2, IFR was dispersed in the PA6 phase and the compatibilizer elevated the interphase adhesion between the PA6 phase and the matrix. Therefore, the tensile strength, elongation at break, and impact strength were increased about 60, 300, and 50% in comparison with C-0, respectively. Table 5 Mechanical Properties of the Blends Prepared by Different Methods sample code tensile stress (MPa) elongation at break (%) impact strength (kJ/m2) C-0 7.1 ± 0.9 9.6 ± 3.5 3.0 ± 0.08 C2-M1 8.3 ± 1.2 27.3 ± 8.3 2.0 ± 0.01 C5-M1 9.0 ± 1.5 23.4 ± 9.3 3.3 ± 0.98 C8-M1 9.0 ± 0.9 23.3 ± 10.2 3.5 ± 0.78 C2-M2 11.9 ± 0.5 28.9 ± 4.9 3.7 ± 0.06 C5-M2 11.7 ± 0.6 24.2 ± 8.2 4.3 ± 0.08 C8-M2 11.9 ± 0.6 29.4 ± 7.1 4.4 ± 0.02 It was observed that the compatibilizer contents exhibited a weak effect on the mechanical properties of LLDPE/PA6/LLDPE-g-MAH/IFR prepared by different methods, due to which the increase of compatibilizer contents caused little change of the mechanical properties. For the blends prepared by method 1, although the increase of LLDPE-g-MAH content reduced the PA6 phase size and contributed to the augment of the mechanical properties to a certain extent, the poor interphase adhesion between IFR and the LLDPE matrix showed a greatly negative impact on mechanical properties, indicating that the mechanical properties were not influenced remarkably along with the increase of LLDPE-g-MAH content. In the blends prepared by method 2, the PA6 phase remained solid when LLDPE-g-MAH was melt-blended with LLDPE/PA6/IFR. Therefore, LLDPE-g-MAH cannot reduce the PA6 phase size. The increase of LLDPE-g-MAH content increased the coating thickness of LLDPE-g-MAH on the PA6 particles surface rather than increasing the interfacial strength. Therefore, the poor effect of the compatibilizer contents on mechanical properties was exhibited. 3 Conclusions Different processing methods were employed to prepare LLDPE/PA6/IFR blends with different microstructures, and the results showed that the microstructure affected the flame retardancy, mechanical properties, and water resistance greatly. Melt-blending IFR with LLDPE/PA6 simultaneously formed the microstructure in which IFR was selectively dispersed in the PA6 phase of LLDPE/PA6/IFR and the interphase adhesion between PA6 and the LLDPE matrix was poor. Although IFR dispersion in the PA6 phase was beneficial for the increase of flame retardancy, poor interphase adhesion deteriorated the mechanical properties and water resistance. When the compatibilizer LLDPE-g-MAH was melt-blended with LLDPE/PA6/IFR simultaneously, a microstructure was formed, indicating that IFR and PA6 particles were respectively dispersed in the LLDPE matrix and that LLDPE-g-MAH strengthened the interphase adhesion between the PA6 phase and the LLDPE matrix rather than IFR particles and the LLDPE matrix. The improvement of the interphase adhesion increased the mechanical properties and water resistance, but IFR dispersion in the LLDPE matrix decreased the flame retardancy sharply. A novel processing method that the IFR was first melt-blended with LLDPE/PA6 and then LLDPE-g-MAH was melt-blended with LLDPE/PA6/IFR between the melt temperatures of LLDPE and PA6 was employed to obtain the microstructure with the dispersion of IFR in the PA6 phase and strong interface adhesion of the PA6 phase with the matrix. The flame retardancy, mechanical properties, and water resistance were improved simultaneously. IFR dispersion in the PA6 phase and high viscosity caused by the compatibilization of LLDPE-g-MAH should be responsible for the improvement of flame retardancy. Strong interface adhesion of the PA6 phase with the matrix and IFR dispersion in the PA6 phase caused good water resistance. Moreover, good mechanical properties were attributed to the strong interface adhesion between the PA6 phase and the LLDPE matrix. 4 Experimental Section 4.1 Materials The materials used in this study were LLDPE (LL6201XR, MI = 50 g/10 min, d = 0.926 g/cm3) supplied by ExxonMobil Corp. and PA6 (33500, relative viscosity of 3.50, d = 1.14 g/cm3) supplied by Xinhui Meida-DSM Nylon Chips Co., Ltd. The IFR system consists of APP and PER, and the weight ratio of APP to PER is 4:1. APP [(NH4PO3)n, purity level > 90%] was supplied by Zhejiang Longyou Gede Chemical Factory (China). PER was supplied by Jinan Taixing Fine Chemicals Co., Ltd. LLDPE-g-MAH (TRD200L, MI = 2 g/10 min, d = 0.92 g/cm3) was supplied by Wujiang Siruda Plastic Industry Co., Ltd. The amount of maleic anhydride in LLDPE-g-MAH was 1 wt %. 4.2 Preparation of Composites All the materials were oven-dried for 12 h at 85 °C before extrusion and injection. The blends were extruded via a corotating twin-screw extruder with a barrel diameter of 20 mm and a barrel-length-to-diameter ratio of 25. Then the extruded blends were molded into sheets of suitable thickness at the injection pressure of 50 MPa via an injection molding machine. The formula of the blend is listed in Table 6. Table 6 Formulation of Blends sample code LLDPE PA6 IFR LLDPE-g-MAH processing method C-0 64 16 20 0 method 1 C2-M1 62 16 20 2 method 1 C5-M1 59 16 20 5 method 1 C8-M1 56 16 20 8 method 1 C2-M2 62 16 20 2 method 2 C5-M2 59 16 20 5 method 2 C8-M2 56 16 20 8 method 2 Two processing methods were employed for preparing the blends. Method 1: all the materials were melt-blended above the melt temperature of LLDPE and PA6 for preparing the blends. The temperatures from hopper to die were 140, 160, 190, 220, and 240 °C. Method 2: two steps were employed to prepare the blends. In the first step, PA6 and LLDPE were melt-blended with IFR to prepare LLDPE/PA6/IFR, and the processing temperature was higher than the melt temperature of LLDPE and PA6. The temperatures from hopper to die were 140, 160, 190, 220, and 240 °C. In the second step, LLDPE/PA6/IFR was melted with LLDPE-g-MAH, and the processing temperature was between the melt temperatures of LLDPE and PA6. The temperatures from hopper to die were 140, 150, 160, 160, and 160 °C. 4.3 Measurement and Characterization LOI was measured according to ASTM D2863-77. The apparatus used was a JF-3 instrument (Chengde, China). The specimens used for the test were of dimensions 120 × 6 × 3 mm3. The vertical test was carried out according to the UL94 test standard. The specimens used for the test were of dimensions 127 × 12.7 × 3 mm3. The sample flammability performed on the cone calorimeter (FTT, UK) test according to ISO 5660 standard procedures. The specimens used for the test were of dimensions 100 × 100 × 3 mm3. The specimen was exposed horizontally at an incident flux of 35 kW/m2. FTIR spectra were recorded on a Bruker Vector 33 spectrometer. LLDPE/PA6/APP (24/56/20) or LLDPE/PA6/PER (27/63/10) in which LLDPE was used as the matrix and PA6 formed dispersed phase was pressed into disks with KBr. Before being pressed, the samples of LLDPE/PA6/APP were extracted by formic acid for 48 h. The samples of LLDPE/PA6/PER were extracted first by formic acid and later by alcohol. The sample of LLDPE/PA6 (85/15) or LLDPE/PA6/LLDPE-g-MAH (80/15/5) was extracted by dimethylbenzene at 120 °C to remove LLDPE and LLDPE-g-MAH, and the residuum was pressed into disks with KBr for the FTIR test. A JEOL 6301F scanning electron microscope was used to investigate the morphology of residue char and molded specimens at an acceleration voltage of 20 kV. The residue char was obtained from the specimen left after the vertical test. The molded specimens were fractured in liquid nitrogen. To determine the water resistance of polymer blends, specimens, of the same size as used for the UL-94 test, were put in distilled water at 70 °C and kept at this temperature for 168 h. The water was replaced every 24 h, according to UL746C. The treated specimens were subsequently dried in a vacuum oven at 80 °C for 72 h, and the weight of the specimens was measured before water immersion and after drying. The mass loss percentages were calculated in the following equation18 where W0 is the initial weight of the specimens before water immersion and W is the remaining weight of the specimens after water immersion and drying. The tensile strength was measured by a tensile tester (LJ1000, Guangzhou Test Instrument Factory, China) according to ASTM D638. The MFI of melting polymer blends was measured by a melt index instrument, and the measurement temperature was 225 °C; load was 2.16 kg. The authors declare no competing financial interest. Acknowledgments The authors gratefully acknowledge the financial support of this work by the National Natural Science Foundation of China (contract number: 51673059), Natural Science Foundation of Education Department of Henan Province (contract number: 17A150009), and Student Research Training Program of HAUST. ==== Refs References Chen M. ; Xu Y. ; Chen X. ; Ma Y. ; He W. ; Yu J. ; Zhang Z. Thermal stability and combustion behaviors of flame retardant polypropylene with thermoplastic polyurethane encapsulated ammonium polyphosphate . High Perform. Polym. 2014 , 26 , 445 –454 10.1177/0954008313517910 . 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==== Front ACS OmegaACS OmegaaoacsodfACS Omega2470-1343American Chemical Society 3145984110.1021/acsomega.8b03473ArticleFabrication of Graphene Nanoplatelet-Incorporated Porous Hydroxyapatite Composites: Improved Mechanical and in Vivo Imaging Performances for Emerging Biomedical Applications Kumar Sunil †Gautam Chandkiram †Mishra Vijay Kumar ‡Chauhan Brijesh Singh §Srikrishna Saripella §Yadav Ram Sagar ∥Trivedi Ritu ‡Rai Shyam Bahadur ∥† Advanced Glass and Glass Ceramics Research Laboratory, Department of Physics, University of Lucknow, Lucknow 226027, Uttar Pradesh, India‡ LSS-101 Laboratory, Endocrinology Division, CSIR-Central Drug Research Institute, Lucknow 226031, Uttar Pradesh, India§Cell and Neurobiology Laboratory, Department of Biochemistry, and ∥Department of Physics, Banaras Hindu University, Varanasi 221005, Uttar Pradesh, India E-mail: [email protected]. Phone: +918840389015.24 04 2019 30 04 2019 4 4 7448 7458 29 12 2018 05 04 2019 Copyright © 2019 American Chemical Society2019American Chemical SocietyThis is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes. Three-dimensional nanocomposites exhibit unexpected mechanical and biological properties that are produced from two-dimensional graphene nanoplatelets and oxide materials. In the present study, various composites of microwave-synthesized nanohydroxyapatite (nHAp) and graphene nanoparticles (GNPs), (100 – x)HAp–xGNPs (x = 0, 0.1, 0.2, 0.3, and 0.5 wt %), were successfully synthesized using a scalable bottom-up approach, that is, a solid-state reaction method. The structural, morphological and mechanical properties were studied using various characterization techniques such as X-ray diffraction (XRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM), and universal testing machine (UTM). XRD studies revealed that the prepared composites have high-order crystallinity. Addition of GNPs into nHAp significantly improved the mechanical properties. Three-dimensional nanocomposite 99.5HAp–0.5GNPs exhibited exceptionally high mechanical properties, for example, a fracture toughness of ∼116 MJ/m3, Young’s modulus of ∼98 GPa, and compressive strength of 96.04 MPa, which were noticed to be much greater than in the pure nHAp. The MTT assay and cell imaging behaviors were carried out on the gut tissues of Drosophila third instars larvae and on primary rat osteoblast cells for the sample 99.5HAp–0.5GNPs that have achieved the highest mechanical properties. The treatment with lower concentrations of 10 μg/mL on the gut tissues of Drosophila and 1 and 5 μg/mL of this composite sample showed favorable cell viability. Therefore, owing to the excellent porous nature, interconnected surface morphology, and mechanical and biological properties, the prepared composite sample 99.5HAp–0.5GNPs stood as a promising biomaterial for bone implant applications. document-id-old-9ao8b03473document-id-new-14ao-2018-03473uccc-price ==== Body 1 Introduction Nanohydroxyapatite (nHAp) is an advanced bioceramic nanocrystalline material suitable for designing artificial bone or implants. Bone engineering is a clinical procedure that replaces missing bone and repairs the bone fracture.1 The living bone has the ability to regenerate the hard and soft tissues but requires a porous scaffold for providing mechanical support to it.2 The biocompatibility, osteoconductivity, high porosity, and mechanical strength are the fundamental criteria for designing a bone substitute material.3 Due to the limited number of donor autograft, allograft does not fulfill the requirement of the implantations; synthetic materials are widely being used as a bone substitute material.4 The synthetic bone material obtained from nHAp and its composite possess a chemical and structural composition similar to the natural bone.5 However, nHAp has been widely used in bone, drug delivery, sensor, and environmental engineering.6 Recently, researchers pay much attention to the fabrication and designing of nHAp nanocomposites with sufficient mechanical properties for biomedical applications. However, the main features of bone implantation materials are 3D porous microstructures with a high amount of nHAp and low secondary phases as an additive.7 It is well reported that the porous scaffolds must have a mechanical strength as close as possible to that of the substituted bone; otherwise, this porous structure is to be destroyed during tissue regeneration and load applications.8 The unadulterated nHAp material cannot fulfill the prerequisites of the embedded bone because of its fragile nature and low mechanical properties which limit their applications.9 Therefore, with the aim of improving the mechanical properties, the various nHAp composites have been developed to enhance the mechanical strength of the pure nHAp, such as HAp–MgO,10 HAp–SrCO3,11 HAp–La2O3,12 HAp–Al2O3,13 and HAp–GO.14 Furthermore, it is also reported that the addition of Fe2O3 into the nHAp considerably improves the mechanical strength.15 The TiO2-added nHAp nanocomposites present improved fracture toughness and mechanical strength than the pure HAp.16,17 The 1.0 wt % CNT-added nHAp composite displays a 120% enhancement in toughness.18 However, the glass-added nHAp composite remarkably enhanced the density and compressive strength of the nHAp that was sintered at 1200 °C.19 Although, nHAp composites with metals, ceramic oxides, and polymers showed not only promising results for enhanced mechanical properties but also the reverse effect toward the biological properties.20,21 The incorporation of the second phase of carbon nonmaterials such as CNT and graphene are thought suitable in a ceramic matrix due to excellent mechanical and biological properties such as a high Young’s modulus and compressive strength along with adequate bioactivity.22,23 The graphene has been frequently used in various biological applications such as biosensing,24,25 drug delivery,26 tissue engineering,27,28 and cell modulating interfaces and cell scaffolds to control the cell growth.29 The high surface area and low density of graphene act in applications as a mechanical and biological modifier.30−32 The graphene combined with nHAp makes low-density nanocomposites that possess high mechanical strength, high surface area, biostability, and excellent biocompatibility.33 The addition of graphene into nHAp could be promising for maintaining the nHAp bioactivity.34 The mechanical strength and biological properties of nHAp–GNP composites have already been widely studied and have been found to be mechanically strong compared to pure nHAp.35,36 Various studies have been carried out on graphene nanoplatelets added to composites, silicon nitride,33 and aluminum37 on enhanced mechanical and biological properties. Recently, graphene is added as an effective additive for toughening the nHAp composite. The HAp–GNP composite prepared by an additive manufacturing technique showed excellent improvement in compressive strength.38 It is all around detailed that the graphene-included nHAp composite upgraded a few mechanical and natural parameters, for example, the versatile modulus, crack sturdiness, and osteoblast cell bond and their expansion.39−41 The nanocomposites with high porosity and interconnectivity of porous structure are essential to enhance the restorability and provide mechanical support to tissue growth.42 The mechanical property of the composites strictly depend on microstructure, porosity, pore size, and formation of secondary phases.5 The incorporation of graphene into the pure nHAp enhanced its mechanical properties significantly and showed a reverse effect for their densities.43 Recently, biocompatibility of graphene and its composites were successfully evaluated on mouse embryonic fibroblast cells as well as on human lung cells.44,45 It is previously reported that the addition of GNPs into the nHAp matrix is promising for improving the mechanical strength and cell imaging.46,47 The nHAp composites coadded with graphene and gold nanoparticles showed more favorable differentiation important in bone regeneration and remodeling of the bone tissue.48,49 The imaging of intracellular structure is an important matter in the biomedical field.50 The fluorescent nanoparticles are more stable in the biological and excellent tissue imaging applications. The nHAp is a nontoxic, self-activated fluorescent material and becomes an excellent bioimaging material that retains their fluorescing nature in a biological environment.51 Comparatively, to develop the better nHAp-based composite, the optimized strategy has been adopted using different concentrations of the GNPs to synthesize various nanocomposites with improved performances. Current efforts have been made to synthesize highly porous crystalline nHAp and its composites, which can be used for bone implant applications. Thus, highly efficient biocompatible nHAp has been successfully prepared via a microwave irradiation technique. However, nHAp–GNP composites were synthesized by using a simple solid-state reaction method. In order to improve the mechanical properties by maintaining the biological properties; different concentrations of GNPs were used as an additive. However, GNPs may have a more exploitable porous structure than other additives. In the previously reported studies, most of the researchers have been focused only on the structural properties of such types of composite materials and did not have a detailed study on their biological properties. In the present study, we propose the synthesis of porous nHAp and its composites with improved mechanical and in vivo imaging performances for biomedical applications using a simple and scalable bottom-up approach, that is, a solid-state reaction method. Enhancement in the mechanical strength, in vivo imaging, and biocompatibility without changing the other properties by standard sintering etiquette was for the first time reported. 2 Results and Discussions 2.1 X-ray Diffraction The crystallographic nature, phase formation, and phase stability of the GNP-reinforced nHAp composites sintered at 1200 °C for 3 h were analyzed using a powder XRD method. All the XRD patterns were matched with standard stoichiometric HAp (JCPDS file no. 240033) and were not found of any peak of the impurities and their byproducts. These XRD patterns also indicate the presence of the utmost of high intensity peaks, which are lying between the 2θ ranges of 25°–45°. The XRD pattern of pure GNPs is shown in Figure S1. Three different characteristic peaks were observed at different 2θ angles, 26.66°, 44.64°, and 54.74°, corresponding to their lattice planes, (002), (100), and (004), which confirmed the purity of the utilized GNPs (JCPDS file no. 030401). The X-ray diffraction patterns of all the biocomposite samples are shown in Figure 1a–e. The various diffraction peaks in the XRD patterns of composite samples are well matched with the pure hexagonal HAp phase and the pure GNPs. The characteristic X-ray diffraction peaks corresponding to planes (002), (100), and (004) of GNPs were observed at different 2θ angles, 26.1°, 42.24°, and 53.14°, which clearly showed the presence of GNPs in nHAp.6 The XRD patterns also revealed that the addition of GNPs does not alter the phase stability of nHAp; however, minor peaks due to GNPs themselves have been altered slightly. Thus, XRD patterns of the nHAp–GNP composites are almost similar. The sharp and highly diffracted peaks revealed a good crystalline nature of the nHAp–GNPs composites.9 The average crystallite size of the composite samples was determined from the XRD pattern and was found to be in the range of 411 to 479 nm with ±0.003 accuracies. Thus the calculated average crystallite sizes of all the samples are enlisted in Table 1. These results were also correlated with the results of the TEM and shown in Figure 2. Figure 1 X-ray diffraction patterns of (a) 99.9HAp–0.1GNP, (b) 99.8HAp–0.2GNP, (c) 99.7HAp–0.3GNP, (d) 99.5HAp–0.4GNP and (e) 99.5HAp–0.5GNP composites sintered at 1200 °C for 3 h. Figure 2 Variations of (a) density with different doping concentrations of GNPs in the composite system (100 – x)HAp–xGNPs (x = 0.1, 0.2, 0.3, 0.5 wt %). (b) Variation of percentage of porosity with increasing doping concentrations of GNPs in the composite system (100 – x)HAp–xGNPs (x = 0.1, 0.2, 0.3, 0.5 wt %). (c) Variations of density and percentage of porosity in the composite system (100 – x)HAp–xGNPs (x = 0.1, 0.2, 0.3, 0.5 wt %). Table 1 Mechanical Properties of Crystalline Nano HAp and nHAp–GNP Composite Samples Sintered at 1200 °C for 3h samples density (g/cm3) average particle size (nm) compressive strength (MPa) Young’s modulus (GPa) fracture toughness(MJ/m3) percentage of porosity nHAp 2.87 ± 0.087 411 ± 12 87 ± 5 89 ± 5 104 ± 5 8.97 ± 0.15 99.9HAp–0.1GNPs 2.79 ± 0.085 432 ± 9 87 ± 5 89 ± 5 105 ± 6 11.43 ± 0.17 99.8HAp–0.2GNPs 2.72 ± 0.088 479 ± 8 91 ± 7 93 ± 6 110 ± 8 13.87 ± 0.25 99.7HAp–0.3GNPs 2.65 ± 0.070 431 ± 6 93 ± 8 95 ± 6 112 ± 5 15.98 ± 0.24 99.5HAp–0.5GNPs 2.50 ± 0.075 454 ± 5 97 ± 7 98 ± 8 116 ± 9 20.87 ± 0.41 natural bone(human cancellous)57−62 1.8–2.54 5–10 0.05–0.1 2.2 Density and Porosity Analysis The calculated values of the density of nHAp and its composites 99.9HAp–0.1GNPs, 99.8HAp–0.2GNPs, 99.7HAp–0.3GNPs, and 99.5HAp–0.5GNPs are listed in Table 1. The variations in the density and percentage of GNPs for all the composite samples are shown in Figure 2a. From a minuscule point of view, a decrease in the density of the fabricated composite samples with increasing concentrations of GNPs may be due to the incorporation of a low density (GNPs, 0.4 g/cm3) by a high density (without sintering nHAp, 2.95 g/cm3). Therefore, the density of the composite sample 99.5HAp–0.5GNPs, x = 0.5 was found to be a minimum of 2.83 g/cm3, while it was found to be a maximum of 3.01 g/cm3 for sintered nHAp, x = 0.0. The porous structure of nHAp samples was affected by a microstructural parameter, such as grain size, grain packing, particle shape, and distribution of grain size, and the amount of additive. Thus, the pore size, fraction, and architectures of the composites were also strongly affected by the mechanical properties of the nHAp. The variations in the percentage of porosity and different concentrations of GNPs are shown in Figure 2b. The increasing concentrations of GNPs for nHAp reduced the densification significantly. Thus; the porosity of the composites is increased by increasing the concentration of GNPs from 8.97 to 20.87%. The variations of density and percentage of porosity with different doping concentrations of GNPs in the composite system (100 – x)nHAp–xGNPs (x = 0.1, 0.2, 0.3, 0.5 wt %) is shown in Figure 2c. The density versus percentage porosity bar graph shows a reverse effect, that is, density is increasing with a decreasing percentage of porosity. Moreover, the addition of GNPs into an nHAp matrix creates a 3D porous structure and exhibits a random grain growth by preventing the grain boundaries.45 Therefore, the high mechanical strength, high fracture toughness, and high 3D porous nature of the composite 99.5HAp–0.5GNPs could suggest the required fluid hauling by this sample and offer superior biological activities. 2.3 Morphological Analysis The surface morphology and porous structure of nHAp and composites of HAp–GNPs, as well as the fractured samples, were analyzed by field emission scanning electron microscopy (FE-SEM). The SEM images of the pure nHAp and composite samples are shown in Figure 3a–e. According to the microstructures, significant changes were observed in the surface morphology with the addition of GNPs. From the SEM images, a more obvious restricted effect was observed for composite 99.8nHAp–0.2GNPs as compared to the composite 99.7nHAp–0.3GNPs. It is observed that the nHAp–GNP composite had open pores on the surfaces as well as inside the material (disc) with a high degree of interconnectivity. The porosity and pore diameter increased with the increasing amount of GNPs because GNPs reduce the agglomeration of nHAp particles during sintering. The overlapping, interconnectivity of the pores, enhanced the porosity of the nHAp–GNP composites. Moreover, as the concentration of GNPs was increased, the grain boundaries and their separation with the pores were seen clearly. The SEM images of the nHAp and its composite samples are shown in Figure 3a–e. Figure 3a reveals a dense surface morphology of nHAp in comparison to the nHAp–GNP composite samples. It also exhibits a uniform grain growth of nHAp along with an average pore size of ∼5 μm; the pores are marked by yellow arrows throughout the SEM image. This nHAp–GNP composite is predictable for the grains growing at a high rate along the path where no GNPs are present. It is also observed that the composite surfaces are highly porous in nature with the increased pore size interconnectivity between GNPs and nHAp. The highly porous nature and interconnectivity between GNPs and nHAp are attributed to a homogeneous dispersion of GNPs into the nHAp matrix (Figure 3b–e). Figure 3 Scanning electron microscopy images of (a) nHAp, (b) 99.9HAp–0.1GNP, (c) 99.8HAp–0.2GNP, (d) 99.7HAp–0.3GNP and (e) 99.5HAp–0.5GNP composites sintered at 1200 °C for 3 h. The different pores are presented by yellow arrows while blue arrows present the mixed and large grains of HAp–GNPs. The elemental analysis was done using EDAX results and is shown in Figure 4a,b. The tentative chemical compositions of nHAp and the 99.5HAp–0.5GNP composite sintered at 1200 °C for 3 h are selected for the recording of the EDAX spectrum. The EDAX recorded grains, as well as the elemental, were marked by the red arrow. The EDAX spectra revealed the presence of different elements of HAp as Ca, P, C, and O peaks; however, a low intensity peak of Na impurity was observed, which might be due to the precursor impurity (Figure 4a). Hence, the incorporation of GNPs into the nHAp was observed in the form of carbon (i.e. “C”) for the composite sample 99.5HAp–0.5GNPs. Thus, an EDAX spectrum clearly confirmed the presence of the used additive. Figure 4 Energy dispersive X-ray analysis spectrums of (a) pure nHAp and (b) 99.5HAp–0.5GNP composites sintered at 1200 °C for 3 h. The insets along with red arrows reveal the selected area of the SEM at which EDAX spectrum was recorded and also present the weight and atomic percentage of different elements. 2.4 Fractured SEM Analysis The fractured SEM images of the samples nHAp, 99.9nHAp–0.1GNPs, 99.8nHAp–0.2GNPs, 99.7nHAp–0.3GNPs, and 99.5nHAp–0.5GNPs are shown in Figure 5a–e and exhibited similar interconnected porous morphology. Moreover, SEM images of the fractured samples show the irregular shape and size of the pores. The addition of GNP content into the nHAp matrix improved the porous microstructures significantly (Figure 5e).5 Figure 5 Scanning electron microscopy images of fractured samples (a) nHAp, (b) 99.9HAp–0.1GNP, (c) 99.8HAp–0.2GNP, (d) 99.7HAp–0.3GNP and (e) 99.5HAp-0.5GNP composites sintered at 1200 °C for 3 h. The red dashed lines show randomly oriented crack lines. When the compressive load is applied to the samples, a random fractured debonding morphology of the composite was observed. Due to the compression load, debonding persuaded the surface roughness of the fractured samples. Thus, there is no straight crack propagation presented in the fractured SEM images. However, the microstructure of these composites showed some randomly oriented grain and grain boundaries, which are found to be prevented by GNP nanosheets that acted as a bridge while initially fine cracks are propagated. It is well reported that the load bearing applications of pure nHAp are limited due to its inherently low mechanical strength and high brittleness as compared to graphene and natural bones.52−54 Thus, the grain boundaries of nHAp are weaker than those of GNPs that randomly overlap each other and make them stronger.55 As 0.1 and 0.2 wt % of GNPs was added to the nHAp, the grain boundaries appeared blurred as shown in Figure 5b,c. The further the relatively large amount of GNPs was increased, the agglomeration of the GNPs particles was observed and was reducing its density (Figure 5e). The observed rough surfaces of the porous composite bodies may favor the cellular adhesion that induces the new bone formation activity.6,16 2.5 Transmission Electron Microscopic (TEM) Analysis The structural and morphological analyses were confirmed on the basis of the TEM results. Figure 6a–c shows the low and high magnification TEM images of the composite sample 99.5HAp–0.5GNPs sintered at 1200 °C for 3 h soaking time with heating at 5 °C/min. Figure 6a presents a low magnification image of the composite 99.5HAp–0.5GNPs and shows a well-interconnected porous network between nHAp and GNPs. The calculated average pore size of ∼4 nm was observed, and the graphene nanoparticles are well dispersed in the nHAp matrix, which is indicated by the dark region specified by yellow arrows. The nanoparticles of the GNPs are uniformly distributed into the porous network of nHAp; the dense nHAp matrix formed a big cluster that enhanced the strength of these composites. The high resolution TEM images are shown in Figure 6b,c. The porous structure of the composite can be seen clearly in these micrographs. However, the agglomeration of the nHAp particles was observed over large nanosheets of GNPs, which makes them very dense in nature (Figure 6b,c).9,10 The high-magnification bright field TEM image shows the large HAp sheets in which GNPs are uniformly embedded with adequate porosity (Figure 6d). The selected area electron diffraction pattern (SAED) is shown in Figure 5e. This pattern clearly exhibits the three different characteristics of diffraction rings corresponding to the lattice planes (000), (002), and (211) of nHAp, which confirmed the highly crystalline nature and are very close to the [11̅00] zone axis of the composite.12 Thus, the obtained results are well consistent with the results of the XRD and SEM. Figure 6 TEM images of the composite sample 99.5HAp–0.5GNPs sintered at 1200 °C for 3 h: (a) low-magnification bright field image, (b) low-magnification bright field image showing the porous interconnected network between HAp and GNPs, (c, d) High-magnification images containing fine grains of HAp with dispersed GNPs, and (e) SAED pattern of 99.5HAp–0.5GNPs. 2.6 Mechanical Behavior After a structural characterization of the synthesized composites, we performed the mechanical tests. For this purpose, samples were subjected to compression by an Instron universal testing machine. The compressive load–displacement curves are shown in Figure 7a–e. The curves show the load bearing capability and were linearly increased with the increasing concentrations of GNPs.10 The load bearing capability of the 0.5 wt % composite sample 99.5HAp–0.5GNPs was found to be ∼3.41 kN, which is almost 3-fold of the pure nHAp (1.33 kN).11 Moreover, in load versus displacement plots, the displacement decreased with the increase of the amount of GNPs, and it was found to be 0.45 mm for the composite 99.5HAp–0.5GNPs instead of 0.60 mm for pure nHAp.12 Thus, a reduction in displacement shows an improvement in the strength of nHAp as well as an enhancement in the load bearing capability of nHAp that resulted from the addition of GNPs into nHAp. The GNPs enhanced the interlocking and resisted crack propagation for these composites.13 Being 2D in nature, graphene can bear the maximum load in both directions, that is, longitudinal and lateral, and therefore, enhanced the load transfer efficiency significantly. The reduced extensibility with improved brittleness may sometime facilitate the material into confined bone defects or enable hard tissue engineering applications.2,5,15,23 The increasing compressive strength of a material revealed that the mechanical strength was significantly enhanced by increasing the concentration of GNPs into the nHAp during sintering.14 The composite 99.5HAp–0.5GNPs achieves the highest value of compressive strength ∼97 MPa, while it is ∼87 MPa for pure nHAp. However, the compressive strength value of the 0.1 wt % GNPs–HAp composite is ∼87 MPa, which is very close to the pure nHAp.15 The compressive strength and elastic modulus of the nHAp–GNPs composites are significantly higher than that of the compressive strength and elastic modulus of the pure nHAp.16 The improvement in mechanical properties suggests the role of the bonding interface between nHAp and GNPs.18 Figure 7 The mechanical characteristics of nHAp and its composites: (a) nHAp, 99.9nHAp–0.1GNPs, 99.8nHAp–0.2GNPs, 99.7nHAp–0.3GNPs, and 99.5nHAp–0.5GNPs. (b) Variations of Young’s modulus with increasing weight percentage of GNPs. (c) Variations of fracture toughness with increasing weight percentage of GNPs. (d) Bar graph showing the variation of compressive strength with increasing weight percentage of GNPs sintered at 1200 °C for 3 h. The homogeneous dispersion of GNPs in the HAp matrix improves the mechanical properties of the composites in different ways. Graphene prevented the migration of the grain boundaries of nHAp that ultimately enhance the compressive strength by decreasing the defect size and increasing the interface area between GNPs and nHAp.19 The interaction between the fine grains of HAp and nanosheets of the GNPs improved the binding strength that enhanced mechanical strength, interlocking, and load transfer capability between nHAp and GNPs.20 The variation of Young’s modulus with the increasing concentration of GNPs is shown in Figure 7b. The increasing weight percent of GNPs in the nHAp matrix enhanced the Young’s modulus value up to ∼98 GPa for the composite sample 99.5HAp–0.5GNPs.21 For the fracture toughness as shown in Figure 7c, the highest value of 116 MJ/m3 was observed for the same composite sample. The incorporation of GNP particles are seen inside the intergranular region that provides a high resistance to crack propagation.22 The values of both fracture toughnesses and Young’s modulus increased with the increasing concentrations of GNPs in the nHAp matrix (Table 1). The compressive strength of the prepared nHAp and its composites are shown in Figure 7d. As increasing the weight percentage of GNPs, the compressive strength was also found to have increased from 87 to 96 MPa. Thus, the overall study concluded that the addition of 0.5 wt % GNPs significantly enhanced the mechanical properties of the nHAp.23 The incorporation of GNPs has the ability to tune the mechanical properties of the different composite materials that were demonstrated for useful applications of bone implants. 2.7 Cell Viability Behavior Qualification of the cell viability examination is necessary for a biomaterial to be considered as an implant/scaffold material. Therefore, among all compositions, the 99.5HAp–0.5GNPs that has achieved the best mechanical properties was examined through this cell viability test. In this test known as the MTT assay, the MTT metabolic activity is measured by using a specific cell type. In the present study, the gut tissues of Drosophila larvae and primary osteoblast cells of a rat are individually used, and cytotoxicity/cell viability results were measured by using MTT [3-(4, 5-dimethyl thiazolyl-2)-2,5-diphenyltetrazolium bromide] for both types of cells. In the case of the toxicity measurement on the gut tissues of Drosophila larvae, various increasing concentrations (10, 30, 50, 100, 500, and 1000 μg/mL) of nHAp and the 99.5HAp–0.5GNP composite were used, as shown in Figure 8a,b. Treatments with 10 μg/mL of nHAp, as well as composite powder samples, exhibited maximum cell viability in their respective groups; however, treatments with the highest concentrations of 500 and 1000 μg/mL of the composite sample and 30 μg/mL of nHAp showed the least cell viability, that is, the highest cytotoxicity. Figure 8 MTT assay of the (a) nHAp and (b) 99.5HAp–0.5GNP composite in gut tissues of Drosophila larvae for a 1 h incubation period at different concentrations. Figure 9a,b presents the rat osteoblast (ROB) cells obtained from calvaria and the cell viability (in terms of optical density, i.e., O.D.) of ROB cells after 24 h of treatment with different concentrations (0, 1, 5, 25, 50, and 100 μg/mL) of the 99.5nHAp–0.5GNP composite. The material’s exposure with lower concentrations of 1 and 5 μg/mL significantly enhanced the cell proliferation as compared to the control. However, higher concentrations 25–100 μg/mL were nonsignificant. It is interesting to note that the exposure with each concentration of the composite 99.5nHAp–0.5GNPs rather than pure nHAp exhibits improved cell viability of the gut tissues of Drosophila larvae. Therefore, the overall cell viability studies demonstrated that there is no cytotoxic effect of the 99.5HAp–0.5GNP composite on any cell type, which may offer the synthesized composite sample for bone implant application. Figure 9 (a) Rat osteoblast (ROB) cells obtained from calvaria and (b) cell viability (in terms of optical density, O.D.) of ROB cells after 24 h of treatment with different concentrations (0, 1, 5, 25, 50, and 100 μg/mL) of the 99.5HAp–0.5GNP composite. The material’s exposure with lower concentrations 1 and 5 μg/mL significantly enhanced the cell proliferation as compared to the control. Recently, it has been reported that the nHAp composites are bioactive fluorescent materials that can be used for bioimaging diagnosis.48 The micrographs that present the fluorescence activities of nHAp and the composite 99.5nHAp–0.5GNP sample in the gut tissues of Drosophila larvae are shown in Figure 10. For the control, the untreated gut tissue facilitates no fluorescence as compared to nHAp-treated tissues.49 However, composite 99.5nHAp–0.5GNPs showed a maximum emission spectrum at the green filter (465–495 nm). The gut tissues also exhibited red fluorescence at the red filter (540–625 nm) with a predominant emission in the green range. Further, there was no clear mechanism that existed regarding the fluorescence of newly synthesized composite 99.5HAp–0.5GNPs, because there was neither Ca2+ nor PO43– that exhibited any known fluorescence in the living cells.50 Hence, the nHAp composite could be attributed to self-activated fluorescing behavior named as “fluorescent HAp” (fHAp).51 In the present study, pure nHAp and composite 99.5HAp–0.5GNPs present improved fluorescence activities in gut tissues of Drosophila larvae as compared to the control. Fluorescence emission in the green channel was found to be stronger in comparison to the red and bright field filters. In the merged channel, the 99.5HAp–0.5GNP composite also shows an improved fluorescence. Moreover, the 99.5nHAp–0.5GNP composite displays a better fluorescence behavior as compared to the pure nHAp. The results demonstrate that the biocompatible HAp–GNPs were performed successfully by in vivo imaging. Thus, nHAp and the HAp–GNP composite have promising fluorescence and wide range applicability for optimization and designing in the research of bioimaging. Figure 10 In vivo fluorescence micrographs present the fluorescence activities of nHAp and the 99.5HAp–0.5GNP composite in gut tissues of Drosophila larvae. 3 Conclusions Highly porous nHAp and its nanocomposites have been successfully synthesized via microwave irradiation and a scalable solid-state reaction method. The maintained structure of the nHAp–GNP composite revealed a strong bonding between nHAp and GNPs in the composite. The strong interaction between nHAp and GNPs is due to the increased interfacial area between these nanoparticles. The GNPs are well distributed into the nHAp matrix, which enhanced the mechanical properties of the composite. The addition of a relatively large concentration of GNPs into the nHAp matrix prevents the nHAp grain growth during sintering of the composites at 1200 °C. All the synthesized composites possess a porous 3D interconnected structure with low density. At this high sintering temperature, the mechanical properties of nHAp were significantly improved by the incorporation of GNPs. The enhanced mechanical properties were observed for the composite sample 99.5HAp–0.5GNPs. It is concluded that only a 0.5 wt % amount of GNPs highly modified the surface morphology and mechanical properties. The biological tests of the nHAp and composite 99.5nHAp–0.5GNPs showed good biocompatibility and cell viability with gut tissues of Drosophila third instar larvae as well as with ROB cells. Therefore, the fabricated composite may have great potential for load bearing and bioimaging applications in bone engineering. 4 Methods and Materials 4.1 Materials Synthesis In the synthesis of nHAp, calcium nitrate tetrahydrate (Ca(NO3)2·4H2O), disodium hydrogen phosphate (Na2HPO4), and sodium hydroxide (NaOH) pellets were purchased from Merck (Merck Pvt. Ltd., Mumbai, India) and used as starting raw materials. To adjust the pH value of the solution, sodium hydroxide pellets were used with continuous stirring. The synthesis of nHAp follows the given chemical reaction below: To synthesize the nHAp, the amount of reactant in the molar ratio of Ca2+/PO43– was adjusted to 1.67. However, Ca(NO3)2·H2O:Na2HPO4 were taken in the molar ratio of 5:3; Ca(NO3)2·H2O (1.0 M) and Na2HPO4 (0.6 M) were separately dissolved in 100 mL of double-distilled water. The Ca(NO3)2·4H2O solution was stirred by a magnetic stirrer; after 45 min of stirring, the Na2HPO4 solution was added to the calcium nitrate solution and continuously stirred for 60 min. Further, the pH value of the solution was adjusted to 12 by adding a suitable amount of sodium hydroxide (NaOH) pellets. The suspension with a certain pH value was kept to a household microwave oven of 600 W with a refluxing system for 15 min; the microwave oven followed a working cycle of 5 s on and 10 s off. After microwave irradiation, the sample was cooled to room temperature.9 The obtained material contained a milky suspension, which was centrifuged and washed with deionized water for several times to remove the NH4+, NO3– ions and other volatile impurities. The white precipitate was dried at 100 °C for 20 h in a hot air oven. The dried HAp slurry was crushed by mortar and pestle to convert the crushed material into a fine powder form. Finally, the dried nHAp powder was calcined at 800 °C for 2 h with a heating and cooling rate of 5 °C/min.10 4.2 Synthesis of nHAp–GNP Composites To synthesize the nHAp–GNP composites, different concentrations of GNP powders (0.1, 0.2, 0.3, and 0.5 wt %) were mixed by dissolving into 200 mL of ethanol. The dissolved mixture was stirred by a magnetic stirrer using zirconia beads for 4 h to produce a homogeneous composite of nHAp–GNPs.11 The obtained slurry was dried in a hot air oven at 80 °C for 6 h. The dried slurry was crushed and sieved, then ball-milled for 10 h to make fine powder for compaction. The nHAp–GNP composite fine powder was then cold-compressed using a hydraulic press machine at 2 ton and was held for 60 s to obtain a cylindrical shape of dimensions (height × diameter) 20 mm × 13 mm.12 The compact green samples were sintered at 1200 °C with a heating and cooling rate of 3 °C/min for 3 h to achieve desired densification. 4.3 Material Characterizations In order to confirm the phase formation of pure nHAp and nHAp–GNP composite powders, the various X-ray diffraction (XRD) patterns were recorded using a Rigaku Miniflex II X-ray diffractometer equipped with a monochromatic Cu-Kα radiation (λ = 0.15418 nm) operated at 40 kV and 40 mA. Data were recorded in the 2θ range from 20°–60° with fixed scanning at 3°/min. XRD patterns of the sintered nHAp and nHAp–GNP composite samples were then compared with standard JCPDS files no. 24–0033 for the determination of different phases and formation of the reaction product as new phases. The crystallite size (d in nanometers) was calculated with the help of Scherrer’s formula 1 where k is a Scherrer constant (k = 0.94), β is the peak width of the diffraction peak profile at full width half maximum (FWHM) in radians, λ is the X-ray wavelength of Cu-Kα radiation (λ = 1.54 nm), and θ denotes the Bragg’s angle in degrees. The densification behavior of pure HAp, as well as nHAp–GNP composite samples, was measured using the Archimedes method. Distilled water was used as a substituting material because it can easily penetrate into the pores of the nHAp–GNP composite samples. The sintered porous pellets of nHAp and nHAp–GNP composite samples were immersed in double-distilled water, and no vapors were seen coming out from them. Then, the dry, soaked, and hanging weights of the pellets were measured using a Shimadzu-made weighing digital balance having readability up to 0.0001 mg. By using the formula below, the density of the sintered samples was calculated 2 where ρ (water) is the density of distilled water (1 g/cm3), Wt (air) is the weight of the specific gravity bottle with the sample in air, and Wt (water) is the weight of the specific gravity bottle with sample and water. The percentage porosity of the HAp samples was measured using the Archimedes principle according to the equation below 3 where the apparent density was calculated by mass per unit volume of the composite samples in g/cm3. The standard theoretical density of pure nHAp was 3.156 g/cm3.,56,57 The surface morphology of the nHAp and nHAp–GNP composite samples, as well as the fractured samples, were recorded using a scanning electron microscope (SEM) attached with an FE-SEM detector (JEOL JSM-6400, Japan). The energy dispersive X-ray analyses (EDS) were also carried out to investigate the elemental composition of the nHAp and HAp–GNP composites. JEOL 2100 field emission gun transmission electron microscope was used to record the TEM images and diffraction patterns. To record the images, a small amount of the composite particle was diluted with isopropyl alcohol. The diluted particle was dropped onto a carbon-coated copper grid and allowed to dry in vacuum for the duration of 30 min. Then TEM samples were placed in the vacuum for 12 h. TEM images were analyzed through the Image J software to determine the size and shape of the particles. The mechanical behavior of the nHAp and HAp–GNP composites were measured using a universal testing machine (UTM Instron 3639) in compression mode at a crosshead speed of 5 mm/min. The samples’ size of 1.3 cm in diameter and 2.0 cm in length was used for mechanical testing on cylindrical pellet samples. The mechanical characterizations were also carried out at 23 °C temperature and 20% humidity atmospheric conditions. The load bearing behavior of the composites was studied by plots of the load–displacement curve until failure. The Young’s modulus was obtained from the slope of the stress–strain curve for each sample. The fracture toughness of the sintered nHAp–GNP composites was measured as the area under the stress–strain curve within the limit of an initial point to the elastic limit. 4.3.1 MTT Assay of HAp and nHAp–GNP Composites 4.3.1.1 On Larval Tissues of Drosophila Drosophila (Oregon R+) obtained from the Bloomington stock center, Indiana, USA, was used in this study. An MTT [3-(4,5-dimethyl thiazolyl-2)-2,5-diphenyltetrazolium bromide] assay was performed to test the cytotoxicity of the nHAp–GNP composite in gut tissues of Drosophila third instar larvae. The nHAp–GNP composites were dissolved in 2% dimethyl sulfa-oxide (DMSO) with increasing concentrations (10, 30, 50, 100, 500, and 1000 μg/mL) in fresh Eppendorf tubes. For cytotoxicity experiment, initially, 10 larvae were teased out in 1× PBS (pH 7.4) and incubated with each composite for 1 h at room temperature. However, for the control, the gut tissues were incubated for 1 h in DMSO only. After the end of incubation, the tissues were washed twice with 1× PBS and incubated with 0.6 mg/mL MTT for 2 h at 37 °C in the dark. After incubation, the tissue was washed twice with 1× PBS and incubated with 200 μL DMSO for another 1 h at 37 °C to dissolve the purple-colored formazan crystal, and the colored solutions were transferred into the distinct wells of a 96-well culture plate. The absorbance intensity was analyzed by the microplate reader (Auto Reader 4011, SPAN Diagnostics Ltd.) at 492 nm with a reference wavelength of 630 nm. All experiments were performed in triplicate, and the cell viability was expressed in percentage in relation to the control. The cells’ viability was determined by the absorption at 570 nm. The absorption of the wells without the sample was considered as blanks. The adsorption of HAp resulted in the highest absorption, which reflected the best cell viability. The effect of the samples on the cell proliferation can be written as the cell viability using the following formula 4 where At is the absorbance of the test sample, and Ac is the absorbance of the cells without HAp treatment. 4.3.1.2 On Rat Osteoblast Cells Drawn from Calvaria The experimental protocol for studying the cell viability of rat osteoblast calvarial cells due to the exposure of prepared materials was adopted exactly from our previously reported studies.12,63 4.3.1.3 Statistical Analysis of the MTT Test For the analysis of biological data, the following statistics were used. The biological data are represented as mean ± standard error of the mean (mean ± S.E.M). The group differences were determined using a one-way analysis of variance (ANOVA) with a Neuman–Keuls post hoc test by Prism version 5.0 software. Moreover, probability values of p < 0.05 are taken to be statistically significant (*P < 0.05), when compared with the control, that is, without the treated cells. 4.3.2 Treatment of nHAp–GNP Composites in Gut Tissues of Drosophila Larvae The gut tissues of Drosophila third instar larvae were dissected in 1× PBS (pH 7.4), and for the control, 10 dissected gut tissues were incubated in 2% DMSO for 2 h at room temperature; the same number of gut tissues was also incubated separately with each nHAp composite concentration 100 μg/mL for 2 h in Maximo slides. The 2% DMSO solvent was used for each of the HAp composites. After the end of incubation, the gut tissues were washed twice with 1× PBS solution for 5 min, and we captured the HAp composite treated with gut tissues fluorescence images of Drosophila using a Nikon Niu upright fluorescence microscope. Supporting Information Available The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.8b03473.Synthesis of nanohydroxyapatite (nHAp), details on the synthesis of nanocomposites via a solid-state route, and XRD analysis of pure graphene nanoplatelets (PDF) (PDF) Supplementary Material ao8b03473_si_001.pdf Author Contributions The present research work was conceived and supervised by C.R.G., and materials synthesis, fabrication, and manuscript writing were performed by C.R.G., S.K., and V.K.M. Biological testing was performed and analyzed by B.S.C., S.S., R.S.Y., S.B.R., V.K.M. and R.T. The authors declare no competing financial interest. Acknowledgments C.G. would like to thank the University Grant Commission, New Delhi, India for providing the financial support under Raman Post-Doctoral Research Award Fellowship (Award no. F5-65/2014 (IC)) and also appreciatively acknowledges the Science and Engineering Research Board, Department of Science and Technology (SERB-DST), New Delhi, Government of India for giving the financial support under Empowerment and Equity Opportunities for Excellence in Science (file no. EEO/2018/000647). Authors highly acknowledge Prof. P.M. Ajayan and Prof. Robert Vajtai, Department of Materials Science and NanoEngineering, Rice University, Houston, Texas, U.S.A. for managing the required facilities to carry out this research work. V.K.M. is thankful to SERB, Government of India for providing financial aid in form of N-PDF (file no. PDF/2015/000915). ==== Refs References Bhatt R. A. ; Rozental T. D. Bone graft substitutes . 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==== Front ACS OmegaACS OmegaaoacsodfACS Omega2470-1343American Chemical Society 3145973410.1021/acsomega.9b00282ArticleEnhanced Electricity Generation and H2O2 Production in a Photocatalytic Fuel Cell and Fenton Hybrid System Assisted with Reverse Electrodialysis Xu Peng †‡Xu Hao ††Key Laboratory for Green & Advanced Civil Engineering Materials and Application Technology of Hunan province and ‡College of Civil Engineering, Hunan University, Changsha 410082, China E-mail: [email protected]. Tel.: +86 18874029590.26 03 2019 31 03 2019 4 3 5848 5851 31 01 2019 18 03 2019 Copyright © 2019 American Chemical Society2019American Chemical SocietyThis is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes. A novel integrating system coupled with photocatalytic fuel cell and Fenton system assisted by reverse electrodialysis (PREC) is proposed. The results demonstrate that H2O2 concentration increased continuously in the reaction process to finally reach 960 mg/L and the current became stable at around 5.2 mA. The salinity-driven potential derived from the high concentration and low concentration cells in the hybrid system was 0.72 and 0.90 V respectively, at the salinity ratio of 50 and 100. The hybrid system has an energy recovery of 16%, a cathodic efficiency of 51%, and the maximum power of 76 W/m2 at a salinity ratio of 50, with a 100 Ω external resistance. It is proved that PREC-Fenton possessed great potential in industrial wastewater treatment. document-id-old-9ao9b00282document-id-new-14ao-2019-00282accc-price ==== Body 1 Introduction Electro-Fenton as one of advanced oxidation processes has been attracting extensive interests with significant advantages such as easy operation and environmental compatibility. It is regarded to be a promising technology for treating refractory-containing industrial wastewater, which is attributed to the production of powerful oxidizing species. However, external electricity supply severely impedes its large-scale applications.1 To overcome the above shortcomings, increasing attention is paid to bioelectro-Fenton system in which the microbial fuel cell (MFC) is combined with electro-Fenton. In this system, bacteria in the anode chamber could oxidize the substrates to generate electrons, which are transported through an external resistor to the cathode chamber, thereby forming H2O2 via the reduction of dissolved oxygen.2,3 Nevertheless, MFC still faces many challenges including complex operation, low potential, long start-up time, and stringent working conditions. Moreover, the potential generated by MFC is very low.4−6 Comparably, photocatalytic fuel cell (PFC), which can decompose organics pollutants by radical reactions and simultaneously generate electricity by the self-bias between photoanode and cathode, may be an useful alternative. In this system, electricity is generated by the flow of photogenerated electrons from photoanode to cathode.7,8 Compared to MFC in which much attention should be paid to maintaining and monitoring the microbial activity, PFC system could be much more easy to develop and operate.9 Additionally, another interesting water treatment technology is reverse electrodialysis (RED), which is built as alternately stacked cation membranes and anion-exchange membranes.10 It is reported that RED is a valid process to generate electricity directly based on the salinity gradient between water and seawater river.11 Consequently, integration of PFC and RED would be an ideal technology to obtain higher electrical energy and contaminant-degrading ability. However, up to date, no reports of combination of PFC and RED have been published. In this study, an integrating system coupled with photocatalytic fuel cell and Fenton system assisted by reverse electrodialysis (PREC) is proposed. The H2O2 production, salinity-driven energy, energy recovery, and energy efficiency were investigated. The integrated system possessed great potential in industrial wastewater treatment. 2 Results and Discussion 2.1 Enhanced H2O2 Production in PREC The formation of H2O2 plays an important role in Fenton reactions, since it controls the evolution of •OH. The H2O2 concentration change in different systems is shown in Figure 1. Clearly, the H2O2 concentration increased with reaction time, reaching approximately 960 mg/L at 24 h (Figure 1a) without further increase under an extended operation time, indicating that the accumulated H2O2 reached a steady state, where its production rate was the same as the decomposition rate. The similar phenomenon has been founded in PFC systems. The possible reason is the generation of H2O2 in the process of electrical reaction, the decrease of H2O2 on cathode, and the self-decomposition of H2O2, which may occur at the same time. Comparatively, the H2O2 yield in the control reactors was very low during the same operation time (Figure 1a). The respective H2O2 concentration were 110, 4.8, and 10.9 mg/L in the absence of salinity gradient in the RED (Control 1), applied current (Control 2), and ultraviolet irradiation on the anode (Control 3). Figure 1 H2O2 production (a) and current (b) in PREC-Fenton under normal and different control conditions (Control 1: both high concentration (HC) and low concentration (LC) solution are 500 mM NaCl solution; Control 2: open circuit; Control 3: no ultraviolet irradiation on photoanode). Along with H2O2 production, the current was investigated. As displayed in Figure 1b, no current could be detected in Control 2 and Control 3. Although current in Control 1 reached 2.8 mA, it was much lower than 5.2 mA in normal condition. These results demonstrated the RED stack and ultraviolet irradiation have significant influence on the increase in current and H2O2 generation in PREC.12 In PREC, the H2O2 generation could be driven and enhanced with the help of an external power derived from salinity gradient energy. Moreover, H2O2 generated in the cathode could be further reduced to •OH for destroyed pollutants in the wastewater, which is important for further applications. 2.2 Salinity-Driven Energy and Electrochemical Analysis The performance of RED stack has very significant influence on the production of H2O2 in PREC. Under the driving effect of concentration gradient, cations and anions in high concentration solution would penetrate through the membranes and migrate into low-concentration solution, resulting in potential difference. In this study, 5 cell membrane pairs were used to investigate the effect of the salinity gradient. Results show that the salinity-driven potential (Δϕjct) created in RED was contrary to the salt concentration of the LC solution. Under the salinity ratios (SR) of 50 and 100, Δϕjct was 0.72 and 0.90 V, respectively. This is consistent with the cell potentials achieved in PREC. When the current densities were lower that 0.5 mA/cm2, the system achieved a smaller maximum cell potential with SR = 50 compared with SR = 100 (Figure 2a). However, the power density at SR = 50 was 76 W/m2, which is much higher than that of 41 W/m2 for SR = 100 (Figure 2b). The reasons for poor performance under larger SR are higher internal resistance and minimal increase in current. There are two key factors associated with internal resistance in RED: solution conductivity in LC compartment and mass transfer limitations at the membrane interface, which significantly increased at bigger SR.13 Figure 2 Polarization (a) and power densities (b) at different SR in PREC-Fenton. Additionally, it could be calculated that at the SR = 50, the energy recovery of PREC was 16%. These results mean that 91.8% of the recovered energy in PREC was derived from the salinity difference. Moreover, a much higher cathodic efficiency (Rcat) of 51% in PREC was calculated compared with previous reported cathodic efficiency of 42%.14 Although H2O2 accumulation is still very low, but the small energy consumption of 0.85 kWh/kg H2O2 still makes the technology very promising. 2.3 Practical Significance and Perspectives Results in this study demonstrated the PREC-Fenton shows great prospects in the fields of electricity production and wastewater treatment, as well as simultaneous H2O2 production. The integrated process has excellent advantageous in following aspects: first, compared with single PFC system, the performance was greatly improved with the help of RED.15 Second, a stable and persistent electric potential difference could be obtained between the two electrodes due the sustainable photocatalytic reaction in the system.16 Third, the PREC process is energy efficient with a lower energy consumption of 0.85 kWh/kg H2O2, indicating its potential for further application in wastewater treatment. 3 Conclusions Electrical power could be derived from salinity gradient in a RED system. In this study, a novel integration of PFC and Fenton process assisted with RED was designed for enhanced H2O2 production. Results demonstrated that H2O2 production increased by adding a 5-pair cells RED stack. The PREC-Fenton with an SR of 50 achieved a maximum power density of 76 W/m2, an energy recovery of 16%, and a cathodic efficiency of 51%. The integrated system possessed great potential in industrial wastewater treatment. 4 Materials and Methods 4.1 Reactor Setup and Operation The schematic diagram of PREC-Fenton reactor is depicted in Figure 3. A novel dual-chamber PREC-Fenton system consisted of an anode and a cathode chamber, and a working volume of 50 mL (5 cm × 5 cm × 2 cm) each, all connected by a RED stack with five cell pairs. Every cell pair of RED stack has an anion-exchange membrane, a cation-exchange membrane, a high concentration (HC) cell, and a low concentration (LC) cell. Membrane spacing was 1.5 mm and was divided by silicon gasket. The HC solution flows from the HC cell beside the cathode to the HC cell beside the anode, whereas the LC solution flows in the opposite direction. A 5 mol/L NaCl was selected as the HC solution and deionized water containing NaCl for three different salinity ratios (SR) of 100 (0.05 M), 50 (0.1 M), and 1 (5 M) was the LC solution. They were continuously injected into the RED stack through a peristaltic pump. Prior to each experiment, the cathode chamber was washed with NaCl solution. Unless otherwise stated, the initial pH in the anode and cathode chambers was 7.0 and 5.0, respectively. Fresh air was bubbled into the catholyte continuously at 16 mL/min. An ultraviolet lamp was used as light source of the PFC. The prepared ZnO/Zn and FeVO4/CF was used as a photoanode and a cathode, respectively. All these experiments were carried out in duplicate for accuracy. Figure 3 Schematic illustration of the PREC-Fenton reactor. 4.2 Electrochemical Analysis and Calculations The solution pH was determined by a pH meter (PH 210). The H2O2 concentration was determined with potassium titanium(IV) oxalate using UV–vis spectrophotometry (Spectronic 20D). A digital multimeter (PISO-813) was used to measure the voltages with a 100 Ω external resistors at an interval of 30 min. Current density was calculated normalized to the cathode area.17 The potentials were determined with the reference of saturated calomel electrode (+0.242 V vs SHE) under the resistances varied from 20 000 to 5 Ω. The junction potential (\|Δφjct\|) across an ion-exchange membrane with different salinity could be calculated as follows18 1 In the equation, R, T, and F are the gas constant, the absolute temperature, and the Faraday constant, respectively. z, a, and t are the ionic charge, the activity, and the transport number, respectively. The superscripts “HC” and “LC” are the HC solution and the LC solution, whereas the subscripts “counter” and “co” indicate the counterions and co-ions, respectively. The energy recovery (RE) is a normalized value between the produced power and the provided power in PREC13 2 In the equation, P, ΔHC, and nsin are the power produced (W), the substrate combustion heat (J/mol), and the supplied substrate (mol), respectively. However, tB and Xin are the time span and the theoretical energy (W), respectively. Cathodic H2O2 efficiency (Rcat) was determined by the ratio of real cumulative H2O2 production and the theoretical amount, which could be calculated as follows19 3 In the equation, n is the mole of H2O2 generated during the time interval of ti–t0. The authors declare no competing financial interest. Acknowledgments This study was supported by the National Natural Science Foundation, China (No. 51708196) and the Natural Science Foundation of Hunan Province (No. 2018JJ3060). ==== Refs References Ben Abdallah F. ; Hmani E. ; Bouaziz M. ; Jaziri M. ; Abdelhedi R. Recovery of hydroxytyrosol a high added value compound through tyrosol conversion by electro-Fenton process . Sep. Purif. Technol. 2017 , 188 , 260 –265 . 10.1016/j.seppur.2017.07.035 . Xu P. ; Xu H. ; Shi Z. A novel bio-electro-Fenton process with FeVO4/CF cathode on advanced treatment of coal gasification wastewater . Sep. Purif. 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==== Front ACS OmegaACS OmegaaoacsodfACS Omega2470-1343American Chemical Society 3145968810.1021/acsomega.9b00170ArticleFe3O4 Nanoparticles Grown on Cellulose/GO Hydrogels as Advanced Catalytic Materials for the Heterogeneous Fenton-like Reaction Chen Yian †§Pötschke Petra †Pionteck Jürgen †Voit Brigitte †§Qi Haisong ‡† Leibniz-Institut für Polymerforschung Dresden e. V. (IPF), Hohe Straße 6, D-01069 Dresden, Germany‡ State Key Laboratory of Pulp and Paper Engineering, South China University of Technology, 510630 Guangzhou, China§ Organic Chemistry of Polymers, Technische Universität Dresden, D-01062 Dresden, Germany* E-mail: [email protected] (P.P.).* E-mail: [email protected] (H.Q.).11 03 2019 31 03 2019 4 3 5117 5125 18 01 2019 20 02 2019 Copyright © 2019 American Chemical Society2019American Chemical SocietyThis is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes. Cellulose/graphene oxide (GO)/iron oxide (Fe3O4) composites were prepared by coprecipitating iron salts onto cellulose/GO hydrogels in a basic solution. X-ray photoelectron spectroscopy (XPS), Fourier-transform infrared, and X-ray diffraction characterization showed that Fe3O4 was successfully coated on GO sheets and cellulose. Cellulose/GO/Fe3O4 composites showed excellent catalytic activity by maintaining almost 98% of the removal of acid orange 7 (AO7) and showed stability over 20 consecutive cycles. This performance is attributable to the synergistic effect of Fe3O4 and GO during the heterogeneous Fenton-like reaction. Especially, the cellulose/GO/Fe3O4 composites preserve their activity by keeping the ratio of Fe3+/Fe2+ at 2 even after 20 catalysis cycles, which is supported by XPS analysis. document-id-old-9ao9b00170document-id-new-14ao-2019-001706ccc-price ==== Body Introduction Dye-contaminated wastewaters from textile, plastic, and paper industries cause worldwide attention in the water environment. The textile industry presents a global pollution problem on account of the accidental or dumping discharge of dye waste–water into waterways.1 To meet stringent environmental regulations, the treatment of dye wastewater is compulsory, enforced, and highly regulated. Advanced oxidation processes (AOPs) are used as an efficient and robust method for the treatment of dye wastewater because of their low toxicity and high degradation performance. AOPs are based on the generation of highly reactive HO• to degrade organic pollutants.2 The HO• radicals are produced by either hydrogen peroxide (H2O2) and/or ozone (O3) whereby ultrasound, light, and temperature can be used to support the process. Oxidation with Fenton- or Fenton-like reactions is a promising process within AOPs, resulting mainly from the generation of HO• radicals that efficiently and nonselectively degrade organic pollutants.3,4 However, there are still many disadvantages when applying such homogeneous Fenton- or Fenton-like reactions, such as the restricted experimental conditions, the accumulation of iron hydroxide sludge, and the nonregeneration of the catalyst.5−7 In order to overcome these disadvantages of the homogeneous reaction, heterogeneous Fenton-like catalysts were developed as a suitable alternative, which at the same time allows the catalyst to be reused in further cycles. The iron species are commonly immobilized in catalyst supports, such as clay,8−12 alumina,13,14 zeolite,15,16 silica,17 and carbonaceous materials.18−21 Therefore, the Fenton or Fenton-like reactions generally occur at the solid–liquid interfaces, where the iron remains substantially in the solid phase either as a mineral or as an adsorbed ion.18,22 In particular, carbonaceous materials, such as activated carbon (AC),20,23,24 carbon nanotubes (CNTs),18,21,25 mesoporous carbon,26 and carbon aerogels,20,27 have been well proven as catalyst carriers to immobilize the iron species because of their special performance as a cocatalyst. Recently, Voitko et al.28 reported that graphene oxide (GO) showed better degradation performance than CNTs and AC, which was attributed to the many oxygen-containing functional groups of the GO plates. Although GO shows great attractiveness to act as a catalyst support, there are very few works reported using GO in heterogeneous Fenton-like catalysts for the degradation of persistent organic pollutants. The main reason is the difficulty in recycling of iron species and the catalyst support. Therefore, embedding iron species onto proper supports to improve their recyclability and stability is an important issue. A step in this direction was taken by Zubir et al.,29−31 who reported in recent years on the use of GO/Fe3O4 nanocomposites to degrade acid orange 7 (AO7). Although the interaction of GO and Fe3O4 leads to the stability and great performance in the degradation of AO7, the use of GO as the catalytic support still limits its convenient application as it is difficult to remove the catalyst after the catalytic reaction. This work proposes a novel approach to use cellulose/GO hydrogels as a catalyst support for catalysis, especially in the Fenton-like reaction system. The iron species can be processed into catalytic composite materials based on film or paper, the so-called “dip catalyst”. A “dip-catalyst” is characterized by its high recyclability and its convenient use, in particular by the fact that it can switch the reaction on and off almost immediately by immersion/removal in the reaction medium. Such catalytic film-based composites can be prepared by dip-coating cellulose/GO hydrogels with iron ions. As proved in our previous work, we have successfully produced cellulose/GO hydrogel with GO sheets uniformly dispersed within the cellulose matrix. The open network structure formed by cellulose provides ideal conditions for the easy diffusion of Fe2+ and Fe3+ into the cellulose/GO hydrogel. Thus, Fe3O4 can immobilize uniformly onto the GO surface and the cellulose chains, while maintaining the structure of the cellulose matrix. Although, the cellulose matrix contains GO and Fe3O4 nanoparticles, it retains the hydrogel structure during the catalyst reaction so that no secondary contamination occurs. About half of the worldwide production of dyes can be classified as azo compounds that have one or more azo groups (−N=N−) in their molecular structure.4,6 Among the azo dyes, AO7 is the most widely used dye in the textile manufacturing industry because of its high stability and low cost.5 Therefore, this dye was used as a model pollutant to study the catalytic performance of cellulose/GO/Fe3O4 composites with respect to oxidation in the heterogeneous Fenton-like reaction. Results and Discussion Preparation and Properties of Cellulose/GO/Fe3O4 Composites The preparation of cellulose/GO/Fe3O4 composites is shown in Figure 1. We used the NaOH/urea aqueous solution in the first step to prepare the cellulose/GO hydrogel because the NaOH/urea aqueous solution was found to facilitate good dispersion of GO in the cellulose matrix, which was sketched in Figure 1b. The proposed generation mechanism of Fe3O4 nanoparticles in cellulose/GO hydrogel is shown in Figure 1c,d. When the cellulose/GO hydrogel was immersed in the FeCl3/FeCl2 solution, Fe3+/Fe2+ were adsorbed around the carboxyl and hydroxyl groups of the cellulose and the GO sheets. Afterward, when NaOH was added into the solution, Fe3+/Fe2+ were hydrolyzed to generate Fe(OH)3/Fe(OH)2. After the condensation of Fe(OH)3/Fe(OH)2, Fe3O4 was immobilized in the cellulose/GO hydrogel. Multiple hydrogen bonds formed in the cellulose/GO/Fe3O4 hydrogel, as shown in Figure 1e, stabilize the Fe3O4 nanoparticle dispersion in the hydrogel. Figure 1 Illustration of cellulose/GO/Fe3O4 hydrogel synthesis: (a) cellulose and GO; (b) cellulose/GO hydrogel; (c) cellulose/GO hydrogel in FeCl3/FeCl2 solution; (d) cellulose/GO/Fe3O4 hydrogel; (e) multiple hydrogen bonds. The results of structural and morphological analysis of cellulose/GO/Fe3O4 composites are shown in Figure 2. The cellulose/GO hydrogel as the catalyst support provides ideal conditions for easy diffusion of Fe2+ and Fe3+ into the hydrogel and their efficient reaction with GO and cellulose. The Fe3O4 nanoparticles formed in NaOH were evenly generated on the surface of GO sheets and cellulose chains (Figure 2a,b). The average size of the Fe3O4 is 10–13 nm, as depicted in Figure 2c. It is noteworthy that due to the strong chemical interaction between the carboxylate ions and iron oxide,32 Fe3O4 nanoparticles were applied directly to the GO surface and cellulose without molecular links, as shown in Figure 1e. Figure 2 (a) Low magnification and (b) high magnification TEM images of Fe3O4 nanoparticles grown on GO sheets. (c) Fe3O4 nanoparticles size distribution as determined from TEM micrographs. (d) Wide scan XPS spectra of cellulose/GO (8%) and cellulose/GO (8%)/Fe3O4. (e) FTIR spectra of cellulose/GO (8%) and cellulose/GO (8%)/Fe3O4. (f) XRD spectra of cellulose/GO (8%) and cellulose/GO (8%)/Fe3O4. The prepared composites were further analyzed using wide scan X-ray spectra to confirm the formation of Fe3O4 nanoparticles in the cellulose/GO hydrogel. The spectra of cellulose/GO (8%)/Fe3O4 shows photoelectron lines at binding energies of 285, 530, and 711 eV, which are attributed to C 1s, O 1s, and Fe 2p, respectively. More detailed XPS information of different composites is shown in Figure S1a. In the high resolution Fe 2p scan (Figure S1b), the binding energy peaks at 724.7 and 711.2 eV correspond to Fe 2p1/2 and Fe 2p3/2, respectively. The generation of Fe3O4 nanoparticles can be confirmed by the disappearance of the charge-transfer satellite of Fe 2p3/2 at about 720 eV.33,34 The formation of Fe3O4 nanoparticles was also confirmed by Fourier-transform infrared (FTIR) spectroscopy (Figure 2e). For cellulose/GO (8%)/Fe3O4 composite, there is an extra band at around 584 cm–1, which is attributed to Fe–O. The crystal structure of Fe3O4 nanoparticles was confirmed by the presence of diffraction peaks at 2θ = 30.3° (220), 35.4° (311), 43.3° (400), 53.6° (422), 57.2° (511), and 62.9° (440) in Figure 2f. All the peaks in the X-ray diffraction (XRD) patterns of Fe3O4 nanoparticles are consistent with reports in the literature.35,36Figure S2a shows the thermal stability of the composite films. The Fe3O4 nanoparticle content can be calculated by the residue of composite films with and without Fe3O4 (Figure S2b). The Fe3O4 nanoparticle content of the different cellulose/GO/Fe3O4 composite was not affected by the content of GO in the cellulose/GO/Fe3O4 composites and is around 12 wt %. Optimization Analysis of the AO7 Degradation Conditions The catalytic performance of cellulose/GO/Fe3O4 composites was assessed on the basis of their reactivity to the oxidation of AO7 as a model pollutant in the heterogeneous Fenton-like reaction. In this part, using cellulose/GO (8%)/Fe3O4 hydrogel as the catalyst, the optimization of the experimental conditions for the AO7 oxidation was performed, including the effects of the initial AO7 dye concentration, temperature, pH, and the initial concentration of H2O2. Figure 3a shows the relationship between the initial AO7 dye concentration and the degradation of AO7. As the AO7 dye content increased, the degradation activity obviously slowed down. The high AO7 concentration leads to inductive effects and the active sites of the cellulose/GO (8%)/Fe3O4 hydrogel were covered by excess AO7. Thus, it can limit the generation of HO• from H2O2 at the active sites of the cellulose/GO (8%)/Fe3O4 hydrogel. Figure 3 (a) Effect of the initial AO7 concentration on the degradation of AO7 by H2O2 catalyzed with the cellulose/GO (8%)/Fe3O4 composite. (b) Effect of temperature on the degradation of AO7 by H2O2 catalyzed with the cellulose/GO (8%)/Fe3O4 composite. (c) Effect of H2O2 content for AO7 oxidation by H2O2 catalyzed with the cellulose/GO (8%)/Fe3O4 composite. (d) Effect of pH on the degradation of AO7 by H2O2 catalyzed with the cellulose/GO (8%)/Fe3O4 composite. Figure 3b illustrates the influence of temperature on the AO7 oxidation. Fast rates could be observed and 64% degradation was reached within 15 min at 338 K. In contrast, it takes 45 min at 298 K. Obviously, high temperature increases the mobility of H2O2 and AO7 to the surface of the cellulose/GO (8%)/Fe3O4 hydrogel, thereby resulting in more HO•, thus accelerating the degradation of AO7. Nevertheless, the temperature of 298 K was selected in further experiments because raising the temperature from 298 to 338 K would result in additional energy costs. The concentration of H2O2 is also a significant factor in generating HO• for AO7 oxidation and its effect is depicted in Figure 3c. As the concentration of H2O2 increased from 5.5 to 22 mM, the AO7 oxidation increased from 80 to 98% (Figure 3c). The increased degradation of AO7 results from the increased HO• concentration. As the concentration of H2O2 increased further to 33 mM, the degradation of AO7 decreased to 82%. The result suggests that the excess H2O2 could lead to a reaction between H2O2 with HO• generating hydroperoxyl radicals (HOO•).37 The degradation performances of AO7 at different pH experimental conditions are shown in Figure 3d. Clearly, the best degradation performances of AO7 are reached at pH = 3. When the pH value was higher than 3, the AO7 degradation decreased. This can be attributed to the decomposition of more H2O2 into water and oxygen without the generation of appreciable amounts of HO•. When the pH value was lower than 3, the degradation of AO7 also decreased. The results suggest that the excess H+ lead to a scavenging effect of HO•, thus reducing the degradation reaction. On the basis of the above results, the optimized experimental condition for AO7 degradation are [AO7] = 0.1 mM, [H2O2] = 22 mM, pH = 3, and T = 298 K. Catalytic Activity of Cellulose/GO/Fe3O4 Hydrogels The degradation of AO7 by H2O2 catalyzed with the cellulose/GO (8%)/Fe3O4 composite can be analytical tracked by the change in the UV–vis absorption spectrum over the course of degradation (Figure 4a). The UV–vis spectrum of the chromophore AO7 is characterized by absorptions at 430 and 484 nm, which are caused by the azo and hydrazine form, respectively. The other two peaks at 230 and 310 nm are caused by the adjacent auxochrome benzene and naphthalene rings, respectively.38 These four characteristic peaks decreased significantly with increasing reaction time, which was attributed to the destruction of the auxochromic and chromophoric structures during the heterogeneous Fenton-like reactions. Figure 4 (a) UV–vis analysis of the degradation of AO7 by H2O2 catalyzed with the cellulose/GO (8%)/Fe3O4 composite. (b) Effect of catalysts on degradation profiles of AO7 ([AO7] = 0.1 mM, [catalyst] = 0.2 g L–1, pH = 3, [H2O2] = 22 mM, and T = 298 K). The catalytic activity of different composites in degrading the AO7 dye is presented in Figure 4b. Insignificant degradation of the AO7 dye was observed in the presence of H2O2 only. The result in the blank conditions of the Fenton-like reaction could be because of the higher oxidation potential of HO• radicals compared to H2O2. In the presence of cellulose/Fe3O4 and cellulose/GO (8%) composites, the AO7 dye was degraded by 64 and 23% after 180 min of reaction, respectively. In the presence of different cellulose/GO/Fe3O4 composites, the degradation degree of AO7 varied within the range of 71–98% after 180 min. The increase in degradation rate is because of the synergistic effects between Fe3O4 nanoparticles and GO sheets. First, GO features aromatic ring structures that are facilitated through π–π interactions and the adsorption of AO7, which contains also aromatic units (Figure 4a). This could provide an increase of AO7 concentration39 near the active sites, and the accumulated AO7 will be oxidized faster by the HO• generated from H2O2 in the direct neighborhood.18 Second, the interactions between GO sheets and Fe3O4 nanoparticles could generate the electron transport between Fe3O4 and GO.40 Third, the partial reduction of GO promotes the regeneration of Fe2+ during the degradation reaction, which enables electron transport to accelerate the redox cycle. In summary, the structural and morphological interactions of cellulose/GO/Fe3O4 composites determine the high catalytic activity for the degradation of AO7. The durability of the cellulose/GO/Fe3O4 catalyst for the degradation of AO7 was also verified (Figure 5a). The degradation of AO7 was monitored for twenty consecutive cycles, with each cycle lasting 3 h. Before the addition of fresh AO7 solution in each cycle, cellulose/GO/Fe3O4 hydrogel was taken out of the solution and washed thoroughly with water. There was only very small decrease in the degradation rate during the five consecutive cycles, and even in the 20th cycle only a small decrease in degradation efficiency was detected, indicating the good stability of the prepared cellulose/GO/Fe3O4 catalyst. Figure 5 (a) Cycling runs in the AO7 degradation using cellulose/GO (8%)/Fe3O4 composites. (b) Comparison of the long-term stability of the catalytic activity of the cellulose/GO/Fe3O4 and cellulose/Fe3O4 composites tested by repeated use for AO7 degradation. Compared to the cellulose/Fe3O4 composites, as shown in Figure 5b, the cellulose/GO (8%)/Fe3O4 composites exhibited excellent catalytic stability over five cycles, with unobvious AO7 degradation efficiency lost. Conversely, the degradation activity of cellulose/Fe3O4 composites declines quickly. While in the first cycle, 64% of AO7 were decomposed, the efficiency of cellulose/Fe3O4 composites decreased to only 14% AO7 degradation in the fifth cycle. The catalytic stability of cellulose/GO/Fe3O4 composites over several cycles (even twenty cycles) resulted from the co-effect between Fe3O4 and GO. The counter-intuitive result was due to the loss of surface passivation of cellulose/GO/Fe3O4 hydrogels. In the system, the surface passivation of cellulose/GO/Fe3O4 hydrogel might be prevented by the effective formation of HO•.41,42 In cellulose/Fe3O4 hydrogels, the ineffective regeneration of Fe2+ during the degradation reaction could bring about the generation of passivated Fe3O4,18,43 resulting in a loss of catalytic activity. XPS analysis was carried out to clarify the possible regeneration of Fe2+ of cellulose/GO/Fe3O4 and cellulose/Fe3O4 composites. The Fe 2p spectra of cellulose/GO (8%)/Fe3O4 composites and cellulose/Fe3O4 composites (see Figure 6) showed the spin–orbit doublets of Fe 2p1/2 at 724.6 eV and Fe 2p3/2 at 711.1 eV. The deconvolution of Fe2+ and Fe3+ confirms the regeneration of Fe2+ by quantifying the changes in the Fe3+/Fe2+ ratio. Table 1 summarized the details of Fe2+ and Fe3+ peaks in regard to their ratio. An apparent increase in the Fe3+/Fe2+ ratio after twenty catalysis cycles was only found in the cellulose/Fe3O4 composites rather than the cellulose/GO/Fe3O4 composites. The increase in the Fe3+/Fe2+ ratio from 2.02 to 4.56 in cellulose/Fe3O4 is due to a proportion of the Fe2+ being oxidized into Fe3+ on the surface of Fe3O4 nanoparticles throughout the catalysis. On the contrary, no increase in the ratio of Fe3+/Fe2+ was observed for the cellulose/GO/Fe3O4 composites. These results plausibly confirmed the ineffective regeneration of Fe2+ in cellulose/Fe3O4 composites during degradation reaction of AO7 and the good regeneration of Fe2+ of cellulose/GO/Fe3O4 composites, which was associated with the presence of synergistic interactions between Fe3O4 nanoparticles and GO during the heterogeneous Fenton-like reaction. In fact, these results are also related to the high performance of cellulose/GO/Fe3O4 composites in removal of AO7 during the long-term stability test (Figure 5). Figure 6 XPS spectra of (a) cellulose/GO (8%)/Fe3O4 composites and (b) cellulose/Fe3O4 composites during 20 cycles. Table 1 Fe3+/Fe2+ Ratio of Cellulose/GO (8%)/Fe3O4 and Cellulose/Fe3O4 Composites during 20 Cycles Fe 2p1/2 Fe 2p3/2 samples cycles Fe3+ (%) Fe2+ (%) Fe3+ (%) Fe2+ (%) Fe3+/Fe2+ ratio cellulose/GO/Fe3O4 0 22.14 8.14 44.75 24.97 2.02 1 21.42 7.36 45.47 25.75 2.02 3 22.56 8.01 44.22 25.21 2.01 5 22.43 7.95 44.23 25.38 2.00 10 22.44 7.75 44.45 25.36 2.02 20 22.98 8.10 43.41 25.51 2.05 cellulose/Fe3O4 0 22.01 7.77 44.77 25.45 2.02 1 21.95 8.45 48.11 21.49 2.34 3 22.43 8.35 49.48 19.74 2.56 5 21.87 6.14 53.98 18.01 3.14 10 24.19 3.60 55.36 16.85 3.89 20 24.90 3.62 57.12 14.36 4.56 As mentioned above, the cellulose/GO/Fe3O4 composites as the catalyst exhibit outstanding durability and recyclability. To explain this phenomenon, Figure 7 presents the proposed mechanism of synergy of Fe3O4 and GO in delivering the excellent and durability performance. First, the degradation of AO7 mainly takes place at the solid–liquid interface, where the generation of HO• radicals is caused by the decomposition of the adsorbed H2O2 by the actives sites of the Fe3O4 nanoparticles coated on the surface of GO (eq 1 and 2). Second, in addition to the active sites (Fe2+/Fe3+) of Fe3O4 nanoparticles, H2O2 could also be decomposed on the surface of GO to generate HO• radicals,44 which result from the donor–acceptor properties of GO sheets45−48 based on an electron-transfer mechanism (eq 3 and 4). Third, π electrons on the surface of GO in turn can generate the electron transfer between Fe3O4 and GO,40 which result from the existence of many semiconducting π-conjugated sp2 carbon domains.49 The feasibility of the electron transfer can be ascribed to the standard reduction potential of GO (−0.19 V),50 which is lower than that of Fe3+/Fe2+ (+0.771 V).21,45 Therefore, the spontaneous reduction from Fe3+ to Fe2+ could be available because the electron donation from the GO sheets to the oxidized active sites. Because the regeneration of Fe2+ by H2O2 (eq 2) was quite slow, this synergistic effect between Fe3O4 nanoparticles and GO is beneficial in speeding up the redox cycles (eq 5).29 The fast regeneration of Fe2+ is greatly promoting the decomposition of H2O2. Finally, the hydrogel structure of cellulose/GO/Fe3O4, which consists of multiple hydrogen bonds, ensures the stability of every component after many catalysis cycles. Overall, the electron transfer in the cellulose/GO/Fe3O4 hydrogel plays the major role in the redox reaction of Fe3O4 nanoparticles, which can achieve the good and long-lasting degradation performance of AO7. 1 2 3 4 5 Figure 7 Proposed mechanism of the cellulose/GO/Fe3O4 hydrogel as the catalyst to degrade AO7. In order to have a rough estimation of the efficiency of cellulose/GO/Fe3O4 hydrogels as the catalyst to degrade AO7, the degradation performance and stability were compared with those reported in the literature. Table 2 contains different catalysts able to degrade AO7 dye at similar operational conditions. Under this condition, most of the recent reports have lower removal percentage of AO7 than the ones reported here. Compared to them, the cellulose/GO/Fe3O4 hydrogel exhibited good degradation performance and excellent recyclability. Only the system described in ref (53) shows similar behavior compared to our cellulose-based system. Moreover, another advantage of the hydrogel as the catalyst is that the cellulose matrix contains GO and Fe3O4 nanoparticles and maintains the hydrogel structure during the catalyst reaction, which will not result in secondary pollution. The “dip-catalyst” can be easily removed from the polluted water after the degradation reaction. In other words, it is easy to start and stop the degradation reaction by taking in and out the whole hydrogel. It is obvious that the cellulose/GO/Fe3O4 hydrogel, with its sustainable feature, convenient fabrication, great catalytic efficiency, good recyclability, and easy removability, is expected to be very useful in AO7 dye removal. Table 2 Degradation Performance and Stability of Different Systems dyes catalyst percentage of removal stability conditions refs AO7 GO/Fe3O4 80.0% in 20 min pH 3; 0.1 mM dye; 22 mM H2O2; 298 K (30) 98.0% in 180 min AO7 BiOI/ZnFe2O4 90.5% in 180 min pH 5; 20 mg L–1 dye; 298 K (51) AO7 pillared saponite clay impregnated with Fe(II)acetylacetonate 91.8% in 180 min pH 5; 0.1 mM dye; 20 mM H2O2; 303 K (52) AO7 BiOI–BiOCl/C3N4 96.6% in 140 min 87.4% in 140 min after the 4th run pH 6; 0.1 mM dye; 20 mM H2O2; 303 K (53) AO7 CdO–ZnO 69% in 140 min pH 7; 20 mg L–1 dye; 298 K (54) AO7 Cd–TiO2 95% in 120 min pH 2; 20 mg L–1 dye; 298 K (55) AO7 Ag–ZnO/CNT 98% in 120 min 95% in 120 min after the 4th run pH 5; 20 mg L–1 dye; 298 K (56) AO7 cellulose/GO (8%)/Fe3O4 hydrogel 97% in 120 min 90% in 180 min after the 20th run pH 3; 0.1 mM dye; 22 mM H2O2; 298 K this work 98% in 180 min Conclusions In conclusion, we have successfully grown Fe3O4 nanoparticles on cellulose/GO hydrogels by a simple, scalable, and facile method. This method produced cellulose/GO/Fe3O4 composites, in which Fe3O4 nanoparticles were uniformly and densely coated on the surface of GO and cellulose chains. The resulting composites show superior and durable catalytic activity in oxidation of AO7 dye compared to cellulose/Fe3O4 composites, which results from the synergistic effect of Fe3O4 and GO. We expect that the composite hydrogels can be also useful for the purification of other dye-contaminated wastewaters. Our method could be further extended to grow other functional materials on cellulose or cellulose/GO composites for advanced applications. Experimental Section Materials The cellulose samples are cotton linters, DP 500, supplied by Hubei Chemical Fiber Group Ltd. (Xiangfan, China). AO7 (orange II; 85%), GO dispersion (4 mg/mL), FeCl2·4H2O (99%), and FeCl3·6H2O (97%) were purchased from Sigma-Aldrich and used as received. Preparation of Cellulose/GO Composite Hydrogels A solution of NaOH/urea/H2O (7:12:81 by weight) was prepared as solvent. The designated amounts of NaOH and urea were added into distilled water and then the solvent was precooled to −12 °C. The designated amount of cellulose was added and dissolved into the solvent under vigorous stirring for 5 min. Then, a calculated amount of GO dispersion (4 mg/mL, Sigma-Aldrich) was added into the cellulose solution under vigorous stirring for 5 min. After degasification, the mixture was cast on a glass plate to give a 400 μm thick gel sheet, which was then immersed into a coagulation bath with 5 wt % H2SO4 for 5 min at room temperature to coagulate and regenerate. It was then immersed in distilled water to remove urea and NaOH. By adjustment of the amount of the GO aqueous dispersion, cellulose/GO composite hydrogels consisting of 100 g water, 4 g cellulose, and different GO contents of 3, 5, and 8 wt %, related to the cellulose amount were obtained. Preparation of Cellulose/GO/Fe3O4 and Cellulose/Fe3O4 Composite Hydrogels The cellulose/GO hydrogel was immersed into an aqueous solution (200 mL) consisting of 8 mmol FeCl3·6H2O and 4 mmol FeCl2·4H2O for 2 h at room temperature. Subsequently, the cellulose/GO hydrogel was added into the NaOH (1 M) solution for another 5 min. The resultant hydrogels were washed with running water and deionized water. Characterization of Morphology and Microstructure of Composite Films For the characterization of the composites, the hydrogels were fixed on glass plates using Scotch tape and dried at ambient temperature to obtain films with a thickness of ca 40 μm. Transmission electron microscopy (TEM) was performed using a LIBRA 200 MC (Carl Zeiss SMT, Oberkochen, Germany) at an accelerating voltage of 200 kV. The films were embedded in epoxy resin, and ultrathin sections with a thickness of about 90 nm were cut with an ultrasonic diamond knife (DIATOME, Switzerland) by using ultramicrotome EM UC/FC 6 of the company Leica (Austria) at room temperature. XRD was performed using a D/MAX-1200 (Rigaku Co., Japan) with a wavelength of 0.154 nm and a Lynx Eye detector at a scanning rate of 2θ = 1° min–1. X-ray photoelectron spectroscopy (XPS) was performed using a Kratos Axis ULTRA X-ray photoelectron spectrometer with monochromatic Al Kα (hν = 1486.6 eV). All XPS spectra were corrected using the C 1s line at 284.6 eV. FTIR spectroscopy in the range of 4000–500 cm–1 was performed on a Spectrum 400 FT-IR/ATR Spectrometer (PerkinElmer, USA). Thermogravimetric analysis (TGA) was done using a TGA Q5000 (TA Instruments, USA) from 30 to 800 °C with an increment of 10 K min–1. Testing of Catalytic Activity First, the cellulose/GO (8%)/Fe3O4 hydrogel was chosen to study in detail the effect of the parameters initial AO7 dye concentration, temperature, pH, and the initial concentration of H2O2 on the catalytic performance. Hydrogel (5 g) in the shape of a thick film of about 120 × 80 × 0.5 mm3 were dipped into 250 mL solution of AO7 and the AO7 degradation was followed with time. The experimental design was chosen by changing one factor and keeping the others constant. The variation of parameters was chosen as follows: AO7 concentration = 0.1, 0.2, 0.3, and 0.4 mM; H2O2 concentration = 5.5, 11, 22, and 33 mM; pH = 2, 2.5, 3, 3.5, and 4; temperature = 298, 318, and 338 K. Then, so as to compare the degradation performance of different cellulose/GO/Fe3O4, cellulose/GO, and cellulose/Fe3O4 hydrogels, the catalytic activity of AO7 was studied using the different hydrogels at constant conditions with AO7 aqueous solutions of 250 mL (experimental conditions: [AO7] = 0.1 mM, [catalyst] = 0.2 g L–1, pH = 3, [H2O2] = 22 mM, and T = 298 K). The initial pH of the AO7 solution was adjusted to 3. The reactions were initiated by adding H2O2 (22 mM) into the suspension. It should be noted that, before the reaction, the solution was stirred in the dark for 30 min to achieve the adsorption equilibrium. The concentration of AO7 was analyzed by spectrophotometer at a wavelength of 484 nm. The UV–vis spectra of the samples were recorded in air atmosphere at room temperature from 200 to 600 nm using a UV–vis spectrophotometer (SPECORD 201 PLUS, Analytik Jena, Germany). Supporting Information Available The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.9b00170.Wide-scan XPS spectra and high-resolution Fe 2p spectra of different nanocomposites and Fe3O4 nanoparticles and TGA curves of different composites and cellulose and residues of cellulose/GO and cellulose/GO/Fe3O4 composites (PDF) Supplementary Material ao9b00170_si_001.pdf Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. The authors declare no competing financial interest. Acknowledgments We would like to thank Mrs. Reuter for TEM observation, Mrs. Pilch for UV–vis spectrophotometer testing as well as Dr. Simon for XPS characterization (all from IPF). Y.C. appreciates the funding from China Scholarship Council (grant 201606240112) for the PhD study at Leibniz-Institut für Polymerforschung Dresden e.V. 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==== Front 9607835 20545 Mol Psychiatry Molecular psychiatry 1359-4184 1476-5578 30733594 10.1038/s41380-019-0364-x nihpa1519504 Article Full recovery of the Alzheimer’s disease phenotype by gain of function of Vacuolar Protein Sorting 35 Li Jian-Guo Chiu Jin Praticò Domenico Alzheimer’s Center at Temple, Lewis Katz School of Medicine, Temple University, Philadelphia, PA 19140 Authors’ contributions J-GL and DP designed the study; J-GL and JC performed the experiments; J-GL and DP analyzed the data and drafted the manuscript. All authors have discussed the results and seen the final version of the paper before submission. Correspondence to: Domenico Praticò, MD, Scott Richards North Star Foundation Chair, Alzheimer’s Research, Alzheimer’s Center at Temple, 1160, Medical Education and Research Building, 3500 North Broad Street, Philadelphia, PA 19140, [email protected], Telephone: 215-707-9380, Fax: 215-707-9890 24 1 2019 07 2 2019 10 2020 07 8 2019 25 10 2630 2640 Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:http://www.nature.com/authors/editorial_policies/license.html#termsDeficit in retromer complex function secondary to lower levels of one of its major components, the vacuolar protein sorting 35 (VPS35), has been reported in Alzheimer’s disease (AD) brains. VPS35 genetic reduction results in increased Aβ levels and synaptic pathology in mouse models of the disease. However, whether restoration of its levels has an effect on the AD-like phenotype which includes Aβ plaques, tau tangles and memory impairments remains unknown. In this paper, we investigated the effect of VPS35 gene delivery into the central nervous system on the development of the neuropathology and behavioral deficits of the triple transgenic (3xTg) mice. Compared with controls, animals over-expressing VPS35 had an amelioration of spatial learning and working memory, which associated with a significant reduction in Aβ levels and deposition and tau phosphorylation. Additionally, the same animals had a significant improvement of synaptic pathology and neuroinflammation. In vitro study confirmed that VPS35 up-regulation by reducing total levels of APP and tau results in a significant decrease in their metabolic products and phosphorylated isoforms, respectively. Our results demonstrate for the first time that VPS35 is directly involved in the development of AD-like phenotype, and for this reason should be considered as a novel therapeutic target for AD. ==== Body Introduction Aging is the strongest risk factor for Alzheimer’s disease (AD), the most common of dementia worldwide which is characterized by a complex pathogenesis and for which unifying mechanisms have been widely investigated by research labs in the field [1,2]. Loss of protein homeostasis is one common feature of the majority of aging organisms, and increasing evidence indicates that alteration in cell systems responsible for protein sorting and trafficking such as the vacuolar protein sorting system, aka retromer complex, may contribute to neurodegeneration in the AD brains by interfering with the removal of the disease-related specific pathologic proteins (i.e., Aβ and hyper-phosphorylated tau) [3–5]. Interestingly, consistent data in the literature show that the development of retromer dysfunction-dependent neuropathology is always secondary to a partial “loss of function” of this system. Thus, deficiency in the complex function resulting from down-regulation of one of its major components, VPS35, has been reported in hippocampi of AD patients; and genetic studies found that its variants increase the risk of developing AD [6,7]. On the other hand, VPS35 genetic reduction results in an increase of Aβ levels, cognitive impairments and synaptic dysfunction in a mouse model of AD-like amyloidosis [8, 9]. Recently, we have assessed the expression of VPS35 and other components of the retromer recognition core in the brains of the Tg2576 mice and reported an age-dependent decrease in the steady state levels of these proteins in the cortex but not in the cerebellum, an area known to be avoided of any AD-like pathology [10]. Taken together, all these studies clearly support the hypothesis that VPS35 is an active player and functionally involved in AD pathogenesis. However, whether restoration of its levels or a more general “gain of function” of the retromer complex system has an effect in vivo on part of the full AD-like phenotype is completely unknown. To test this hypothesis, in the current study we assessed the effect of VPS35 over-expression in the brains of 3xTg mice which are known to develop Aβ plaques, tau tangles and memory impairments. Material and Methods Injection of AAV-VPS35 into Neonatal Mice 3xTg mice harboring a human mutant PS1 (M146V) knock-in, and mutant amyloid precursor protein (APP; KM670/671NL) and tau (P301L) transgenes, and 3xTg wild‐type (WT) are the animals used in this study. The AAV‐VPS35 with a specific neuronal promoter (synpasin-1) and the AAV-empty vector were purchased from a commercial vendor (Vector Biosystems Inc., Malvern, PA). The injection procedures were performed as described previously [11, 12, 13]. Briefly, 2μl (3.5 × 1013 genome particles/ml) were bilaterally injected into the cerebral ventricle of newborn mice using a 10μl Hamilton syringe. A total of 41 pups were used for the study, ten WT and ten 3xTg mice were injected with AAV‐ VPS35; whereas eleven WT and ten 3xTg mice were injected with empty vector (Ctrl). Animals were then followed until they were 12 months old, when they first underwent behavioral testing, and two weeks later euthanized. All animal procedures were approved by the Institutional Animal Care and Usage Committee, in accordance with the U.S. National Institutes of Health guidelines. Behavioral Tests All animals were pre-handled for 3 days prior to testing. They were tested in a randomized order, and all tests were conducted by an experimenter blinded to the treatment or genotype. Y‐Maze The Y‐maze apparatus consisted of 3 arms 32cm long × 10cm wide with 26cm walls (San Diego Instruments, San Diego, CA). Testing was always performed in the same room and at the same time to ensure environmental consistency, as previously described [14, 15]. Morris water maze To perform the Morris water maze, we used a white circular plastic tank (122 cm in diameter, walls 76 cm high), filled with water maintained at 22°±2°C, and made opaque by the addition of a nontoxic white paint, as previously described [14,15]. Briefly, mice were trained for four consecutive days to find a Plexiglas platform submerged in water from four different starting points. Mice were assessed in the probe trial, which consisted of a free swim lasting for 60 s without the platform, 24 h after the last training session. Animals’ performances were monitored using Any‐Maze™ Video Tracking System (Stoelting Co., Wood Dale, IL). Immunoblot Analyses Primary antibodies used in this paper are summarized in the Table. Proteins were extracted in enzyme immunoassay buffer containing 250mM Tris base, 750mM NaCl, 5% NP‐40, 25mM EDTA, 2.5% sodium deoxycholate, 0.5% sodium dodecyl sulfate, and an EDTA‐free protease and phosphatase inhibitors cocktail tablet (Roche Applied Science, Indianapolis, IN), sonicated, and centrifuged at 45,000 rpm for 45 minutes at 4°C, and supernatants were used for immunoblot analysis, as previously described [16–18]. Briefly, total protein concentration was determined by using a BCA Protein Assay Kit (Pierce, Rockford, IL), samples were electrophoretically separated according to the molecular weight of the target molecule, and then transferred onto nitrocellulose membranes (Bio‐Rad). They were blocked with Odyssey blocking buffer for 1 hour, and then incubated with primary antibodies overnight at 4°C. After 3 washing cycles with T‐TBS, membranes were incubated with IRDye 800CW‐ or IRDye 680CW‐labeled secondary antibodies (LI‐COR Bioscience, Lincoln, NE) at 22°C for 1 hour. Signals were developed with Odyssey Infrared Imaging Systems (LI‐COR Bioscience). Actin was always used as an internal loading control. Biochemical Analyses Mouse brain homogenates were sequentially extracted first in radioimmunoprecipitation assay (RIPA) for the Aβ 1–40 and 1–42 soluble fractions, then in formic acid for the Aβ 1–40 and 1–42 insoluble fractions, and then assayed by a sensitive sandwich enzyme‐linked immunosorbent assay (ELISA) kit (Wako Chemicals, Richmond, VA) as previously described [16–18]. The assay for measuring the insoluble sarkosyl-soluble tau fraction was performed as previously described [16–18]. Immunohistochemistry Primary antibodies used are summarized in the Table. Immunostaining was performed as reported previously by our group [16–18]. Briefly, serial coronal sections were mounted on 3‐aminopropyl triethoxysilane‐coated slides. Every eighth section from the habenular to the posterior commissure (8–10 sections per animal) was examined using unbiased stereological principles. The sections for testing Aβ (4G8 antibody) were deparaffinized, hydrated, and pretreated with formic acid (88%) and subsequently with 3% H2O2 in methanol. The sections for testing total tau (HT7 antibody), and phospho‐tau epitopes, were deparaffinized, hydrated, subsequently pretreated with 3% H2O2 in methanol, and then treated with citrate (10mM) or IHC‐Tek Epitope Retrieval Solution (IHC World, Woodstock, MD) for antigen retrieval. Sections were blocked in 2% fetal bovine serum before incubation with primary antibody overnight at 4°C. Next, sections were incubated with biotinylated anti‐mouse immunoglobulin G (Vector Laboratories, Burlingame, CA) and then developed by using the avidin‐biotin complex method (Vector Laboratories) with 3,3′‐diaminobenzidine as a chromogen. Light microscopic images were used to calculate the area occupied by the immunoreactivities by using the software Image‐Pro Plus for Windows version 5.0 (Media Cybernetics, Bethesda, MD). Thioflavin-S Staining. The staining was performed as previously described [19]. Briefly, brain sections were deparaffinized and hydrated with the clearing agent xylene and a series of grade ethanol. After washed 3 times with PBS, the brain sections were incubated in filtered 1% Thioflavin S (Sigma-Aldrich, St. Louis, MO) for 8 minutes at room temperature. The tissues were washed twice in 70% ethanol, washed in PBS and mounted under a coverslip with anti-fading mounting media. The images were captured using the Nikon TiE fluorescent microscope (Nikon Instruments Inc., Melville, NY). Real-Time quantitative reverse transcription-PCR amplification RNA was extracted and purified using RNeasy mini kit (Qiagen, Valencia, CA), as previously described [20, 21]. Briefly, 1μg of total RNA was used to synthesize cDNA in a 20μl reaction using the RT2 First Strand Kit for RT-PCR (SuperArray Bioscience, Frederick, MD). Human APP and Tau genes were amplified by using the proper primers obtained from Super-Array Bioscience (Valencia, CA), and β-actin was always used as an internal control gene to normalize for the amount of RNA. Quantitative real-time RT-PCR was performed by using StepOnePlus Real-Time PCR Systems (Applied Biosystems, Foster City, CA). One microliter of cDNA was added to 10μl of SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA). Each sample was run in triplicate, and analysis of relative gene expression was done by StepOne software v2.1. Cells and treatment Neuro-2 A neuroblastoma (N2A) cells stably expressing human APP carrying the K670 N, M671L Swedish mutation (N2A-APPswe) were cultured in Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum, 100 U/mL streptomycin (Cellgro, Herdon, VA) and 400 mg/mL G418 (Invitrogen, Carlsbad, CA) at 37°C in the presence of 5% CO2. The cells were cultured to 80% to 90% confluence in six-well plates and then transfected with VPS35 plasmid (Addgene, Cambridge, MA), as previously described [20]. After 48 hrs transfection, media were collected for Aβ 1–40 measurement and cell lysates harvested after treatment with RIPA buffer for Western blotting analyses. Immunofluorescence studies were performed as previously described [21]. Briefly, N2A-APPswe cells transient transfected with control vector and VPS35 plasmid were plated on glass cover slips and the following day fixed in 4% paraformaldehyde for 15 min at RT. After rinsing 3 times with PBS, cells were incubated in a blocking solution (3% normal serum / 0.1 % TX-100) for 10 min at RT and then with the primary antibody against VPS35 for 1 hour at room temperature. After 3 times washings with PBS, cells were incubated for 1 h with a secondary Alexa 448- conjugated antibody. Cover slips were mounted using VECTASHIELD mounting medium (Vector Laboratories, Burlingame, CA) and analyzed with an Olympus BX60 fluorescent microscope (Olympus, Center Valley, PA). To control for cell viability after transfection, we always assayed for levels of LDH release in the supernatant collected at the end of the transfection. No differences in the levels of LDH between control and transfected cells were observed under our experimental condition (data not shown). Data Analysis One‐way analysis of variance and then Bonferroni multiple comparison tests were performed using Prism 5.0 (GraphPad Software, La Jolla, CA). All data are always presented as mean ± standard error of the mean. Significance was set at p < 0.05. Results VPS35 gene transfer ameliorates cognition in 3xTg mice. To assess the effect of VPS35 gene transfer on behavioral responses, mice were initially tested in the Y-maze paradigm. First, we observed that there was no difference in the general motor activities among the four groups of mice and between the ones receiving empty vector or treated with AAV-VPS35, as we found that the total number of arm entries was not different (Figure 1A). On the other hand, we observed that compared with wild type mice (WT), 3xTg had a significant reduction in the percentage of alternations, which was rescued in the 3xTg mice receiving the AAV-VPS35 (Figure 1B). Next, mice underwent the Morris water maze testing paradigm, which involve a visible platform training followed by hidden platform testing with four probe trials per day. During the four days training of the test, no differences were observed among the four groups and all mice in each group reached the training criterion and were proficient swimmers. By contrast, in the probe trial compared with WT the 3xTg mice had a reduction in the number of entries to platform, and the time spent in the platform quadrant, but an increase in the latency to the first entry to the platform, which were improved in the 3xTg receiving the AAV-VPS35 (Figure 1C–E). Finally, although compared with WT the 3xTg mice spent more time in the opposite quadrant and the treatment partially reduced this measure, the results never reached a statistical significant difference (Figure 1F). No differences for both tests were observed when males and females were analyzed separately (data not shown). No significant effects for both paradigms were observed in the WT mice receiving the AAV-VPS35 treatment (Figure 1A–F). VPS35 gene transfer decreases brain Aβ levels and deposition Mice were euthanized two weeks after the behavioral testing, and brains assayed first of all for the expression levels of the product of the gene that was transferred. As shown in figure 2, compared with 3xTg mice receiving empty vector the ones treated with AAV-VPS35 had a significant increase in the steady state level of the protein. Histochemical analysis of brain sections from the same mice confirmed a higher immune-reactivity for this protein in the treated mice (Figure 2C,D). Coincidental with this increase we observed that also the other protein component of the retromer recognition core, VPS26b, but not VPS29, was significantly increased (Figure 2A, B). Next, we investigated the effect of VPS35 over-expression on the AD-like amyloidotic aspect of the phenotype of this model by assessing Aβ/APP levels and metabolism. Compared with 3xTg controls, mice treated with AAV-VPS35 had a significant reduction of Aβ 1–40 and Aβ1–42 in both the RIPA-soluble as well as the formic acid-soluble fraction of these peptides (Figure 3 A, B). Confirming these data, we observed that Aβ immuno-reactive areas and the Thioflavin-S positive areas in the brains of the same animals were also significantly decreased when compared with 3xTg controls (Figure 3 C–E). Because we observed those changes in Aβ peptides levels, next we investigated the metabolism of its precursor protein, APP, in an attempt to identify potential mechanisms responsible for this effect. To this end, we assessed levels of APP, α-secretase (ADAM10), β-secretase (BACE1) and the four components of the γ-secretase complex by western blot analysis. Compared with controls, 3xTg receiving the AAV-VPS35 had a significant reduction in the steady state levels of APP, sAPPα and sAPPβ (Figure 3 F,G). By contrast, we did not observe any significant changes in the level of ADAM10, BACE1 and the four components of the γ-secretase complex, PS1, Nicastrin, Pen 2, and APH1, between the two groups of mice (Figure 3 F, G). Since we observed a reduction in APP at the protein levels, we also assessed its mRNA levels, which, as shown in figure 3G, were not different between the two groups of mice (Figure 3 H). VPS35 brain over-expression affects tau phosphorylation Next, we evaluated the effect of brain VPS35 over-expression on total tau levels and its phosphorylation at different epitopes in the two groups of mice. As shown in figure 4, we observed that levels of total soluble tau protein were significantly reduced in the 3xTg mice over-expressing VPS35. Interestingly, this was also the case when the levels of sarkosyl-soluble tau fraction (the insoluble fraction) was assessed (Figure 4 A,B). In addition, compared with controls, mice over-expressing VPS35 had a significant decrease in tau phosphorylated at epitopes Ser2020/Thr205, Thr181, Ser396/Ser404, Ser396, as respectively recognized by the AT8, AT270, PHF1, and PHF13 antibodies, but not changes were observed for tau phosphorylated at epitope Thr231and, as recognized by the AT180 antibody (Figure 4 A,B). Histochemical staining analyses confirmed a decrease in the immune-reactivity for total tau and the same phosphorylated tau epitopes in brain sections of mice over-expressing VPS35 when compared with controls (Figure 4 D,E). Since we observed a decrease in total tau at the protein levels, we also assessed its mRNA levels, which, as shown in figure 4C, were not different between the two groups of mice (Figure 4C). VPS35 brain overexpression modulates synaptic integrity and neuroinflammation It is known that the memory impairments the AD-like phenotype is typically associated with altered markers of synaptic proteins. For this reason, next we investigated whether VPS35 gene transfer had any effect on it. Compared with controls, 3xTg overexpressing VPS35 had a significant elevation in the steady state levels of synaptophysin (SYP), which was confirmed by immunohistochemistry analysis (Figure 5 A–D). By contrast, no significant differences between the two groups were observed for post-synaptic density protein-95 (PSD-95) and the microtubule associated protein 2 (MAP2) (Figure 5A, B). Finally, compared with controls 3xTg, mice over-expressing VPS35 had a statistically significant decrease in the steady state level of GFAP, a marker of astrocytes activation, which was also confirmed by immunohistochemistry analyses (Figure 5 E–H). However, no significant differences were observed between the two groups when the steady state levels of Iba1, a marker of microglia cells activation, were assessed (Figure 5 E, F). In Vitro Study: VPS35 effects on Aβ formation and APP processing To further confirm the effect of VPS35 on Aβ formation and APP metabolism, we transfected the N2A-APPswe cells with VPS35 plasmid for 48 hrs, supernatants collected and assayed for Aβ 1–40, while cells lysates used for Western blot analyses. Compared with controls, we observed that the neuroblastoma cells transfected with VPS35 had a significant increase in steady state levels of VPS35 and VPS26b, but not VPS29 (Figure 6A, B). The increase in VPS35 was also confirmed by immunofluorescence analyses (Figure 6C). As shown in Figure 6D, we found that compared with vehicle controls, conditioned media from cell over-expressing VPS35 had significantly lower Aβ 1–40 peptide levels. In addition, we observed that the over-expression of VPS35 was accompanied by a significant decrease in the steady state levels of APP, sAPPα and sAPPβ (Figure 6 E, F). Discussion In the current study we show for the first time that over-expression of VPS35, the main component of the retromer recognition core, in the central nervous system (CNS) of the 3xTg mice rescues their behavioral deficits, results in a significant reduction of the Aβ and tau neuropathology, lowers neuroinflammation and ameliorates synaptic pathology. The retromer system is a highly conserved multimeric protein complex present in all eukaryotic cells whose activity is essential for regulating the recycling and retrieval of several protein cargos from the endosome to the trans-Golgi network or the cell surface [22, 23]. In recent years, molecular and genomic studies have provided strong evidence that aberrant regulation of endosomal protein sorting and trafficking secondary to a dysfunction of the retromer complex system is implicated in the pathogenesis of several neurodegenerative diseases [24, 25]. From a structural point of view, the retromer complex is a multi-modular protein assembly, in which each module can vary depending on the specific function. However, the different modules are unified by having the same cargo recognition core formed by three main VPS proteins: VPS35, VPS26 and VPS29 and a membrane–targeting dimer of the sortin nexin which binds and transports different cargos [26]. Today, we know that VPS35 is the single most critical protein of the whole retromer assembly, and indeed knocking down VPS35 is sufficient to cause retromer complex dysfunction. Importantly, previous studies have shown that lower levels of VPS35 can ultimately affect the formation of the complex by influencing expression of the other two retromer core proteins (VPS29 and VPS26) suggesting a loss of stability of the complex as a functional unity [27]. The first implication of the complex in AD pathogenesis originated form a molecular profiling study showing that VPS35 and VPS26 were significantly down-regulated in the hippocampi of patients with late-onset of the disease. Later, it was reported that retromer deficiency associates with some of the disease processes since VPS35 deficient mice have cognitive deficits, synaptic dysfunction and higher Aβ levels. However, a strong biologic link between retromer dysfunction and AD was provided by human genetic studies showing that variants of genes encoding for VPS35 significantly increase the risk to develop the disease [28]. Intriguingly, analysis of all the literature available so far consistently demonstrates that retromer/VPS35-dependent pathology typically derives from a reduction or partial loss and not a gain of function of this complex [25]. To this end, whether up-regulating or restoring its levels in the CNS have any direct biological effect on the pathophysiology of AD remains unknown. To fill this gap, we implemented a genetic approach aimed at over-expressing VPS35 in the CNS of the 3xTg mice, which develop Aβ and tau neuropathology together with behavioral deficits. First, we demonstrated that VPS35 gene transfer had the effect of rescuing working memory in the 3xTg mice, as shown in the Y-maze, which measure spontaneous alternation of rodents once placed in the maze [29]. Thus, while as expected 3xTg controls had less alternation that the WT mice, the 3xTg mice receiving the VPS35 gene had a significant improvement of this measure, which was undistinguishable from WT. These changes could not be ascribed to an alteration of the general motor activities secondary to the injection or the treatment since no differences were observed among the four groups of mice when the number of entries in each arm of the maze was assessed. In addition, the same treatment resulted in a significant amelioration of their spatial learning and memory as documented in the Morris water maze [30]. Thus, in the probe trial compared with WT the 3xTg mice receiving empty vector had a lower number of platform entry and time spent in the platform zone together with an increase in the latency to first entry, and all of these parameters were improved in the 3xTg mice receiving the AAV-VPS35. Consistent with the efficacy of our experimental protocol, we observed that indeed the steady state levels of VPS35 protein were significantly up-regulated in the brains of mice receiving the active treatment even after 12 months. This result was in accordance with our previous studies in which we implemented a similar gene transfer approach [11–13]. Over-expression of VPS35 in the brains of the 3xTg mice manifested with a significant reduction of Aβ peptides levels both in the RIPA- and formic acid-soluble fractions, which was also reflected by a significant decrease in the amount of Aβ immuno-reactive deposits as assessed by measuring the brain amyloid burden. These deposits were thioflavin-S positive, which were also reduced after the treatment. Having observed those significant changes in amyloidosis, next we assessed the effect of the treatment on the metabolic processing of APP. While we did not find any changes in the levels of the major proteases involved in its cleavage, we observed that mice over-expressing VPS35 had a significant reduction in the steady state levels of APP which was associated with a similar decrease in sAPPα and sAPPβ. Interestingly no changes were observed for the mRNA levels of APP, suggesting a post-translational regulation of APP secondary to VPS35 over-expression. Importantly, by using neuronal cell lines stably expressing the human APP Swedish mutant we were able to reproduce the same results in terms of Aβ formation and APP metabolism after transient transfection with VPS35 plasmid. Taken together these findings support the hypothesis that restoration of the retroer complex system function resulted in decreased time spent by the APP in the endosome and therefore in less time for its own proteolytic processing and generation of Aβ peptides. Accumulation of highly phosphorylated tau protein and formation of intracellular fibrillary tangles is the second most common feature of AD neuropathology. For this reason, next we investigated the effect of the treatment on this aspect of the phenotype. Compared with 3xTg mice controls, we observed that VPS35 brain over-expression resulted in a significant decrease in total soluble tau, its insoluble fraction and tau phosphorylation levels at specific epitopes [31]. Since learning and memory tasks are known to be directly modulated not only by the amount of tau pathology but also by synaptic integrity, we measured the levels of some important synaptic proteins. Compared with control 3xTg mice, we found that VPS35 brain over-expression resulted in a significant elevation in the levels of synaptophysin, a marker of pre-synaptic protein integrity, underscoring the biochemical substrate for the behavioral amelioration we initially observed in the treated mice [32, 33]. Having found significant effect of our treatment on behavior, neuropathology and synapse, next we wanted to assess whether VPS35 gene transfer also affected neuroinflammatory responses. In this regard, we assayed the levels of two markers of inflammatory cells activation, GFAP for astrocytes and Iba1 for microglia, which have been reported to be elevated in AD reflecting dysregulated inflammatory reactions [34–37]. Under our experimental condition and coincidental with the lowering effect observed for bot Aβ and tau neuropathology we observed that compared with controls, brains from mice over-expressing VPS35 had a significant reduction in astrocyte activation. In summary, our study is the first demonstration that restoration of VPS35 levels and function directly rescue the AD-like phenotype of a mouse model with learning and memory impairments, Aβ deposits, and tau neurofibrillary tangles. Collectively, the findings presented in our paper further support an important functional role of the retromer recognition core which by modulating APP levels and its metabolic fate can ultimately ameliorates the AD-associated behavioral deficits, synaptic integrity and neuroinflammation. We conclude that VPS35 should be considered as a potentially promising therapeutic target for AD treatment. It is of great interest to note that, since there are reports showing an association of VPS35 mutations with the development of Parkinson’s disease (PD) [38], our results would have much broader clinical and translational implications. Thus, one could envision a scenario where restoring VPS35 levels would be beneficial not only for AD but also for PD. Additionally, small pharmacologic chaperones have been recently identified and characterized as novel compounds that selectively bind and stabilize the retromer complex and by doing so increase VPS35 levels in hippocampal neurons [39]. This fact together with our findings let us speculate that we are now well positioned from a therapeutic point of view to target VPS35 with a pharmacological probe in vivo. This approach would be certainly a departure from the current ones aiming at enzyme(s) directly responsible for Aβ biosynthesis, since it would target a common cellular mechanism (i.e., protein sorting and trafficking) which is widely considered at the core of AD pathogenesis. Acknowledgments Domenico Praticò is the Scott Richards North Star Charitable Foundation Chair for Alzheimer’s Research. This study was supported in part by grants from the National Institute of Health (AG056689, AG055707), and the Scott Richards North Star Charitable Foundation. Conflict of interest The authors have no conflicting financial interest to disclose. Figure 1. VPS35 gene transfer rescues behavioral deficits of 3xTg mice. (A) Number of total arm entries for 3xTg mice (3xTg) and wild‐type mice (WT) treated with AAV‐VPS35 (VPS35) or AAV‐empty vector control (Ctrl). (B) Percentage of alternations between 3xTg and WT mice receiving AAV‐VPS35 or AAV‐empty vector (p<0.05). (C) Morris water maze, probe trial for the same four groups of mice, number of entries to the platform area; (D) latency to first entry to the platform area; (E) time spent in the platform quadrant; (F) time spent in the opposite quadrant. Values represent mean ± standard error of the mean (p<0.05). (WT-Ctrl: n=11; WT‐VPS35: n =10; 3xTg-Ctrl, n=8; 3xTg-VPS35, n=8). Figure 2. Brain VPS35 overexpression modulates retromer recognition core levels in the 3xTg mice. (A) Representative western blot analysis of VPS35, VPS26b, VPS29 proteins in brain cortex homogenates from 3xTg mice receiving AAV-VPS35 (VPS35) or empty vector control (Ctrl). (B) Densitometry of the immunoreactivities shown in the previous panel (p<0.05, n=6). (C). Representative images of brain cortex sections from mice receiving AAV-VPS35 (VPS35) or empty vector (Ctrl) immuno-stained with VPS35 antibody (scale bar: 100μm). (D) Quantification of the integrated optical density for the immunoreactivity to the same antibody shown in panel C. Values represent mean ± standard error of the mean (p<0.05). Figure 3. Brain VPS35 overexpression lowers Aβ peptides levels and deposition in the 3xTg mice. (A, B) Radioimmunoprecipitation assay (RIPA)‐soluble and formic acid (FA)‐extractable Aβ1‐40 and Aβ1‐42 levels in cortex of 3xTg mice receiving empty vector (Ctrl) or AAV-VPS35 (VPS35) were measured by sandwich enzyme‐linked immunosorbent assay (p<0.05, Ctrl, n=8; VPS35, n=8). (C) Representative images of brain sections from mice receiving AAV-VPS35 (VPS35) or empty vector (Ctrl) stained with Thioflavin-S. (D) Representative images of brain sections from mice receiving AAV-VPS35 (VPS35) or empty vector (3xTg) immuno-stained with 4G8 antibody to detect Aβ immunoreactivity (scale bar: 500 μm). (E) Quantification of the area occupied by Aβ immunoreactivity in brains from mice receiving AAV-VPS35 (VPS35) or empty vector (3xTg) (p<0.05). (F) Representative Western blots of amyloid precursor protein (APP), sAPPα, sAPPβ, BACE1, ADAM‐10, APH‐1, Nicastrin, Pen‐2, and PS1 in cortex homogenates from 3xTg mice receiving AAV-VPS35 (VPS35) or empty vector (Ctrl). (G) Densitometric analyses of the immunoreactivities to the antibodies shown in the previous panel (p<0.05). (H) APP mRNA levels measured by RT-PCR in brain cortex from 3xTg mice receiving AAV-VPS35 (VPS35) or empty vector (Ctrl). Values represent mean ± standard error of the mean (Ctrl, n=6; VPS35, n=6). Figure 4. Brain VPS35 overexpression reduces tau phosphorylation and pathology in the 3xTg mice. (A) Representative Western blots of total soluble tau (HT7), insoluble tau (HT7), and phosphorylated tau at residues Ser202/Thr205 (AT8), Thr231/Ser235 (AT180), Thr181 (AT270), Ser396/Ser404 (PHF1), and Ser396 (PHF13) in brain cortex homogenates from 3xTg mice receiving empty vector (Ctrl) or AAV‐VPS35 (VPS35). (B) Densitometric analyses of the immunoreactivities to the antibodies shown in the previous panel (p<0.05, n=6). (C) Tau mRNA levels measured by RT-PCR in brain cortex from 3xTg mice receiving AAV-VPS35 (VPS35) or empty vector (Ctrl). (D) Representative immuno-histochemical staining images for HT7, AT270 and PHF13 positive areas in brain sections of 3xTg mice receiving empty vector control (Ctrl) or AAV‐VPS35 (VPS35) (scale bar: 100 μm). (E) Quantification of the integrated optical density for the immunoreactivity to the same antibody shown in the previous panel (p<0.05). Values represent mean ± standard error of the mean (Ctrl, n=6; VPS35, n=6). Figure 5. Brain overexpression of VPS35 affects synaptic integrity and neuroinflammation in the 3xTg mice. (A) Representative western blot analysis of synaptophysin (SYP), post-synaptic density-95 (PSD-95), microtubule associated protein2 (MAP2) in brain cortex homogenates from 3xTg mice receiving empty vector (Ctrl) or AAV-VPS35 (VPS35). (B) Densitometric analyses of the immunoreactivities presented in the previous panel (p<0.05). (C) Representative images of brain sections from mice receiving AAV-VPS35 (VPS35) or empty vector (3xTg) immuno-stained with SYP antibody (scale bar: 100 μm). (D) Quantification of the integrated optical density for the immunoreactivity to the same antibody shown in panel C (p<0.05). (E) Representative Western blot analyses of glial fibrillary acidic protein (GFAP) and Iba1 in brain cortex homogenates from 3xTg mice receiving empty vector (Ctrl) or AAV-VPS35 (VPS35). (F) Densitometric analyses of the immunoreactivities presented in the previous panel (p<0.05). (G) Representative images of brain sections from mice receiving AAV-VPS35 (VPS35) or empty vector (3xTg) immuno-stained with GFAP antibody (scale bar: 100 μm). (H) Quantification of the integrated optical density for the immunoreactivity to the same antibody shown in the previous panel (p<0.05). Values represent mean ± standard error of the mean (Ctrl: n=6; VPS35, n=6). Figure 6. Over-expression of VPS35 in N2A-APPswe cells affects Aβ formation and tau phosphorylation. (A) Representative western blot analysis of VPS35, VPS26b, VPS29 proteins in N2A-APPswe cells transfected with VPS35 plasmid (VPS35) or vector control (Ctrl). (B) Densitometry of the immunoreactivities shown in the previous panel (p<0.05, n=6). (C) Representative images of immunofluorescence analysis of cells transiently transfected with VPS35 cDNA and incubated with primary antibody for VPS35 (green). (D) Aβ1‐40 levels in conditioned media from cell transfected with empty vector (Ctrl) or VPS35 plasmid (VPS35) were measured by sandwich enzyme‐linked immunosorbent assay (p<0.05, Ctrl, n=6; VPS35, n=6). (E) Representative Western blots of amyloid precursor protein (APP), sAPPα and sAPPβ in N2A-APPswe cells transfected with VPS35 plasmid (VPS35) or vector control (Ctrl). (F) Densitometric analyses of the immunoreactivities to the antibodies shown in the previous panel. Values represent mean ± standard error of the mean (Ctrl: n=6; VPS35, n=6; p<0.05). Table 1. Antibodies used in the study. Antibody Host Application Source Catalog Number 4G8 Mouse IHC Covance SIG-39220 ADAM10 Rabbit WB Millipore AB19026 APH1 Rabbit WB Millipore AB9214 APP Mouse WB Millipore MAB348 BACE1 Rabbit WB IBL 18711 GFAP Mouse WB, IHC Santa Cruz sc-33673 HT7 Mouse WB, IHC Thermo MN1000 Iba1 Mouse WB Santa Cruz sc-32725 MAP2 Rabbit WB Millipore AB5622 Nicastrin Rabbit WB Cell Signaling 3632 Pen2 Rabbit WB Invitrogen 36–7100 PHF1 Mouse WB Dr. P. Davies Gift PHF13 Mouse WB, IHC Cell Signaling 9632 PS1 Rabbit WB Cell Signaling 3622S PSD95 Mouse WB Thermo MA1–045 sAPPα Mouse WB IBL 11088 sAPPβ Mouse WB IBL 10321 SYP Mouse WB, IHC Santa Cruz sc-12737 VPS26b Rabbit WB Proteintech 15915–1-AP VPS29 Goat WB AbCam ab10160 VPS35 Goat WB, IHC, IF AbCam ab10099 β-Actin Mouse WB Santa Cruz sc-47778 WB: Western blot; IHC: Immunohistochemistry; IF: Immunofluorescence ==== Refs References 1. Alzheimer’s Association . Alzheimer’s disease facts and figures . Alzheimer Dement 2017 ; 2017 ; 325 –373 . 2. 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==== Front ACS OmegaACS OmegaaoacsodfACS Omega2470-1343American Chemical Society 10.1021/acsomega.8b00153ArticleSolid-to-Solid Crystallization of Organic Thin Films: Classical and Nonclassical Pathways Wei Zhixian †Fan Jihui †Dai Chenghu ‡Pang Zhiyong ‡Han Shenghao ‡†School of Physics, State Key Laboratory of Crystal Materials and ‡School of Microelectronics, State Key Laboratory of Crystal Materials, Shandong University, Jinan 250100, P. R. China* E-mail: [email protected] (Z.P.).* E-mail: [email protected] (S.H.).25 06 2018 30 06 2018 3 6 6874 6879 24 01 2018 14 06 2018 Copyright © 2018 American Chemical Society2018American Chemical SocietyThis is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes.ACS Omega2019, 4, DOI: 10.1021/acsomega.9b01801 The solid-to-solid crystallization processes of organic molecules have been poorly understood in view of the complexity and the instability of organic crystals. Here, we studied the crystallization of a π-conjugated small molecular semiconductor, bis-(8-hydroxyquinoline) copper (CuQ2), by annealing the thin films at different temperatures. We observed a classical film-to-nanorods crystallization at 80 °C, a coexistence of classical and nonclassical nucleation and particle growth at 120 °C, and a nonclassical crystal growth at 150 °C. We found that the growth of the crystals followed the following processes: particle nucleation, particle growth, particle migration, nondirectional particle attachment, and structure reconstruction. We notice that the growth of CuQ2 particles follows an outside-to-inside process. More interestingly, our experiments suggest that the submicron CuQ2 particles are able to migrate dozens of micrometers at 150 °C. document-id-old-9ao8b00153document-id-new-14ao-2018-001539ccc-priceThis article was retracted on July 19, 2019 (ACS Omega2019, DOI: 10.1021/acsomega.9b01801). ==== Body 1 Introduction Crystal growth process is basic and essential in the preparation of high-quality single crystals or low-dimensional materials and contributes to the study of the intrinsic properties of functional materials.1−3 Classical crystallization theory assumes that the growth units are individual atoms, ions, or molecules.4−7 However, the recently discovered nonclassical solid-to-solid crystallization, involving aggregation of nanoparticles or prenucleation clusters, challenges the classical growth mechanisms.8−10 For example, the classical and nonclassical crystallization of zinc oxide nanoparticles and the nonclassical crystallization of copper hydroxide acetate have been recently reported.11,12 Compared with inorganic materials, the crystallization processes of organic crystals are more complex in view of the complexity of the soft organic molecules and the instability of the organic crystals.13,14 Organic crystals have enhanced performance, and, more interestingly, offer some novel properties not commonly seen in amorphous films, such as self-healing ability,15 superelasticity,16,17 terahertz emission,18 ambipolar charge-transport behavior,19,20 ferroelectricity,21 and multiferroics.22 Recently, nonclassical solid-to-solid crystallizations of [Ni(quinolone-8-thiolate)2] and hydroxyapatite have been observed.23,24 However, in view of the complexity and the instability of organic crystals, the solid-to-solid crystallization process of organic molecules is still poorly understood. 8-Hydroxyquinoline-based complexes are an interesting and versatile class of materials in view of their excellent thermal stability, adequate charge-carrier mobility, and high luminescence efficiency.25−28 Since the first tris-(8-hydroxyquinoline) aluminum (AlQ3)-based organic light-emitting diodes (OLEDs) reported by Tang et al. in 1987,29 8-hydroxyquinoline-based complexes have been widely used in OLEDs, organic photovoltaics, organic field-effect transistor, and other organic electronic or optoelectronic devices.30,31 More interestingly, 8-hydroxyquinoline-based complexes have shown great potential in the new research field of organic spintronics owing to their extremely long spin relaxation times.32,33 In this work, we study the solid-to-solid crystallization of a π-conjugated small molecular semiconductor, bis-(8-hydroxyquinoline) copper (CuQ2, C18H12N2O2Cu), by one-step heat treatment. Figure 1 shows the molecular structure of CuQ2. CuQ2 is not only an optoelectronic material but also an organic magnet with spontaneous spin polarization that is attractive for organic spintronics.34 Interesting crystallization process has been found recently in CuQ2-based materials, for example, the single-crystal-to-single-crystal transformation in CuQ2–tetracyanoquinodimethane.35−37 However, the crystallization process in pure CuQ2 is poorly understood. Here, we observed a classical film-to-nanorods crystallization at low temperature (80 °C) and a nonclassical film-to-crystal crystallization at high temperature (120–150 °C). High-quality CuQ2 crystals have been grown by annealing the thin films at 150 °C. The crystallization mechanism of CuQ2 crystals was discussed, and, more interestingly, we found that the submicron CuQ2 particles were able to migrate dozens of micrometers at 150 °C. Figure 1 Molecular structure of CuQ2. 2 Results and Discussion Figure 2 shows the scanning electron microscopy (SEM) images of CuQ2 thin films before and after heat treatment. The as-evaporated CuQ2 samples were thin films with nanograins distributed randomly on the surface. The surface morphology of the samples had no obvious change, as the heat treatment temperature was below 80 °C. As the heat treatment temperature increased to 80 °C, film-to-nanorods transformation occurred. From the SEM image of CuQ2 films, after heat treatment at 80 °C for 2 h, randomly distributed nanorods with their lengths about 1 μm can be clearly seen. The CuQ2 nanorods have smooth surfaces and no signs of particle attachment or grain agglomerates were found, exhibiting that the growth units are individual molecules. The CuQ2 molecules acquired energy from the annealing process, migrated on the surface under the van der Waals force, hydrogen bonding, and π–π bond stacking interactions,38,39 and then piled up in specific direction on the base of the “seed nanorods”, i.e., nanograins, indicating that the mechanism of the film-to-nanorods transformation is a classical crystallization mechanism rather than a nonclassical particle-attachment pathway. Figure 2 SEM images of (a) the as-evaporated CuQ2 thin films and the samples after heat treatment at (b) 80 °C, (c) 120 °C, and (d) 150 °C. The insets in (a–c) show images with higher resolutions. The inset in (d) shows the image with lower magnification. The yellow circles show that the diameters of the empty areas can be up to ∼100 μm. The CuQ2 shows different crystallization behaviors at high temperatures. From the SEM images of CuQ2 films, after heat treatment at 120 and 150 °C for 2 h, crystalline particles rather than nanorods can be found, indicating that the CuQ2 grains (120 and 150 °C) have different molecular packing modes compared with the rods (80 °C). This is confirmed by the X-ray diffraction (XRD) spectra of the CuQ2 sample (Figure S2), in which the samples annealed at 120 and 150 °C have different diffraction peaks than those of the samples annealed at 80 °C. When annealed at 120 and 150 °C, three new peaks emerged at 8.5, 11.9, and 13°, which correspond to (100), (002), and (002̅) facets of the β phase. This means that the CuQ2 crystal transforms from the α-phase to the β-phase when the temperature is above 120 °C. The CuQ2 migrated, nucleated, and grew into particles. As shown in Figure 2c, most of the particles have regular or irregular hexagonal structures from the top view, indicating an oriented growth perpendicular to the Si substrate. In the plane of the substrate, the particles have no obvious orientation. The hexagonal particles distributed densely on most of the areas of the Si substrate, and their diameters are about 2 μm or less. Surrounding these particles are empty areas where nearly no CuQ2 pieces were left. The distance between two particles is about several hundred nanometers. There are also areas where no hexagonal particles but only CuQ2 pieces nucleated (dashed-line ellipses), showing the inhomogeneity of the nucleation process. For the samples heated at 150 °C, crystals having sizes of several microns were obtained. The smooth surfaces and regular shapes reflect that the quality of the crystals grown directly from thin films is quite high. Surrounding the crystals are large empty areas where most of the CuQ2 molecules have migrated out to the crystals close by. From the SEM image with lower magnification, as shown in inset of Figure 2d, it can be seen that the diameters of the empty areas can be up to 100 μm, indicating that the migration distance of the CuQ2 is extremely long (dozens of microns), not only far longer than the well-known Ostwald ripening by the migration of molecules in surface science,5,40 but also longer than the reported long-range migration of organic nanoparticles (∼1.6 μm) during the nonclassical growth of molecular crystal.23 As the CuQ2 films annealed at 180 °C and above, the CuQ2 molecules volatilized and nothing was left on the substrate. For a better understanding of the nucleation process of the CuQ2 particles, Figure 3 shows the SEM image of the CuQ2 samples at different nucleation stages. The samples were obtained by annealing the CuQ2 thin films at 120 °C for different times. At the first stage, the substrate was covered by cuboid nanograins and no particle nucleation can be observed. With the increase in the heat treatment time, hexagonal particles (dashed-line squares) nucleated in stage #2 grew from nanometers to about 1–2 μm. At the same time, the shape of the CuQ2 nanograins, especially those that surround the hexagonal particles, changed from regular cuboid to irregular cuboid and further to irregular pieces. This phenomenon was more obvious in stages #3 and #4. The change in the shape of nanograins revealed molecular migration processes of CuQ2 molecules, indicating a classical crystallization pathway in this period. We notice an interesting phenomenon that almost all of the hexagonal particles have a cavity on the topside. It reveals that the growth of the particles is from outside to inside. At first, the CuQ2 molecules migrate to the outside of the particles, diffuse across the surface, and adjust their positions according to the crystal orientation, resulting in an outside-first growth process of the particles. It is also found that the bigger particles have smaller cavities, confirming the outside-to-inside process during particle growth. This process is also reflected by the neat side faces of the hexagonal particles, no matter how small the particles are. Besides the classical molecular migration, attached particles have also been found from the beginning of the particle growth. As labeled by circles in stages #2–5 in Figure 3, aggregation and attachment of two or more CuQ2 particles are clearly shown, exhibiting a coexistence of classical and nonclassical crystal growth pathways at 120 °C. From stage #2 to stage #5, the number of the particles increases significantly; however, the sizes of the particles are limited to about 1–2 μm and there is no obvious increase. At the same time, the amount of the CuQ2 grains (pieces) decreases substantially to an extremely small value in stage #5, exhibiting that classical molecular migration is the main crystal growth mechanism at this temperature. For the nonclassical crystallization pathway via particle migration, attachment, and structure reconstruction, the progress is much slower than that in the classical pathway because particle migration needs more energy than molecular migration. The yellow circles label some particles that are formed by particle migration and attachment of proximate particles. These particles have amalgamated into big particles, although their irregular shapes exhibit that the structure reconstruction process has not yet finished. As labeled by the blue circles, there are also “particle groups” that are composed of two or more CuQ2 particles. These particles are still at the stage of particle aggregation or attachment. Different from the previous observation of “oriented attachment” crystallization pathway that the nanoparticles adjust their orientation and attach to a large (compared with the particle) crystal, in this experiment, the attached particles have similar sizes and no evidence of oriented attachment has been found. The in-plane orientations of the attached particles seem to be random. The dashed-line circle labels a particle formed from three attached particles. It can be clearly seen that the particles are distorted to adjust their orientation, revealing that the particles reconstruct their structures after particle attachment.41,42 Figure 3 SEM images of the CuQ2 samples at different nucleation stages. The samples are obtained by annealing the CuQ2 thin films at 120 °C for different times. The SEM images in Figure 4 shows the crystallization process of CuQ2 at 150 °C. First, CuQ2 particles were nucleated via molecular migration (20 min). Different from the particles that nucleated at 120 °C, the CuQ2 particles that nucleated at 150 °C have complete single crystalline shapes and no cavity can be found on their topside (Figure 4), showing that the CuQ2 molecules acquired enough energy at 150 °C and had the ability of completing the molecular migration and structure adjusting process at short time. Once nucleated, nearby particles moved from their original positions and attached to each other (40 min). Figure 4d shows an aggregate of CuQ2 particles. It can be clearly seen that the aggregate is formed by nondirectional attachment of several microparticles. The aggregates underwent a structure reconstruction process and then grew into big particles (∼3 μm, yellow circles). Subsequently, the new formed big particles agglomerated into larger particle groups (60 min) and then underwent the structure reconstruction process again. As shown in Figure 4f, in this stage, the attachment of big particles is also nondirectional. The irregular curve labeled by the red arrow shows a particle boundary under structure reconstruction. Different from the previous observation that organic crystal grows via oriented attachment of small particles to a large crystal, in this experiment, the crystals are formed via nondirectional attachment of particles with similar sizes and large-scale structure reconstruction. Figure 4g shows the crystals growth by above process (120 min). The crystals have sizes of several microns and complete crystalline shapes, showing that the qualities of the crystals grown from this process are quite high. We note that the CuQ2 particles or crystals have excellent migration ability. As labeled by the dash–dot circles, it can be seen that the diameters of the empty areas can be up to dozens of microns, indicating that the migration distance of the CuQ2 is extremely long. Figure 4 Images of CuQ2 samples heat-treated at 150 °C for (a, b) 20 min; (c, d) 40 min; (e, f) 60 min; and (g, h) 120 min. The left column and the right column have different magnifications. To clarify the classical and nonclassical solid-to-solid crystallization pathway, the statistical processing of the images has been performed. Figure 5a shows the quantity of CuQ2 crystal nucleus of different stages at 120 °C (Figure 3). The quantity of CuQ2 crystal nucleus of different stages increased drastically with annealing time. These results are in accordance with our analysis of coexistence of classical and nonclassical processes. On the contrary, as shown in Figure 5b, the quantity of CuQ2 crystal nucleus of different annealed stages at 150 °C decreased fast with an increase in time. Furthermore, the average sizes of different stages became bigger and reached 8 μm after 120 min, confirming the existence of the nonclassical crystallization pathway. Figure 5 (a) Quantity of CuQ2 crystal nucleus for different stages at 120 °C. The data are collected from Figure 3. (b) The quantity of CuQ2 crystals and average sizes for different stages at 150 °C. The data are collected from the left column of Figure 4. 3 Conclusions In summary, the solid-to-solid crystallization process of CuQ2 thin film has been investigated by heat treatment. It is found that the film shows different crystallization behaviors at different temperatures: at a low temperature (80 °C), the film grows into nanorods via classical Ostwald ripening; at a middle temperature (120 °C), the film grows into crystals via particle nucleation, particle growth, particle migration, particle attachment, and structure reconstruction. The growth of hexagonal particles is from outside to inside. Classical Ostwald ripening and nonclassical particle-attachment pathways have both been found at this stage; at a high temperature (150 °C), the particles grow into high-quality crystals via several times nondirectional particle-attachment and structure reconstruction processes. It is found that the CuQ2 particles have the ability of migrating dozens of micrometers. Our results may facilitate the understanding of the crystallization of solid-state organic materials and further the development of molecular crystalline materials with controlled structures and properties. 4 Experimental Details CuQ2 was purchased from J&K Scientific in powder form and used as received without further purification. The energy dispersive spectrometer (EDS) analysis of the purchased CuQ2 powder as well as the evaporated thin films have been performed and shown in Supporting Information. The CuQ2 thin films were deposited on precleaned Si substrates by thermally evaporating pure CuQ2 powder at a base pressure of 2.0 × 10–4 Pa in a home-made thermal evaporator. The film thickness was in situ monitored using a quartz crystal thickness monitor. In our experiments, the thickness of the CuQ2 thin films was about 200 nm. The vacuum-deposited CuQ2 thin films were thermally annealed under flowing argon gas for 2 h using the crystal growth apparatus. Heat was applied by electronic resistance furnace, and the temperature rate can be controlled accurately. The flowing rate of high-pure and dry argon gas (99.999%) was 0.4–0.6 L/min, and the working gas pressure was 0.35 Pa. Moreover, to avoid the ambient adverse interference, the annealing chamber was washed by high-pure and dry argon (99.999%) gas with 2.0 L/min for 30 min prior to annealing. The annealing temperature was set for 2 h at temperatures 80, 120, and 150 °C. The surface morphology imaging of the samples were performed by a high-resolution scanning electron microscope (S-4800). Supporting Information Available The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.8b00153.SEM side view of samples; XRD of samples and calculated XRD from α- and β-phase CuQ2; EDS of purchased substance and CuQ2 thin films on Si substrate (PDF) Supplementary Material ao8b00153_si_001.pdf The authors declare no competing financial interest. Acknowledgments The authors are grateful for financial support from the Natural Science Foundation of China (11374184) and the Shandong Provincial Natural Science Foundation, China (ZR2018MF030). ==== Refs References Trasi N. S. ; Baird J. A. ; Kestur U. S. ; Taylor L. S. Factors Influencing Crystal Growth Rates from Undercooled Liquids of Pharmaceutical Compounds . J. Phys. Chem. B 2014 , 118 , 9974 –9982 10.1021/jp504450h .25076138 Davey R. J. ; Schroeder S. L. M. ; ter Horst J. H. Nucleation of Organic Crystals-A Molecular Perspective . Angew. Chem., Int. 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==== Front 04104626011NatureNatureNature0028-08361476-46873146277110.1038/s41586-019-1511-xnihpa1535907ArticleFatty acids and cancer-amplified ZDHHC19 promote STAT3 activation through S-Palmitoylation Niu Jixiao 16Sun Yang 16Chen Baoen 16Zheng Baohui 1Jarugumilli Gopala K. 1Walker Sarah R. 25Hata Aaron N. 3Mino-Kenudson Mari 4Frank David A. 2Wu Xu 11 Cutaneous Biology Research Center, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA2 Department of Medical Oncology, Dana Farber Cancer Institute, and Department of Medicine, Harvard Medical School, Boston, MA3 Massachusetts General Hospital Cancer Center, and Department of Medicine, Harvard Medical School, Charlestown, MA4 Department of Pathology, Massachusetts General Hospital, Harvard Medical School, Boston, MA5 Present address: Department of Molecular, Cellular, and Biomedical Sciences, University of New Hampshire, Durham, NH6 These authors contributed equallyAuthor contributions X.W conceived the concepts, designed the experiments and supervised the studies. J.N. designed and performed STAT3 palmitoylation, dimerization, ZDHHCs related biochemistry and cell biology experiments with the help with B.C., S.Y. and S.R.W.. J.N. and S.Y. performed cancer cell biology and tumorsphere experiments. B.C. performed palmitoylation assay, confocal imaging and Ras palmitoylation experiments. S.Y. and J.N. designed and performed in vivo animal experiments. S.Y. performed STAT3 disulfide assays, cancer stem cell analysis, bioinformatics, IHC analysis of LSCC tissue microarray. B.Z and G.K.J. synthesized the chemical probes. B.Z. identified STAT proteins from mass spec studies. A.N.H contributed to the LSCC PDX model. M.M-K contributed to pathology studies of LSCC patient samples. D.A.F. and S.R.W. contributed to experimental design and studies of STAT3 signaling. STAT3 palmitoylation has been independently reproduced by J.N, S.Y and B.C. for multiple times. J.N., S.Y., B.C. and X.W. analyzed the data; J.N. S.Y, B.C and X.W wrote the manuscript with input from all co-authors. Correspondence and requests for materials should be addressed to [email protected] 7 2019 28 8 2019 9 2019 28 2 2020 573 7772 139 143 Reprints and permissions information is available at www.nature.com/reprintsUsers may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:http://www.nature.com/authors/editorial_policies/license.html#termsSignal transducer and activator of transcription 3 (STAT3) plays a critical role in regulating cell fate, inflammation and immunity1,2. Cytokines and growth factors activate STAT3 through kinase-mediated tyrosine phosphorylation and dimerization3,4. It remains unknown whether other factors could promote STAT3 activation through different mechanisms. Here we show that STAT3 is posttranslationally S-palmitoylated at the Src Homology 2 (SH2) domain, promoting its dimerization and transcriptional activation. Fatty acids could directly activate STAT3 by enhancing its palmitoylation, in synergy with cytokine stimulation. We further identified ZDHHC19 as a palmitoyl acyltransferase (PAT) regulating STAT3. Cytokine stimulation enhances STAT3 palmitoylation by promoting ZDHHC19–STAT3 association mediated by Grb2 SH3 domain. Silencing ZDHHC19 blocks STAT3 palmitoylation and dimerization, impairing cytokine and fatty acid-induced STAT3 activation. Importantly, ZDHHC19 is frequently amplified in multiple human cancers, including in 39% of lung squamous cell carcinomas (LSCCs). High ZDHHC19 levels correlate with high nuclear STAT3 in patient samples. In addition, ZDHHC19 knockout in LSCC cells significantly blocks STAT3 activity, and inhibits fatty acid-induced tumorsphere formation and high-fat diet (HFD)-induced tumorigenesis in vivo. Taken together, we reveal that fatty acid and ZDHHC19-mediated palmitoylation are additional signals regulating STAT3, linking deregulation of palmitoylation to inflammation and cancer. ==== Body STAT3 is an important regulator of immunity, inflammation, and tumorigenesis1,2. Previously, JAK or Src-mediated tyrosine phosphorylation is considered as a major regulatory mechanism of STAT33,4. Posttranslational S-palmitoylation or S-fatty acylation attaches fatty acids, such as palmitate, to the cysteine residues of a protein, and regulates diverse functions of proteins under physiological and pathological conditions5,6. Using chemical reporters of protein palmitoylation7,8, we identified that STAT3 is palmitoylated (Extended Data Fig. 1a), consistent with other proteomic studies using acyl exchange methods in adipocytes9. Further studies using chemical reporters in multiple cell lines confirmed that ectopically expressed and endogenous STAT3 is indeed S-palmitoylated through thioester bonds, which are cleavable by hydroxylamine (NH2OH) treatment (Fig. 1a and Extended Data Fig. 1b, c. Original scans of all blots in Supplementary Fig. 1). In addition, we detected S-palmitoylation of STAT1 (α and β), STAT5B and STAT6, but not of STAT2 and 4 (Extended Data Fig. 1d–h), indicating that palmitoylation among STAT family members is different. Moreover, STAT3 can be efficiently labeled by palmitoylation (Alk-C16) or stearoylation (Alk-C18) probes, but much less efficiently labeled by probes with C14 or C20 chain length (Fig. 1b), suggesting that palmitoyl (C16) and stearoyl (C18) are the major acyl groups for STAT3 modification. Interestingly, nuclear STAT3 was highly palmitoylated (~75%) compared with cytoplasmic STAT3 (~20%) as showed by acyl-PEG exchange (APE) assay, which enables the separation and semi-quantification of acylated protein (Fig. 1c, d). In pulse-chase experiments, we found that the half-life of STAT3 palmitoylation turnover was ~1.8 hours, while the total protein remained stable during the assay period (Fig. 1e, and Extended Data Fig. 1i), suggesting that STAT3 palmitoylation is a dynamic process. Furthermore, the SH2 domain deletion (ΔSH2) and the C-terminal truncation (STAT31−585) mutants cannot be palmitoylated, suggesting that the palmitoylation sites might be located in SH2 domain or C-terminus (Extended Data Fig. 1j, k). Mutating the evolutionarily conserved C687 and C712 located in this region to serine, singly (C687S, C712S) or in combination (C687/712S, or 2CS) could markedly block palmitoylation (Fig. 1f, and Extended Data Fig. 1l–n), suggesting that C687 and C712 are required for STAT3 palmitoylation. We did not observe significant changes between STAT3 wild type (WT) and 2CS mutant in thermal stability and the pattern of disulfide bond formation, suggesting that these mutations might not alter overall folding of STAT3 significantly (Extended Data Fig. 1o–r). As JAK-kinase phosphorylation site Y705 is located near C687 and C712, we tested whether phosphorylation and palmitoylation could influence each other. We observed that IL-6 or interferon-γ (IFN-γ) stimulation markedly enhanced, and the selective JAK1/2 inhibitor ruxolitinib decreased STAT3 palmitoylation (Fig. 2a–c, Extended Data Fig. 2a). Moreover, the enhanced palmitoylation following IL-6 stimulation was attenuated by C687S mutation (Extended Data Fig. 2b). Interestingly, the phosphorylation-deficient, dominant-negative STAT3 mutant (DN-STAT3, Y705F) showed decreased palmitoylation levels compared to the WT, but the mutation did not completely abolish its palmitoylation (Fig. 2d). Taken together, these results suggest that cytokine-induced STAT3 phosphorylation can enhance, but is not required for its palmitoylation. Palmitoylation has been known to regulate protein membrane localization and trafficking10,11. However, we did not observe membrane localized STAT3. Interestingly, palmitoylation-deficient STAT3 2CS mutant can still be phosphorylated at Y705 at similar levels as WT upon IL-6 stimulation, but showed significantly decreased nuclear localization (Extended Data Fig. 2c, d). In addition, STAT3 2CS showed no significant change on K685 acetylation compared to WT, with or without the expression of acetyltransferase p300/CBP12 (Extended Data Fig. 2e). Surprisingly, STAT3 2CS mutant showed markedly lower homodimerization (Fig. 2e) or STAT1–STAT3 heterodimerization, but not JAK1–STAT3 heterodimerization (Extended Data Fig. 2f, g). Blocking STAT3-SH2 dimerization using 5, 15-diphenylporphyrin (5, 15-DPP) has no effect on STAT3 palmitoylation (Extended Data Fig. 2h, i). Taken together, palmitoylation might regulate STAT3 dimerization, which is not due to alternation of its phosphorylation and acetylation. It has been shown that all SH2 domains harbor putative lipid-binding pockets13, which could potentially accommodate fatty acyl chain binding and enhance dimerization. We generated mutants with reduced size of the hydrophobic pocket, which might limit the potential lipid binding in STAT3 SH2 domain (Extended Data Fig. 2j). Indeed, STAT3 A651V mutant showed significantly reduced palmitoylation levels and homodimerization (Extended Data Fig. 2k, l). Therefore, it is possible that palmitoylation could enhance STAT3 dimerization through engaging the lipid-binding pocket in SH2 domain. To test the function of STAT3 palmitoylation, we re-expressed STAT3 WT and 2CS mutant in STAT3–/– mouse embryonic fibroblast (MEF) cells. Consistently, the STAT3 2CS mutant lost palmitoylation in basal and IL-6-induced conditions (Extended Data Fig. 2m), and blocked IL-6-induced transcriptional activation and expression of STAT3 target genes (BCL2, BCL2L1 and MMP9) (Extended Data Fig. 2n–q) in both MEF and HEK293A cells, functioning as a dominant negative mutant. As cytokine-stimulation can promote STAT3 palmitoylation, palmitoylation may serve as a positive feedback response for cytokine stimulation, and promote STAT3 dimerization and activation. HFD has been shown to increase free fatty acid levels, and linked to STAT3 activation and inflammation in animals14,15. We found that HFD notably increased STAT3 palmitoylation and phosphorylation levels compared to normal-fat diet (NFD) in mouse lung and liver tissues (Fig. 2f, Extended Data Fig. 3a, b), indicating that HFD may enhance STAT3 activation by promoting its phosphorylation and palmitoylation. In addition, palmitic acid and stearic acid could directly activate STAT3 reporter, consistent with the finding that STAT3 can be palmitoylated and stearoylated (Extended Data Fig. 3c). Indeed, exogenous palmitic acid could increase STAT3 palmitoylation levels in a dose-dependent manner (Fig. 2g, h). Compared to IL-6, palmitic acid induced relatively moderate STAT3 nuclear localization (Extended Data Fig. 3d), and delayed transcriptional activation in STAT3 reporter and target gene expression analyses (Extended Data Fig. 3e, f). Similar to IFN-γ and IL6, palmitic acid alone could induce STAT3 dimerization in disuccinimidyl glutarate (DSG) crosslinking assay (Fig. 2i). Depalmitoylation by hydroxylamine treatment or C687S mutation could almost abolish STAT3 homodimerization in response to palmitic acid (Extended Data Fig. 3g,–h, and Fig. 2j), suggesting that palmitoylation contributed to palmitic acid-induced STAT3 dimerization. Interestingly, combining IL-6 and palmitic acid further increased STAT3 transcriptional activity compared to IL-6 or palmitic acid treatment alone in STAT3–/– MEF cells expressing STAT3 WT. However, such effects were lost in cells expressing the STAT3 2CS mutant (Extended Data Fig. 3i, j). Taken together, palmitic acid could activate STAT3 and synergize with cytokines to enhance STAT3 transcriptional activity through palmitoylation. HFD could also induce cytokine secretion16, therefore, it is possible that a positive feedback loop composed of STAT3 phosphorylation and palmitoylation may lead to strong activation of STAT3 in response to fatty acid and cytokine stimulation. In screens for potential palmitoyl acyltransferases (PATs) regulating STAT3, we identified ZDHHC5, 18 and 19 as potential primary hits17. In reconfirmation assays, ZDHHC19 showed consistent STAT3 palmitoylation activities and binding to STAT3 (Extended Data Fig. 4a–f). We generated the catalytically inactive mutant of ZDHHC19 (C142S) by mutating active site cysteine residue to serine18 (Extended Data Fig. 4g, h), which failed to promote STAT3 palmitoylation under basal conditions or with IL-6 stimulation (Fig. 3a–c). Consistently, ectopic expressing ZDHHC19 WT, but not C142S mutant, could significantly induce the activation of STAT3 reporter (Extended Data Fig. 4i). Furthermore, ZDHHC19 knockdown by shRNA blocked STAT3 palmitoylation and its target gene expression, which can be rescued by re-expressing shRNA-resistant mouse WT ZDHHC19, but not C142 mutant (Fig. 3d, Extended Data Fig. 4j). Loss of ZDHHC19 also abolished STAT3 target gene expression induced by IL-6, palmitic acid, or their combination (Extended Data Fig. 4k–m). Taken together, our results suggest that ZDHHC19 is the major palmitoylating enzyme regulating STAT3 palmitoylation, and is required for palmitic acid-induced STAT3 activation. We explored the mechanisms of how cytokine stimulation promotes STAT3 palmitoylation, and found that ZDHHC19 contains a highly conserved SH3 binding site around Pro18 and Pro20. ZDHHC19 could interact with the SH3 domain of Grb2, a key adaptor protein, which assembles signaling complexes in JAK-STAT signaling (Extended Data Fig. 4n, o)19. ZDHHC19 SH3 binding motif mutant (P18A) showed significantly reduced interaction with Grb2 and STAT3 (Extended Data Fig. 4p), suggesting that Grb2 may recruit ZDHHC19 to STAT3 through its SH3 domain. Furthermore, we found that IL-6 stimulation significantly increased (Extended Data Fig. 4q), while JAK inhibitor ruxolitinib blocked the complex formation of STAT3, Grb2 and ZDHHC19 (Fig. 3e). Consistently, Grb2 knockdown substantially blocked cytokine stimulation-induced STAT3 palmitoylation (Fig. 3f). Taken together, upon cytokine stimulation, Grb2 may serve as a critical adaptor protein, recruiting ZDHHC19 through its SH3 domain to the membrane complex, allowing efficient ZDHHC19–STAT3 association and STAT3 palmitoylation. Although Grb2 is known to be involved in Ras signaling20, ZDHHC19 does not regulate HRAS and NRAS palmitoylation21. Oncogenic HRAS (G12V) and NRAS (G12V or Q61R), or their palmitoylation did not affect STAT3 transcriptional activities either (Extended Data Fig. 4r–v). To explore the roles of ZDHHC19 and ZDHHC19-mediated STAT3 palmitoylation in pathogenesis, we found that ZDHHC19 is located in 3q26–29, a region frequently amplified in multiple human cancers, especially in lung squamous cell carcinoma (LSCC) in bioinformatics analyses (Extended Data Fig. 5a)22. Among the 56 genes in the focal 3q29 amplicon, ZDHHC19 expression has the strongest correlation with STAT3 target genes (BCL2 and BCL2L1) expression (Extended Data Fig. 5b–d). In addition, ZDHHC19 showed co-occurrence instead of mutual exclusivity with known oncogenes (PIK3CA, PRKCI and WWTR1 etc.) and tumor suppressors (XRN1, AIM2 and PYHIN1) in LSCC (Extended Data Fig. 5e–g). Consistently, ZDHHC19 expression is significantly higher in LSCCs, compared to adenocarcinoma (Extended Data Fig. 5h, i). In addition, high ZDHHC19 expression correlated significantly with poor patient survival (Extended Data Fig. 5j, k). The expression of STAT3 target genes (BCL2 and BCL2L1) is positively and significantly correlated with ZDHHC19 expression levels in two LSCC patient datasets (Extended Data Fig. 5l–o). We further carried out immunohistochemistry (IHC) studies of 131 tumor samples from a cohort of 85 LSCC patients, and confirmed that high ZDHHC19 protein levels showed significant correlation with nuclear STAT3 staining (Figure 4a, Extended Data Fig. 6, Supplementary Table 1). Consistently, in LSCC cell line HCC95, IL-6 stimulation markedly increased endogenous STAT3 palmitoylation and ZDHHC19–STAT3 interaction, which could be blocked by JAK inhibitor ruxolitinib (Extended Data Fig. 7a, b). Knocking down ZDHHC19 in multiple LSCC cell lines (HCC95, KNS-62 and/or SK-MES) reduced STAT3 palmitoylation and its nuclear localization (Extended Data Fig. 7c–f). Furthermore, we found that knockdown or knockout of ZDHHC19 markedly inhibited cell proliferation, colony formation, and cell migration of LSCC cells (Extended Data Fig. 7g–o). Interestingly, ectopic expression of the constitutive active form of STAT3 (STAT3C)23 could rescue the inhibitory effects of ZDHHC19 knockdown in colony formation of KNS62 cells, suggesting that ZDHHC19 regulates LSCC cell clonal growth through STAT3 (Extended Data Fig. 7p). LSCC cells can form tumorspheres when cultured at three-dimensional conditions, which enrich cancer stem-like cells and recapitulate many aspects of tumorigenesis ex vivo24. Consistently, we observed tumorsphere formation in multiple LSCC cell lines, with high expression of “stemness” markers (SOX2, OCT4, NANOG, ALDHA1 and CD133) (Extended Data Fig. 8a–d). Interestingly, palmitic acid significantly enhanced tumorsphere formation of KNS62 cells (sphere number, size, and the stem cell frequency) in limiting dilution assays, suggesting that fatty acid could promote LSCC “stemness” (Fig. 4b, c). In addition, loss of STAT3 or ZDHHC19 significantly blocked tumorsphere formation under basal conditions or with palmitic acid stimulation (Extended Data Fig. 8e–h). Interestingly, ectopic expression of STAT3C could rescue the inhibitory effects of ZDHHC19 knockdown in tumorsphere formation (Extended Data Fig. 8i), suggesting that ZDHHC19–STAT3 signaling axis plays an important role in maintaining LSCC “stemness”. To validate the roles of ZDHHC19 in LSCC in vivo, we established a xenograft model of HCC95 (control or CRISPR/Cas9-mediated ZDHHC19 knockout) in mice, fed with NFD or HFD. HFD promoted body weight gain, and accelerated tumor growth of control HCC95 cells. ZDHHC19 knockout significantly blocked HFD-induced tumor growth (Fig.4d, and Extended Data Fig. 9a–c), and decreased HFD-induced STAT3 palmitoylation, nuclear localization and target genes expression (BCL2, BCL2L1 and MMP9), and reduced Ki67-positive proliferative tumor cells (Extended Data Fig. 9d–g, Fig. 4e). Similar results were obtained in xenograft model with shRNA-knockdown of ZDHHC19 (Extended Data Fig. 9h–i). Furthermore, we tested a human LSCC patient-derived xenograft (PDX) model with high ZDHHC19 expression (PDX7 in Extended Data Fig. 10a, Supplementary Table 2). Consistently, HFD significantly promoted PDX tumor growth (Fig.4f, Extended Data Fig. 10b, c), correlated with the upregulation of STAT3 target gene expression, increased STAT3 palmitoylation and nuclear localization (Extended Data Fig. 10d–f). We isolated primary cells from the PDX tumor, and found that palmitic acid could markedly enhance tumorsphere formation in primary LSCC cells, which could be significantly inhibited by ZDHHC19 knockdown (Fig.4g, Extended Data Fig. 10g, h). Taken together, our data suggest that ZDHHC19-mediated STAT3 palmitoylation plays an important role in LSCC and HFD-related tumorigenesis in vivo. In summary, we demonstrate that S-palmitoylation of STAT3 is critical for its homodimerization, nuclear localization, and transcriptional activity. STAT3 palmitoylation and phosphorylation regulate STAT3 through a positive feedback signaling network, contributing to fatty acid-induced activation of STAT3 and inflammation. HFD and obesity have been linked to multiple cancers25. Indeed, smoking and high intakes of saturated fat have been shown as significant risk factors for human LSCC26. HFD enhances stemness and tumorigenicity of intestinal progenitors27, and the fatty acid translocase CD36 promotes tumor metastasis by regulating fatty acid uptake into cells28. Our findings of the fatty acid–ZDHHC19–STAT3 signaling axis might provide new mechanistic insights linking palmitoylation, inflammation and cancer (Extended Data Fig. 10i). Although ZDHHC19 is the major PAT regulating STAT3 in LSCC, other PATs, such as ZDHHC5, have also been implicated in lung cancer29. Taken together, these studies highlight the importance of protein palmitoylation in pathogenesis. METHODS Reagents Tris(2-carboxyethyl) phosphine hydrochloride (TCEP) (C4706), Copper(II) sulfate (496130), Tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amide (TBTA) (678937), Palmitic acid (P5585), Myristic acid (M3128), Palmitoleic acid (P9417), stearic acid (S4751), oleic acid (O1008), arachidic acid (A3631), PEG-maleimide 10 kDa (712469), N-Ethylmaleimide (NEM) (E3876), DL-Dithiothreitol (D9779) and Formamide (F9037), Anti-FLAG® M2 Magnetic Beads (M8823), Phosphatase inhibitor cocktail (P0044) and Crystal Violet (C0775) were purchased from Sigma-Aldrich. Biotin Picolyl Azide (1167), Alkynyl Palmitic acid (Alk-C16) (1165), Alkynyl Myristic Acid (Alk-C14) (1164) and Alkynyl Stearic Acid (Alk-C18) (1166) were purchase from Click Chemistry Tools. Disuccinimidyl glutarate (DSG) (20593), Streptavidin Agarose beads (SA10004), Trizol reagent (15596026) and 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) (M6494) were purchased from Thermo Fisher. PEG-maleimide 5 kDa (146–109) was purchased from Layson Bio. 2-bromohexadec-15-ynoic acid (16-BYA) and Alkynyl arachidic acid (Alk-C20) were synthesized in-house (>98% of purity). Human Recombinant IL-6 (200–06) was purchased from Pepro tech. Human Recombinant IFN-γ (01–172) was purchased from Millipore. Protein A/G XPure Agarose Resin (P5030–1) was purchased from UBPBio. Laemmli (SDS-Sample Buffer, Reducing, 6X) (BP-111R) was purchased from Boston BioProducts. cOmplete EDTA-free protease inhibitors cocktail (05892791001) and Liberase DL (05401160001) were purchased from Roche. Polyethylenimine (PEI) (23966–1) was purchased from Polyscience. Corning® Matrigel® Basement Membrane Matrix (354234) was purchased from Corning. Antibody Myc-tag (sc-40), GFP-tag (sc-9996) and GAPDH (sc-47724) were purchased from Santa Cruz Biotechnology. Flag-tag (F1804) and α-Tubulin (T9026) were purchased from Sigma-Aldrich. Flag-tag (14793S), HA-tag (3724S), phospho-STAT3 (Tyr705) (9145S), STAT3 (9139S), Grb2 (3972), Lamin B1(68591S), HDAC1 (5356S), Ki67 (9027S), Anti-Rabbit HRP (7074S), Anti-Mouse HRP (7076S) were purchased from Cell Signaling. STAT3 (MA1–13042), Streptavidin-HRP (N100), Alexa Fluor 488 Goat anti-Rabbit (A11008) and Alexa Fluor 594 Goat anti-Mouse (A11032) were purchased from Thermo Fisher Scientific. ZDHHC19 (26–021) was purchased from Prosci. ZDHHC19-HRP (orb473136) was purchased from Biorbyt. β-Actin (ab6276) was purchased from ABCAM. Mouse TrueBlot® ULTRA Anti-Mouse Ig HRP (18-8817-33) and Rabbit TrueBlot® ULTRA Anti-Rabbit Ig HRP (18-8816-33) were purchased from Rockland. Plasmids Full-length human STAT3 cDNA was obtained from DF/HCC DNA Resource Core at Harvard Medical School, and was subcloned into pCMV-3Tag-6 vector (Agilent). The Myc -tagged STAT3 constructs (Myc-STAT3-WT and Myc-STAT3-Y705F) used in this study are the same as previously described30. All the mutants (STAT3 C687S, C712S, C687/712S; ZDHHC19 C142S, P18A) were generated by site-directed mutagenesis using the QuickChange II Site-Directed Mutagenesis kit (Agilent) following manufacturer’s instructions. The STAT3 SH2 deletion, N- and C-terminally truncated variants (STAT31−585 and STAT3586−770) were cloned into pCMV-3Tag-6 vector according to the previous report31. The constructs of STAT1α Flag pRc/CMV and STAT1β Flag pRc/CMV were gifts from Jim Darnell (Addgene plasmids #8691 and #8704). The EGFP STAT1 plasmid was a gift from Alan Perantoni (Addgene plasmid #12301). The HA-JAK1 plasmid was generously provided by Lixin Rui at University of Wisconsin School of Medicine and Public Health, Madison, WI). The Flag-tagged STAT2 plasmid was generously provided by Dr. Tom Maniatis (Mount Sinai School of Medicine, New York, NY). The other constructs (STAT3, STAT4, STAT5B, STAT3C and STAT3-specific m67-luciferase reporter) used in this study are the same as previously described32,33. The HA-ZDHHC constructs were gifts from Dr. Masaki Fukata (National Institute for Physiological Sciences, Japan). The Myc-tagged ZDHHC19 plasmid was purchased from GenScript (Piscataway, NJ). Cell culture Human MDA-MB-231, HCC95, SK-MES-1, KNS-62 HCC827, , Phoenix and HEK293A cell lines were obtained from ATCC (Manassas, VA) or Cell Line Depository at Massachusetts General Hospital (MGH) Cancer Center. U3A and STAT3 null (STAT3–/–) mouse embryonic fibroblasts (MEF) cells were provided by Dr. David A. Frank. All the cells were cultured in high-glucose Dulbecco’s Modified Eagle Media (DMEM) (Life technologies) with 10% (v/v) fetal bovine serum (FBS) (Thermo/Hyclone, Waltham, MA), 100 units/mL penicillin and 100 μg/mL streptomycin (Life technologies) at 37°C with 5% CO2. STAT3–/–MEF cells were grown in DMEM containing 10% FBS in the presence of 100 units/mL penicillin and100 μg/mL streptomycin at 37°C with 5% CO2. ATCC cell lines were characterized by Short Tandem Repeat (STR) profiling. None of cell lines used in this paper are listed in the database of commonly misidentified cell lines maintained by ICLAC. Generation of stable cell lines Two different shRNAs targeting human ZDHHC19 (ZDHHC19 MISSION shRNA3: TRCN0000134807Sequence CCGGCCAGCAACTGGTATTTAACAACTCGAGTTGTTAAATACCAGTTGCTGGTTTTTTG and ZDHHC19 MISSION shRNA5: TRCN0000137220 CCGGGCCAGCAACTGGTATTTAACACTCGAGTGTTAAATACCAGTTGCTGGCTTTTTTG) were purchased from Sigma (St. Louis, MO). Each shRNA was cloned into the pLKO.1 lentiviral vector. Viruses were produced following the manufacturer’s instructions. Briefly, a 10-cm dish of 80% confluent HEK293T cells was transfected with 10 μg of the transfer plasmid, 5μg pVSVG, 7.5 μg psPAX2, 500 μL of jetPRIME buffer, and 25 μL of jetPRIME transfection reagent (Polyplus). Media was changed after overnight incubation. After 48 hours, viral supernatants were filtered through a 0.45 μm low protein binding membrane (Millipore) and used immediately. Transduction was performed in HCC95 cells, followed by selection with 1 μg/mL puromycin for 2 weeks. ZDHHC19 knockdown efficiency was analyzed by Western blot. For generation of ZDHHC19 knockout stable cells, sgRNAs targeting human ZDHHC19 were cloned into pLentiCRISPR v2 (a gift from Feng Zhang’s lab). Viruses were produced by following the published protocols from Zhang lab. The empty vector pLentiCRISPR v2 was taken as control. After infection, cells were selected with 1 μg/mL puromycin. ZDHHC19 knockout efficiency was analyzed by western blot. The mixed cell populations were used for the experiments. For generation of STAT3 expression stable cell lines, Flag-tagged STAT3 WT or C687/712S mutant was cloned into pBABE-hygro retroviral vector. Retroviruses were produced in the Pheonix packaging cell lines. Transduction of HEK293A stable cells was performed in 6-well plate and selected under 200 μg/mL hygromycin B for 3 days. Flag-STAT3 expression was analyzed by western blot using anti-FLAG M2 (F1804, Sigma Aldrich, 1:5000). Immunoblotting and immunoprecipitation Cells were lysed with RIPA buffer supplemented with protease inhibitors (Roche) and phosphatase inhibitors (Roche). Lysates were denatured by heating for 5 minutes at 95 °C and loaded onto 4–12% Bis-Tris polyacrylamide gel. NuPAGE MOPS or MES running buffer (Invitrogen) was used for the SDS-PAGE. The proteins were subsequently transferred to polyvinylidene fluoride (PVDF) membranes (Millipore). The membranes were blocked and incubated with primary antibodies and secondary HRP-conjugated antibodies, and developed by exposure to film. Antibody and dilutions used in the studies: anti-FLAG M2 (1:2000), anti-HA (1:1000), anti-GFP (1:1000), anti-STAT3 (1:1000), anti-pSTAT3 (1:1000), anti-ZDHHC19 (1:500), anti-GAPDH (1:2000), anti-c-Myc (1:1000), streptavidin-HRP antibody (1:5000). Anti-rabbit and anti-mouse IgG, HRP-linked antibodies were diluted 1:5000. For co-immunoprecipitation experiments, cells were lysed in IP buffer (20 mM Tris-HCl, pH 7.0, 250 mM NaCl, 3 mM EDTA, 3 mM EGTA, 0.5% NP-40) supplemented with protease inhibitors (Roche) and phosphatase inhibitors (Roche). 1 μg of indicated antibody was added into lysates and incubated with protein G-Sepharose (life technologies) at 4°C for overnight. Sepharose-enriched immunocomplexes were resolved on SDS-PAGE, transferred to PVDF membrane and analyzed with immunoblotting. Mass spectrometry analysis Proteins were precipitated and washed by methanol. The precipitated pellets were then dissolved in suspension buffer containing 4% (w/v) SDS, 50 mM Tris-HCl, pH 7.4, 150 mM NaCl and 10 mM EDTA and further diluted with. Brij97 buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 10 mM EDTA, 1% Brij97). Diluted proteins were incubated with streptavidin agarose beads for 1.5 hours at room temperature on a rotator. The captured proteins were incubated for 40 min in dark with 500 μL 8M urea, 50 μL 500 mM TCEP and 50 μL of 400 mM iodoacetamide and digested on beads. The supernatants were collected for LC-MS/MS analysis as described previously34. Labeling, Click reaction and streptavidin pull down Cells were labeled with DMSO or probe for the indicated time. Cells were lysed in lysis buffer (50 mM TEA-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 0.2% SDS, cOmplete EDTA-free protease inhibitors) followed by Click reaction with biotin-azide. Proteins were precipitated with 9 volumes of 100% methanol for 2 hours at −80°C and recovered by centrifugation at 14,000 × g for 10 min. The pellets were dissolved in 100 ml suspension buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 2% SDS) and then diluted to 10 fold immunoprecipitation buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 0.5% NP40).Labeled cellular proteins were enriched using streptavidin agarose (Life technologies) at room temperature with rotation for 3 hours. Protein-bound streptavidin agarose beads were washed three times with immunoprecipitation buffer and bound proteins were eluted with elution buffer (10 mM EDTA pH 8.2, 95% formamide) for 10 min at 95°C. Samples were processed with western blot analysis. Fractionation assay Fractionation of cytoplasmic proteins was performed by using the Subcellular Protein Fractionation Kit (Cat# 78840, Thermofisher). The nucleus was lysed with lysis buffer (50 mM TEA, pH7.3, 150 mM NaCl, 4% SDS, 1× protease inhibitor cocktail (Roche, EDTA free), 1,500 units/ml benzonase nuclease, 2 mM PMSF). When the solution was clear, EDTA was added to the final concentration of 5 mM. The cytoplasmic proteins were diluted with the lysis buffer containing 5 mM EDTA before APE assay. Acyl-PEG exchange (APE) assay The assay was performed as reported35. Briefly, Cells were lysed in lysis buffer (50 mM TEA-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 2% SDS) with protease and phosphatase inhibitors (Roche) followed by incubation with 25 mM TCEP at 55°C for 1 hour. Free cysteine residues were alkylated with 50 mM NEM for 1 hour at room temperature. The mixture was subjected to Methanol/Chloroform protein precipitation and then incubated with NH2OH to cleave palmitoylation thioester bonds at 37°C for 1 hour. Proteins were precipitated by Methanol/Chloroform and incubated with 2 mM PEG at room temperature. After 1 hour, proteins were precipitated again by Methanol/Chloroform and then re-suspended with 1× SDS loading buffer and boiled at 95°C for 3 min and subjected to western blotting analysis. DSG crosslinking assay Cells were lysed with RIPA buffer supplemented with protease and phosphatase inhibitors (Roche). 20-fold molar excess of DSG was added into cell lysates. Allow the sample to react on ice for 2 hours. Unreacted DSG was quenched with 100 mM Tris-HCl, pH 7.4 for 10–15 minutes at room temperature. Samples were then boiled with 6× SDS loading buffer at 95°C for 3 min and processed with western blot analysis. Disulfide detection assay (PEG-maleimide gel shift) Disulfide detection assay was conducted according to the protocol as previously described36. Briefly, the cells were lysed in lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 2% SDS) with protease and phosphatase inhibitors (Roche). Free cysteine residues were alkylated with 50 mM NEM for 1 hour at room temperature. The cell lysates were incubated with 50 mM DTT at 37°C for 1 hour. The proteins were precipitated by Methanol/Chloroform and incubated with 2 mM PEG-maleimide (10 kDa) at room temperature for 2 hours. The proteins were precipitated again by Methanol/Chloroform and then re-suspended with 1× SDS loading buffer and boiled at 95°C for 3 min and subjected to western blot analysis. Transfection and infection Expression constructs were transfected into cells using jetPRIME transfection reagent, Lipofectamine 2000 or Polyethylenimine (PEI) following previous report37. Retrovirus and lentivirus were prepared using Phoenix and 293T cells respectively. RNA extraction and quantitative real-time PCR (qPCR) Total RNA was extracted from cells using the TRIZOL (Invitrogen, Carlsbad, CA). Message RNA was converted to first-strand cDNA using high capacity cDNA reverse transcription kit (Thermo, Cat#4368814) and followed by real-time PCR reaction using LightCycler®480 SYBR green Master according to the manufacturer’s directions. Real-time PCR analyses were performed in triplicate by LightCycler®480 PCR machine (Roche). GAPDH was used for normalizing gene expression. Gene expression was calculated using comparative CT method (ΔΔCT method). Data from triplicates experiments were pooled and plotted as shown. The sequences of gene specific primers used for PCR are shown bellow: Human BCL2 Forward: GCTACCGTCGTGACTTCGC, Reverse: CCCCACCGAACTCAAAGAAGG; Mouse BCL2 Forward: ATGCCTTTGTGGAACTATATGGC, Reverse: GGTATGCACCCAGAGTGATGC; Human BCL2L1 Forward: GGTCGCATTGTGGCCTTTTTC, Reverse: TGCTGCATTGTTCCCATAGAG; Mouse BCL2L1 Forward: GACAAGGAGATGCAGGTATTGG, Reverse: TCCCGTAGAGATCCACAAAAGT; Human MMP9 Forward: GGGACGCAGACATCGTCATC, Reverse: TCGTCATCGTCGAAATGGGC; Mouse MMP9 Forward: CTGGACAGCCAGACACTAAAG, Reverse: CTCGCGGCAAGTCTTCAGAG; Human GAPDH Forward: TGCACCACCAACTGCTTAGC, Reverse: GGCATGGACTGTGGTCATGAG; Mouse GAPDH, Forward: AGGTCGGTGTGAACGGATTTG, Reverse: TGTAGACCATGTAGTTGAGGTCA; Human 18S rRNA Forward: GTAACCCGTTGAACCCCATT, Reverse: CCATCCAATCGGTAGTAGCG; Human Nanog, Forward: CCCCAGCCTTTACTCTTCCTA, Reverse: CCAGGTTGAATTGTTCCAGGTC; Human OCT4, Forward: GGGAGATTGATAACTGGTGTGTT, Reverse: GTGTATATCCCAGGGTGATCCTC; Human Sox2 Forward: TACAGCATGTCCTACTCGCAG, Reverse: GAGGAAGAGGTAACCACAGGG; Human CD133 Forward: GAGAAAGTGGCATCGTGCAA, Reverse: CACGTCCTCCGAATCCATTC; Human ALDH1 Forward: GCCATAACAATCTCCTCTGCT, Reverse: CATGGAAACCGTACTCTCCC. Transwell migration assay The assay was performed as previously reported38. Briefly, cells were transfected with indicated plasmids and seeded into Trans-well inserts (Corning) in 24-well culture plates. After 16 hours, the cells were fixed and stained by crystal violet solution (1XPBS, 0.05% w/v crystal violet, 1% formaldehyde, 1% methanol). The migrated cells were photographed under Zeiss microscope and counted from 5 randomly selected fields. Data from triplicates experiments were pooled and plotted as shown. Luciferase assay Cells were transfected with luciferase construct harboring m67 sequence and Renilla control construct. After 48 hours of transfection, cells were lysed with passive lysis buffer. The activities of Firefly and Renilla luciferase in the lysates were measured with the dual Luciferase Assay System (Promega, Madison, WI). The luminescence was measured using EnVision Multilabel Reader (Perkin Elmer, Waltham, MA). The relative luminescence was calculated by dividing Firefly to Renilla luminescence and normalized to control. Data from triplicates experiments were pooled and plotted as shown. Cell proliferation assay Cells were either mock-transfected or transfected with the indicated plasmid. After 24 hours of transfection, cells were seeded into 96-well plate. The cell viability was determined at the indicated time points. 100μL fresh media and 20μL MTT were added into each well. The plates were incubated at 37°C for 2 hours. After incubation, 100μL MTT solvent (methanol) was added into each well. Absorbance values (OD = 590nm) of stained cells were measured using Perkin Elmer EnVision Multilabel Reader. Data from triplicates experiments were pooled and plotted as shown. Immunofluorescence Cells were cultured for 48 hours on glass coverslips in 6-well plate and fixed with 4% paraformaldehyde in PBS for 10 min at room temperature, followed by permeabilization with 0.1% Triton X-100 for 5 min at room temperature, incubation with primary antibody for one hour, wash with 1% Triton X-100, and incubation with Alexa Fluor-conjugated secondary antibodies for one hour in the dark. The coverslips were stained with Hoechst 33342 (H3570, Invitrogen, 1:3000) before being mounted with VECTASHIELD mounting medium (H-1000, Vector laboratories). Images were captured by Leica TCS-NT 4D confocal microscope. Z stacks were collected with a spacing of 1 μm. Antibody and dilutions used in the studies: anti-Flag (14793S, Cell signaling, 1:100), anti-Lamin B1 (68591S, Cell signaling, 1:100), anti-STAT3 (MA1–13042, Thermo Fisher Scientific, 1:100). Alexa Fluor 488 Goat anti-Rabbit (A11008, Life technologies, 1:5000), Alexa Fluor 594 Goat anti-Mouse (A11032, Life technologies, 1:5000). The fluorescence-intensity profile along the Z-axis from confocal Z-stacks were shown. The fluorescence intensity of nuclear localized STAT3 was quantified using the confocal software to define the selected ROI (Region of Interest) area based on nuclear DAPI signal. More than 200 cells were quantified in at least three independent experiments. Immunohistochemistry (IHC) For paraffin embedded tumor tissue, slides were de-paraffinized and rehydrated using standard procedures. After antigen retrieval (10mM Sodium citrate, 0.05% Tween 20), the slides were processed using EXPOSE Mouse and Rabbit specific HRP/DAB Detection IHC kit (Abcam Cat#80436) according to the manufacturer’s instruction. In brief, the slides were quenched to block endogenous peroxidase and blocked with buffer. The anti-STAT3 (1:2000) or anti-Ki67 (1:500) antibody was incubated for overnight in a humidified chamber. The slides were developed with DAB Chromogen at optimized time. The images were captured using Zeiss microscope. Ki67 positive cells were counted from 5 randomly selected fields and plotted as shown. IHC staining of STAT3 was quantified by nuclear localization. Score: 0 (negative), 1 (few nuclei), 2 (10%), 3 (10–50%), 4 (>50%); IHC staining of ZDHHC19 was quantified by Score: 0 (<10%), 1 (10–25%), 2 (25–50%), 3 (50–75%), 4 (>75%). Tumorsphere formation assay Stem like cells were enriched from HCC95, KNS62 and HCC827 by culturing in serum-free DMEM-F12 medium (Life Technologies, Grand Island, NY) containing 50 μg/ml insulin (Sigma-Aldrich St. Louis, MO), 0.4% Albumin Bovine Fraction V (Sigma-Aldrich St. Louis, MO), N-2 Plus Media Supplement (Life Technologies, Grand Island, NY), B-27 Supplement (Life Technologies, Grand Island, NY), 20 ng/mL EGF (STEMCELL technologies, Cambridge, MA) and 10 ng/ml basic FGF (STEMCELL technologies, Cambridge, MA) in ultra-low attachment plates (Corning, Corning, NY). The images were captured using Zeiss microscope. Spheres were counted from 5 randomly selected fields and plotted as shown. in vitro limiting dilution assay Tumor-initiating cell frequencies were calculated using the extreme limiting dilution analysis (ELDA) algorithm as previously described39. In brief, the cancer cells were serially diluted to obtain the following final cell concentrations: 1 (288 wells), 10 (192 wells), 100 (96 wells), 1000 (24 wells) cells/well in 96 well ultralow attachment plates (Corning, CLS3474–24EA). After 2 weeks of incubation, the plates were removed from the incubator and the tumorsphere formation was determined using microscope. The cancer cell initiating frequency and significance was determined using Extreme Limiting Dilution Analysis online software (http://bioinf.wehi.edu.au/software/elda/). Images were captured using Zeiss microscope. Cellular thermal shift assay Cellular thermal shift assay was conducted according to the protocol as previously described40. In brief, HEK293A cells were transfected with STAT3 plasmids and analyzed by intact cell-based and cell lysate-based thermal shift assay at 36 hours post-transfection. For intact cell-based assay, HEK293A cells were collected and washed with PBS buffer for three times before being distributed into different 0.2 mL PCR tubes (~1 million cells per tube). The cells were denatured at the indicated temperatures for 3 min on PCR instrument, following freeze-thaw twice using liquid nitrogen. The samples were centrifuged, and the supernatants were analyzed by Western blot. For cell lysate-based assay, HEK293A cells were collected and harvested with lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 1% Triton X100). After denaturation at the indicated temperatures for 3 min, the lysates were centrifuged, and the supernatants were analyzed by Western blot. Patient samples PDX tumor (PDX7) bearing NSG mouse was purchased from the Jackson Laboratory (PDX LIVETM model TM00231 lung squamous cell carcinoma). The Jackson Laboratory received coded patient tumor samples, with all personal identifier removed, under a Jackson IRB approved PDX Resource Protocol (http://tumor.informatics.jax.org/mtbwi/pdxDetails.do?modelID=TM00231). PDX1 to PDX6 tumor samples were obtained from Dr. A. N. Hata’s lab at Massachusetts General Hospital Cancer Center, the PDX samples are collected in compliance with all relevant ethical regulations for human research participants with patient consent following Protocol 02–180 and 13–416 approved by Dana Farber/Harvard Cancer Center (DF/HCC) IRB. For tissue microarray, one sample set was purchase from US Biomax (OD-CT-RsLug02–001) with 32 cases of lung squamous cell carcinoma tissue. All human tissues are collected under HIPPA approved protocols with the donor being informed completely and with their consent. The US Biomax Inc. states to follow standard medical care and protect the donors’ privacy. Other three slides with 57 cases were obtained from Dr. Mari Mino-Kenudson of Department of Pathology, Massachusetts General Hospital (MGH). The tissues are collected from surgically removed tumors with patient consent following protocol (2009P001838) approved by MGH IRB. All information regarding the patient samples is listed in >Supplementary Table 1 and 2. All samples are de-identified by removing patient information and assigned a code, and the researcher who analyzed the samples could not trace back to patient personal information. Animal studies SCID mice were maintained at the Massachusetts General Hospital (MGH) animal facility. All animal studies were conducted in accordance with the Guide for the Care and Use of Laboratory Animals from NIH and a protocol (2013N000065) approved by MGH Institutional Animal Care and Use Committee (IACUC). In none of the experiments did xenograft tumor size surpass 1.5 cm in any two dimensions, and no animal had severe loss of body weight (>15%) or evidence of infections or wounds (endpoints that permitted by IACUC). 2×106 HCC95 WT or ZDHHC19 knockout stable cells, and shRNA control cells or shRNA targeting ZDHHC19 (sh3) stable cells were injected subcutaneously into mouse flanks. The mice were fed with autoclaved regular chow diet (normal fat diet, NFD, composed of 10% fat based on caloric content) or high-fat diet (HFD, composed of 60% fat based on caloric content) (Research Diet Cat#D12492, D12450J New Brunswick, NJ) for one week before cancer cell transplantation and their specific diet were maintained until the experiments were terminated. Group assignment and tumor monitoring were carried out in a double-blinded manner. Tumor volume was assessed by caliper measurement using the formula (width2× length/2) (mm3). After four weeks, the mice were euthanized, and the primary tumors were isolated for further experiments. The animal tissues used for HFD-induced STAT3 palmitoylation studies were prepared by The Jackson Laboratory (Bar Harbor, ME). Briefly, 10 C57BL/6 mice were randomly divided equally into two groups and fed with NFD or HFD at The Jackson Laboratory. The mice were sacrificed at Day 14. All harvested samples were shipped on dry ice and stored at −80°C for further analysis. Establishment of patient-derived xenografts in mice PDX tumor (PDX7) bearing NSG mouse was purchased from the Jackson Laboratory (PDX LIVETM model TM00231 lung squamous cell carcinoma). Once the tumors were established, the mice were euthanized, and the tumors were dissected and dissociated. 2–3 mm PDX tumor fragments mixed with Matrigel were loaded into syringe with 14 gauge and subcutaneously implanted into both flanks of SCID mice. When the tumors were established at the week 3 post-inoculation, normal fat diet or high fat diet were administered. Tumor volume was determined by caliper measurement using the formula (width2× length/2) (mm3). Isolation of primary cancer cells from PDX tumor Tumor tissue collected from PDX mouse was minced into small pieces and enzymatically dissociated into single cells with 0.28 Unit/ml Liberase DL (Roche #05401160001) for 1–2 hours at 37°C. Cells were then washed with Hank’s balanced salt solution containing 2% FBS. The single cell suspension was then cultured in advanced DMEM-F12 medium supplemented with 10% FBS, 50 μg/ml insulin, 20 ng/mL EGF, 10 ng/mL basic FGF, 6 mmol/L glutamine, 1% of penicillin/streptomycin and 40 ng/mL dexamethasone. The isolated cells were cultured in tumorsphere media mentioned in Tumorsphere formation assay to determine stem like cell enrichment. Statistical analysis Results were presented as mean ± s.e.m of at least three independent experiments. Measurements at single time points were analyzed by ANOVA and if significant, further analyzed by a two-tailed Student’s t-test. Time courses were analyzed by repeated measurements (mixed model) ANOVA with Bonferroni post-tests. Survival analysis was estimated by the Kaplan-Meier method. Survival in different groups was compared using the log-rank test with GraphPad Prism, version 6.0 (GraphPad Software, San Diego, CA). The Pearson’s correlation coefficient (r) was used to measure the strength of the association between two variables. P < 0.05 was considered to indicate statistical significance. Data availability The data supporting the findings of this study are available within the paper. Uncropped raw images from western blots are shown in Supplementary Information. Source Data for all graphs are available online. Tumor sample information is shown in Supplementary table. All other data are available from the corresponding author upon reasonable request. Extended Data Extended Data Figure 1. STAT3 is S-palmitoylated at evolutionarily conserved cysteine residues and mutation of STAT3 palmitoylation sites does not affect disulfide formation and protein stability. (a) HEK293A cells were incubated for 4 hours with DMSO or 50 μM clickable probes. Cell lysates were reacted with azide-biotin for enrichment of labeled proteins with streptavidin beads and identified by mass spectrometry. The peptide spectral counts were shown in the table. (b) HEK293A cells were labeled with 50 μM chemical probe (Alk-C16) for 4 hours. Cell lysates were reacted with biotin-azide and precipitated with streptavidin beads and subjected to western blot using anti-STAT3 antibody. Endogenous STAT3 palmitoylation was analyzed by western blot. (c) HEK293A cells were transfected with Myc-STAT3 WT and labeled with 50 μM chemical probe Alk-C16 for 4 hours, cell lysates were subjected to Streptavidin blot following anti-Myc IP and subsequent Click reaction. (d) HEK293A cells were transfected with empty vector or GFP-STAT1 and labeled with 50 μM chemical probe Alk-C16 for 4 hours. After Click reaction, cell lysates were subjected to streptavidin blot showing detection of STAT1. (e) Similar analysis as panel (c) was performed in HEK293A cells transfected with Flag-STAT1α and Flag-STAT1β. (f) HEK293A cells were transfected with Flag-STAT3 or Flag-STAT5 and labeled with 50 μM chemical probe Alk-C16 for 4 hours, Cell lysates were reacted with biotin-azide and precipitated with streptavidin beads and subjected to western blot using anti-Flag antibody. (g-h) Similar analysis as panel (e) was performed in HEK293A cells transfected with Flag-STAT3, Flag-STAT2, Flag-STAT4 and Flag-STAT6. (i) HEK293 cells were pulse labeled with 50 μM chemical probe (Alk-C16) for 4 hours and subsequently chased by the addition of an excess of 50 μ M BSA-conjugated palmitic acid. Cells were harvested at the indicated time point and subjected to analysis of STAT3 palmitoylation. (j) Schematic representation of Flag-STAT3 and Flag-STAT3 truncation mutant constructs. (k) HEK293A cells were transfected with vector control, Flag-tagged wild type (WT), SH2 deletion (ΔSH2), STAT3 1–585 or STAT3 586–770 mutant, and labeled with 50 μM chemical probe (Alk-C16) for 4 hours. STAT3 palmitoylation levels were analyzed by Click reaction and streptavidin bead pull-down, followed by western blotting using anti-Flag antibody. (l) Alignment of STAT3 protein sequence among different species. (m) HEK293A cells were transfected with Flag-tagged wild type (WT), C687S, C712S or C687/712S (2CS) mutant of STAT3, and total cell lysates were subjected to Acyl-PEG exchange (APE) assay. Samples were analyzed by western blot using anti-Flag antibody. The upper band indicated the palmitoylated STAT3. (n) The palmitoylated STAT3 in panel (m) were quantified using Image J. The data are presented as mean ± s.e.m., n = 3 biologically independent experiments. P value is determined by two-tailed t-test. (o) HEK293A cells were transfected with Flag-tagged wild type (WT), C687S, C712S or C687/712S (2CS) mutant of STAT3, and total cell lysates were reduced with DTT. Samples were analyzed by western blot using anti-Flag antibody. (p) HEK293A cells were transfected with Flag-tagged wild type (WT) or C687/712S (2CS) mutant of STAT3, and free thiols of protein were blocked by NEM. Disulfide bonds were reduced by DTT. Maleimide-PEG was applied to attach at the reduced thiols. Samples were analyzed by western blot using anti-Flag antibody. (q) HEK293A cells were transfected with Flag-tagged wild type (WT), C687S, C712S or C687/712S (2CS) mutant of STAT3, and total cell lysates were heated at indicated temperature. Samples were analyzed by western blot using anti-Flag antibody. Soluble fractions were plotted versus temperatures. (r) HEK293A cells were transfected with Flag-tagged wild type (WT), C687S, C712S or C687/712S (2CS) mutant of STAT3, and cells were heated at indicated temperature. Total cell lysates were analyzed by western blot using anti-Flag antibody. Soluble fractions were plotted versus temperatures. In (b-i, k, o-r), the experiments were independently repeated at least 3 times with similar results. For gel source data, see Supplementary Figure 1. Extended Data Figure 2. STAT3 palmitoylation promotes STAT3 nuclear translocation and synergically enhances STAT3 signaling activity with phosphorylation. (a) HEK293A cells were pretreated with ruxolitinib (1 μM) for 0.5 hour and then labeled with 50 μM chemical probe (Alk-C16) for 2 hours with the incubation of IFNγ (1 ng/ml). Endogenous STAT3 palmitoylation was analyzed by Click reaction and streptavidin beads pull-down and subjected to western blotting. (b) HEK293A cells were transfected with Flag-STAT3 WT or Flag-STAT3 WT C687S and then labeled with 50 μM chemical probe Alk-C16 for 2 hours with the incubation of IL-6 (20 ng/ml). STAT3 palmitoylation was analyzed by western blot following Click reaction and streptavidin beads pull down. (c) HEK293A cells were transfected with Myc-STAT3-WT, Myc-STAT3-C687S or Myc-STAT3-Y705F and treated with IL-6 (20ng/ml, 1 hour). Whole cell lysates were immunoprecipitated with anti-Myc antibody, and followed with immunoblotting using indicated antibodies. (d) Confocal microscopy showing changes in subcellular localization of STAT3 WT and C687/712 mutant in HEK293A stable cell lines. Cells were stained with anti-Flag antibody (green), anti-Lamin B1 antibody (red) and DAPI (blue). Lamin B1 displays the nuclear membrane. The red line indicates the position of the Z stack. Western blot showing STAT3 WT and C687/712 mutant were expressed at comparable levels in HEK293A stable cell lines. The levels of nuclear-localized STAT3 were quantified by measuring the Flag (STAT3) fluorescence intensity in the nucleus using the confocal software to define the selected ROI (Region of Interest) based on nuclear DAPI signal. The data are presented as mean ± s.e.m., n = 225 or 300 cells in each group, respectively. P value is determined by two-tailed student’s t-test. Scale bars represent 20 μm in all images. (e) HEK293A cells were transfected with Flag-STAT3-WT, Flag-STAT3-C687/712S or Flag-STAT3-K685S with or without CBP. Whole cell lysates were analyzed by western blot using indicated antibodies. (f) HEK293A cells were co-transfected with Flag-STAT3-WT or Flag-STAT3–2CS along with GFP-STAT1. Whole cell lysates were analyzed by anti-Flag IP followed by immunoblotting using the indicated antibodies. (g) Similar analysis as panel (f) was performed in HEK293A cells transfected with Flag-STAT3 and HA-JAK1 followed by anti-HA IP. (h) HEK293A cells were co-transfected with Flag-STAT3 and Myc-STAT3 and pretreated with 5, 15-DPP (10 μM) and then treated with IL-6 (20 ng/ml, 1hour). Whole cell lysates were analyzed by anti-Flag IP followed by immunoblotting using the indicated antibodies. (i) HEK293A cells were transfected with Flag-STAT3-WT treated with 5, 15-DPP alone or along with IL-6 (20 ng/ml, 1 hour) and labeled with 50 μM chemical probe (Alk-C16). STAT3 palmitoylation was analyzed by Click reaction and streptavidin beads pull-down and subjected to western blotting. (j) The acyl chain binding pocket according to the STAT3 crystal structure (PDB 4E68), hydrophobic amino acids are shown in yellow. (k) Mutational analysis of selected residues involved in STAT3 palmitoylaiton. (l) HEK293A cells were transfected with Myc tagged and Flag tagged STAT3 as indicated. Whole cell lysates were immunoprecipitated with anti-Myc antibody, and followed with immunoblotting using indicated antibodies. (m) STAT3 null (STAT3–/–) mouse embryonic fibroblast (MEF) cells were transfected with vector control, Flag-tagged wild type (WT) or C687/712S (2CS) mutant of STAT3. Cells were labeled with 50 μM Alk-C16 probe for 2 hours with the incubation of IL-6 (20 ng/ml). Western blotting using anti-Flag antibody in the streptavidin bead pull-down samples indicates the palmitoylation levels of STAT3. (n) STAT3−/− MEF cells were co-transfected with STAT3 reporter construct (m67-luciferase reporter) and Renilla luciferase control construct, and a vector control, a Flag-tagged STAT3 wild type (WT) or C687/712S mutant. Cells were then treated with IL-6 (20 ng/ml) for 8 hours. Luciferase activity was obtained from triplicated experiments and normalized to the Renilla luciferase. Relative fold induction was plotted as shown. (o) Similar experiment as (n) was performed in HEK293A cells. (p) STAT3−/− MEF cells were transfected with vector control, Flag-tagged STAT3 wild type (WT) or C687/712S mutant. Cells were treated with IL-6 (20 ng/ml) for 8 hours. The mRNA levels of STAT3 target genes (BCL2, BCL2L1 and MMP9) were analyzed by qRT-PCR. (q) Similar experiment as (p) was performed in HEK293A cells. In (n-q), the data are presented as mean ± s.e.m., n = 3 biologically independent samples. P value is determined by two-tailed student’s t-test. In (a-c, e-i, k-m), the experiments were independently repeated at least 3 times with similar results. For gel source data, see Supplementary Figure 1. Extended Data Figure 3. Palmitic acid promotes STAT3 nuclear localization and activity through induction of dimerization in synergy with cytokine stimulation. (a) C57BL/6 mice were fed with normal fat diet (10 kcal% fat, NFD) or high fat diet (60 kcal% fat, HFD) for 2 weeks. STAT3 palmitoylation and p-Y705 levels were analyzed by APE and western blot in mouse lung tissues. (b) Similar experiment as (a) was performed in liver tissue. (c) U3A cells were treated with various fatty acids at 100 μM or IL-6 (20 ng/ml) for 6 hours. Luciferase activity was measured and relative fold induction from triplicate experiments. (d) Confocal microscopy showing changes in subcellular localization of STAT3 in HEK293A cells treated with palmitic acid (PA, 50 μM, overnight) and/or IL-6 (20 ng/ml, 1 hour). Cells were stained with anti-STAT3 antibody (red) and DAPI (blue). The yellow line indicates the position of the Z stack. The levels of nuclear-localized STAT3 were quantified by measuring the STAT3 fluorescence intensity in the nucleus using the confocal software to define the selected ROI (Region of Interest) based on nuclear DAPI signal. Scale bars represent 20 μm in all images. The data are presented as mean ± s.e.m., n = 217, 246, 261, 288 cells in each group, respectively. P value is determined by two-tailed student’s t-test. (e) HEK293A cells were transfected with STAT3-luciferase reporter with Renilla control constructs and treated with 20 ng/ml IL-6 or 100 μM PA for indicated time points. Luciferase activity was measured and relative fold induction from triplicate experiments was plotted with mean ± s.e.m., n = 3 biologically independent samples. (f) HEK293A cells were treated with 20 ng/ml IL-6 or 100 μM palmitic acid, the expression of indicated genes was quantified with qPCR. (g) HEK293A cells were transfected with Flag-STAT3 along with or without Myc-STAT3 WT and treated with 100 μM palmitic acid for 2 hours. Whole cell lysates were analyzed by anti-Flag IP followed by immunoblotting using the indicated antibodies. (h) Similar experiments as in (g) were performed in HEK293A cells with or without NH2OH treatment. (i) HEK293A cells were co-transfected with m67-luciferase reporter and Renilla reporter constructs along with vector control, Flag-tagged STAT3 wild type (WT) or C687/712S mutant. After 48 hours, cells were treated with palmitic acid (100 μM, 8 hours) and/or IL-6 (20 ng/ml, 8 hours). Normalized luciferase activity from triplicate experiments was plotted as shown. (j) HEK293A cells were transfected with vector control, Flag-tagged STAT3 wild type (WT) or C687/712S mutant. Cells were treated with IL-6 (20 ng/ml) for 8 hours. The mRNA levels of STAT3 target gene BCL2 were analyzed by qRT-PCR. Relative fold change (normalized to GAPDH) was shown from triplicate experiments. In (c, f, i, j), the data are presented as mean ± s.e.m., n = 3 biologically independent samples. P value is determined by two-tailed student’s t-test. In (a, b, g, h), the experiments were independently repeated at least 3 times with similar results. For gel source data, see Supplementary Figure 1. Extended Data Figure 4. ZDHHC19 mediates STAT3 palmitoylaiton and Ras is not involved in ZDHHC19 mediated STAT3 signaling. (a-b) HEK293A cells were co-transfected with construct encoding Flag-STAT3 and HA-ZDHHCs. After 48 hours, cells were labeled for 4 hours with 50 μM palmitoylation probe Alk-C16. Cells lysates were reacted with azide-biotin and subjected to Streptavidin beads pull-down. STAT3 palmitoylation was shown by western blot. (c) HEK293A cells were transfected with Flag-STAT3 alone or along with HA-ZDHHC19, HA-ZDHHC 5 and HA-ZDHHC 18 and labeled with 50 μM probe Alk-C16 for 4 hours following analysis of STAT3 palmitoylation. (d) HEK293A cells were transfected with Flag-STAT3 alone or along with HA-ZDHHC19, HA-ZDHHC 5 and HA-ZDHHC 18. Whole cell lysates were analyzed by anti-Flag IP followed by immunoblotting using the indicated antibodies. (e) HEK293A cell were transfected as in panel (d). Total cell extracts were analyzed by western blot after IP with anti-HA antibody. (f) HEK293A cells were transfected with Flag-STAT3-WT or Flag-STAT3-K685S with or without ZDHHC19. Palmitoylation of STAT3 was analyzed as in panel (c). (g-h) HEK293A cells were transfected with HA-ZDHHC19-WT or inactive mutant C142S (CS) and labeled with 50 μM Alk-C16 or 16C-BYA. Cell lysates were subjected to streptavidin beads pull-down. STAT3 palmitoylation was showed by western blot. (i) HEK293A cells were transfected with STAT3-luciferase reporter and Renilla control, co-transfected with ZDHHC19-WT and ZDHHC19-C142S as shown treated with IL-6 (20 ng/ml) and/or palmitic acid (PA, 100 μM) for 8 hours. Luciferase activity was measured and relative fold induction from triplicate experiments. (j) HEK293 cells were co-transfected with ZDHHC19-shRNA and ZDHHC19-WT or ZDHHC19- C142S, the expression of indicated genes was quantified with qRT-PCR. (k-m) KNS-62 control cells and ZDHHC19 KO stable cells were treated with IL-6 (20 ng/ml) and/or palmitic acid (100 μM) for 8 hours. The mRNA levels of STAT3 target genes (BCL2, BCL2L1 and MMP9) were analyzed by qRT-PCR. (n) Schematic representation of Grb2 wild type and SH3-domain truncation mutants and alignment of ZDHHC19 SH3 binding motif sequences. (o) HEK293A cells were co-transfected with Myc-tagged ZDHHC19 and GFP-tagged Grb2 WT, N terminal SH3 deletion (ΔN-SH3) or C terminal SH3 deletion (ΔC-SH3). Whole cell lysates were subjected to immunoprecipitation using anti-Myc antibody, followed by western blotting using the indicated antibodies. (p) HEK293A cells were co-transfected with Myc-tagged ZDHHC19 WT or P18A mutant, Flag-tagged STAT3 and GFP-tagged Grb2 following Co-IP assay. (q) HEK293A cells were treated with IL-6 (20 ng/ml) and analyzed by anti-STAT3 Co-IP assay followed by western blotting using the indicated antibodies. (r-s) HA tagged ZDHHC19 was co-transfected with HA tagged HRAS (r) or NRAS (s) into HEK293A cells. After 24 hours, cells were incubated with fresh media containing 10% dialyzed FBS for 2 hours and subsequently labeled with 50 μM Alk-16C probe for 4 hours. Cells lysates were reacted with biotin-azide and subjected to SDS-PAGE. Streptavidin blot was used to detect HRAS (r) or NRAS (s) palmitoylation as previously described. Comparable protein loading was confirmed by anti-HA and β-Actin western blotting. (t-u) Depalmitoylase inhibitor Palmostatin B (10 μM) treatment or overexpression of depalmitoylase ABHD17A has no effect on the expression of STAT3 target genes (BCL2, BCL2L1 and MMP9) in HEK293A cells expressing oncogenic HRAS G12V (t) or NRAS G12V (u). (v) Treatment with different concentrations of Palmostatin B (1 μM, 5 μM and 10 μM) has no effect on the expression of STAT3 target genes (BCL2, BCL2L1 and MMP9) in melanoma SK-MEL-2 cells containing NRAS Q61R mutation. In (i-m, t-v), the data are presented as mean ± s.e.m., n = 3 biologically independent samples. P value is determined by two-tailed student’s t-test. In (a-h, o-s), the experiments were independently repeated at least 3 times with similar results. For gel source data, see Supplementary Figure 1. Extended Data Figure 5. ZDHHC19 gene is amplified and highly expressed in lung squamous cell carcinoma and correlated with poor clinical outcomes. (a) Alteration frequency and oncoprint diagram of ZDHHC19 gene alteration summary in cancer patients (11,413) obtained from cBioPortal (TCGA). (b) The regions of the genome that are significantly amplified across a set of lung squamous cell carcinoma samples from TCGA were identified using GISTIC2 from Gene Pattern (Broad Institute). ZDHHC19 was confirmed as one of the genes in the amplified 3q29 region. (c) Genes in amplified 3q29 region are correlated with STAT3 target genes BCL2L1 and BCL2 cross two Geo DataSets (GSE28571 (n = 100 samples) and GSE73403 (n = 69 samples)). 28 genes are analyzed. P value is calculated by two-tailed Pearson correlation. (d) Venny diagram of genes in amplified 3q29 region significantly correlated with STAT3 target genes BCL2L1 and BCL2 cross two Geo DataSets (GSE28571 (n = 100 samples) and GSE73403(n = 69 samples)). 36 genes in GSE28571 and 30 genes in GSE73403 are analyzed. P value is determined by two-tailed Pearson correlation. (e) The plots of p/q value of oncogenes showing co-occurrence or mutual exclusivity with ZDHHC19 in lung squamous cell carcinoma from TCGA (n = 501 samples). 231 genes (162 co-occurrence and 69 mutual exclusivity) are analyzed. p and q value are determined by Fisher’s exact test and FDR-test. (f) The plots of p/q value of tumor suppressors showing co-occurrence or mutual exclusivity with ZDHHC19 in lung squamous cell carcinoma from TCGA (n = 501 samples). 180 genes (115 co-occurrence and 65 mutual exclusivity) are analyzed. p and q value are determined by Fisher’s exact test and FDR-test.. (g) Gene list of oncogenes or tumor suppressors showing significant co-occurrence or mutual exclusivity with ZDHHC19 in lung squamous cell carcinoma (n = 501 samples). p and q value are determined by Fisher’s exact test and FDR-test. (h) Comparison of ZDHHC19 expression in lung squamous cell carcinoma (Lung Squ) and lung adenocarcinoma (Lung Adeno) patient samples obtained from cBioPortal TCGA dataset (n = 1097 samples). P values were determined by two-tailed student’s t-test. (i) The expression level of ZDHHC19 in human lung cancer cell lines was grouped in 4 subtypes: Lung squamous cell carcinoma cell lines (Lung squ) (n = 29 samples); Lung large cell carcinoma cell lines (Large lung) (n = 14 samples); lung adenocarcinoma cell lines (Lung adeno) (n = 53 samples); and, Lung small cell lung carcinoma cell lines (Small lung) (n = 53 samples). Data were obtained from Cancer Cell Line Encyclopedia database (CCLE, www.broadinstitute.org/ccle) and graphed by GraphPad Prism. P value is determined by two-tailed student’s t-test. (j) Kaplan-Meier curves of 68 lung squamous cell carcinoma patients after stratification by the median level of ZDHHC19 were used for depicting survival time. Patient data were derived from GSE 73403 and analyzed by GraphPad Prism. P values is determined by Log-rank (Mantel-Cox) test. (k) Kaplan-Meier curves of lung squamous cell cancer patients after stratification by the median level of ZDHHC19 were used for depicting Survival time. Patient data were analyzed by http://kmplot.com. P values is determined by Log-rank (Mantel-Cox) test. (l) Pearson analysis of gene expression data from lung cancer patients (GSE 73403 dataset (n = 69 samples)) was used for depicting the correlation between BCL2 and ZDHHC19. Data were graphed and analyzed by GraphPad Prism software. (m) Similar analysis as panel (l) of gene expression data from lung cancer patients (GSE 73403 dataset (n = 69 samples)) was used for depicting the correlation between BCL2L1 and ZDHHC19. (n) Pearson analysis of gene expression data from lung cancer patients (GSE 28571 dataset (n = 100 samples)) was used for depicting the correlation between BCL2 and ZDHHC19. (o)Similar analysis of gene expression data from lung cancer patients (GSE 28571 dataset (n = 100 samples)) was used for depicting the correlation between BCL2L1 and ZDHHC19. 100 samples were graphed and analyzed by GraphPad Prism software. In (l-o), center line indicates a line of best fit through the data of two variables in Pearson correlation coefficient model, and the dotted lines indicates 95% confidence band. Extended Data Figure 6. ZDHHC19 expression correlates with STAT3 nuclear localization in lung squamous cell carcinoma. (a) Immunohistochemistry staining showing correlation of ZDHHC19 expression with STAT3 nuclear localization. 131 biologically independent samples were analyzed. (b) Statistical analysis of Pearson correlation between immunohistochemistry staining scores of ZDHHC19 expression and STAT3 nuclear localization. (c) Summary of lung SCC patient tissue samples and the scores of ZDHHC19 expression and STAT3 nuclear localization. Extended Data Figure 7. ZDHHC19 mediated STAT3 palmitoylaiton facilitates LSCC tumor cell growth, colony formation and migration in vitro through induction of STAT3 activity. (a) HCC95 lung squamous cell carcinoma cells were pretreated with ruxolitinib (1 μM) for 30 minutes and then labeled with 50 μM probe Alk-C16 for 2 hours with the incubation of IL-6 (20 ng/ml). Endogenous STAT3 palmitoylation was analyzed by Click reaction and streptavidin beads pulldown, and followed by western blotting. (b) HCC95 cells were treated as in panel (a). Whole cell lysates were analyzed by anti-STAT3 IP followed by western blotting using the indicated antibodies. (c) Lung squamous cell carcinoma cell lines HCC95 and KNS-62 were transfected with shRNA control or shZDHHC19 (3 and 5) and labeled with 50 μM Alk-C16 probe for 4 hours, Cell lysates were reacted with biotin-azide and precipitated with streptavidin beads. Endogenous STAT3 palmitoylation was determined by western blot. (d) SK-MES-1 cells were transfected with ZDHHC19-shRNA and labeled with 50 μM palmitoylation probe Alk-C16 for 4hours. Cells lysates were reacted with azide-biotin and subjected to streptavidin beads pull-down. STAT3 palmitoylation was shown by western blot. (e) HCC95 shRNA control cells and Sh3 ZDHHC19 stable cells were pretreated with ruxolitinib (1 μM) for 30 minutes and then labeled with 50 μM probe Alk-C16 for 2 hours with the incubation of IL-6 (20 ng/ml). Endogenous STAT3 palmitoylation was determined by western blot. (f) Confocal immunofluorescence imaging showed STAT3 localization in HCC95 Sh3 ZDHHC19 stable cells and vector control cells. Cells were stained with anti-STAT3 antibody (green) and DAPI (blue). The levels of nuclear-localized STAT3 were quantified by measuring the STAT3 fluorescence intensity in the nucleus using the confocal software to define the selected ROI (Region of Interest) based on nuclear DAPI signal. The scale bar represents 20 μm. The data are presented as mean ± s.e.m., n = 275, 320 cells. P value is determined by two-tailed student’s t-test. (g) Cell proliferation was determined in HCC95 Sh3 ZDHHC19 stable cells or shRNA control cells. The data are presented as mean ± s.e.m., n = 3 biologically independent samples. P value is determined by two-way ANOVA followed by Bonferroni’s test. (h) Cell proliferation was determined in HCC95 cells with ZDHHC19 CRISPR knockcout or control. The data are presented as mean ± s.e.m., n = 6 biologically independent samples. P value is determined by two-way ANOVA followed by Bonferroni’s test. (i) Colony formation of HCC95 cells with ZDHHC19 CRISPR knockcout or control. The colony number was quantified. The data are presented as mean ± s.e.m., n = 5 biologically independent samples. P value is determined by two-tailed student’s t-test.. (j) Cell proliferation showing KNS62 cells with ZDHHC19 knockdown or control shRNA. The data are presented as mean ± s.e.m., n = 12 biologically independent samples. P value is determined by two-way ANOVA followed by Bonferroni’s test. (k) Cell proliferation was determined in KNS62 cells with ZDHHC19 CRISPR knockcout or control. The data are presented as mean ± s.e.m., n = 6 biologically independent samples. P value is determined by two-way ANOVA followed by Bonferroni’s test. (l) Colony formation of HCC95 cells with ZDHHC19 CRISPR knockcout or control. The colony number was quantified. The data are presented as mean ± s.e.m., n = 6 biologically independent samples. P value is determined by two-tailed student’s t-test. (m) Cell proliferation was determined in SK-MES-1 cells with ZDHHC19 CRISPR knockcout or control. The data are presented as mean ± s.e.m., n = 12 biologically independent samples. P value is determined by two-way ANOVA followed by Bonferroni’s test. (n) Colony formation of SK-MES-1 cells with ZDHHC19 CRISPR knockcout or control. The colony number was quantified. The data are presented as mean ± s.e.m., n = 6 biologically independent samples. P value is determined by two-tailed student’s t-test.. (o) The migration ability of HCC95 Sh3 ZDHHC19 stable cells or shRNA control cells was measured using the transwell migration assay. Numbers of invading cells were quantified. The data are presented as mean ± s.e.m., n = 5 biologically independent samples. P value is determined by two-tailed student’s t-test.(p) Crystal staining showing cell growth of KNS62 ZDHHC19 knockdown or control stable cells transfected with STAT3C or vehicle control. In (a-e, p), the experiments were independently repeated at least 3 times with similar results. For gel source data, see Supplementary Figure 1. Extended Data Figure 8. STAT3 palmitoylaiton through ZDHHC19 involves in maintaining the cancer stem cell niche. (a) Photomicrographs of HCC95, KNS62, and HCC827 parental adherent cells (left) and tumorspheres (right) in low-adherence culture. The experiment was independently repeated at least 3 times with similar results. (b) qRT-PCR analysis of the expression level of stem cell markers in tumorsphere of HCC95 cells compared to parental HCC95 cells. Fold change was normalized to 18S rRNA. The data are presented as mean ± s.e.m., n = 3 biologically independent samples. P value is determined by two-tailed student’s t-test. (c-d) Similar experiments were performed in KNS62 and HCC827 cells. The data are presented as mean ± s.e.m., n = 3 biologically independent samples. P value is determined by two-tailed student’s t-test. (e) HCC95 empty vector control cells and STAT3 or ZDHHC19 knock out stable cells were cultured in low-attachment plates with 25 μM palmitic acid for 7 days. Phase contrast photomicrographs showing tumorsphere formation. Numbers of spheres were counted from 5 randomly selected fields. The data are presented as mean ± s.e.m., n = 5 biologically independent samples. P value is determined by two-tailed student’s t-test. (f) KNS62 empty vector control cells and ZDHHC19 shRNA knockdown stable cells were cultured in low-attachment plates with 25 μM palmitic acid for 5 days. Fluorescent (calcein AM staining) and phase contrast photomicrographs showing tumorsphere formation. The data are presented as mean ± s.e.m., n = 12 biologically independent samples. P value is determined by two-tailed student’s t-test. (g) SK-MES-1 empty vector control cells and ZDHHC19 shRNA knockdown stable cells were cultured in low-attachment plates with 25 μM palmitic acid for 5 days. Fluorescent (calcein AM staining) and phase contrast photomicrographs showing tumorsphere formation. The data are presented as mean ± s.e.m., n = 12 biologically independent samples. P value is determined by two-tailed student’ t-test. (h) KNS62 empty vector control cells and ZDHHC19 shRNA knockdown stable cells were cultured in low-attachment plates with 25 μM palmitic acid for 5 days. The cells were trypsinized and seeded in plate again. Fluorescent (calcein AM staining) photomicrographs showing secondary tumorsphere formation after 5 days. The data are presented as mean ± s.e.m., n = 4 biologically independent samples. P value is determined by two-tailed student’s t-test. (i) KNS62 empty vector control cells and ZDHHC19 shRNA knockdown stable cells transfected with vehicle control or constitutive active STAT3C were cultured in low-attachment plates for 5 days. Fluorescent (calcein AM staining) photomicrographs showing tumorsphere formation. Tumorsphere numbers were quantified. The data are presented as mean ± s.e.m., n = 4 biologically independent samples. P value is determined by two-tailed student’s t-test. Extended Data Figure 9. High-fat diet induced tumor growth of lung squamouse cell carcinoma in vivo is dependent on ZDHHC19 mediated STAT3 palmitoylaiton. (a) Representative image of the xenograft tumors isolated from HCC95 empty vector control and ZDHHC19 knock out xenografts as indicated. (b) Weight of the tumors was measured and plotted. The data are presented as mean ± s.e.m., n = 10 biologically independent samples. P value is determined by two-tailed student’s t-test. (c) Body weight change of mouse fed with normal or high fat diet. Data were normalized to original weight. The data are presented as mean ± s.e.m., n = 5 mice. (d) STAT3 palmitoylation were analyzed by APE and western blotting in human-derived cell tumor tissues. The palmitoylated STAT3 were quantified using Image J. The data are presented as mean ± s.e.m., n = 3 biologically independent samples. P value is determined by two-tailed student’s t-test. (e) Representative images of immunohistochemical (IHC) staining of Ki-67 in paraffin-embedded xenograft tumor tissues harvested from the indicated groups. The scale bar represents 200μm. Numbers of Ki-67 positive cells were counted from 5 randomly selected fields. The data are presented as mean ± s.e.m., n = 10 biologically independent samples. P value is determined by two-tailed student’s t-test. (f) Representative images of immunohistochemical (IHC) staining of STAT3. (g) qRT-PCR analysis of the expression level of STAT3 target genes (BCL2, BCL2L1 and MMP9) was performed in tumor tissues harvested at the end of the experiment. Fold change was normalized to 18S rRNA. The data are presented as mean ± s.e.m., n = 10 biologically independent samples. P value is determined by two-tailed student’s t-test. (h) HCC95 shRNA control and Sh3 ZDHHC19-stable cell lines were injected into mouse. Representative image of the xenograft tumors isolated from the indicated groups. (i) Tumor growth was monitored and readout by the tumor volume. The data are presented as mean ± s.e.m., Control n = 10 biologically independent samples, shZDHHC19 n = 9 biologically independent samples. P value is determined by two-way ANOVA followed by Bonferroni’s test. (j) Weight of the tumors was measured, and the data are presented as mean ± s.e.m., Control n = 10 biologically independent samples, shZDHHC19 n = 9 biologically independent samples. P value is determined by two-tailed student’s t-test. (k) qRT-PCR analysis of the expression level of STAT3 target genes in HCC95 shRNA control or Sh3 ZDHHC19 stable xenografts. The data are presented as mean ± s.e.m., Control n = 10 biologically independent samples, shZDHHC19 n = 9 biologically independent samples. P value is determined by two-tailed student’s t-test. (l) Representative images of immunohistochemical (IHC) staining of STAT3 and Ki-67 in paraffin-embedded xenograft tumor tissues harvested from the indicated groups. Ki-67 positive cells were quantified as in panel (e). The data are presented as mean ± s.e.m., n = 5 biologically independent samples. P value is determined by two-tailed student’s t-test. Extended Data Figure 10. High fat diet facilitates tumor growth in lung squamous cell carcinoma PDX model. (a) qRT-PCR analysis of the expression level of ZDHHC19 was performed in a series of patient-derived xenograft (PDX) tumor tissues (PDX1-PDX7). Fold change was normalized to 18S rRNA. The data are presented as mean ± s.e.m., n = 3 technical replicates. (b) Representative image of the xenograft tumors isolated from PDX mouse with normal fat diet (NFD) or high fat diet (HFD). (c) Weight of the tumors was measured and the data are presented as mean ± s.e.m., NFD n = 8 biologically independent samples, HFD n = 10 independenlly samples. P value is determined by two-tailed student’s t-test. (d) STAT3 palmitoylation was analyzed by APE and western blotting in PDX tumor tissues. The palmitoylated STAT3 was quantified using Image J. The data are presented as mean ± s.e.m., n = 5 biologically independent samples. P value is determined by two-tailed student’s t-test. (e) qRT-PCR analysis of the expression level of STAT3 target genes (BCL2, BCL2L1 and MMP9) was performed in tumor tissues harvested at the end of the experiment. Fold change was normalized to 18S rRNA. The data are presented as mean ± s.e.m., NFD n = 8 biologically independent samples. HFD n = 10 biologically independent samples. P value is determined by two-tailed student’s t-test. (f) Representative images of immunohistochemical (IHC) staining of STAT3 and ZDHHC19 in paraffin-embedded PDX tumor tissues harvested from the indicated groups. (g) Transcriptional level of ZDHHC19 was shown the knockdown efficiency in primary PDX cells by qRT-PCR. The data are presented as mean ± s.e.m., n = 3 biologically independent samples. P value is determined by two-tailed student’s t-test. (h) Representative images of tumor sphere formation using primary tumor cells isolated from lung squamous cell carcinoma PDX tumor tissues. (i) Graphic scheme showing fatty acids and amplified ZDHHC19 promote STAT3 activation through S-Palmitoylation. In (f, h), the experiments were independently repeated at least 3 times with similar results. Supplementary Material 1 2 3 4 Acknowledgments This work was supported by Samuel M. Fisher Memorial–MRA (Melanoma Research Alliance) Established Investigator Award, the Idea Award from Prostate Cancer Research Program, U.S Department of Defense (W81XWH-17-1-0361), and grants from National Institutes of Health (R01CA181537, R01DK107651–01 and R01CA238270–01) to X.W and (R01CA160979) to D.A.F. We thank Dr. Tom Maniatis (Mount Sinai School of Medicine, New York, NY) for the expression vector of STAT2, Dr. Masaki Fukata (National Institute for Physiological Sciences, Japan) for the expression vectors of ZDHHC proteins, the Confocal Imaging Core at Cutaneous Biology Research Center of Massachusetts General Hospital with the Shared Instrumentation Grant (1S10RR027673–01), and the Taplin Mass Spec Core at Harvard Medical School for proteomic studies. Competing interests The authors declare no competing interests. Figure 1. STAT3 is S-palmitoylated at evolutionarily conserved cysteine residues. (a) Myc-STAT3 is palmitoylated in HEK293 and MDA-MB-231 cells. Streptavidin blots showed detection of STAT3 S-palmitoylation band (Palm-STAT3) after metabolic labeling with 50 μM chemical reporter of palmitoylation (Alk-C16) with or without hydroxylamine treatment. (b) Analysis of STAT3 fatty acylation using different chemical reporters of fatty acylation (Alk-C14 to Alk-C20). STAT3 fatty acylation levels (Acylated-STAT3) were analyzed by streptavidin bead pull--down, and followed by western blotting. (c) Analysis of STAT3 palmitoylation in cytoplasm and nucleus by APE assay, following fractionation. HDAC1 and α-Tublin are nuclear and cytoplasmic fraction controls, respectively. STAT3-PEG bands indicated palmitoylated STAT3. (d) Quantification of STAT3 palmitoylation percentage in cytoplasm and nucleus in APE assays using Image J. All The data are presented as mean ± s.e.m., n = 3 biologically independent samples. P value is determined by two-tailed student’s t-test. (e) Determination of the half-life of STAT3 palmitoylation turnover from pulse-chase results. Plots were fitted with one-phase exponential decay curve in GraphPad. The data are presented as mean ± s.e.m., n = 4 biologically independent samples. (f) Palmitoylation levels of Flag-STAT3 wild type (WT), C687S, C712S and C687/712S (2CS) mutant analyzed by metabolic labeling with Alk-C16, Click reaction and streptavidin bead pull-down, and followed by western blotting. Palm-STAT3 band indicated palmitoylated STAT3. In a, b, f, the experiments were independently repeated at least 3 times with similar results. For gel source data, see Supplementary Figure 1. Figure2. A signaling relay involving STAT3 phosphorylation and palmitoylation promotes STAT3 dimerization in response to cytokine and fatty acids. (a) Flag-STAT3 palmitoylation levels were analyzed by APE assay and western blotting upon IL-6 stimulation with or without hydroxylamine treatment. STAT3-PEG bands indicated palmitoylated STAT3. (b) Quantification of STAT3 palmitoylation percentage from APE assays in (a), n = 3 biologically independent samples. (c) Palmitoylation and Y705 phosphorylation of endogenous STAT3 in HEK293 cells, treated with IL-6 and/or JAK inhibitor ruxolitinib. Palmitoylation of STAT3 (Palm-STAT3) is detected by chemical reporter (Alk-C16) labeling, Click reaction, followed by Streptavidin pull-down and western blotting. (d) HEK293A cells were transfected with Flag-tagged wild type (WT) or Y705F mutant. The Palmitoylation levels (Palm-STAT3) of STAT3 WT or Y705F mutant were analyzed same as in (c). (e) Co-immunoprecipitation (Co-IP) assay to detect homodimerization of STAT3 WT or palmitoylation-deficient C687/712S (2CS) mutant in HEK293A cells treated with IL-6. Whole cell lysates were analyzed by anti-Flag immunoprecipitation followed by immunoblotting using the indicated antibodies (f) Percentage of STAT3 palmitoylation in mouse lung and liver tissues fed with normal-fat diet (NFD) or high-fat diet (HFD) were analyzed by APE assay, n = 5 animals. . (g) HEK293A cells were transfected with Flag-STAT3 and treated with BSA-conjugated palmitic acid (PA) at the indicated doses. STAT3 palmitoylation levels (indicated by STAT3-PEG bands) were analyzed by the APE assay. (h) Quantification of STAT3 palmitoylation percentage in (g). n = 3 biologically independent samples. . (i) Detection of endogenous STAT3 dimerization using disuccinimidyl glutarate (DSG) crosslinking assay in HEK293A cells, treated with IFN-γ, IL-6 or BSA-conjugated palmitic acid (PA, 100μM). (j) Co-IP assay to detect homodimerization of STAT3 WT or palmitoylation-deficient C687S mutant in HEK293A cells, treated with BSA-conjugated palmitic acid (PA, 100μM). Whole cell lysates were analyzed by anti-Flag IP followed by immunoblotting using the indicated antibodies. In c-e, i, j, the experiments were independently repeated at least 3 times with similar results. For gel source data, see Supplementary Figure 1. All the data in bar graph (b, f, h) are presented as mean ± s.e.m. P value is determined by two-tailed student’s t test. Figure 3. ZDHHC19 mediates STAT3 palmitoylation. (a) Palmitoylation of Flag-STAT3 was analyzed by APE assay and western blotting, with co-transfection of vector control, ZDHHC19 wild type (WT), or the catalytically inactive C142S mutant. STAT3-PEG bands indicated palmitoylated STAT3. (b) Quantification of STAT3 palmitoylation percentage in (a). The data are presented as mean ± s.e.m., n = 3 biologically independent samples. P value is determined by two-tailed student’s t-test. (c) Palmitoylation of Flag-STAT3 in cells co-transfected with vector control, ZDHHC19 WT or C142S mutant, then treated with or without IL-6. Palm-STAT3 bands indicated palmitoylated STAT3, detected by Alk-C16 labeling, Click reaction, followed streptavidin pulldown and western blotting. (d) Palmitoylation of endogenous STAT3 in HEK293 cells stably transfected with shRNA targeting human ZDHHC19, then re-expressed mouse HA-ZDHHC19 wild type (WT) or C142S mutant. STAT3 palmitoylation (Palm-STAT3) was analyzed the same as in (c). (e) Complex formation of GFP-Grb2, Myc-ZDHHC19 and Flag-STAT3 in HEK293 cells upon IL-6 stimulation and/or JAK inhibitor ruxolitinib treatment. Whole cell lysates were subjected to Co-IP using anti-Flag antibody, following western blotting using indicated antibodies. (f) Palmitoylation of endogenous STAT3 in HEK293A cells transfected with shRNA control or shRNA targeting Grb2 (sh2 or sh4), and cells were treated with or without IL-6. STAT3 palmitoylation (Palm-STAT3) was analyzed the same as in (c). In c-f, the experiments were independently repeated at least 3 times with similar results. For gel source data, see Supplementary Figure 1. Figure 4. ZDHHC19 is amplified in lung squamous cell carcinoma (LSCC) and promotes tumorigenesis in vitro and in vivo. (a) Representative images of IHC staining of STAT3 and ZDHHC19 in paraffin-embedded LSCC samples, n = 131 biologically independent samples from 85 patients (detailed information in Supplementary Table 1). Analysis was done once for each sample. (b) Representative images of tumorsphere formation of KNS62 cells stably infected with shRNA control or shRNA targeting ZDHHC19 (shZDHHC19) under limiting dilution conditions. Cells were treated with vehicle control or palmitic acid (PA, 100μM). The numbers of wells with tumorsphere/total wells were labeled in the panels in indicated conditions. (c) Quantification of KNS62 stem cell frequency in (b). The center line is the log-active cell fraction and the dotted lines give the 95% confidence interval. P values are determined by two-tailed student’s t-test. (d) Xenograft tumor growth of HCC95 wild type (WT) control or with CRISPR/Cas9 knockout of ZDHHC19. Animals were fed with normal-fat diet (NFD) or high-fat diet (HFD) for 4 weeks, n = 10 biologically independent tumors per group. (e) Quantification of the percentage of nuclear-localized STAT3 in tumor samples in (d) by IHC staining. n = 10 biologically independent tumor samples per group. (f) Tumor growth curve of LSCC patient-derived xenograft (PDX) model under NFD or HFD. n = 8 (in NFD) or 10 (in HFD) biologically independent tumors. (g) Quantification of tumorsphere numbers from PDX tumor cells infected with shRNA control (pLKO) or shRNA-targeting ZDHHC19 (shZDHHC19), and then treated with vehicle control or palmitic acid (PA, 100 μM), n = 6 biologically independent samples. 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==== Front Cell Cycle Cell Cycle KCCY kccy20 Cell Cycle 1538-4101 1551-4005 Taylor & Francis 31345079 1647024 10.1080/15384101.2019.1647024 Research Paper Downregulated lncRNA CRNDE contributes to the enhancement of nerve repair after traumatic brain injury in rats M. YI ET AL. CELL CYCLE Yi Min Dai Xingping Li Qiuxia Xu Xia Chen Yanyi http://orcid.org/0000-0003-0229-4471 Wang Dongsheng Integrated Traditional Chinese and Western Medicine, Xiangya Hospital, Central South University, Changsha, PR China CONTACT Dr. Dongsheng Wang [email protected] 2019 29 7 2019 18 18 23322343 3 6 2019 10 7 2019 14 7 2019 © 2019 Informa UK Limited, trading as Taylor & Francis Group 2019 Informa UK Limited, trading as Taylor & Francis Group ABSTRACT Objective: Long non-coding RNAs (lncRNAs) have recently been demonstrated to be involved in craniocerebral disease, but their expression in traumatic brain injury (TBI) is still unearthed. Therefore, we aimed to elucidate the effect of lncRNA CRNDE on TBI. Methods: Firstly, CRNDE expression was determined in serum of TBI patients and healthy controls. The TBI rat model was established based on Feeney’s freefall impact method. The modeled rats were injected with siRNA against CRNDE, and the rats’ neurobehavioral function were measured. Besides, expression of inflammatory factors, size, shape and number of hippocampal neurons, neuron apoptosis, Beclin I, LC3-I, LC3-II, glial fibrillary acidic protein (GFAP), BrdU, nerve growth factor (NGF), nestin, and neuronal nuclei (NeuN) expression were detected through different methods. Results: In TBI, CRNDE was found to be upregulated. Downregulated CRNDE improved neurobehavioral function, repressed expression of neuroinflammatory factors, elevated number of Nissl bodies, as well as restricted neuronal apoptosis and autophagy in TBI rats. Besides, downregulated CRNDE also promoted expression of GFAP, BrdU, NGF, nestin, and NeuN, thus induced the differentiation of neurons and the directional growth and regeneration of nerve fibers. Conclusion: Altogether, we found that silencing of CRNDE might be able to promote the nerve repair after TBI in rats. KEYWORDS Long non-coding RNA CRNDE Traumatic brain injury nerve repair This study was supported by Provincial science and technology innovation platform and talent plan(420010087). ==== Body Introduction Traumatic brain injury (TBI) is a main reason of death and morbidity, especially at both ends of the age range, which brings enormous costs to society [1]. Statistics have shown increased incidence, mortality, and disability of TBI in recent years [2]. As reported, primary mechanical injury and secondary injury are two well-known mechanisms of TBI [3]. As one highly complex disease, patients suffering TBI present different degrees of contusion, hemorrhage, and hypoxia [4]. To be specific, TBI causes secondary biochemical changes which contributes to neurological dysfunction, delayed neuroinflammation, as well as nerve cell death [5]. Nowadays, although many efforts have been made, there is no treatment to alleviate or prevent nerve dysfunction after TBI [6]. Researchers are now turning to biomarkers, objective molecular characteristics of injury, as a platform for finding more sensitive and specific approaches for TBI treatment and diagnosis. Long non-coding RNAs (lncRNAs) are vital in multiple kinds of pathophysiological processes of brain diseases [7]. Moreover, lncRNAs are proved to be closely related to nervous system development and neurodegenerative diseases [8,9]. Colorectal neoplasia differentially expressed (CRNDE) transcripts are described as one lncRNA and positioned on chromosome 16, exerting oncogenic functions in diverse cancers [10]. In addition, CRNDE is known to be highly expressed in multiple kinds of cancerous diseases, particularly in cancers of the brain [11]. Furthermore, up-regulation of CRNDE was demonstrated to promote glioma cell viability, migration, and invasion [12]. Song H et al. also proposed that highly expressed CRNDE was found in medulloblastoma, knowing as the most frequent malignant brain tumor in children [13]. Thus, we speculate that CRNDE may exert affect in TBI. Herein, we initially detected CRNDE expression in clinical serum sample from patients with TBI and rat serum and hippocampal tissues. Furthermore, the effects of CRNDE on nerve repair were fully investigated using siRNA-mediated silencing. Materials and methods Ethics statement The study was permitted by the Ethics Committee of Xiangya Hospital, Central South University and an informed consent form were signed with the patient’s family. The experimental animal program has been permitted by the Laboratory Animal Ethics Committee. It conformed to the principles of animal protection, animal welfare, and ethics, as well as the relevant regulations of the national ethics of laboratory animal welfare. Study subjects A total of 43 patients with TBI (male, n = 25; female, n = 18; mean age of 63.7 ± 4.9 years; American Society of Anesthesiologists grade II or Ⅲ) diagnosed in Xiangya Hospital, Central South University from June 2017 to June 2018 were collected as the case group. All patients had preoperative Glasgow Coma Scale (GCS) score of 6–10 points. Patients were included if they had no obvious history of heart, lung, liver, kidney disease before operation; they had no long-term history of psychotropic drugs and alcoholism; they undergo craniotomy under general anesthesia with drug sedation required during the perioperative period. Patients were excluded if they had dysfunction of blood coagulation; they had GCS score less than four points; they need deep sedation due to pregnancy, coma, and elevated intracranial pressure; they died within 72 h after operation. Besides, 32 healthy physical examiners (male, n = 15; female, n = 17; mean age of 54.7 ± 7.8 years) were randomly selected as the control group. There was no statistical significance in age, gender, and state of an illness between the two groups. TBI model establishment A sum of 55 healthy Sprague-Dawley rats (aged about 6–8 weeks and weighed 180–200 g) obtained from Henan Experimental Animal Center were selected for our study. The feeding conditions shall be carried out according to the standard of the animal at clean level. All rats were assigned into a sham group (n = 10) and a TBI group (n = 45) randomly. The rat model of TBI was established according to Feeney’s freefall impact method [14,15]. Rats were fixed on brain stereotactic instrument with prone position, and the bumper was placed on the exposed right dural. Rats were anesthetized with 1% pentobarbital sodium (40 mg/kg) via an intraperitoneal injection. And then, 40 g weight was dropped from 25 cm height along the peripheral catheter, and the bone window was sealed with bone wax after impact. Rats in the sham group only received cranial window opening which was then sealed with bone wax. After modeling, the rats were placed in the cage according to the label and fed with adequate food and water at room temperature. Animal treatment A total of 40 rats were randomly selected from 42 rats with successful modeling and assigned into the following groups: TBI group (TBI rats without any other treatment), si-negative control (NC) group (TBI rats injected with si-NC plasmid of CRNDE), si-CRNDE-1 group (TBI rats injected with CRNDE interference plasmid 1) and si-CRNDE-2 group (TBI rats injected with CRNDE interference plasmid 2). The transfection plasmids were provided by GenePharma Co. Ltd., Shanghai, China (Shanghai, China). In the sham group, rats received no treatment. Measurement of spontaneous activity The spontaneous activity of rats was analyzed by Smart-128 small animal behavior analysis system (Panlab, Barcelona, Spain). After 30 min of the last administration for rats in each group, a single rat was placed in an observation box (42 cm × 42 cm × 30 cm) with a camera placed in at the top. The activities of rats were tracked by video, and the tracks of rats were recorded spontaneously, and the distance of activities was calculated. The total activity distance of rats within a short period of time was used to evaluate the index of spontaneous activity. Measurement of inhibitory avoidance (IA) On the 5th day of administration, the rats were placed in the bright room of the IA training instrument with their back facing the isolation door. When the rats were facing the lift door, the door was raised with the darkroom exposed. When all 4 feet of the rat entered the dark room, the lift door will be closed, the rat foot will be given a single click (0.4 m, 2 s), and then the rat will be taken out. Twenty-four hours later, the IA memory of the rats was measured, and the time (latent period) of staying in the bright room before entering the darkroom was recorded. Morris water maze test Morris water maze test [16] was used for assessing the learning and memory capacity and evaluating the degree of memory impairment in rats in the second week of cultivation. The test was conducted in rats of each group at the 2nd and 4th week after operation, respectively. Each test began by placing the rats in the water close to and facing the wall of the pool in one of the four entry points. The times across the original platform within 90 s was recorded. After behavioral examination, 2 mL orbital blood was taken and then the rats were euthanized with their brain tissues collected. Afterwards, the tissues were fixed by 4% paraformaldehyde, dewaxed by routine gradient alcohol and embedded by paraffin. The coronal sections were continuously cut at the optic intersection, and the sections were 4 μm thick. Enzyme-linked immunosorbent assay (ELISA) The rats were euthanized to collect serum with brain tissue separated and preserved. According to the instructions of ELISA kits, the serum levels of tumor necrosis factor (TNF)-α (SBJ-R0040), interleukin (IL)-1β (SBJ-R0024), IL-6 (SBJ-R0755) and IL-10 (SBJ-R0786) (SenBeiJia Biological Technology Co., Ltd. Nanjing, China) were detected. The optical density (OD) values measured at 450 nm by a microplate reader (BS-1101, Detie Experimental equipment Co., Ltd., Nanjing, China). Nissl’s staining The paraffin-embedded sections were treated with xylene, absolute ethyl alcohol, alcohol (95%, 80%, and 70%), and rinsed by distilled water. Afterwards, the sections were stained with 1% tar violet or 1% thionine for 10 to 60 min, dehydrated with alcohol, absolute ethyl alcohol, and cleared with xylene. After that, the sections were sealed with 1, 3- diethyl-8-phenylxanthine (DPX) and observed under a microscope. The Nissl body was purple, and the nucleus was lilac. Terminal deoxyribonucleotidyl transferse (TDT)-mediated biotin-16-dutp nick-end labeling (TUNEL) assay After 24-h transfection, the cells were fixed with 2%formaldehyde and permeated with 0.1% Triton X-100 (Solarbio Science & Technology Co., Ltd., Beijing, China). According to the instructions, the cells were incubated with 50 μl TUNEL reaction mixture (Roche, Basel, Switzerland) in the dark. The results were observed under an inverted fluorescence microscope (Leica, Microsystems AG, Wetzlar, Germany). Apoptosis rate = the apoptotic positive cells number/total cell count × 100%. Immunofluorescence assay The tissue sections were fixed by 4% paraformaldehyde, treated with 0.2% TritonX-100, sealed by 3% BSA at 4°C, and next, incubated with fluorescence primary antibody GFAP (ab33922, 1:500, Abcam, Shanghai, China) and BrdU (ab6326, 1:200, Abcam, Shanghai, China). After that, the sections were incubated with fluorescence secondary antibody (1:500) in the dark for 2 h, and reacted with DAPI (ab104139, 1:100, Abcam, Shanghai, China) in darkness for 10 min. The sections were sealed and observed under an inverted fluorescence microscope. Transmission electron microscope The fresh hippocampal tissue was fixed in 2.5% glutaraldehyde solution, fixed with 1% osmic acid (Rongbai biological technology Co., Ltd., Shanghai, China), and washed with 0.1 M buffer solution for 5 min. After that, the tissues were dehydrated by gradient ethanol, soaked by 90% ethanol acetone, 90% acetone and 100% acetone (each for 5 min), by a mixture of acetone and epoxy resin (1:1) for 2 h, embedded, polymerized for 10 h and made into ultrathin sections (70 nm). Subsequently, the sections were stained by uranyl acetate and acetate leaching, respectively, for 10 min with the ultrastructure of hippocampal CA1 region observed by a transmission electron microscopy (HTT700, Hitachi, Tokyo, Japan). Reverse transcription quantitative polymerase chain reaction (RT-qPCR) The extraction of total RNA was carried out using the Trizol kit (16,096,020, Thermo Fisher Scientific Inc., Waltham, Massachusetts, USA), and 5 µg of total protein was reversely transcribed into complementary DNA (cDNA) based on the RT-qPCR kit (ABI Company, Oyster Bay, NY). The primers (Invitrogen, Shanghai, China) used are listed in Table 1. Glyceraldehyde phosphate dehydrogenase (GAPDH) was used as the internal parameter with the 2−ΔΔCt [17] used for data analysis.10.1080/15384101.2019.1647024-T0001 Table 1. Primer sequences. Primer sequences (5ʹ-3ʹ) CRNDE Forward: 5ʹ-CGCGCCCGCGCGGCGGAGGA-3ʹ Reverse: 5ʹ-AGTATGAATTGCAGACTTTGCA-3’ GAPDH Forward: 5ʹ-TCCCATCACCATCTTCCA-3’ Reverse: 5ʹ-CATCACGCCACAGTTTTCC-3’ Western blot analysis Brain tissues were extracted, lysed and centrifuged at 12,000 r/min to remove tissue fragment or cell debris. After that, a bicinchoninic acid kit (Beyotime Biotechnology, Shanghai, China) was utilized for protein concentration determination. Next, 50 µg protein was collected and separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred onto a polyvinylidene fluoride membrane. Afterwards, the membrane was sealed with 5% skimmed milk and incubated with the diluted primary antibodies: Beclin I (ab62557, 1:500), LC3-I (ab48394, 1:1000), LC3-II (ab51520, 1:3000), NGF (ab6199, 1:1000), Nestin (ab6142, 1:1000), NeuN (ab236870, 1:1000) (all from Abcam, Shanghai, China), and GAPDH (1:500, ab8245, Abcam, Cambridge, UK). On the following day, the membrane was further incubated with horseradish peroxidase (HRP)-conjugated secondary antibody. After that, the protein bands were viewed with an enhanced chemiluminescence reagent (BB-3501, Amersham Company, Piscataway, NJ, USA), which were imaged on Bio-Rad image analysis system (Bio-Rad Laboratories, Hercules, CA, USA). Image analysis was followed using Quantity One v4.6.2 software. The target proteins were quantified as relative gray values against the internal reference GAPDH. Statistical analysis All experimental data were processed with SPSS 21.0 statistical software (IBM Corp., Armonk, NY, USA). All data are consistent with normality distribution and variance homogeneous test. Measurement data are reported as mean ± standard deviation. Differences between two groups were compared using independent t-test, while among multiple groups, using one-way analysis of variance, followed by Tukey’s post hoc test. A P value <0.05 was described as statistically significant. Results CRNDE is highly expressed in patients with TBI In order to elucidate the relationship between CRNDE and TBI, the expression of CRNDE in serum of the patients with TBI was determined. The results revealed (Figure 1A) that expression of CRNDE elevated in the serum of patients with TBI. Besides, the CRNDE expression in serum and hippocampal tissues of TBI rats was also detected, and the results also revealed highly expressed CRNDE (P < 0.05) (Figure 1B and C). All the above results suggested that CRNDE was highly expressed in TBI.10.1080/15384101.2019.1647024-F0001 Figure 1. CRNDE was highly expressed in TBI. Notes: A, The expression of CRNDE in serum of TBI patients was detected by RT-qPCR; B, The expression of CRNDE in serum of TBI rats was detected by RT-qPCR; C, The expression of CRNDE in hippocampal tissues of TBI rats was detected by RT-qPCR; All data are measurement data and presented as mean ± standard deviation. Differences between two groups were compared using independent sample t-test; * compared with the control or sham groups, P < 0.05; In panel A, control group, n = 32; case group, n = 43; In panel B and C, sham group, n = 10; TBI group, n = 10. Silencing of CRNDE improves neurobehavioral function in TBI rats Subsequently, we evaluated the interference efficiency of CRNDE after interfering through detecting the CRNDE expression in cells by RT-qPCR. Following that, two of them which have good interference efficiency were selected for subsequent experiments (Figure 2A). Besides, behavioral changes of rats were detected, and the results showed that the total distance of spontaneous activity prolonged (Figure 2B), the 24-h memory latency (Figure 2C) and the number of crossing the platform (Figure 2D) decreased in rats with TBI. In contrast to the si-NC group, the total distance of spontaneous activity shortened, and the 24-h memory latency and the number of crossing the platform increased in the si-CRNDE-1 and the si-CRNDE-2 groups (all P < 0.05).10.1080/15384101.2019.1647024-F0002 Figure 2. Silencing of CRNDE improves neurobehavioral function in TBI rats. Note: A, Interference efficiency detection results; B, The total distance of spontaneous activity was observed in each group of rats; C, The 24-h memory latency was detected in each group of rats; D, Morris water maze test was used to count the number of crossing the platform in each group of rats; All data are measurement data and presented as mean ± standard deviation. * compared with the sham group, P < 0.05; # compared with the si-NC group, P< 0.05; n = 10. Silencing of CRNDE inhibits the expression of neuroinflammatory factors in TBI rats After TBI, the blood-brain barrier was destroyed and the pro-inflammatory factors in the vascular system were expressed with the inflammatory cells aggregated, which released a large number of inflammatory factors, thus aggravating the secondary TBI. Therefore, we would like to explore whether CRNDE could inhibit pro-inflammatory factors with the help of ELISA. The results suggested that expression of pro-inflammatory factor TNF-α, IL-1β, and IL-6 in serum of TBI rats increased, while the expression of IL-10 decreased in rats with TBI. TNF-α, IL-1β, and IL-6 expression declined in serum of rats in the si-CRNDE-1 and the si-CRNDE-2 groups, while the IL-10 expression increased relative to the si-NC group (P< 0.05) (Table 2). These results implied the neuroprotective effect of silencing of CRNDE on rats with TBI may be related to the inhibition of neuroinflammation.10.1080/15384101.2019.1647024-T0002 Table 2. Serum levels of TNF-α, IL-1β, IL-6, and IL-10 in each group were detected by ELISA. Sham TBI si-NC si-CRNDE-1 si-CRNDE-2 TNF-α 154.12 ± 20.68 245.23 ± 28.41* 247.01 ± 27.59 181.32 ± 15.97# 189.65 ± 14.46# IL-1β 34.32 ± 10.23 85.15 ± 21.30* 88.07 ± 17.09 62.81 ± 10.12# 60.12 ± 9.89# IL-6 331.19 ± 45.54 765.44 ± 56.18* 770.54 ± 61.09 542.09 ± 56.64# 550.54 ± 61.09# IL-10 51.19 ± 4.09 25.44 ± 2.18* 26.04 ± 2.09 39.09 ± 2.64# 40.54 ± 2.09# Note: All data are measurement data and presented as mean ± standard deviation. * compared with the sham group, P < 0.05; # compared with the si-NC group, P < 0.05. Silencing of CRNDE increases the number of Nissl bodies and inhibits neuronal apoptosis in rats with TBI According to Nissl’s staining, the morphological changes of nerve cells in the injured area were observed under a common optical microscope, and the quantitative analysis of the number of neurons under a high magnification microscope was carried out with computer software. The results revealed that in the sham group, the cells in the cortex were arranged orderly and the shape was regular; the Nissl body was blue and clearly visible with the cell numbers of (79.78 ± 5.63). In the TBI and si-NC groups, the cells in the injured area were disordered and sparse with decreased Nissl body, the cell structure was not clear, and the number of nerve cells was less than that in the sham group. In the si-CRNDE-1 group and the si-CRNDE-2 group, the cell arrangement was disordered, the cell volume was slightly smaller, the shape was irregular, the Nissl body was decreased, but the number of cells was more than that in the si-NC group (all P< 0.05) (Figure 3A and B). At the same time, TUNEL assay was used to detect neuronal apoptosis in each group. The results showed that the neuronal apoptosis was higher in rats with TBI; the neuronal apoptosis weakened in the si-CRNDE-1 and the si-CRNDE-2 groups versus the si-NC group (P < 0 05) (Figure 3C and D). These results revealed that silencing of CRNDE may increase the number of Nissl bodies and inhibit neuronal apoptosis in rats.10.1080/15384101.2019.1647024-F0003 Figure 3. Silencing of CRNDE increases the number of Nissl bodies and inhibits neuronal apoptosis in rats with TBI. Note: A, Observation of the size and morphology of hippocampal neurons in rats by Nissl staining (× 200); B, Statistical analysis of the number of neurons observed by Nissl staining in panel A; C, Detection of neuronal apoptosis by TUNEL assay (× 400); D, Statistical analysis of the number of neuronal apoptosis in panel C; All data are measurement data and presented as mean ± standard deviation. * compared with the sham group, P < 0.05; # compared with the si-NC group, P< 0.05; n = 10. Silencing of CRNDE increases GFAP and BrdU protein expression in brain tissues in TBI rats Evidence has shown that GFAP, as the intermediate filament protein of astrocyte cytoskeleton, is a new marker of astrocytes which are involved in the repair and regeneration of nerve injury [18]. The increased expression of BrdU positive cells contributes to the induction of the differentiation process of endogenous neural stem cells and promote neural repair after TBI in rats [19,20]. So, immunofluorescence assay was applied for GFAP and BrdU protein expression evaluation in brain tissues. The results revealed that GFAP and BrdU protein expression elevated in rats with TBI; GFAP and BrdU protein expression enhanced in the si-CRNDE-1 and the si-CRNDE-2 groups relative to the si-NC group (P < 0 05) (Figure 4A and B). The results showed that GFAP and BrdU expression could be promoted after TBI, and downregulated CRNDE could further decrease GFAP and BrdU protein expression in brain tissues, which might promote the nerve repair of rats after TBI.10.1080/15384101.2019.1647024-F0004 Figure 4. Silencing of CRNDE increases the expression of GFAP and BrdU protein in brain tissues in TBI rats. Note: A, Detection of GFAP protein expression in brain tissues by immunofluorescence assay (× 200); B, Detection of BrdU protein expression in brain tissues by immunofluorescence assay (× 200); All data are measurement data and presented as mean ± standard deviation. * compared with the sham group, P < 0.05; # compared with the si-NC group, P< 0.05; n = 10. Silencing of CRNDE reduces the autophagy of neuronal cells in rats with TBI The neuronal cells were observed under a transmission electron microscopy (Figure 5A), in the sham group, neuronal cells had normal morphology and structure, complete mitochondrial structure and abundant endoplasmic reticulum; In the TBI group, the ultrastructure of the cells was disordered, mitochondria were seriously damaged with obvious swelling, rough endoplasmic reticulum structure was not clear, and a large number of vacuolar degeneration could be seen in the matrix. After silencing of CRNDE, in contrast to the TBI group, the ultrastructural damage of neuronal cells was alleviated, a few vacuolar degeneration in the matrix and slight damage of mitochondria were observed. At the same time, the expression of autophagy-related factors Beclin-1, and LC3-II/I was detected by western blot analysis (Figure 5B). Expression of Beclin-1 and LC3-II/I increased in rats with TBI. In comparison to the si-NC group, Beclin-1 and LC3-II/I protein expression declined in the si-CRNDE-1 and the si-CRNDE-2 groups (P < 0.05). The above results clarified that downregulated CRNDE reduces the autophagy of neuronal cells in TBI rats.10.1080/15384101.2019.1647024-F0005 Figure 5. Silencing of CRNDE reduces the autophagy of neuronal cells in TBI rats. Note: A, Ultrastructural changes of neurons in different groups observed by a transmission electron microscopy (× 10,000); B, The expression of autophagy-related factors Beclin-1, and LC3-II/I was detected by western blot analysis; All data are measurement data and presented as mean ± standard deviation. * compared with the sham group, P < 0.05; # compared with the si-NC group, P< 0.05; n = 10. Silencing of CRNDE elevates the expression of NGF, nestin, and NeuN in brain tissues in TBI rats TBI can trigger the expression of many kinds of nutritive factors in the injured tissues. Therefore, expression of NGF, nestin, and NeuN in the brain tissues of each group was determined. The results indicated that expression of NGF, nestin and NeuN protein increased in rats with TBI; and the expression of NGF, nestin and NeuN protein increased in the si-CRNDE-1 group and the si-CRNDE-2 groups relative to those in the si-NC group (P < 0 05) (Figure 6A and B). These results presented that silencing of CRNDE may enhance the expression of many kinds of nutrient factors, thus promote the differentiation of neurons and induce the directional growth and regeneration of nerve fibers.10.1080/15384101.2019.1647024-F0006 Figure 6. Silencing of CRNDE elevates the expression of NGF, Nestin, and NeuN in brain tissues in TBI rats. Note: A, grey value analysis of NGF, Nestin, and NeuN in brain tissues; B, protein expression of NGF, Nestin and NeuN was detected by western blot analysis; All data are measurement data and presented as mean ± standard deviation. * compared with the sham group, P < 0.05; # compared with the si-NC group, P< 0.05; n = 10. Discussion TBI, characterized by a vast array of evolving neuropathologies, is now regarded as a major health issue and a well-known pathogenic factor for the later development of neurodegenerative disorders [21]. Therefore, effective therapies are needed to reduce TBI-related disabilities. It is reported that lncRNAs have been revealed to present strong expression in adult brains and are proved to be implicated in not only neural differentiation but also synaptic plasticity [22–24]. In our study, we tried to figure out the regulatory role of CRNDE in nerve repair after TBI to find a new treatment target for TBI. The present study made conclusion that silencing of CRNDE could promote the nerve repair after TBI in rats. For investigation, we firstly tested CRNDE expression in serum of TBI patients and healthy controls as along with serum and hippocampal tissues of TBI rats. The results in our study found that CRNDE was highly expressed in TBI. In recent years, evidence has revealed that abnormal regulation of lncRNA is common in heterogeneous tumors, which can effectively affect the malignant cells by promoting growth, thereby resulting in unrestrained tumor growth [25]. It is worth noting that the increased expression of CRNDE usually occurs in cancers derived from normal expressed cell types or organs, strongly suggesting that CRNDE is of great significance in the carcinogenesis of human tumors [11]. Although CRNDE was initially found in colorectal cancer, it was later found that CRNDE was up-regulated in other tumors, especially in brain tumors [13], which was partly in line with our result. After model establishment of TBI via Feeney’s freefall impact method, the underlying mechanisms of CRNDE in nerve repair in TBI rats were further explored. Our study pointed out that silencing of CRNDE could inhibit expression of neuroinflammatory factors, neuronal apoptosis, autophagy of neuronal cells as well as increased expression of GFAP and BrdU and expression of nutrition factors. Interestingly, neuroinflammation could result in neurological disorders and tissue damage that the TBI involves, and evidence corroborated that the initiation of neuroinflammation after TBI is essential in the loss of neurological function [26,27]. In addition, the elevation of proinflammatory cytokines, immune cell proteases, as well as oxidative stress can lead to excess tissue damage, and thus inducing neuronal cell death [28,29]. The increasing reactive astrocytes as presented by an increased GFAP is recognized as a emblematical sign of injury to the central nervous system [30]. It is reported that neurotropic factors (BDNF and GDNF), play a positive role in development and regeneration of neurons, in fact, these factors can promote survival of neurons following a mechanic damage as in the TBI [31,32]. Previous evidence has been clarified that CRNDE is highly expressed in multiple cancers associated with blood and brain, and CRNDE is obviously increased in gliomas, which is related to a neurodegenerative process [11]. Remarkably, Lin et al. also pointed out that CRNDE was expressed in the process of neural differentiation from human-induced pluripotent stem cells [33]. Specifically, overexpressed CRNDE might induce inflammation to modulate tumorigenesis in glioma [34]. Furthermore, up-regulation of CRNDE triggered WI-38 cell injuries with obviously suppressed cell viability, enhanced cell apoptosis as well as elevated inflammatory cytokines levels [35], which was partly consistent with our study. Finally, we recognized that CRNDE, a lncRNA, was overexpressed in TBI, and silencing of CRNDE could inhibit expression of neuroinflammatory factors, neuronal apoptosis and autophagy of neuronal cells using a series of assays, thus promoting nerve repair. The underlying mechanisms contributing to the oncogenic phenotype of CRNDE in TBI remain to be revealed. We provided evidence that CRNDE might serve as a potent therapeutic target for TBI treatment. Acknowledgments We would like to acknowledge the reviewers for their helpful comments on this paper. Disclosure statement The authors declare that they have no conflicts of interest. ==== Refs References [1] Youssef MRL , Galal YS. Causes and outcome predictors of traumatic brain injury among emergency admitted pediatric patients at Cairo University Hospitals[J]. J Egypt Public Health Assoc 2015;90 (4 ):139–145. [2] Mondello S , Schmid K , Berger RP , et al The challenge of mild traumatic brain injury: role of biochemical markers in diagnosis of brain damage. Med Res Rev. 2014;34 (3 ):503–531.23813922 [3] Xu SY , Liu M , Gao Y , et al Acute histopathological responses and long-term behavioral outcomes in mice with graded controlled cortical impact injury. 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==== Front Cell Cycle Cell Cycle KCCY kccy20 Cell Cycle 1538-4101 1551-4005 Taylor & Francis 31305205 1642068 10.1080/15384101.2019.1642068 Research Paper Loss of exosomal MALAT1 from ox-LDL-treated vascular endothelial cells induces maturation of dendritic cells in atherosclerosis development H. LI ET AL. CELL CYCLE Li Hongqi ab* Zhu Xiang a* Hu Liqun ab Li Qing c Ma Jian d Yan Ji eb a Department of Gerontology, Affiliated Anhui Provincial Hospital, Anhui Medical University, Hefei, China b Anhui Institute of Cardiovascular Disease, Hefei, China c The Central Laboratory of Medical Research Center, Affiliated Anhui Provincial Hospital, Anhui Medical University, Hefei, China d Department of Cardiology, Shanghai Sixth People’s Hospital, Shanghai Jiaotong University, Shanghai, China e Department of Cardiology, Affiliated Anhui Provincial Hospital, Anhui Medical University, Hefei, China CONTACT Ji Yan [email protected] * Co-first authors 2019 29 7 2019 18 18 22552267 1 2 2019 30 6 2019 2 7 2019 © 2019 Informa UK Limited, trading as Taylor & Francis Group 2019 Informa UK Limited, trading as Taylor & Francis Group ABSTRACT Objectives: Maturation of dendritic cells (DCs) contributes to atherosclerosis (AS) development. Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is a long non-coding RNA (lncRNA) that is involved in tumorigenesis. This study was designed to explore the role of exosomes from oxidized low-density lipoprotein (oxLDL)-treated vascular endothelial cells (VECs) in regulating DCs maturation in AS, and to elucidate whether MALAT1 was involved in this process. Methods: Human umbilical VECs (HUVECs) were treated with or without ox-LDL, after which exosomes were isolated and then co-cultured with immature DCs (iDCs). The phenotypic profile and cell endocytosis in DCs were examined to assess the degree of maturation of DCs. The interaction between MALAT1 and NRF2 protein in DCs was evaluated using RNA pull-down assay and RNA immunoprecipitation. A mouse model of AS was eatablished by feeding ApoE knockout (ApoE−/-) mice with a high-fat diet for 12 weeks. Results: The ox-LDL-HUVECs-Exos exhibited lower MALAT1 expression when compared with HUVECs-Exos. Furthermore, exosomes from ox-LDL-treated MALAT1-overexpressing-HUVECs (ox-LDL-HUVECs-ExosLv-MALAT1) released elevated expression of MALAT1 to iDCs, which interacted with NRF2 and activated NRF2 signaling, and thereby inhibited ROS accumulation and DCs maturation. Further in vivo experiments showed that a decrease in MALAT1 content in mouse VECs-Exos might be associated with mouse AS progression. Conclusion: Loss of exosomal MALAT1 from ox-LDL-treated VECs induces DCs maturation in atherosclerosis development. KEYWORDS MALAT1 NRF2 dendritic cells atherosclerosis exosomes Natural Science Foundation of Anhui Province10.13039/5011000039951808085MH281 Central Guidance for Local Science and Technology Development Program Special Funds2016080802D113 Open Project of Anhui Provincial Cardiovascular InstituteKF2018014 This study was supported by grants from the Open Project of Anhui Provincial Cardiovascular Institute [KF2018014]; the Natural Science Foundation of Anhui Province [1808085MH281]; the Central Guidance for Local Science and Technology Development Program Special Funds [2016080802D113]. ==== Body Introduction Atherosclerosis (AS) is a chronic inflammatory and autoimmune disease with increased morbidity and mortality globally [1,2]. Dendritic cells (DCs) are the most potent antigen-presenting cells in the immune system and are hyperactive in atherosclerotic plaques [1,3]. DCs are present in immature forms in the arterial wall under physiological conditions and become activated following capturing antigens during atherogenesis [3,4]. DCs contribute to atherogenesis and have been identified as a major target for the control of this harmful immune response in AS [3,5]. The nuclear factor erythroid 2-related factor (NRF2) has antioxidant and anti-inflammatory effects in AS. Recent data revealed that NRF2 deficiency promotes features of plaque instability in hypercholesterolemic mice [6]. Furthermore, NRF2 activation exerts anti-atherosclerosis effects [7] and attenuates oxidized low-density lipoprotein (oxLDL)-induced endothelial cell injury [8]. In addition, NRF2 is involved in the regulation of the activation [9], maturation [10], and immune tolerance of DCs [11]. Moreover, inhibition of NRF2 in DCs in glioma-exposed microenvironment enhances DCs maturation and the subsequent T cells activation [12]. However, it has not been reported whether the NFR2 signaling pathway participates in the development and progression of AS by mediating DCs immune tolerance. Long non-coding RNA (lncRNA) are important regulators of gene expression and are crucial mediators in various diseases, including AS [13–15]. One prominent lncRNA known as metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) has been widely shown to be involved in various cancers [16–18]. For AS, it was demonstrated that MALAT1 knockdown promotes AS progression [19]. Recent data has shown that MALAT1 overexpression induces tolerogenic DCs and immune tolerance in heart transplantation and autoimmune disease [20]. However, whether MALAT1 affects the immune tolerance of DCs in the setting of AS is still uncertain. Exosomes are small vesicles delivered by many cells of the organism and have recently been recognized as important mediators of intercellular communication by transmitting and exchanging donor cell-specific proteins, mRNA, small noncoding RNA including lncRNA, and so on [21]. It has been widely demonstrated that ox-LDL is involved in the AS development by inducing oxidative stress and endothelial dysfunction [15,22]. In addition, MALAT1 has been shown to activate NRF2 signaling in HUVECs [23]. Accordingly, this study explored the role of MALAT1 expressed in exosomes from oxLDL-treated vascular endothelial cells (VECs) in regulating DCs maturation in the context of AS. Furthermore, we investigated whether the underlying mechanisms involved NRF2 signaling. Materials and methods Human sample collection This study was conducted in accordance with the protocol approved by the Clinical Research Ethics Committee of Affiliated Anhui Provincial Hospital, Anhui Medical University. AS patients (AS group, n = 25, mean age 65.3 ± 8.8 years, 14 male) and healthy participants (Normal group, n = 20, mean age 55.6 ± 10.1 years, 12 male) who underwent physical examinations during the same period were enrolled in this study. AS was diagnosed if brachial-ankle pulse wave velocity (baPWV) >1400 cm/s. All subjects with other complications were excluded, including valvular heart disease, severe arrhythmia, diabetes, malignant tumor, and severe liver and kidney dysfunction. Whole blood from each participant was exsanguinated, cooled at 4ºC for 1 h, and then centrifuged at 3000 rpm for 10 min. The resulting supernatant was sera that were stored at −80ºC for subsequent experiments. Cell culture and oxLDL treatment Human umbilical vein endothelial cells (HUVECs) and mouse VECs were purchased from Procell (Wuhan, China). HUVECs and mouse VECs were cultured in VECs-specific complete medium (Procell). oxLDL (50 mg/L) was added into HUVECs and mouse VECs for 24 h of incubation. Isolation and identification of serum- or VECs-derived exosomes Isolation of serum-derived exosomes was performed using miRCURY Exosome Serum/Plasma Kit according to the manufacturer’s instructions. Isolation of VECs-derived exosomes was performed using miRCURY Exosome Cell/Urine/CSF Kit (QIAGEN, Germany) according to the manufacturer’s instructions. Briefly, samples were centrifuged at 300 × g for 10 min and the resulting cell supernatant was then centrifuged again at 2,000 × g for 10 min to discard dead cells. The supernatant was subject to additional centrifugation at 10,000 × g for 30 min to discard cell debris. Afterward, the supernatant was centrifuged again at 100,000 × g for 70 min. The resultant exosome pellets were resuspended in PBS and prepared for subsequent analysis. For identification, total protein was extracted from exosomes using Total exosome RNA and protein isolation kit (Invitrogen, USA). The protein expression of exosomal surface markers TSG101 and CD63 were examined by western blot. Generation of iDCs from monocytes To prepare immature dendritic cells (iDCs), human peripheral blood CD14+ monocytes were isolated using magnetic beads (Miltenyi Biotec, USA). CD14+ cells were cultured in complete RPMI1640 media containing recombinant human granulocyte- macrophage colony-stimulating factor (rhGM-CSF; 100 ng/mL) and recombinant human interleukin-4 (rhIL-4; 50 ng/mL) for 5 days. The resulting non-adherent cells were collected and used as iDCs. RNA extraction and qRT-PCR analysis Total RNA from exosomes was extracted using Total exosome RNA and protein isolation kit (Invitrogen). Total RNA from DCs was extracted using TRIzol reagent (Invitrogen). RNA was reverse transcribed to cDNA using the PrimeScript RT reagent Kit (Takara Bio Company, Shiga, Japan). Relative MALAT1 expression was detected using an SYBR Green Kit (Takara Bio Company) on an ABI PRISM 7500 Sequence Detection System (Applied Biosystems, USA). The relative quantification of gene expression was calculated by the 2−ΔΔCt method. GAPDH was used as an internal control. The primers were as follows: MALAT1 (human)-F, 5’- GGGTGTTTACGTAGACCAGAACC-3’; MALAT1 (human)-R, 5’ CTTCCAAAAGCCTTCTGCCTTAG- −3’; MALAT1 (mouse)-F, 5’‐GTTACCAGCCCAAACCTCAA‐3’’; MALAT1 (mouse)-R, 5’-CGATGTGGCAGAGAAATCAC-3’; GAPDH (human)-F, 5’- GCACCGTCAAGGCTGAGAAC 3’; GAPDH (human)-R, 5’ – ATGGTGGTGAAGACGCCAGT −3’; GAPDH (mouse)-F, 5’-TTTG −3’; GAPDH (mouse)-R, 5’- TGTAGACCATGTAGTTGAGGTCA-3’. FCM analysis Flow cytometry (FCM) analysis was performed to detect the phenotypic profile of DCs and to examine the FITC-Dextran endocytosis in DCs. For detection of DCs cell surface markers, cells were washed with PBS for three times and then incubated with PE-anti-CD80 (BD Biosciences), PE-anti-CD86 (eBioscience), and PE-anti-HLA-DR (eBioscience) for 30 min in the dark. The cell mixture was analyzed on a FACSCalibur flow cytometer (BD Biosciences). Endocytosis was measured as the cellular uptake of FITC-dextran. Briefly, FITC-Dextran (0.5 mg/mL) was added into DCs (approximately 3 × 105 cells per sample) for 2 h of incubation at 4°C and 37°C, respectively. Afterwards, cells were washed with cold (4°C) PBS three times to remove excess dextran and subjected to FCM analysis. The quantitative uptake of FITC-dextran by the cells was determined using FCM analysis. Inactive intake in each group was excluded by subtracting the fluorescence intensity at 4°C. Values are presented as fold induction (median intensity values) relative to uptake by untreated cells. Detection of reactive oxygen species (ROS) content ROS content in DCs was determined using the Reactive Oxygen Species Assay Kit (YEASEN, Shanghai, China) according to the manufacturer’s instructions. ROS content in mouse sera was determined using the Mouse ROS ELISA Kit (Wuhan EIAab Science Co. Ltd, China) according to the manufacturer’s instructions. Enzyme-linked immunosorbent assay (ELISA) The levels of IL-12, IL-6, IL-10, and TGF-β in mouse sera were measured using their commercial ELISA kits (R&D Systems) according to the manufacturer’s instructions. RNA pull-down assay The interaction between MALAT1 and NRF2 protein was determined by RNA pull-down assay. Briefly, the DNA probe complementary to MALAT1 was synthesized and biotinylated by GenePharma Co., Ltd (Shanghai, China). RNA pull-down assay was performed using the Pierce™ Magnetic RNA-Protein Pull-Down Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. The RNA-binding protein complexes were washed and eluted and subjected to western blot analysis. RNA immunoprecipitation (RIP) RIP was conducted to verify the binding between MALAT1 and NRF2. RIP was performed using the RNA-Binding Protein Immunoprecipitation Kit (Millipore) according to the manufacturer’s instructions. The cells were lysed and the cell lysis solutions were incubated with NRF2 antibody or isotype control IgG. RNA-protein complexes were immunoprecipitated with protein A agarose beads and RNA was extracted by using TRIzol (Invitrogen). qRT-PCR was performed to quantify the MALAT1. Cell infection and transfection To stably express MALAT1 in HUVECs, the lentiviral pcDNA3.1-YFP-puro-MALAT1 expression vector (Lv-MALAT1) and pcDNA3.1-YFP-puro vector control (Lv-ctrl) were designed and synthesized by GenePharm Co. (Shanghai, China). The Lv-MALAT1 and Lv-ctrl was added to HUVECs and the infected cells were selected by puromycin (1.0 µg/mL, Sigma). To overexpress MALAT1 in DCs, the full-length MALAT1 cDNA fragments were cloned into the pcDNA 3.1 plasmid (Invitrogen, USA), generating pcDNA3.1- MALAT1. An empty pcDNA3.1 vector was used as the control. DCs were transfected with the plasmids using LipofectamineTM 3000 (Invitrogen) according to the manufacturer’s instructions. To knockdown MALAT1 in DCs, si-MALAT1-1, si-MALAT1-2, and scramble control siRNA (si-Ctrl) were designed and synthesized by GenePharma (Shanghai, China). The sequences were as follows: si-MALAT1-1, sense: 5’-CACAGGGAAAGCGAGUGGUUGGUAA-3’, si-MALAT1-2, sense: 5’-GAUCCAUAAUCGGUUUCAA-3’, antisense: 5’-UUGAAACCGAUUAUGGAUC-3’. DCs were transfected with these siRNAs using Lipofectamine™ RNAiMAX Transfection Reagent (Invitrogen) according to the manufacturer’s instructions. The knockdown or overexpression efficiency was examined by qRT-PCR analysis 48 h post-transfection. Western blot Cell lysates were prepared in protein extraction reagent (Pierce Biotechnology, IL) containing protease inhibitor (Pierce Biotechnology). Proteins were then separated by 10% SDS-PAGE and transferred to PVDF membranes (Bio-Rad, USA). After being blocked with 5% nonfat dry milk, the membrane was then incubated with the primary antibody against NRF2, HO-1, and NQO1 (all from Santa Cruz Biotechnology, USA), at 4°C overnight, and incubated with horseradish peroxidase-conjugated secondary antibodies at room temperature for 1 h. Blots were developed using an enhanced chemiluminescence kit (ECL kit, Pierce Biotechnology, IL) and band intensity was quantified with Quantity One software. GAPDH or tubulin served as the loading control. Nuclear NRF2 detection Nuclear and cytosolic proteins were extracted using the Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime) according to the manufacturer’s instructions. Detection for NRF2 protein expression in nuclear lysates was performed by western blot as described above. Lamin B1 served as the nuclear loading control. Animals Specific pathogen-free (SPF) ApoE knockout (ApoE−/-) mice were purchased from Changzhou Cavans Experimental Animal Co., Ltd. (Changzhou, China). All mice were kept under constant temperature and humidity with 12 h light-dark cycles, and had free access to food and water at a temperature of 25°C ± 1°C and humidity of 50%. The animal experiment was approved by the Ethics Committee of the Affiliated Anhui Provincial Hospital, Anhui Medical University. Animal experiments Mice were randomly divided into five groups (n = 10/each group): Control, AS, AS+PBS, AS+VECs-Exos, and AS+ox-LDL-VECs-Exos. The ApoE−/- mice were fed with a high-fat diet containing 21% fat and 0.15% cholesterol for 12 weeks to establish a mouse model of AS. The mice in the control group received an ordinary diet instead. One week before the completion of AS modeling, mice in the AS+PBS, AS+VECs-Exos, and AS+ox-LDL-VECs-Exos group received an intravenous injection of either PBS (control), exosomes from mouse VECs (VECs-Exo; 1.2 μg/g), or exosomes from ox-LDL-treated mouse VECs (ox-LDL-VECs-Exos; 1.2 μg/g), respectively, twice for a week. At the end of the twelfth week, when Exos had been injected for one week, these animals were sacrificed and their serum samples were prepared for detection of MDA, ROS, IL-10, IL-12, IL-6, and TGF-β. The aortas were cut into sections for histological examination. Histology Oil red O staining was performed in the aorta to analyze vascular lipid deposition and plaque area. Hematoxylin and eosin (HE) staining in aortic arch was used to analyze the gross morphology of tissue cells. Briefly, the aorta sections were fixed in 4% buffered paraformaldehyde, embedded in paraffin, and then sectioned at 4 μm thickness. The resulting sections were prepared for HE and Oil red O staining according to standard protocols. All sections were evaluated using a light microscope (Olympus BH-2; Olympus Corporation, Japan). Statistical analysis All statistical analyses were performed using SPSS version 16.0 (SPSS, Inc., Chicago, USA). Values are presented as the mean ± standard deviation (SD) from three independent experiments. p < 0.05 was considered to indicate a statistically significant difference. The unpaired Student’s t-test was used to analyze differences between the two groups. One-way analysis of variance (ANOVA) was used to analyze differences among two or three groups. Results MALAT1 expression is decreased in exosomes from AS-sera and ox-LDL-HUVECs Exosomes were isolated from sera from normal and AS humans, also from HUVECs treated with PBS or ox-LDL. Western blot analysis confirmed enrichment of the exosomal surface markers TSG101 and CD63 (Figure 1(a)). Importantly, the qRT-PCR analysis showed that MALAT1 expression was significantly decreased in AS-exosomes when compared with the exosomes from normal humans (Figure 1(b)). Furthermore, we also observed a notable lower MALAT1 expression in exosomes derived from ox-LDL-treated HUVECs than that in the exosomes from PBS-treated HUVECs (Figure 1(c)).10.1080/15384101.2019.1642068-F0001 Figure 1. Exosomal MALAT1 expression was decreased in AS-sera and ox-LDL-HUVECs. Exosomes were isolated from sera from normal individuals (Normal group) and AS patients (AS group), also from HUVECs treated with PBS (PBS group) or ox-LDL (ox-LDL group). (a) The protein expression of exosomal surface markers TSG101 and CD63 in isolated exosomes in each group were examined by western blot. (b) Exosomal MALAT1 expression in the Normal and AS group was detected by qRT-PCR analysis. (c) Exosomal MALAT1 expression in the PBS- and the ox-LDL- treated HUVECs was detected by qRT-PCR analysis.p < 0.05 vs. Normal (b) or PBS (c). Exogenous overexpression of MALAT1 from ox-LDL-HUVECs-Exos inhibits DCs maturation The iDCs were treated with LPS to induce oxidative stress injury. Data revealed that LPS treatment significantly decreased cell endocytosis activity evidenced by lower cellular uptake of FITC-dextran in iDCs co-cultured with LPS (Figure 2(e)). Furthermore, LPS treatment notably increased expression of DCs markers CD80, CD86, and HLA-DR (Figure 2(f)). Reduction of internalization ability is an early signal of DC maturation. Thus, these data indicated that LPS promoted DCs maturation. Importantly, HUVECs-Exos treatment significantly increased cellular uptake of FITC-dextran in iDCs (Figure 2(e)) and decreased expression of DCs markers CD80, CD86, and HLA-DR (Figure 2(f)), suggesting that HUVECs-Exos attenuated the LPS-induced DCs maturation. Furthermore, ox-LDL-HUVECs-Exos showed weaker anti-DCs maturation effects when compared with HUVECs-Exos group (Figure 2(e,f)).10.1080/15384101.2019.1642068-F0002 Figure 2. Exogenous overexpression of MALAT1 from ox-LDL-HUVECs-Exos inhibited DCs maturation. (a) The overexpression efficiency of MALAT1 in Lv-MALAT1-transfected HUVECs was confirmed by qRT-PCR. (b) MALAT1 expression was upregulated in exosomes derived from Lv-MALAT1-transfected HUVECs. (c) The HUVECs-Exos were labeled with the lipophilic fluorescent dye DiO (green) and co-incubated with DCs transfected with mCherry plasmid (red). The intake of exosomes by iDCs was analyzed under a laser confocal microscope. Scale bar: 25 μm. (d) The iDCs were co-cultured with PBS (control of exosomes), exosomes derived from control HUVECs (Exos), exosomes derived from ox-LDL-treated HUVECs (ox-LDL-Exos), exosomes derived from ox-LDL-treated HUVECs that have been transfected with Lv-Ctrl (ox-LDL-ExosLv-ctrl), exosomes derived from ox-LDL-treated HUVECs that have been transfected with Lv-MALAT1 (ox-LDL-ExosLv-MALAT1). Relative MALAT1 expression in DCs was detected by qRT-PCR analysis. The iDCs were co-cultured with LPS (2 μg/mL, 30 min), PBS, or the exosomes mentioned in (d), and (e) endocytosis of DCs was measured by FCM analysis as the cellular uptake of FITC-dextran. (f) The expression of DCs cell surface markers CD80, CD86, and HLA-DR were measured by FCM analysis. p < 0.05 vs. Lv-ctrl (A-B) or iDCs+PBS or iDCs (D-F); #p < 0.05 vs. iDCs+Exos (D) or LPS+iDCs+PBS (E-F); $p < 0.05 vs. iDCs+ox-LDL-ExosLv-ctrl (d) or LPS+iDCs+Exos (E-F); &p < 0.05 vs. LPS+iDCs+ox-LDL-ExosLv-ctrl (E-F). Exos are important mediators of intercellular communication by transmitting donor cell-specific proteins and RNA. Notably, consistent with the decreased MALAT1 in ox-LDL-HUVECs-Exos (Figure 1(c)), MALAT1 expression was also downregulated in the iDCs co-cultured with ox-LDL-HUVECs-Exos when compared with iDCs co-cultured with HUVECs-Exos (Figure 2(d)). These data indicated that iDCs co-cultured with ox-LDL-HUVECs-Exos absorbed lower MALAT1 when compared with the iDCs co-cultured with HUVECs-Exos. Thus, we may suggest that, the mechanism underlying the ox-LDL-HUVECs-Exos-mediated weaker inhibitory effect on DCs maturation might be associated with lower MALAT1 expression. To address this, MALAT1 was overexpressed in HUVECs foolwoed by treatment with ox-LDL, and exosomes were isolated from HUVECs and then co-cultured with iDCs. The overexpression efficiency of MALAT1 in Lv-MALAT1-transfected HUVECs was confirmed by qRT-PCR (Figure 2(a)). Furthermore, MALAT1 expression was upregulated in exosomes derived from Lv-MALAT1-transfected HUVECs (Figure 2(b)). The intake of exosomes by iDCs was confirmed under a laser confocal microscope (Figure 2(c)). Furthermore, we observed an increased MALAT1 in iDCs co-cultured with ox-LDL-HUVECs-ExosLv-MALAT1 when compared with the ox-LDL-HUVECs-ExosLv-ctrl group (Figure 2(d)). More importantly, compared with ox-LDL-HUVECs-ExosLv-ctrl group, ox-LDL-HUVECs-ExosLv-MALAT1 significantly increased higher cellular uptake of FITC-dextran in iDCs (Figure 2(e)), and decreased expression of DCs markers CD80, CD86, and HLA-DR (Figure 2(f)). These data indicated that exogenous overexpression of MALAT1 from ox-LDL-HUVECs-Exos inhibited DCs maturation. Exogenous overexpression of MALAT1 from ox-LDL-HUVECs-Exos activates NRF2 signaling and thereby inhibits ROS accumulation Recent evidence indicates that ROS production promotes DCs maturation and activation [24]. NRF2 is the master regulator of anti-oxidative responses. Thus, we then elucidated the effect of exogenous overexpression of MALAT1 from ox-LDL-HUVECs-Exos on NRF2 signaling and ROS accumulation in iDCs. To this end, MALAT1 was overexpressed in ox-LDL-HUVECs, and exosomes were isolated from ox-LDL-HUVECs and then co-cultured with iDCs. Data revealed that LPS downregulated protein levels of NRF2 and NRF2 signaling downstream genes including HO-1 and NQO1 (Figure 3(a)). Furthermore, LPS treatment decreased NRF2 nuclear translocation, a key step of NRF2 signaling activation (Figure 3(b)). These data indicated that LPS significantly inhibited NRF2 signaling. Importantly, HUVECs-Exos treatment attenuated the LPS-mediated inhibition of NRF2 signaling. ox-LDL-HUVECs-Exos showed weaker attenuation when compared with HUVECs-Exos group (Figure 3(a,b)). Importantly, compared with ox-LDL-HUVECs-ExosLv-ctrl, ox-LDL-HUVECs-ExosLv-MALAT1 significantly upregulated protein expression of NRF2, HO-1, and NQO1 (Figure 3(a)) and increased NRF2 nuclear translocation (Figure 3(b)). These data indicated that upregulation of MALAT1 expression in exosomes from Lv-MALAT1-transfected ox-LDL-HUVECs activated NRF2 signaling in DCs. In addition, compared with ox-LDL-HUVECs-ExosLv-ctrl group, ox-LDL-HUVECs-ExosLv-MALAT1 significantly decreased ROS content (Figure 3(c)). Thus, our findings suggested that exogenous overexpression of MALAT1 from ox-LDL-HUVECs-Exos activated NRF2 signaling and thereby inhibited ROS accumulation in DCs.10.1080/15384101.2019.1642068-F0003 Figure 3. Exogenous overexpression of MALAT1 from ox-LDL-HUVECs-Exos activated NRF2 signaling, and thereby inhibited ROS accumulation. The iDCs were co-cultured with LPS (2 μg/mL, 30 min), PBS or the indicated exosomes, the protein levels of NRF2, HO-1, and NQO1 in total cell lysates (a), nuclear NRF2 in the nuclear fraction lysates (b) were evaluated by western blot. (c) ROS content was detected by the ROS kit. p < 0.05 vs. iDCs; #p < 0.05 vs. LPS+iDCs+PBS; $p < 0.05 vs. LPS+iDCs+Exos; &p < 0.05 vs. LPS+iDCs+ox-LDL-ExosLv-ctrl. MALAT1 interacts with NRF2 and activates NRF2 signaling in DCs Next, we explored the mechanisms underlying the MALAT1-mediated activation of NRF2 signaling. Results of RNA pull-down assay showed that NRF2 was abundantly detected in the pull-down complex of MALAT1 (Figure 4(a)). Furthermore, results of RIP assay further confirmed the binding between MALAT1 and NRF2, as indicated by abundantly expressed MALAT1 when using the NRF2 antibody as compared to using the nonspecific antibody (IgG control) (Figure 4(b)). To verify how MALAT1 expression in DCs regulated NRF2, we overexpressed and silenced MALAT1 in DCs to examine the effect of MALAT1 expression on NRF2 signaling. Data revealed that MALAT1 overexpression significantly upregulated protein levels of NRF2, HO-1, and NQO1 (Figure 4(c)) and increased NRF2 nuclear translocation (Figure 4(d)). In contrast, MALAT1 knockdown exerted the opposite effects (Figure 4(e,f)). These findings indicated that MALAT1 upregulation in DCs activated NRF2 signaling, whereas MALAT1 downregulation in DCs inhibited NRF2 signaling.10.1080/15384101.2019.1642068-F0004 Figure 4. MALAT1 interacted with NRF2 and activated NRF2 signaling. (a) The interaction between MALAT1 and NRF2 protein was evaluated by RNA pull-down assay. (b) The interaction between MALAT1 and NRF2 protein was further validated by RIP assay. Effect of MALAT1 overexpression on the protein expression of NRF2, HO-1, and NQO1 in total cell lysates (c) as well as nuclear NRF2 in the nuclear fraction lysates (d) was evaluated by western blot. Effect of MALAT1 knockdown on the protein expression of NRF2, HO-1, and NQO1 in total cell lysates (e) as well as nuclear NRF2 in the nuclear fraction lysates (f) was evaluated by western blot. p < 0.05 vs. IgG (b) or Vector (c), or si-Ctrl (e). MALAT1 expression in mouse VECs-Exos is associated with AS Finally, we verified the in vivo role of mouse VECs-Exos treatment in AS progression in AS mice. As shown in Figure 5(a), the AS mice displayed obvious formation of atheromatous plaques in comparison with the control mice. Furthermore, the aorta of AS mice showed obvious atherosclerotic plaque, a large amount of porridge-like amorphous substance in the lipid pool, loose and structurally disordered smooth muscle layer of the plaque, and inflammatory cells infiltration (Figure 5(b)). Moreover, serum levels of oxidative stress indexes including MDA content and ROS content (Figure 5(d)) and pro-inflammatory cytokines (IL-12 and IL-6) (Figure 5(e)) were significantly higher in the AS group compared with the control group. In contrast, serum levels of anti-inflammatory cytokines (IL-10 and TGF-β) were lower in the AS group than that in the control group (Figure 5(e)). These data indicated that the mouse model of AS was successfully established.10.1080/15384101.2019.1642068-F0005 Figure 5. MALAT1 expression in mouse VECs-Exos was associated with AS. (a) Representative images of oil red O staining in the aorta. Scale bar: 250 μm. (b) Representative images of HE staining in the aortic arch. Scale bar: 250 μm. (c) Exosomal MALAT1 expression in mouse sera. (d) Levels of oxidative stress indexes including MDA and ROS. (e) Levels of cytokines including IL-10, TGF-β, IL-6, and IL-12 in mouse sera. *p < 0.05 vs. Control, #p < 0.05 vs. AS+PBS, &p < 0.05 vs. AS+VECs-Exos. We also found that mouse VECs-Exos treatment alleviated AS progression, as evidenced by less atheromatous plaques and inflammatory cells infiltration (Figure 5(a,b)), decreased serum levels of oxidative stress indexes (Figure 5(d)) and pro-inflammatory cytokines (IL-12 and IL-6) (Figure 5(e)), as well as increased anti-inflammatory cytokines (IL-10 and TGF-β) (Figure 5(e)). Furthermore, compared with the AS+VECs-Exos group, the mice in the AS+ox-LDL-VECs-Exos group showed more atheromatous plaques and inflammatory cells infiltration (Figure 5(a,b)), as well as increased serum levels of oxidative stress indexes (Figure 5(d)) and pro-inflammatory cytokines (IL-12 and IL-6) (Figure 5(e)), as well as increased anti-inflammatory cytokines (IL-10 and TGF-β) (Figure 5(e)). As shown in Figure 5(c), MALAT1 expression was significantly decreased in the AS group compared with the control group, which was consistent with human data (Figure 1(b)). Furthermore, serum MALAT1 expression was significantly higher in the AS+VECs-Exos group than that in the AS+PBS group. Moreover, serum MALAT1 expression was decreased in the AS+ox-LDL-VECs-Exos group when compared with the AS+VECs-Exos group. Taken togenther, these results indicated that a decrease in MALAT1 content from mouse VECs-Exos was associated with AS progression. Discussion DCs maturation contributes to atherogenesis [3,5]. DCs have functional differences between their immature and mature status. Compared with mature DCs (mDCs), iDCs possess higher phagocytosis and are weaker in antigen presentation and feeble in immunostimulation [3,4]. Furthermore, it is well accepted that iDCs possess tolerogenic and anti-inflammatory properties [3]. Our previous study has demonstrated that captopril treatment inhibits DCs maturation and maintains their tolerogenic property, which is closely associated with their anti-atherosclerosis activity [3]. In this study, our results revealed that exogenous overexpression of MALAT1 from ox-LDL-HUVECs-Exos inhibited DCs maturation, suggesting the potential anti-atherogenesis effect of MALAT1. MALAT1 has been reported to be less expressed in the atherosclerotic plaques [25]. Furthermore, MALAT1 knockdown promotes AS progression in the MALAT1-deficient ApoE−/- mice compared with the MALAT1-wild-type ApoE−/- mice [19]. These findings indicated the potential protective role of MALAT1 in AS. Several studies have shown that MALAT1 play different roles through exosomes as a medium of transmission. For example, exosomal MATAL1 from human adipose-derived stem cells promoted ischemic wound healing [26] and traumatic brain injury recovery [27]. Exosomal MALAT1 derived from oxLDL-treated HUVECs promoted M2 macrophage polarization [28]. Delivery of MALAT1 mediated by breast cancer cells-secreted exosomes induced cell proliferation in breast cancer [21]. Our in vivo assay showed that MALAT1 expression from AS mouse sera-derived exosomes showed an opposite trend to AS progression, indicating that a decrease in MALAT1 content from mouse VECs-Exos was associated with AS progression. Thus, the above-mentioned findings support our notion that MALAT1 has potential anti-atherogenesis effect in AS. We next investigated the underlying mechanism by which increased MALAT1 expression from ox-LDL-HUVECs inhibited DCs maturation. As one of the master regulators of anti-oxidative responses, NRF2 plays critical roles in the regulation of activation [9], maturation [10], and immune tolerance [11] of DCs. Furthermore, NRF2 activation exerts anti-atherosclerosis effects [7] and attenuates ox-LDL-induced endothelial cell injury [8]. Our results showed that exogenous overexpression of MALAT1 from ox-LDL-HUVECs-Exos interacted with NRF2 and activated NRF2 signaling in DCs, and thereby inhibited ROS accumulation. Recent evidence indicates that ROS production promotes maturation and activation of DCs [24]. Hence, we may suggest that exogenous overexpression of MALAT1 from ox-LDL-HUVECs inhibited DCs maturation by interacting with NRF2 and activating NRF2 signaling. Although Chen et al. [29] have found that MALAT1 interacted with NRF2 and inhibited NRF2 downstream gene expression, studies revealing the positive regulation of NRF2 by MALAT1 have been reported. For example, Zeng et al. [23] demonstrated that MALAT1 downregulated NRF2-negative regulator KEAP1 to activate NRF2 signaling in HUVECs. Recent data also revealed that antagonism of MALAT1 downregulated NRF2 in multiple myeloma cells [30]. Consistent with this, our results showed that MALAT1 interacted with NRF2 and activated NRF2 signaling in DCs. Evidence indicates that endothelial cell-derived microvesicles or exosomes can regulate DCs maturation in vascular wall [31]. DCs are present in their immature forms in non-diseased arteries and become activated during atherogenesis. Some DCs cluster with T cells directly within atherosclerotic lesions, while others migrate to lymphoid organs to activate T cells [3,4]. The interaction between endothelial cell-derived microvesicles/exosomes and DCs was complicated and requires further investigation [31–33]. In the present study, our in vitro results showed that exogenous overexpression of MALAT1 from ox-LDL-HUVECs-Exos inhibited DCs maturation. Further assays in AS model mice demonstrated that mouse VECs-Exos treatment alleviated AS progression. In addition, a decrease in MALAT1 content in mouse VECs-Exos might be associated with mouse AS progression. However, whether the mechanism underlying the protective effect of VECs-Exos on AS was associated with MALAT1-mediated regulation of DCs maturation remains to be further studied. 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==== Front Cell Cycle Cell Cycle KCCY kccy20 Cell Cycle 1538-4101 1551-4005 Taylor & Francis 31345099 1647025 10.1080/15384101.2019.1647025 Research Paper The protective role of microRNA-140-5p in synovial injury of rats with knee osteoarthritis via inactivating the TLR4/Myd88/NF-κB signaling pathway X. HUANG ET AL. CELL CYCLE http://orcid.org/0000-0003-0591-9254 Huang Xiaoqiang a Qiao Feng b Xue Peng b a Orthopaedics Department, Honghui Hospital, Xi'an Jiaotong University, Xi’an, PR China b Orthopaedics Department of Integrated Traditional Chinese and Western Medicine, Honghui Hospital, Xi'an Jiaotong University, Xi’an, PR China CONTACT Xiaoqiang Huang [email protected] 2019 29 7 2019 18 18 23442358 27 6 2019 4 7 2019 14 7 2019 © 2019 Informa UK Limited, trading as Taylor & Francis Group 2019 Informa UK Limited, trading as Taylor & Francis Group ABSTRACT Objective: Recently, many studies have revealed the effect of microRNAs (miRNAs) in knee osteoarthritis (KOA). This study aims to explore the role of miR-140-5p in protective effects and mechanisms of synovial injury of rats with KOA via regulating the TLR4/Myd88/NF-κB signaling pathway. Methods: The models of KOA Wistar rats were established by operation of anterior cruciate ligament transection. Rats were injected with agomir NC or miR-140-5p agomir. MiR-140-5p expression in KOA synovial tissues and synoviocytes was evaluated by reverse transcription quantitative polymerase chain reaction (RT-qPCR). The synoviocytes were transfected with mimics NC sequence and miR-140-5p mimics sequence. The expression of TLR4/Myd88/NF-κB signaling pathway-related proteins was measured by RT-qPCR and western blot analysis. The proliferation and apoptosis of synoviocytes in rats with KOA were evaluated by a string of experiments. The expression levels of inflammatory factors in KOA synovial tissues and synoviocytes were detected. Results: MiR-140-5p was down-regulated in KOA synovial tissues and synoviocytes. Upregulation of miR-140-5p could inhibit the inflammation reaction and the apoptosis of synoviocytes as well as promote proliferation of synoviocytes of rats with KOA. Furthermore, upregulated miR-140-5p could inactivate the TLR4/Myd88/NF-κB signaling pathway in rats with KOA. Conclusion: This study suggests that upregulated miR-140-5p could protect synovial injury by restraining inflammation reaction and apoptosis of synoviocytes in KOA rats via TLR4/Myd88/NF-κB signaling pathway inactivation. KEYWORDS MicroRNA 140 5p TLR4/Myd88/NF κB signaling pathway Knee osteoarthritis ==== Body Introduction Knee osteoarthritis (KOA) is a kind of prevalent joint disease, in which over 250 million people were involved around the world [1]. A study which was based on population revealed that the all-cause mortality of patients with KOA or hip OA was 55% higher than ordinary people [2]. According to the data of a recent research, the prevalence of KOA in Chinese was higher than that in Caucasians [3]. The risk factors of KOA have been demonstrated in previous studies, such as meniscectomy [4], obesity, physically demanding work and traumatic knee injury [5]. However, the early examination of OA is still an unresolved issue, which is of great significance [6]. A non-coding small RNA is called microRNA (miRNA), which generally contains about 20 nucleotides and has the ability to modulate some target genes [7]. In recent years, several miRNAs have been confirmed in human diseases, and some extant studies have unraveled that miRNAs were implicated in the progression of KOA, such as miR-9 [8] and miR-29a [9]. Nevertheless, there is little known about the correlation between miR-140-5p and KOA. As one of the miRNAs, miR-140-5p has been proved to be related to several human diseases, including breast cancer [10], pulmonary arterial hypertension [11] and multiple sclerosis [12]. Besides, several studies have provided evidence that miR-140-5p was involved in OA [13–15]. The toll-like receptor 4s (TLR4) is one of the TLRs, which is of great importance in detecting the invasion of pathogens, and nuclear factor kappa-B (NF-κB) is a common transcription factor that was related to immune and inflammation [16]. According to available literature, the TLR4/Myd88/NF-κB signaling pathway was implicated in experimental traumatic brain injury and was testified to have the ability to relieve inflammatory injury [17]. There was another research unraveled that the TLR4/Myd88/NF-κB signaling pathway was involved in osteoporosis [18]. However, the relation among miR-140-5p, the TLR4/Myd88/NF-κB signaling pathway and KOA has not been studied yet. Thus, this study was designed to determine the role of miR-140-5p in protective effects and mechanisms of synovial injury of rats with KOA through the TLR4/Myd88/NF-κB signaling pathway, and we speculated that upregulated miR-140-5p could protect the synovial injury by restraining inflammation reaction and apoptosis of synoviocytes in rats with KOA via the TLR4/Myd88/NF-κB signaling pathway. Materials and methods Ethics statement Animal experiments were strictly in accordance with the Guide to the Management and Use of Laboratory Animals issued by the National Institutes of Health. The protocol of animal experiments was approved by the Institutional Animal Care and Use Committee of Xi’an Honghui Hospital, Yanliang District. Study subjects A total number of 55 healthy Wistar rats (aging 3 weeks, weighing 160–180 g) were obtained from Hunan SJA Laboratory Animal Co., Ltd. (Hunan, China). The rats were adaptively fed for 1 week for the following experiments, the temperature of feeding environment was 18–26°C, relative humidity was 40–70%, the noise was below 85 dB, ammonia concentration was below 22 PPm, and ventilated at 8–12 times/h. Establishment of animal models and grouping The Wistar rats were grouped into control group (15 rats) and experimental group (40 rats), the models of experimental group were established by operation of anterior cruciate ligament transection (ACLT). After weighed before the operation, the rats were injected by 0.1 mL/kg anesthetic, placed on the operating table, their patellae were rightly touched and inner 5 mm from patellae was conducted with longitudinal incision until the knee joint cavities were exposed, the tissues of rats in the control group were layer sutured, then the rats were raised in cages after they were completely awake. Rats in the experimental group were conducted with patella dislocation and flexion of knees, the anterior cruciate ligaments were divided and the medial meniscuses were excised, then the joint cavities were closed and the incisions were layer sutured. The rats were disinfected and raised in cages after awaking, without fixation of the limbs. The 30 rats of successful models were grouped into three groups: the ACLT group (10 rats), the ACLT + agomir negative control (NC) group (10 rats) and the ACLT + miR-140-5p agomir group (10 rats), and 10 rats were set in the control group. Rats in the ACLT + agomir NC group and the ACLT + miR-140-5p agomir group were injected in the knee joint cavities, respectively, with 0.5 mL agomir NC and miR-140-5p agomir (200 nM, purchased from Guangzhou RiboBio Co., Ltd., Guangdong, China) after 3 days, 1 week, 2 weeks, 3 weeks and 4 weeks, respectively. After 4 weeks from the operation, 10 rats were adopted from each group and euthanized, the knee joints were collected, part of which were conducted with X-Ray imaging analysis, and the other part were made into frozen sections and paraffin sections, with their total RNA and total proteins were extracted for histology and molecular biological detection. Joint imaging assessment The rats were weighed and injected with 1% pentobarbital sodium solution (Beijing Mairuida Technology Co., Ltd., Beijing, China), after muscular tension and conjunctival reflex were disappeared, the rats were fixed on the plates with their limbs stretched, then conducted with X-Ray photographs, the iconography data were collected. Preparation and observation of frozen sections The synovial tissues were fixed in 4% paraformaldehyde at 4°C overnight and rinsed by phosphate-buffered saline (PBS) 3 times, 15 min/time. The tissues were placed in 30% sucrose solution at 4°C overnight, embedded and sectioned by a freezing microtome (Thermo Fisher Scientific Inc., Waltham, MA, USA) at a thickness of 35 μm, paved on slides treated with polylysine, and sealed by neutral resins. The agomir NC and miR-140-5p agomir injected in the rats were observed by a fluorescence microscope (Olympus Optical Co., Ltd, Tokyo, Japan). Hematoxylin-eosin (HE) staining The specimens were fixed by 10% formaldehyde, then embedded by paraffin and sectioned into 4 μm sections. The roasted sections successively dewaxed in xylene I and xylene II for 10 min; the dewaxed tissues were successively soaked in absolute ethanol I, absolute ethanol II, 95% ethanol, 80% ethanol and 70% ethanol, each for 2 min, and rinsed by PBS for 2 times, 5 min/time. Next, the tissues were stained with hematoxylin for 3 min, washed by tap water for 3 min, developed by 1% hydrochloric alcohol for 2 s, washed by tap water for 2 min, then successively soaked in 50%, 70% and 80% ethanol for 2 min and immersed in eosin for 5 s, and washed by tap water for 3 min. Afterward, the tissue sections were successively soaked in 90% ethanol, absolute ethanol I and absolute ethanol II for 3 min, and successively immersed in xylene I and xylene II for 5 min; sealed by neutral resins and conducted with microscopic examination. Quantitative analysis was carried on by Mankin score method. Electron microscopic observation The synovial tissues were fixed in 40 g/L glutaraldehyde for 1 h and washed by 0.1 mol/L PBS (pH 7.4) for 3 times, 5 min for each time, fixed by 10 g/L osmium tetroxide for 1.5 h and rinsed by 0.1 mol/L PBS (pH 7.4) for 3 times, 5 min for each time. Subsequently, the tissues were conducted with dehydration by gradient ethanol, soaked in the mixed liquor of acetone and equal Epon812 for 3 h, embedded by Epon812, and polymerized at 60°C for 48 h. The tissues were sectioned and stained by 40 g/L uranyl acetate for 20 min and 27 g/L lead nitrate for 20 min, then observed under an electron microscope (JEOL, Tokyo, Japan). Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining The paraffin-embedded sections were dewaxed and dehydrated, incubated by pepsase (0.25–0.5% hydrochloric acid solution) at 37°C for 25 min, added with 50 μL TUNEL reactive mixed solution (Beijing Zhongshan GoldenBridge Biotechnology Co. Ltd., Beijing, China) and incubated in a wet box at 37°C for 60 min. Subsequently, the sections were added with 50 μL peroxidase (POD) (Beijing Zhongshan GoldenBridge Biotechnology Co. Ltd., Beijing, China) and incubated in a wet box at 37°C for 30 min, then added with diaminobenzidine (DAB) agent and the color was observed by a microscope. The developing was stopped, and the sections were placed in hematoxylin for 2 min and washed for 2 min, dipped by 95% ethanol, absolute ethanol Ⅰ-Ⅱ for 3–5 min, xylene Ⅰ-Ⅱ for 3–5 min, and sealed in neutral resins. The results were analyzed under a light microscope. Fields of view in each group were randomly adopted under a high power lens (× 400) and the number of apoptotic cells, as well as the total cells, were counted (graded by double-blinded method). Enzyme-linked immunosorbent assay (ELISA) Blood from arteria cruralis of anesthetic rats was placed for 1 h and centrifuged, then the serum samples were collected, the synovial tissues were ground into homogenate and centrifuged, the supernatant of each group was collected and subpackaged in aseptic Eppendorf (EP) tubes. Eight standard substances were prepared according to the construction of ELISA kit of IL-1β and TNF-α (Raybiotech, GA, USA), and the 8th well was the blank control group. The standard substances and samples (100 μL) were, respectively, added into 96-well plates, incubated at 37°C for 2 h, added with 100 μL primary antibody and incubated for 1 h. Each well was added with 100 μL secondary antibody, then each well was added with 100 μL chromogenic reagent and incubated for 30 min. Every well was added with 50 μL stop solution to stop the reaction. The absorbance value and concentration of each well were measured, and the standard curve was graphed. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) The total RNA was extracted from specimens and cells by Trizol kit (Invitrogen, Carlsbad, CA, USA). The primers were designed by Takara Biotechnology Co., Ltd. (Osaka, Japan) (Table 1). The RNA was reversely transcripted into cDNA according to the construction of PrimeScript RT kit (Takara Biotechnology Co., Ltd., Dalian, China). The reaction solution was conducted with RT-qPCR by using ABI PRISM® 7300 system (Applied Biosystems, Massachusetts, USA) according to the direction of SYBR® Premix Ex TaqTM II kit (Takara Biotechnology Co., Ltd., Dalian, China). U6 was taken as the internal reference of the relative expression of miR-140-5p, and β-actin was taken as the internal reference of TLR4, Myd88, NF-κB, Bcl-2, Bax, IL-1β and TNF-α. The relative transcriptional levels of mRNA were calculated by 2−△△Ct method [19]. 10.1080/15384101.2019.1647025-T0001 Table 1. Primer sequence. Gene Primer sequence (5’-3’) miR-140-5p U6 TLR4 Myd88 NF-κB Bcl-2 Bax IL-1β TNF-α F: 5’- ACACTCCAGCTGGGAGGCGGGGCGCCGCGGGA- 3’ R: 5’- CTCAACTGGTGTCGTGGA- 3’ F: 5’- CTCGCTTCGGCAGCACA- 3’ R: 5’- AACGCTTCACGAATTTGCGT- 3’ F: 5’- ACAAACGCCGGAACTTTTCG- 3’ R: 5’- GTCGGACACACACAACTTAAGC- 3’ F: 5’- TTGCCAGCGAGCTAATTGAG- 3’ R: 5’- ACAGGCTGAGTGCAAACTTG- 3’ F: 5’-TGTCTGCACCTGTTCCAAAG- 3’ R: 5’-TCAGCATCAAACTGCAGGTG- 3’ F: 5’-ACTTCTCTCGTCGCTACCGTCG- 3’ R: 5’- CCCTGAAGAGTTCCTCCACCACC- 3’ F: 5’- TGGGCTGGACACTGGACTTC- 3’ R: 5’- CTTCCAGATGGTGAGTGAGGC- 3’ F: 5’- GACTTCACCATGGAACCCGT- 3’ R: 5’- GGAGACTGCCCATTCTCGAC- 3’ F: 5’-TTACAGGAAGTCCCTCACCCTC- 3’ R: 5’- CCCAGAGCCACAATTCCCTT- 3’ β-actin F: 5’-TTACAGGAAGTCCCTCACCCTC- 3’ R: 5’-CCCAGAGCCACAATTCCCTT-3’ Note: F, forward; R, reverse; miR-140-5p, microRNA-140-5p; TLR4, toll-like receptor 4; NF-κB, nuclear factor kappa-B; IL-1β, interleukin (IL)-1β; TNF-α, tumor necrosis factor-α. Western blot analysis The total proteins of synovial tissues and cells were extracted. The protein concentration of each sample was evaluated and adjusted by deionized water, insuring the loading quantities were in accordance. The samples and loading buffer were mixed up and boiled at 100°C for 5 min, electrophoresis separation was conducted, the proteins were transferred onto the nitrocellulose membrane, which was then sealed by 5% nonfat dry milk at 4°C overnight. The proteins were added with primary antibodies: TLR4 (Abcam, Cambridge, UK, 2 μg/mL), Myd88 (diluted at 1: 1000, Cell Signaling Technology, MA, USA), NF-κB p65 (Abcam, Cambridge, UK, 0.5 μg/mL), Bcl-2 and Bax (diluted at 1: 500, both from Proteintech, CHI, USA), β-actin (diluted at 1:1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA), incubated overnight and washed by PBS for 3 times, 5 min/time. The proteins were added with IgG secondary antibody (1: 1000, Wuhan Boster Biological Technology Co., Ltd., Hubei, China) which was marked by horseradish peroxidase (HRP), and incubated at 37°C for 1 h. The membrane was soaked into enhanced chemiluminescent (ECL) reaction reagent (Pierce, Rockford, IL, USA) for 1 min. The results were observed after exposure, development and fixation. β-actin was taken as internal reference, and the protein maker was obtained from Piercenet (#854,785), the Western blot image was analyzed by ImageJ2x software (National Institutes of Health, MD, USA). Cell isolation, culture and appraisal The synovial tissues separated from normal rats and rats with KOA were soaked in PBS containing double-antibody for 5 min, washed by PBS for 3 times and placed in the culture dishes. The tissues were cut into small pieces and trypsinized by 6 mL collagenase Ⅱ (4 mg/mL) containing 10% FBS for 3 h, centrifuged with the supernatant discarded, then added with 2 mL Dulbecco’s modified Eagle medium (DMEM) culture solution and centrifuged with the supernatant discarded, added with 4 mL DMEM solution and mixed up, then added with 1 mL PBS (20% final concentration) and incubated with the adherent cells discarded. The cells were trypsinized and passaged by 0.25% trypsin after the primary cells were formed into monolayer, cells in the logarithmic growth phase were adopted for the experiment. The growth of the cells was observed under an inverted microscope. Purity identification of synoviocytes: the third passage cells were seeded on the 24-well plates (1 × 105 cells/well), the medium was discarded after the cells covered the wells, and the cells were fixed with goat blood for 1 h, incubated by vascular cell adhesion molecule (VCAM)-1 (1: 500, Abcam Inc., Cambridge, UK) at 4°C overnight, then the cells were rinsed by PBS for 3 times, incubated by fluorescence secondary antibody for 1 h, stained by 4’,6-diamidino-2-phenylindole 2 hci (DAPI) for 10 min and observed under a fluorescence microscope, the results were preserved. Cell grouping and transfection The synoviocytes were grouped into the normal group (normal synoviocytes without any treatment), the ACLT group (KOA synoviocytes without any transfection), the ACLT + mimics NC group (KA synoviocytes transfected with mimics NC), the ACLT + miR-140-5p mimics group (KOA synoviocytes transfected with miR-140-5p mimics). The cells were transfected with miR-140-5p mimics and mimics NC (Shanghai GenePharma Co., Ltd., Shanghai, China) according to the instructions of Lipofectamine 2000 kit (Invitrogen Inc., Carlsbad, CA, USA), then the transfected cell was incubated at 37°C and 5% CO2. The culture solution was changed after 24 h, the transfection efficiency was observed under an inverted fluorescence microscope. Colony formation assay The fourth passage monolayer cells of each group in the logarithmic growth phase were trypsinized into single cell by 0.25% trypsin with the single cell rate was above 95%, the cell concentration was adjusted using DMEM containing 10% FBS. The cells were seeded into 6-well plates at 100 cells/well and incubated in 5% CO2 at 37°C for 2–3 weeks. The colony formation rate was calculated as the number of colonies/the number of seeded cells × 100%. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-h-tetrazolium bromide (MTT) assay The synoviocytes in each group were detached by trypsin and the cell concentration was adjusted to 1 × 105 cells/mL by DMEM, then the cells were seeded onto 96-well plates at 200 μL/well, and incubated at 37°C and 5% CO2 in an incubator for 24 h, the medium was discarded after the cells became adherent. Next, each well was appended with 20 μL MTT solution (5 μg/mL, Sigma-Aldrich Chemical Company, St Louis, MO, USA) for the 4-h incubation. With supernatant discarded, each well was supplemented with dimethyl sulfoxide (Sigma-Aldrich Chemical Company, St Louis, MO, USA) at 200 μL/well. After the crystals were fully dissolved, the optical density (OD490) value was analyzed by a microplate reader (Thermo Fisher Scientific Inc., Waltham, MA, USA). Hoechst 33,342 staining The cells of each group were added with 2 mL DMEM containing 10% FBS (both from Gibco, Carlsbad, CA, USA), and added with Hoechst 33,342 dye (5 μg/mL, Biolab technology Co., Ltd., Beijing, China), incubated at 37°C without light exposure for 90 min, then added with 1 mL DMEM containing 10% FBS and photographed under a fluorescence microscope. Flow cytometry The cell apoptosis was measured by AnnexinV-APC/propidium iodide (PI) double staining: cells in each group were centrifuged at 1000 rpm and 4°C for 5 min and collected. Binding buffer was 4 times diluted by deionized water (4 mL binding buffer+12 mL deionized water). Subsequently, the cells were washed by precooled PBS twice, and centrifuged at 1000 rpm for 5 min, with supernatant discarded, the cells were resuspended by 250 μL binding buffer, and the cell concentration was adjusted to 1 × 106 cells/well; cell suspension (5 mL) was added into 5 mL flow tubes, then was added with 5 μL Annexin V-APC and 5 μL PI solution (both from BD Biosciences, Franklin Lakes, NJ, USA) and incubated without light exposure for 15 min. PBS (400 μL) was added into the reaction tubes, which was then injected in flow cytometry, the results were analyzed by a computer. Statistical analysis All data analyses were conducted using SPSS 21.0 software (IBM-SPSS, Inc, Chicago, IL, USA). The measurement data conforming to the normal distribution were expressed as mean ± standard deviation. One-way analysis of variance (ANOVA) was used for comparisons among multiple groups. Tukey’s multiple comparisons test was used for pairwise comparisons after the ANOVA. p value < 0.05 was indicative of a statistically significant difference. Results MiR-140-5p was down-regulated in synovial tissues of rats with KOA According to the results of RT-qPCR (Figure 1a), in comparison to the control group, the expression of miR-140-5p was declined in the ACLT group, the ACLT + agomir NC group and the ACLT + miR-140-5p agomir group (all P < 0.05). miR-140-5p expression in the ACLT + miR-140-5p agomir group was elevated contrasted to the ACLT group (P < 0.05). There was no evident difference in the miR-140-5p expression between the ACLT group and the ACLT + agomir NC group (P > 0.05). 10.1080/15384101.2019.1647025-F0001 Figure 1. MiR-140-5p is down-regulated in synovial tissues of rats with KOA. a, the expression of miR-140-5p in synovial tissues was detected by RT-qPCR; b, the results of fluorescence microscopy observation (400 ×), * P < 0.05 vs the control group, # P < 0.05 vs the ACLT group. N = 10, the measurement data conforming to the normal distribution were expressed as mean ± standard deviation. ANOVA was used for comparisons among multiple groups. Tukey’s multiple comparisons test was used for pairwise comparisons after ANOVA. The outcomes of fluorescence microscopy observation of frozen sections implied that there were many green fluorescence could be observed in the synovial tissues of the ACLT + agomir NC group and the ACLT + miR-140-5p agomir group (Figure 1b), indicating that agomir NC and miR-140-5p agomir have been successfully injected into the rats. Upregulated miR-140-5p inhibits KOA development in synovial tissues of rats The images of rats’ knees under the X-Ray are shown in Figure 2a: there was no obvious osteoporosis in the control group, the joint space was obvious and no bone destruction could be found. Compared with the control group, rats in the ACLT group and the ACLT + agomir NC group were with local hyperostosis of knee joint, changes of alignment and narrowing of joint space, which suggested an evident KOA. Relative to the ACLT group, rats in the ACLT + miR-140-5p group were with local sclerotin reduction and clear joint space of knees, indicating a decline of KOA level. 10.1080/15384101.2019.1647025-F0002 Figure 2. Upregulated miR-140-5p inhibits KOA development in synovial tissues of rats. a, X-Ray iconography images; b, results of HE staining (× 400) and Mankin score; c, results of electron microscope observation (15,000 ×). N = 10, * P < 0.05 vs the control group, # P < 0.05 vs the ACLT group. The measurement data conforming to the normal distribution were expressed as mean ± standard deviation. ANOVA was used for comparisons among multiple groups. Tukey’s multiple comparisons test was used for pairwise comparisons after ANOVA. The outcomes of HE staining unraveled that (Figure 2b): under a light microscope, there were regular cellular morphology of joint synoviocytes and neat arrangement in the control group; while there were disorder arrangement, partial necrosis and exfoliation, tissue edema and interstitial osteoporosis of synoviocytes in the ACLT group and the ACLT + agomir NC group. With complete tissue structures, the synoviocytes edges were slightly irregular in the ACLT + miR-140-5p agomir group. Mankin score illustrated that in contrast to the control group, the Mankin scores of the ACLT group, the ACLT + agomir NC group and the ACLT + miR-140-5p agomir group were observably heightened (all P < 0.05). The Mankin score of the ACLT + miR-140-5p agomir group was apparently reduced, which was relative to the ACLT group (P < 0.05). No evident difference could be found in the Mankin score between the ACLT group and the ACLT + agomir NC group (P > 0.05). The outcomes of electron microscope observation suggested that (Figure 2c) there were complete cytomembrane, less cytoplasm, many collagen tissues and no obvious vascular proliferation of the rats’ joint synoviocytes in the control group; while there were cellular atrophy and apparent accumulation of dense fibrous substances of synoviocytes in the ACLT group and the ACLT + agomir NC group. In the ACLT + miR-140-5p agomir group, the atrophy of synoviocyte surfaces was obvious, plasma membranes were complete, and the lysosomes, cytoplasm and organelles were visible. Upregulated miR-140-5p suppresses synoviocytes apoptosis in synovial tissues of rats with KOA The results of TUNEL staining suggested that (Figure 3a) the synoviocytes apoptosis in synovial tissues was accelerated in the ACLT group, the ACLT + agomir NC group and the ACLT + miR-140-5p agomir group, which was compared with the control group (P < 0.05). In comparison to the ACLT group, the synoviocytes apoptosis in synovial tissues in the ACLT + miR-140-5p agomir group was attenuated (P < 0.05). 10.1080/15384101.2019.1647025-F0003 Figure 3. Upregulated miR-140-5p suppresses synoviocytes apoptosis in synovial tissues of rats with KOA. a, results of TUNEL staining (400 ×); b, the mRNA expression of Bax and Bcl-2 was detected by RT-qPCR; c, protein bands of Bax and Bcl-2; d, the statistical results of expression of Bax and Bcl-2 in the synovial tissues in each group by Western blot analysis, * P < 0.05 vs the control group, # P < 0.05 vs the ACLT group. N = 10, * P < 0.05 vs the control group, # P < 0.05 vs the ACLT group. The measurement data conforming to the normal distribution were expressed as mean ± standard deviation. ANOVA was used for comparisons among multiple groups. Tukey’s multiple comparisons test was used for pairwise comparisons after ANOVA. According to the outcomes of RT-qPCR and Western blot analysis (Figure 3b-d), the expression of Bcl-2 in the ACLT group, the ACLT + agomir NC group and the ACLT + miR-140-5p agomir group was decreased, while the expression of Bax was heightened in contrast to the control group (all P < 0.05). The expression of Bcl-2 in the ACLT + miR-140-5p agomir group was enhanced and the expression of Bax was reduced relative to the ACLT group (all P < 0.05). Upregulated miR-140-5p represses inflammation reaction of rats with KOA Results of ELISA and RT-qPCR revealed that (Figure 4a-b) the expression levels of IL-1β and TNF-α of rats’ serum and synovial tissues in the ACLT group, the ACLT + agomir NC group and the ACLT + miR-140-5p agomir group were elevated compared with the control group (all P < 0.05). The expression levels of IL-1β and TNF-α in the ACLT + miR-140-5p agomir group were considerably reduced relative to the ACLT group (all P < 0.05). 10.1080/15384101.2019.1647025-F0004 Figure 4. Upregulated miR-140-5p represses inflammation reaction of rats with KOA. a, the expression of IL-1β and TNF-α in the serum and synovial tissues of each group was detected by ELISA; b, the expression of IL-1β and TNF-α of synovial tissues was detected by RT-qPCR; * P < 0.05 vs the control group, # P < 0.05 vs the ACLT group. N = 10, * P < 0.05 vs the control group, # P < 0.05 vs the ACLT group. The measurement data conforming to the normal distribution were expressed as mean ± standard deviation. ANOVA was used for comparisons among multiple groups. Tukey’s multiple comparisons test was used for pairwise comparisons after ANOVA. Upregulated miR-140-5p inhibits activation of TLR4/Myd88/NF-κB pathway in synovial tissues of rats with KOA According to the outcomes of RT-qPCR and western blot analysis (Figure 5a-c), the expression of TLR4, Myd88 and NF-κB of rats’ synovial tissues in the ACLT group, the ACLT + agomir NC group and the ACLT + miR-140-5p agomir group was elevated, which was compared with the control group (all P < 0.05). The expression of TLR4, Myd88 and NF-κB of rats’ synovial tissues in the ACLT + miR-140-5p agomir group was lowered relative to the ACLT group (all P < 0.05). 10.1080/15384101.2019.1647025-F0005 Figure 5. Upregulated miR-140-5p inhibits activation of TLR4/Myd88/NF-κB pathway in synovial tissues of rats with KOA. a, the expression of TLR4, Myd88 and NF-κB in synovial tissues was detected by RT-qPCR; b, protein bands of TLR4, Myd88 and NF-κB in synovial tissues; c, the statistical results of expression of TLR4, Myd88 and NF-κB in the synovial tissues in each group by Western blot analysis; * P < 0.05 vs the control group, # P < 0.05 vs the ACLT group. N = 10, * P < 0.05 vs the control group, # P < 0.05 vs the ACLT group. The measurement data conforming to the normal distribution were expressed as mean ± standard deviation. ANOVA was used for comparisons among multiple groups. Tukey’s multiple comparisons test was used for pairwise comparisons after ANOVA. The cultured cells were rats’ synovioblasts As shown in Figure 6a, synovioblasts became adherent and extended growth protuberances after 24 h, spindle dendritic growth of cells and thin or short protoplasmic protuberances could be observed under the microscope, which were of characteristics of fibroblasts; cells were clustered after 72-h culture; cells were connected into a whole piece under a microscope after 120-h culture. 10.1080/15384101.2019.1647025-F0006 Figure 6. The cultured cells were rats’ synovioblasts.a, chart of primary synovioblasts (100 ×); b, appraisal charts of synovioblasts (200 ×), respectively, were expression of VCAM-1 in synovioblasts, nucleus of synovioblasts and VCAM-1 was expressed in all the cells; c, the expression of miR-140-5p was detected by RT-qPCR; d, the results of fluorescence microscope observation (100 ×), * P < 0.05 vs the control group, # P < 0.05 vs the ACLT group. The experiment was repeated for three times. The measurement data conforming to the normal distribution were expressed as mean ± standard deviation. ANOVA was used for comparisons among multiple groups. Tukey’s multiple comparisons test was used for pairwise comparisons after ANOVA. Purity appraisal of synovioblasts (Figure 6b): VCAM-1 was the symbolic protein of synovioblasts, which was mainly expressed in the cytoplasm. The third passage cells were constructed with VCAM-1 immunofluorescence staining, more than 98% of fibroblasts expressed VCAM-1 positively and all distributed in the cytoplasm, suggesting that the cultured cells were rats’ synovioblasts with extremely high purity, which were met the demands of following experiments. MiR-140-5p was down-regulated in synoviocytes of rats with KOA According to the results of RT-qPCR (Figure 6c), in comparison to the normal group, the expression of miR-140-5p was declined in the ACLT group, the ACLT + mimics NC group and the ACLT + miR-140-5p mimics group (all P < 0.05). miR-140-5p expression in the ACLT + miR-140-5p mimics group was elevated contrasted to the ACLT group (P < 0.05). There was no evident difference in the miR-140-5p expression between the ACLT group and the ACLT + mimics NC group (P > 0.05). The outcomes of fluorescence microscopy observation implied that there were many green fluorescence could be observed in the synoviocytes of the ACLT + mimics NC group and the ACLT + miR-140-5p mimics group (Figure 6d), indicating that mimics NC and miR-140-5p mimics have been successfully transfected in the synoviocytes of the rats. Upregulated miR-140-5p induces proliferation of synoviocytes of KOA rats The colony formation ability of synoviocytes in each group was detected by colony formation assay (Figure 7a-b), outcomes of which unraveled that the colony formation ability of synoviocytes in the ACLT group, the ACLT + mimics NC group and the ACLT + miR-140-5p mimics group was evidently reduced in comparison to that of the normal group (all P < 0.05), the colony formation ability of synoviocytes in the ACLT + miR-140-5p mimics group was considerably heightened relative to the ACLT group (P < 0.05). There was no obvious difference in colony formation ability between the ACLT group and the ACLT + mimics NC group (P > 0.05). 10.1080/15384101.2019.1647025-F0007 Figure 7. Upregulated miR-140-5p induces proliferation of synoviocytes of KOA rats. a, the results of colony formation assay; b, comparison of colony cells among each group; c, comparison of cell proliferation ability among each group, * P < 0.05 vs the control group, # P < 0.05 vs the ACLT group. The experiment was repeated for three times. The measurement data conforming to the normal distribution were expressed as mean ± standard deviation. ANOVA was used for comparisons among multiple groups. Tukey’s multiple comparisons test was used for pairwise comparisons after ANOVA. The proliferation ability of synoviocytes in each group was measured by MTT assay (Figure 7c), the results suggested that the proliferation ability of synoviocytes in the ACLT group, the ACLT + mimics NC group and the ACLT + miR-140-5p mimics group was obviously lowered in contrast to that of the normal group, the proliferation ability of synoviocytes in the ACLT + miR-140-5p mimics group was apparently advanced contrasted to the ACLT group (P < 0.05). There was no evident difference in proliferation ability between the ACLT group and the ACLT + mimics NC group (P > 0.05). Upregulated miR-140-5p inhibits apoptosis of synoviocytes of rats with KOA Cell apoptosis was evaluated using Hoechst 33,342 staining (Figure 8a), the results of which implied that the number of apoptotic cells in the ACLT group, the ACLT + mimics NC group and the ACLT + miR-140-5p mimics group was observably enhanced in comparison to the normal group (all P < 0.05); while the number of apoptotic cells in the ACLT + miR-140-5p mimics group was decreased relative to the ACLT group (P < 0.05). 10.1080/15384101.2019.1647025-F0008 Figure 8. Upregulated miR-140-5p inhibits apoptosis of synoviocytes of rats with KOA. a, cell apoptosis was detected by Hoechst 33,342 staining; b, cell apoptosis was detected by flow cytometry and comparison of apoptosis rate; c, the expression of Bcl-2 and Bax was detected by RT-qPCR; d, protein bands of Bcl-2 and Bax; E, the statistical results of protein expression of Bcl-2 and Bax in each group by Western blot analysis; * P < 0.05 vs the normal group, # P < 0.05 vs the ACLT group. The experiment was repeated for three times. The measurement data conforming to the normal distribution were expressed as mean ± standard deviation. ANOVA was used for comparisons among multiple groups. Tukey’s multiple comparisons test was used for pairwise comparisons after ANOVA. The outcomes of AnnexinV-APC/PI double staining (Figure 8b) suggested that the apoptosis rate of synoviocytes in the ACLT group, the ACLT + mimics NC group and the ACLT + miR-140-5p mimics group was evidently elevated compared with the normal group (all P < 0.05), while the apoptosis rate of synoviocytes in the ACLT + miR-140-5p mimics group was lowered relative to the ACLT group (P < 0.05). There was no apparent difference in the apoptosis rate of synoviocytes between the ACLT group and the ACLT + mimics NC group (P > 0.05). The results of RT-qPCR and western blot analysis (Figure 8c-e) indicated that the expression of Bcl-2 in the ACLT group, the ACLT + mimics NC group and the ACLT + miR-140-5p mimics group was decreased, while the expression of Bax was heightened in contrast to the normal group (all P < 0.05). The expression of Bcl-2 in the ACLT + miR-140-5p mimics group was enhanced and the expression of Bax was reduced relative to the ACLT group (both P < 0.05). There was no obvious difference in the expression of Bcl-2 and Bax between the ACLT group and the ACLT + mimics NC group (P > 0.05). Upregulated miR-140-5p represses inflammation reaction of synoviocytes in rats with KOA Results of ELISA and RT-qPCR revealed that (Figure 9a-c) the expression levels of IL-1β and TNF-α of synoviocytes in the ACLT group, the ACLT + mimics NC group and the ACLT + miR-140-5p mimics group was elevated compared with the normal group (all P < 0.05). The expression levels of IL-1β and TNF-α in the ACLT + miR-140-5p mimics group was considerably reduced relative to the ACLT group (both P < 0.05). There was no evident difference in the expression levels of IL-1β and TNF-α between the ACLT group and the ACLT + mimics NC group (P > 0.05). 10.1080/15384101.2019.1647025-F0009 Figure 9. Upregulated miR-140-5p represses inflammation reaction of synoviocytes in rats with KOA. a, the expression of IL-1β and TNF-α in the serum of synoviocytes in each group was detected by ELISA; b, the expression of IL-1β and TNF-α of synoviocytes was detected by RT-qPCR; * P < 0.05 vs the normal group, # P < 0.05 vs the ACLT group. The experiment was repeated for three times. The measurement data conforming to the normal distribution were expressed as mean ± standard deviation. ANOVA was used for comparisons among multiple groups. Tukey’s multiple comparisons test was used for pairwise comparisons after ANOVA. Upregulated miR-140-5p represses activation of TLR4/Myd88/NF-κB pathway in synoviocytes of rats with KOA According to the outcomes of RT-qPCR and western blot analysis (Figure 10a-c), the expression of TLR4, Myd88 and NF-κB of synoviocytes in the ACLT group, the ACLT + mimics NC group and the ACLT + miR-140-5p mimics group was elevated, which was compared with the normal group (all P < 0.05); while the expression of TLR4, Myd88 and NF-κB of synoviocytes in the ACLT + miR-140-5p mimics group was lowered relative to the ACLT group (all P < 0.05). No apparent difference could be found in the expression of TLR4, Myd88 and NF-κB of synoviocytes between the ACLT group and the ACLT + mimics NC group (P > 0.05). 10.1080/15384101.2019.1647025-F0010 Figure 10. Upregulated miR-140-5p represses activation of TLR4/Myd88/NF-κB pathway in synoviocytes of rats with KOA. a, the expression of TLR4, Myd88 and NF-κB was detected by RT-qPCR; b, protein bands of TLR4, Myd88 and NF-κB; c, the statistical results of expression of TLR4, Myd88 and NF-κB in the cells of each group by Western blot analysis; * P < 0.05 vs the normal group, # P < 0.05 vs the ACLT group. The experiment was repeated for three times. The measurement data conforming to the normal distribution were expressed as mean ± standard deviation. ANOVA was used for comparisons among multiple groups. Tukey’s multiple comparisons test was used for pairwise comparisons after ANOVA. Discussion OA is the commonest chronic joint disease which usually accompanied by arthralgia as well as disability [20]. It has been testified that miRNAs, which were characterized as small non-coding RNAs, played a role of leading molecules in the RNA silencing [21]. Moreover, there were several recent studies have unraveled that miR-140-5p was implicated in some human diseases, such as ovarian cancer [22] and non-small cell lung cancer [23]. Nevertheless, there is little known about miR-140-5p and the TLR4/Myd88/NF-κB signaling pathway in KOA. Hence, this study was focused on the impacts of miR-140-5p as well as the TLR4/Myd88/NF-κB signaling pathway on KOA, and we have found from the results of this research that upregulation of miR-140-5p could protect synovial injury by inhibiting inflammation reaction and apoptosis of synoviocytes in rats with KOA through the TLR4/Myd88/NF-κB signaling pathway inactivation. One of the findings of our study illustrated that miR-140-5p was poorly expressed in KOA synovial tissues and synoviocytes. Similar to this result, Hao Yang et al. have assessed in their study that miR-140-5p performed a poor expression in hepatocellular carcinoma tissues [24]. As for miR-140-5p expression in cells, it has been demonstrated that the expression of miR-140-5p was down-regulated in the CD4+ T cells of patients with multiple sclerosis [12]. Another essential result of this study is that upregulation of miR-140-5p could evidently inhibit the expression of inflammation-related factors, such as IL-1β and TNF-α in KOA synovial tissues and synoviocytes, indicating an attenuation of inflammation in the rats with KOA. It is consistent with this result that a recent study has provided evidence to prove that the overexpression of miR-140-5p was able to relieve lipopolysaccharide-induced human intervertebral disc inﬂammation and degeneration by restraining the expression of inﬂammation cytokines, such as TLR4, TNF-α, IL-1β and IL-6 [25], which has contributed to the role of miR-140-5p in inflammation of joint diseases. What’s more, we have also found that the upregulation of miR-140-5p was able to inhibit the activation of the TLR4/Myd88/NF-κB signaling pathway in KOA synovial tissues and synoviocytes of rats. Similarly, the relation between upregulated miR-140-5p and the TLR4/Myd88/NF-κB signaling pathway has been identified in a present study that overexpression of miR-140-5p could suppress the TLR4/Myd88/NF-κB signaling pathway [26]. The biological function of miR-140-5p has been unraveled in this research that the upregulation of miR-140-5p could promote the proliferation of synoviocytes of KOA rats. In line with this outcome, Xin et al. have revealed that overexpressed miR-140-5p could elevate the proliferation of human dental pulp stem cells [27]. The effects of miR-140-5p have been unearthed in another existing literature, in which the authors illustrated that miR-140-5p could suppress the proliferation of gastric cancer by modulating YES1 [28]. Besides, we have demonstrated in this study that the upregulation of miR-140-5p has the capacity of inhibiting the apoptosis of synoviocytes in rats with KOA. Similar to this result, a recent research has identified that the overexpression of miR-140-5p could decelerate the apoptosis of human primary chondrocytes by regulating fucosyltransferase 1 [14], which has also confirmed the role of miR-140-5p in OA. Except for that, Zhang et al. have also reported in their study that the overexpression of miR-140-5p could induce the cell apoptosis in colorectal cancer [29]. All of these studies have contributed to proving the mechanism and function of miR-140-5p and the TLR4/Myd88/NF-κB signaling pathway in human diseases. To sum up, our study provides evidence that the upregulation of miR-140-5p could alleviate the inflammation reaction of rats with KOA. Furthermore, the upregulation of miR-140-5p has the ability to suppress the apoptosis and induce the proliferation of synoviocytes, resulting in a protective impact on the synovium of KOA rats. These outcomes would pave a new way of KOA therapy. However, more efforts such as enlarge the experimental specimens are needed to be carried on to further clarify the function mechanisms of miR-140-5p in the development of KOA. Acknowledgments We would like to acknowledge the reviewers for their helpful comments on this paper. Availability of data and material Not applicable Authors’ contributions Guarantor of integrity of the entire study: Xiaoqiang Huang Study design: Feng Qiao Experimental studies: Peng Xue Manuscript editing: Feng Qiao, Peng Xue Manuscript reviewing: Xiaoqiang Huang Consent for publication Not applicable Disclosure statement No potential conflict of interest was reported by the authors. 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==== Front Cell Cycle Cell Cycle KCCY kccy20 Cell Cycle 1538-4101 1551-4005 Taylor & Francis 31438762 1652037 10.1080/15384101.2019.1652037 Research Paper Down-regulation of microRNA-429 alleviates myocardial injury of rats with coronary heart disease Q. YANG ET AL. CELL CYCLE Yang Qin a Li Jingrong b Zhang Hao b Zuo Heping b Zhang Qinglong b Cheng Jinglin c a Emergency Department, Attending doctor, The Second Affiliated Hospital of Anhui Medical University, Hefei, Anhui, China b Emergency Department, The second Affiliated Hospital of Anhui Medical University, Hefei, Anhui, China c Emergency Department, Chief physician, The second Affiliated Hospital of Anhui Medical University, Hefei, Anhui, China CONTACT Jinglin Cheng Email [email protected] 2019 23 8 2019 18 19 25502565 9 7 2019 19 7 2019 22 7 2019 © 2019 Informa UK Limited, trading as Taylor & Francis Group 2019 Informa UK Limited, trading as Taylor & Francis Group ABSTRACT In recent years, the impact of microRNAs (miRNAs) on coronary heart disease (CHD) has been identified. This study was aimed to investigate the regulative role of microRNA (miR-429) in myocardial injury of rats with CHD. Expression of miR-429 in CHD patients and healthy people was detected by reverse transcription quantitative polymerase chain reaction (RT-qPCR). The CHD rat models were injected with normal saline, mimics negative control (NC), miR-429 mimics, inhibitors NC and miR-429 inhibitors twice a week, for 4 weeks. Levels of inflammatory factors, oxidative stress indices as well as apoptosis of cardiomyocytes were determined by a series of assays. Expression of miR-429 was up-regulated in CHD patients. Reduced miR-429 could decline the expression of oxidative stress-related factors and inflammation-related factors, and inhibit the apoptosis of cardiomyocytes in rats with CHD. Moreover, the down-regulation of miR-429 could promote the expression of CrkL and repress activation of the MEK/ERK signaling pathway. This study reveals that restrained miR-429 could exert a protective impact on myocardial injury of rats with CHD by suppressing oxidative stress, inflammation reaction and apoptosis of cardiomyocytes. The function mechanisms may relate to the up-regulation of CrkL and inhibition of the MEK/ERK signaling pathway. KEYWORDS Coronary heart disease MicroRNA-429 CrkL MEK/ERK signaling pathway coronary gensini score myocardial injury oxidative stress inflammatory factors apoptosis None ==== Body Introduction Coronary heart disease (CHD) is the primary cause of death in developed countries and is a major cause of morbidity in developing countries. Three quarters of worldwide deaths that resulted from CHD happened in countries with low and middle income [1]. Some slicing algorithms of risk of CHD have been set, which were on the basis of distinct risk factors, and established in epidemiological researches, such as depression [2], diabetes [3], pulse pressure [4] and multiplex sibling history of CHD [5]. A non-coding small RNA is called as microRNA (miRNA), which generally contains about 20 nucleotides and has the ability to modulate some target genes [6]. In recent years, various miRNAs have been identified in human diseases, and some existing researches have reported that miRNAs were implicated in the development of CHD, such as miR-21 [7], miR-210 [8], and miR-224 [9]. Nevertheless, there remains little known about the relation between miR-429 and CHD. Performed as one of the miRNAs, miR-429 has been proved to be associated with some human diseases, including hepatocellular carcinoma [10], non-small cell lung cancer [11] and prostate cancer [12]. The v-crk sarcoma virus CT10 oncogene homolog (avian)-like (CrkL) is one of the CRK adapter proteins, which consists of two spliced subtypes of CRK and is known as a critical molecule in chronic myeloid leukemia [13]. The relation between miR-429 and CrkL has been uncovered in a recent study that miR-429 could function as a tumor inhibitor in cervical cancer by targeting CrkL [14]. In addition, the extracellular signal-regulated kinase (ERK) signaling cascade is a central mitogen-activated protein pathway that contributes to the modulation of some cellular processes. The impact of mitogen-activated protein kinase/ERK kinase (MEK) has also been reported in a recent study, which was determined as a regulator of gliogenesis in the developing brain [15], and the MEK/ERK signaling pathway inactivation has been demonstrated to be able to repress the progression of human disease, such as transient global cerebral ischemia [16]. Moreover, there was a study revealed that miR-429 could suppress the tumor development by targeting CrkL in hepatocellular carcinoma through the inhibition of the Raf/MEK/ERK signaling pathway [17]. However, the correlation among CHD, miR-429 and its target gene CrkL has not been studied yet. Thus, this study was performed to determine the role of miR-429 in myocardial injury of CHD via regulating CrkL, and we inferred that reduced miR-429 could play a protective role in myocardial injury of CHD through the modulation of CrkL. Materials and methods Ethics statement Written informed consents were obtained from all patients prior to the study. The protocols of this study were approved by the Ethic Committee of The second Affiliated Hospital of Anhui Medical University and based on the ethical principles for medical research involving human subjects of the Helsinki Declaration. Animal experiments were strictly in accordance with the Guide to the Management and Use of Laboratory Animals issued by the National Institutes of Health. The protocol of animal experiments was approved by the Institutional Animal Care and Use Committee of The second Affiliated Hospital of Anhui Medical University. Study subjects A total number of 50 CHD patients (aging 40–75 years old with a mean age of 60.12 ± 3.28 years) who received resection in the department of emergency of The second Affiliated Hospital of Anhui Medical University from January 2015 to January 2018 were collected, 34 males and 16 females. All the patients were diagnosed with CHD by coronary angiography. Fifty healthy people (aging 40–75 years old with a mean age of 60.21 ± 4.33 years) were collected as a control group, 27 males and 23 females. The patients were excluded from this study if they had valvular heart disease, acute or chronic infective disease, hematological diseases, tumor, peripheral vascular disease, liver and kidney dysfunction, systemic immunologic disease, chronic obstructive pulmonary disease, non-ischemic myocardial disease and diseases resulted from other factors, such as thoracalgia, severe dysfunction of vital organs, acute pericarditis, acute myocarditis, congenital heart disease, immune system disease, connective tissue disease, pulmonary embolus and cerebrovascular disease. There was no statistical difference in the gender and age of patients in the two groups (P > 0.05). Collection of general information and detection of serum parameters General information of patients with CHD such as gender, age, and history of smoking, hypertension, and diabetes was collected. Fasting body weight (kg), body height (m), and body mass index (BMI) of the patients were examined. BMI = body weight/body height2 (kg/cm2). Then, the systolic blood pressure (SBP) and diastolic blood pressure (DBP) of the patients were evaluated. Meanwhile, fasting blood (15 mL) was obtained, of which 5 mL for detection of blood sugar and lipid, 5 mL for detection of C-reactive protein (CRP), creatine kinase MB (CK-MB) and troponin I (cTnI), and 5 mL for reverse transcription quantitative polymerase chain reaction (RT-qPCR) experiment. Fasting blood (5 mL) of all the experimental subjects was placed in anticoagulation test tubes by the clinical lab of The second Affiliated Hospital of Anhui Medical University, then the levels of blood sugar and lipid were determined, including serum total cholesterol (TC), triacylglycerol (TG), low-density lipoprotein cholesterin (LDL-C), high-density lipoprotein cholesterin (HDL-C) and fasting blood glucose (FPG). Fasting blood (5 mL) was additionally collected and centrifuged, the supernatant was preserved in a − 80°C refrigerator and then batched for inspection. The cTnI in serum was determined using double-sandwich enzyme-linked immunosorbent assay (BIOMERIENX Co., Ltd., France), CK-MB in serum was detected by continuous-flow ultraviolet spectrophotometry (Ransom Holding Co. USA), and CRP was evaluated by fluorescence immunoassay (the chemiluminescence apparatus and matched kit were from Shanghai Flash Spectrum Biotechnology Co., Ltd., Shanghai., China), the detections were under the guide of instructions. Detection of coronary arteriography and genisini score Coronary arteriography detections on all the patients were conducted by the emergency physicians in the department of emergency in The second Affiliated Hospital of Anhui Medical University. All the segments of the coronary artery could be developed through the femoral or radial artery, the degrees of coronary stenosis were measured by Gensini score. 1. Scores according to the most severe degree of coronary stenosis: 0 score, no stenosis; 1 score, ≤25%; 2 score, 26%-50%; 4 score, 51%-75%; 8 score, 76%-90%; 16 score, 91%-99%; 32 score, 100%. 2. Related coefficient according to different stenosis sites: left main coronary artery, × 5; anterior descending and circumflex proximal branches: × 2.5; anterior descending middle branch, × 1.5; anterior descending and circumflex distal branches, right coronary artery, the first and second diagonal branches and left ventricle postramus, × 1.0; the others: × 0.5. 3. Gensini score = the total of degree of coronary artery stenosis × relative coefficient of every coronary artery. Animal grouping and establishment of CHD rat models A total of 140 clean Sprague Dawley (SD) rats (aged 7 d, weighing 230–250 g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China) and raised in quiet standard animal rooms with free access to food and water, the room temperature was 20.5 ± 1.2°C and humidity was 50.3 ± 3%. The steps of model establishment in each group were: the rats were fed with high-fat fodder during the first 8 w, then were continuously intraperitoneally injected with 30 U/kg pituitrin for 3 d. Rats in the control group were fed with normal fodder during the first 8 w, then were intraperitoneally injected with equal distilled water. After the establishment, three rats in the control group as well as the model group were randomly selected to be conducted with detection of heart and coronary artery tissues, the successful model establishment was decided by the pathology of coronary artery and heart. The 100 successfully established SD rat models were grouped into five groups: the CHD group (intravenously injected with normal saline), the mimics negative control (NC) group (intravenously injected with miR-429 mimics NC), the miR-429 mimics group (intravenously injected with miR-429 mimics), the inhibitors NC group (intravenously injected with miR-429 inhibitors NC), the miR-429 inhibitors group (intravenously injected with miR-429 inhibitors), 20 rats in each group, and 20 rats were set as the control group (intravenously injected with normal saline). The treatment of rats in each group was twice a week for 4 w. The specific recipes were: 5 nmol mimics NC, miR-429 mimics, inhibitors NC and miR-429 inhibitors were mixed up with 250 μL normal saline, then were intravenously injected in the rats by insulin needles. Evaluation criterion of successful models The successful rat models were with decreased weight, activity, and diet, slow response, dim fur and cyanosis of claws and lips. The changes of myocardial pathology were: swelling and disorder of cardiac myocytes, interstitial fibrosis of myocardium, partial areas were with evident focal necrosis and nuclear atrophy. Detection of cardiac echocardiography in small animals After the rats were treated with injection for 4 w, small animal ultrasonic imaging system (VisualSonics Inc., Toronto, Canada) was applied to measure the left ventricular end systolic diameter and left ventricular end diastolic diameter, then the score of left ventricular ejection fraction (LVEF) and the left ventricular fractional shortening (LVFS) were calculated. The mean of more than three consecutive cardiac cycles was adopted. Detection of related indicators After the rats were treated with injection for 4 w, blood of right common carotid artery of random rats in each group was centrifuged (3600 r/min, 10 min), the supernatant was preserved at −20°C, the blood biochemical indexes (TC, TG, LDL-C, and HDL-C) were evaluated by a fully automatic biochemical analyzer (Bio-Rad Laboratories, Hercules, CA, USA). The expression of serum interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, CK-MB and cTnI was determined by enzyme-linked immunosorbent assay (ELISA) kits. Kits of IL-1β, IL-6, and TNF-α were purchased from Well Biological Science Co., Ltd. (Changsha, China), the kit of CK-MB was obtained from Cusabio Biotech Co., Ltd (Wuhan, China), kit of cTnI was acquired from Shenzhen Bio-Wonderful Technology Co., Ltd. (Shenzhen, China). The contents of superoxide dismutase (SOD) and malondialdehyde (MDA) were determined by chemical colorimetry on the basis of the instructions of SOD and MDA activity detection kits (NanJing JianCheng Bioengineering Institute, Nanjing, China). Colorimetry was conducted at 550 nm (SOD) and 532 nm (MDA) on a 722 grating spectrophotometer, the optical density (OD) was evaluated, and activity of SOD as well as the content of MDA was calculated. Observation of rats’ cardiac mass index Ten random rats in each group were conducted with blood collection from the right common carotid artery, then their hearts were separated, washed by normal saline and dried by filter paper. The heart mass (HM) and left ventricular mass (LVM) were, respectively, weighed by an electronic scales, the heart mass index (HMI) and left ventricular mass index (LVMI) were, respectively, calculated. HMI = HM/BM, LVMI = LVM/BM. Collection of heart tissues and hematoxylin-eosin (HE) staining Ten rats in each group were randomly euthanized and executed thoracotomy, the complete hearts were cut from the roots of the ascending aorta, then were washed by cold normal saline for 15 s, the atrial and pericardial tissues were removed. The myocardial tissues were harvested, one part of the myocardial tissues was conducted with HE staining, electron observation and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) after fixation. The rest myocardial tissues were preserved at −80°C for RT-qPCR experiment and Western blot analysis. The paraffin specimen of ventricle and myocardium were continuously sectioned along the ventricular long axis at a thickness of 4 μm, and the sections were attached on the positive charge adhesion glass slides and toasted at 60°C for 4 h. The glass slides were soaked in xylene for 10 min and dewaxed twice, then, respectively, rinsed once by graded ethanol at 100%, 95%, 90% and 70%, each time for 5 min. The sections were washed by sterile water for 2 min and stained by hematoxylin solution for 5 min with redundant dye liquor washed away, followed by color separation by hydrochloric acid ethanol for 30 s. The sections were soaked in sterile water for 15 min and stained by eosin for 2 min, then dehydrated by ethanol, respectively, at 70%, 90%, 95% and 100%, each for 2 min, soaked in xylene for 2 min × 2 times, sealed by neutral resins, observed and recorded by a microscope (Nikon Co., Ltd., Tokyo, Japan). Observation of transmission electron microscope (TEM) The myocardial tissues of rats in each group were cut into 1 mm3 pieces, then fixed by 2% glutaraldehyde for 3 h, washed by phosphate buffered solution (PBS) for 3 times, and fixed by 1% osmium acid for 2 h, dehydrated by graded ethanol and embedded by epoxy resin 618. Then, the pieces of myocardial tissues were sectioned by EM UC7 ultramicrotome (Leica Co., Ltd., Germany), double stained by uranium lead, observed and photographed by an H-7650 transmission electron microscope (Hitachi Co., Ltd., Tokyo, Japan). Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining The paraffin specimens of ventricle and myocardium in each group were toasted at 60°C for 4 h, then stained according to the directions of TUNEL kit (Roche, Basel, Switzerland). The paraffin sections were conventionally dewaxed to water and successively soaked in 100%, 95%, 85% and 70% ethanol, each for 5 min, rinsed by PBS for 2 times, then added with protease K working fluid (2 μL 50 × protease K + 98 μL PBS) reacted at 37°C for 15–30 min, soaked in confining liquid (3% H2O2 insoluble in methanol), blocked for 10 min and each specimen was added with 50 μL Streptavidin-horseradish peroxidase (HRP) working solution and reacted in the dark for 30 min, rinsed by PBS for 3 times and added with 50–100 μL diaminobenzidine (DAB) working solution, reacted for 10 min, washed by PBS for 3 times, then observed and photographed under a light microscope, it was positive that the nuclei were stained to brown. Five fields were randomly selected in each section, and the number of positive cells was recorded, the apoptosis index (AI) = the number of apoptotic nuclei/total nuclei × 100%. RT-qPCR The total RNA of plasma of patients with CHD and healthy people as well as myocardial tissues of rats in each group were extracted by Trizol (Invitrogen, Carlsbad, CA, USA). The OD value and concentration of RNA were detected by ultraviolet spectrophotometry, RNAA260nm/A280nm at a range of 1.8–2.0 indicated a good purity. Then, the RNA was reversely transcripted to cDNA using miRcute miRNA First-Strand cDNA Synthesis Kit (Tiangen Biotech Co., Ltd., Beijing, China), RT-qPCR reaction was conducted on an ABI7500 fluorescence quantitative PCR instrument by SYBR premix Ex TaqTM Ⅱ PCR Kit (TaKaRa Biotech Co., Ltd., Dalian, China). The reaction was conducted by Prism® 7500 (Applied Biosystems, Inc., MA, USA). The PCR primers were designed and synthetized by Invitrogen, Carlsbad (CA, USA) (Table 1). U6 was selected as the internal reference of miR-429, and glyceraldehyde phosphate dehydrogenase (GAPDH) was selected as the internal reference of Bax, Bcl-2, caspase-3, IL-1β, IL-6, TNF-α, and CrkL. The data were analyzed by 2−ΔΔCt method [18].10.1080/15384101.2019.1652037-T0001 Table 1. Primer sequence. Gene Primer sequence (5ʹ→3ʹ) miR-429 F: GGGGGTAATACTGTCTGGT R: TGCGTGTCGTGGAGTC Bax F: AAGCTGAGCGAGTGTCTCCGGCG R: GCCACAAAGATGGTCACTGTCTGCC Bcl-2 F: CTCGTCGCTACCGTCGTGACTTCG R: CAGATGCCCGTTCAGGTACTCAGTC caspase-3 F: AGAGCTGGACTGCGGTATTGAC R: GAACCATGACCCGTCCCTTG IL-1β F: ACGGGTTCCATGGTGAAGT R: CCTCTCAAGCAGAGCACAGA IL-6 F: GATTGTATGAACAGCGATGAT R: AGAAACGGAACTCCAGAAGACC TNF-α F: GAAAGCATGATCCGAGATGT R: CAGGAATGAGAAGAGGCTGA CrkL F: ATCCCAGAACCTGCTCACG R: CAATGTCACCAACCTCCAAT GAPDH F: GAGTCAACGGATTTGGTCGT R: GACAAGATTCCCGTTCTCAG U6 F: GCTTCGGCAGCACATATACTAAAAT R: CGCTTCACGAATTTGCGTGTCAT Note: F, forward; R, reverse; miR-429, microRNA-429; IL-1β, interleukin (IL)-1β; TNF-α, tumor necrosis factor-α; GAPDH, glyceraldehyde phosphate dehydrogenase. Western blot analysis The protein expression of Bax, Bcl-2, caspase-3, CrkL, MEK, and ERK in myocardial tissues was evaluated using Western blot analysis. The total protein of myocardial tissues of rats in each group was extracted, and the protein concentration was determined by bicinchoninic acid (BCA) kit (Beyotime Biotechnology Co., Shanghai, China), then the proteins were transferred onto the membrane and sealed by skim milk powder. The proteins were added with primary antibodies Bax (1: 1000), Bcl-2 (1: 1000), caspase-3 (1: 500), CrkL (1: 50,000), MEK (1: 1000) and ERK (1: 10,000) (all from Abcam, Cambridge, MA, USA) and incubated overnight for more than 16 h, and the relative secondary antibodies were incubated. The proteins were then conducted with film development exposure, GAPDH (diluted at 1: 1000, Millipore Co., Ltd., MA, USA) was taken as the internal reference. The gray value was analyzed by Image J (National Institutes of Health, Maryland, USA) and the gray values of target bands and internal reference bands were selected for statistical analysis. Luciferase activity assay The binding sites of CrkL and relative miR-429 were confirmed by online prediction software http://www.targetscan.org, the primers were designed and synthetized by 3ʹ-untranslated region (3ʹUTR) sequence of CrkL, the restriction enzyme cutting sites of restriction enzyme Hind III and Spe I were introduced in the forward and reverse primers, and the mutation sequences of the binding sites were designed, the target sequence fragments were synthetized by Genscript Biomart Co., Ltd. (Nanjing, China). The obtained target products and pMIR-REPORT™ Luciferase carrier vectors were digested by restrictive endoenzyme Hind III and Spe I, and the products of trypsinization were recycled, connected by T4 DNA ligase and transformed with competent cells of Escherichia coli DH5α, then the plasmids were extracted, the right recombinant plasmids were appraised by enzyme digestion and sequence analysis. The 293T cells were seeded in 24-well plates at 1 × 105 cells/well. After the cells were cotransfected with recombinant plasmids and miR-429 mimics according to the groups for 48 h, the medium was discarded, each well was added with 100 μL cell lysis buffer of luciferase kit, after 30 min, cell lysis buffer (20 μL) was added with 100 μL LARⅡ, the fluorescence value (A) was measured, then added with 100 μL Stop&Glo reagent, the fluorescence value (B) was measured; A was taken as the internal reference, the luciferase activity C = B/A. Statistical analysis All data analyses were conducted using SPSS 21.0 software (IBM Corp. Armonk, NY, USA). The measurement data conforming to the normal distribution were expressed as mean ± standard deviation. The unpaired t-test was performed for comparisons between two groups and one-way analysis of variance (ANOVA) was used for comparisons among multiple groups, and the Fisher’ s exact test was used for pairwise comparisons. The receiver operating characteristic (ROC) curve was used to evaluate the clinical efficiency in the diagnosis of CHD. Pearson correlation analysis was used to assess the relation between miR-429 and clinical indexes of patients with CHD. p value <0.05 was indicative of statistically significant difference. Results Comparison of general data between CHD patients and healthy controls We compared the general data of the subjects, there was no statistically significant difference between the two groups in age, gender, BMI, smoking, alcohol consumption, diabetes, hypertension, TG, TC, LDL-C and FPG (all P > 0.05), while relative to the healthy people, patients with CHD have higher levels of CRP, CK-MB, cTnI and HDL-C (Table 2, all P < 0.05).10.1080/15384101.2019.1652037-T0002 Table 2. Comparison of general data between CHD patients and healthy controls. Variables Healthy controls (N = 50) CHD patients (N = 50) P value Age (years old) 60.21 ± 4.33 60.12 ± 3.28 0.054 Male/female (cases) 27/23 34/16 0.218 BMI (kg/m2） 21.47 ± 2.38 22.19 ± 3.01 0.188 Smoking (yes/no) 28/22 32/18 0.541 Alcohol consumption (yes/no) 23/27 26/24 0.689 Diabetes (yes/no) 17/33 24/26 0.222 Hypertension (yes/no) 20/30 29/21 0.109 CRP (mg/L) 0.89 ± 0.20 4.58 ± 0.23 < 0.001 CK-MB (ng/mL) 2.59 ± 0.12 5.85 ± 0.52 < 0.001 cTnI (ng/mL) 0.10 ± 0.04 0.35 ± 0.04 < 0.001 TG (mmol/L) 1.68 ± 0.55 1.61 ± 0.40 0.992 TC (mmol/L) 4.31 ± 0.61 4.22 ± 0.54 0.437 LDL-C (mmol/L) 2.25 ± 0.58 2.51 ± 0.75 0.055 HDL-C (mmol/L) 1.22 ± 0.21 1.07 ± 0.12 < 0.001 FPG (mmol/L) 4.96 ± 0.68 4.89 ± 0.88 0.657 Note: BMI, body mass index; CRP, C-reactive protein; CK-MB, creatine kinase MB; cTnI, troponin I; TG, triacylglycerol; TC, cholesterol; LDL-C, low-density lipoprotein cholesterin; HDL-C, high-density lipoprotein cholesterin; FPG, fasting blood glucose. The measurement data were analyzed by Fisher’s exact test, and the independent sample t-test was performed for comparisons between two groups. Mir-429 is highly expressed in patients with CHD The expression of miR-429 in the two groups was detected using RT-qPCR, the results of which indicated that (Figure 1(a)): in comparison to the healthy people, miR-429 performed a high expression in the plasma of patients with CHD (P < 0.05). Then, the diagnostic efficiency of miR-429 to CHD patients was measured by ROC curve, the outcomes (Figure 1(b)) implied that the area under the curve (AUC) was 0.806, sensitivity was 0.860 and specificity was 0.700, showing that the expression of miR-429 in peripheral blood exerted a good predictive efficiency in the occurrence of CHD.10.1080/15384101.2019.1652037-F0001 Figure 1. MiR-429 is highly expressed in patients with CHD. a, expression of miR-429 in healthy people (N = 50) and CHD patients (N = 50) was detected by RT-qPCR; b, the diagnostic efficiency of miR-429 to CHD was analyzed by ROC curve; the measurement data were analyzed by t-test. Correlation between mir-429 and clinical variables of CHD patients In order to analyze the relation between miR-429 expression and clinical variables of CHD patients, Pearson correlation analysis was used to assess the correlation among miR-429 expression, Gensini score (r = 0.680, P < 0.001), CK-MB (r = 0.702, P < 0.001) and cTnI (r = 0.671, P < 0.001), the results suggested that (Figure 2) miR-429 expression was in positive relation with Gensini score, CK-MB and cTnI (all P < 0.05).10.1080/15384101.2019.1652037-F0002 Figure 2. Correlation between miR-429 and clinical variables of CHD patients a, the correlation between miR-429 expression and Gensini score of CHD patients (N = 50) was assessed by Pearson correlation analysis; b, the relation between miR-429 expression and CK-MB of CHD patients (N = 50) was measured by Pearson correlation analysis; c, the relation between miR-429 expression and cTnI of CHD patients (N = 50) was detected by Pearson correlation analysis. Down-regulated miR-429 improved cardiac function of rats with CHD LVEF as well as LVFS were calculated by a small animal ultrasonic imaging system, and we found that (Figure 3(a)) contrasted to the normal group, the LVEF and LVFS in the CHD group were considerably reduced (P < 0.05). No obvious difference could be observed in the tendency of LVEF and LVFS between the CHD group and the mimics NC group (P > 0.05). In contrast to the mimics NC group, the LVEF and LVFS were further declined in the miR-429 mimics group (P < 0.05). Compared to the inhibitors NC group, the LVEF and LVFS were remarkably heightened in the miR-429 inhibitors group (P < 0.05).10.1080/15384101.2019.1652037-F0003 Figure 3. Down-regulated miR-429 improved cardiac function of rats with CHD. a, ultrasonic cardiogram detection of LVEF and LVFS of rats in each group (N = 20); b, HMI and LVMI of rats in each group (N = 10), * P < 0.05 vs the normal group; # P < 0.05 vs the mimics NC group; & P < 0.05 vs the miR-429 inhibitors group. The data were all measurement data, and expressed as mean ± standard deviation. One-way ANOVA was used for comparisons among multiple groups, LSD-t method was used for pairwise comparisons after one-way ANOVA. Ventricular mass indexes were applied to testify the impacts of HMI and LVMI in rats with CHD, and we found that (Figure 3(b)) the HMI and LVMI of rats in the CHD group were noticeably elevated relative to that in the normal group (P < 0.01). There was no apparent difference in HMI and LVMI among the CHD group, the mimics NC group and the inhibitors NC group (P > 0.05). In contrast to the mimics NC group, HMI and LVMI in the miR-429 group were further increased in the miR-429 mimics group (P < 0.05). HMI and LVMI were reduced in the miR-429 inhibitors group, which was compared with the inhibitors NC group (P < 0.05). Down-regulated miR-429 attenuates blood lipids and myocardial injury in rats with CHD Related indexes of rats in each group were determined using fully automatic biochemical analyzer and ELISA, the outcomes unraveled that (Figure 4) contrasted to the normal group, the expression of TC, TG, LDL-C, CK-MB, and cTnI was heightened, while HDL-C was declined in the CHD group (all P < 0.05). There was no evident difference in TC, TG, LDL-C, HDL-C, CK-MB and cTnI among the CHD group, the mimics NC group and the inhibitors NC group (all P > 0.05). Contrasted to the mimics NC group, the expression of TC, TG, LDL-C, HDL-C, CK-MB, and cTnI was further elevated, while the expression of HDL-C was further lowered in the miR-429 mimics group (all P < 0.05). In comparison to the inhibitors NC group, expression of TC, TG, LDL-C, HDL-C, CK-MB, and cTnI was reduced, while the expression of HDL-C was increased in the miR-429 inhibitors group (all P < 0.05).10.1080/15384101.2019.1652037-F0004 Figure 4. Down-regulated miR-429 attenuates blood lipids and myocardial injury in rats with CHD. a, expression of TC, TG, LDL-C and HDL-C of rats in each group (N = 10); b, expression of CK-MB of rats in each group (N = 10); c, expression of cTnI of rats in each group (N = 10), * P < 0.05 vs the normal group; # P < 0.05 vs the mimics NC group; & P < 0.05 vs the miR-429 inhibitors group. The data were all measurement data, and expressed as mean ± standard deviation. One-way ANOVA was used for comparisons among multiple groups, LSD-t method was used for pairwise comparisons after one-way ANOVA. Down-regulated miR-429 alleviates inflammation of rats with CHD Expression of IL-1β, IL-6 and TNF-α was detected using ELISA and RT-qPCR, and we found that (Figure 5) the expression of IL-1β, IL-6 and TNF-α was elevated in the CHD group, which was compared with the normal group (all P < 0.05). No notable difference could be observed in the expression of IL-1β, IL-6 and TNF-α in the CHD group, the mimics NC group and the inhibitors NC group (all P > 0.05). Relative to the mimics NC group, the expression of IL-1β, IL-6 and TNF-α was further heightened in the miR-429 mimics group (all P < 0.05). In comparison to the inhibitors NC group, the expression of IL-1β, IL-6 and TNF-α was declined in the miR-429 inhibitors group (all P < 0.05).10.1080/15384101.2019.1652037-F0005 Figure 5. Down-regulated miR-429 alleviates inflammation of rats with CHD. a, mRNA expression of IL-1β, IL-6 and TNF-α of rats in each group (N = 10); b, protein expression of IL-1β, IL-6 and TNF-α of rats in each group (N = 10), * P < 0.05 vs the normal group; # P < 0.05 vs the mimics NC group; & P < 0.05 vs the miR-429 inhibitors group. The data were all measurement data, and expressed as mean ± standard deviation. One-way ANOVA was used for comparisons among multiple groups, LSD-t method was used for pairwise comparisons after one-way ANOVA. Down-regulated miR-429 attenuates oxidative stress of rats with CHD The contents of SOD and MDA of rats in each group were examined by colorimetric method, the outcomes revealed that (Figure 6) in contrast to the normal group, the activity of SOD was lowered and the content of MDA was elevated in the CHD group (P < 0.05). There was no observable difference in the expression of SOD and MDA among the CHD group, the mimics NC group and the inhibitors NC group (P > 0.05). Relative to the mimics NC group, the activity of SOD was lowered and the content of MDA was elevated in the miR-429 mimics group (P < 0.05). The activity of SOD was heightened and the content of MDA was reduced in the miR-429 inhibitors group, which was compared with the inhibitors NC group (P < 0.05).10.1080/15384101.2019.1652037-F0006 Figure 6. Down-regulated miR-429 attenuates oxidative stress of rats with CHD. a, expression of SOD of rats in each group (N = 10); b, expression of MDA of rats in each group (N = 10), * P < 0.05 vs the normal group; # P < 0.05 vs the mimics NC group; & P < 0.05 vs the miR-429 inhibitors group. The data were all measurement data, and expressed as mean ± standard deviation. One-way ANOVA was used for comparisons among multiple groups, LSD-t method was used for pairwise comparisons after one-way ANOVA. Down-regulated miR-429 mitigates the development of CHD in rats The pathological changes of myocardial tissues of rats in each group were evaluated by HE staining, and we found that (Figure 7(a)) the cardiomyocytes in the normal group had clear boundaries and outline with a normal arrangement and there was no rupture and swelling, and no necrocytosis and atrophy. While there were swelling and disordered arrangement of cardiomyocytes, myocardial interstitial fibrosis, nuclear atrophy and scattered myocardial infarctions in the CHD group. The pathological changes of the mimics NC group and the inhibitors NC group were in line with the CHD group. There were multitudinous cardiomyocytes with swelling, disorder and unclear outlines, together with myocardial interstitial fibrosis, nuclear atrophy and more myocardial infarctions in the miR-429 mimics group. While there were alleviated welling, ordered arrangement and clear outlines of cardiomyocytes, and attenuated myocardial interstitial fibrosis, less nuclear atrophy as well as myocardial infarctions in the miR-429 inhibitors group.10.1080/15384101.2019.1652037-F0007 Figure 7. Down-regulated miR-429 mitigates the development of CHD in rats. a, HE staining of rats’ myocardial tissues in each group (× 100, N = 10); b, results of electron microscopy observation of rats’ myocardial tissues in each group (× 5000, N = 10). The changes of ultrastructure of myocardial tissues in CHD rats were observed by an electron microscope, the outcomes revealed that (Figure 7(b)) there were ordered myocardial fibers and clear light and shade of myocardial tissues; regularly shaped mitochondrion, complete cell membranes, dense and regular ridges, integral nuclear membranes and evenly distributed chromatin in the myocardium of rats in the normal group. The myocardial fibers of myocardial tissues were disorderly arranged in the CHD group, together with partial myofilament rupture and absence, sarcomere contracture, rupture and condensation of mitochondrion, degradation of edges and dilatation of sarcoplasmic reticulum of the cardiomyocytes. The changes of ultrastructure in the mimics NC group and the inhibitors NC group were in accordance with the CHD group. Relative to the mimics NC group, there were further disordered arrangement of myocardial fibers of myocardial tissues, condensation, and swelling of mitochondrion, rupture of most cell membranes, degradation of edges, much karyopyknosis and aggravated chromatin margination of the cardiomyocytes of rats in the miR-429 mimics group. Contrasted to the inhibitors NC group, there were orderly arranged myocardial fibers of myocardial tissues, regular cardiomyocytes morphology, order arrangement of sarcoplasmic reticulum, well-formed sarcomere, mild swelling of partial mitochondrion, fuzzy structures of edges and mild dilatation of sarcoplasmic reticulum in the miR-429 inhibitors group. Down-regulated miR-429 suppresses apoptosis of myocardial tissues According to the outcomes of TUNEL staining (Figure 8(a)), the number of apoptotic cardiomyocytes in the CHD group was apparently increased in contrast to the normal group (P < 0.05). No visible difference in the number of apoptotic cardiomyocytes could be observed among the CHD group, the mimics NC group and the inhibitors NC group (P > 0.05). Relative to the mimics NC group, the number of apoptotic cardiomyocytes in the miR-429 mimics group was further increased (P < 0.05). The number of apoptotic cardiomyocytes in the miR-429 inhibitors group was reduced, which was compared with that of the inhibitors NC group (P < 0.05).10.1080/15384101.2019.1652037-F0008 Figure 8. Down-regulated miR-429 suppresses apoptosis of cells in myocardial tissues. a, positive expression of apoptotic cells and the number of positive cells of rats’ myocardial tissues in each group, which were detected by TUNEL staining (N = 10); b, Protein bands of Bax, Bcl-2 and caspase-3 of rats’ myocardial tissues in each group; c, protein expression of Bax, Bcl-2 and caspase-3, which was detected by Western blot analysis (N = 10); d, expression of Bax, Bcl-2 and caspase-3 of rats’ myocardial tissues in each group, which was detected by RT-qPCR (N = 10), * P < 0.05 vs the normal group; # P < 0.05 vs the mimics NC group; & P < 0.05 vs the miR-429 inhibitors group. The data were all measurement data, and expressed as mean ± standard deviation. One-way ANOVA was used for comparisons among multiple groups, LSD-t method was used for pairwise comparisons after one-way ANOVA. Expression of Bax, Bcl-2 and caspase-3 was determined by RT-qPCR and Western blot analysis, we found that (Figure 8(b–d)) in contrast to the normal group, the expression of Bax and caspase-3 was evidently elevated and expression of Bcl-2 was declined in the CHD group (all P < 0.05). There was no significant difference in the expression of Bax, Bcl-2 and caspase-3 among the CHD group, the mimics NC group and the inhibitors NC group (all P > 0.05). Contrasted to the mimics NC group, the expression of Bax and caspase-3 was ulteriorly elevated and expression of Bcl-2 was reduced in the miR-429 mimics group (all P < 0.05). In comparison to the inhibitors NC group, the expression of Bax and caspase-3 was significantly lowered and expression of Bcl-2 was heightened in the miR-429 inhibitors group (all P < 0.05). Down-regulated miR-429 promotes cell expression and represses activation of the MEK/ERK signaling pathway Expression of miR-429 of rats’ myocardial tissues in each group was evaluated using RT-qPCR, the results indicated that (Figure 9(a)) miR-429 expression in the CHD group was apparently elevated in comparison to that in the normal group (P < 0.05), and there was no evident difference in miR-429 expression among the CHD group, the mimics NC group and the inhibitors NC group (P > 0.05). Relative to the mimics NC group, the expression of miR-429 was further heightened in the miR-429 mimics group (P < 0.05). MiR-429 expression in the miR-429 inhibitors group was considerably reduced, which was contrasted to the inhibitors NC group (P < 0.05).10.1080/15384101.2019.1652037-F0009 Figure 9. Down-regulated miR-429 promotes CrkL expression and represses activation of the MEK/ERK signaling pathway (N = 10). a, expression of miR-429 and CrkL of rats in each group by RT-qPCR; b, protein bands of CrkL, MEK and ERK of rats’ myocardial tissues in each group; c, protein expression of CrkL, MEK and ERK, which was detected by Western blot analysis (N = 10), * P < 0.05 vs the normal group; # P < 0.05 vs the mimics NC group; & P < 0.05 vs the miR-429 inhibitors group. The data were all measurement data, and expressed as mean ± standard deviation. One-way ANOVA was used for comparisons among multiple groups, LSD-t method was used for pairwise comparisons after one-way ANOVA. Expression of CrkL of rats’ myocardial tissues in each group was measured by RT-qPCR and Western blot analysis, we found that (Figure 9(a–c)) the expression of CrkL in the CHD group was decreased relative to the normal group (P < 0.05). There was no observable difference in the expression of CrkL among the CHD group, the mimics NC group and the inhibitors NC group (P > 0.05). In comparison to the mimics NC group, the expression of CrkL was ulteriorly declined in the miR-429 mimics group (P < 0.05). In contrast to the inhibitors NC group, the expression of CrkL was elevated in the miR-429 inhibitors group (P < 0.05). Expression of MEK and ERK was detected using Western blot analysis, the outcomes of which implied that (Figure 9(b–c)) contrasted to the normal group, expression of MEK and ERK in the CHD group was apparently elevated (P < 0.05), and there was no evident difference in expression of MEK and ERK among the CHD group, the mimics NC group and the inhibitors NC group (P > 0.05). Relative to the mimics NC group, the expression of MEK and ERK was further heightened in the miR-429 mimics group (P < 0.05). Expression of MEK and ERK in the miR-429 inhibitors group was considerably reduced, which in contrast to the inhibitors NC group (P < 0.05). Crkl is a target gene of miR-429 The binding sites of CrkL and relative miR-429 as well as their sequences in 3ʹ-UTR were confirmed by an online prediction software Target Scan (Figure 10(a)). To testify that it was the binding sites predicted by miR-429 that resulted in the changes of luciferase activity, we, respectively, designed mutation sequence which was without miR-429 binding sites and the wild sequence to insert in the reporter plasmid. The 293T cells were co-transfected with miR-429 mimics and wild recombinant plasmid (Wt-miR-429/CrkL) or mutation recombinant plasmid (Mut-miR-429/CrkL) by dual luciferase reporter gene assay, according to the results, there was no evident impacts of miR-429 mimics on the luciferase activity of the Mut-miR-429/CrkL plasmids, while miR-429 mimics resulted in a broad reduction of luciferase activity in the Wt-miR-429/CrkL plasmids (P < 0.05) (Figure 10(b)).10.1080/15384101.2019.1652037-F0010 Figure 10. CrkL is a target gene of miR-429. a, the binding sites of CrkL and relative miR-429 were predicted by Target Scan; b, the results of dual luciferase reporter gene assay, this experiment was repeated for three times. * P < 0.05 vs the Wt + NC group. Discussion CHD remains one of the leading causes of death in the world [19]. In recent years, it has been testified that miRNAs, which were characterized as small non-coding RNAs, played the role of leading molecules in the RNA silencing [20]. Additionally, there were several recent researches have confirmed the role of miR-429 in some human diseases, such as gastric cancer [21] and hepatocellular carcinoma [22]. Nevertheless, there is little known about miR-429 and CrkL in CHD. Thus, this research was focused on the effects of miR-429 and its target gene CrkL on CHD, and we have found from the results that the down-regulation of miR-429 could play a protective role in myocardial injury of CHD by suppressing oxidative stress, inflammation reaction and apoptosis of cardiomyocytes in rats with CHD by targeting CrkL. One of the findings in this study illustrated that miR-429 was highly expressed in the serum of CHD patients. In accordance with this result, a previous study has confirmed that miR-429 performed a high expression in human prostate cancer [12]. Another research has also revealed that miR-429 was up-regulated in hepatocellular carcinoma tissues [10]. One more vital result of this study was that the down-regulation of miR-429 could apparently improve cardiac function and attenuate myocardial injury. There was little known about the relation between miR-429 and cardiac and cerebral vascular diseases, but we can still find the impacts of miR-429 on biological functions or pathological injury in other human diseases. For example, an extant literature has unearthed that the overexpressed miR-429 could alleviate intestinal barrier function in diabetes via restraining the expression of occludin [23]. What is more, Xiao et al. have unraveled in their study that inhibited miR-429 could attenuate oxygen-glucose deprivation/reoxygenation-induced neuronal injury through GATA-binding protein 4 [24]. We have also found in this study that restrained miR-429 could relieve the inflammation in CHD by repressing the expression of inflammation-related factors IL-1β, IL-6, and TNF-α. It is in line with this finding that the down-regulation of miR-429 was found to be able to reduce the lipopolysaccharide-induced pulmonary inflammatory response [25]. More than the above findings, our research has demonstrated that reduced miR-429 could alleviate the oxidative stress by modulating oxidative stress factors, such as up-regulating the expression of SOD and repressing the expression of MDA. Similar to this finding, a recent research has discovered that miR-141 and miR-200a, which were also the members of miR-200 family, functioned as a controller of the oxidative stress response by targeting p38α in the progression of ovarian tumor [26]. Another result of this study revealed that the down-regulation of miR-429 could suppress the apoptosis of cardiomyocytes. Consistent with this result, up-regulated miR-429 was proved to have the ability of promoting apoptosis in esophageal carcinoma via targeting Bcl-2 as well as SP-1 [27]. It has been also demonstrated that overexpressed miR-429 could induce apoptosis in hepatitis B virus-related hepatocellular carcinoma [28]. Besides, there was another essential finding that CrkL is a target gene of miR-429 and down-regulated miR-429 was able to promote the expression of CrkL and represses activation of the MEK/ERK signaling pathway. A similar result could also be found in previous research, in which the authors have provided evidence to prove that the repression of miR-429 contributed to the up-regulation of CrkL expression [14]. Additionally, it has been verified that miR-429 could restrain the progression of hepatocellular carcinoma through targeting CrkL by inactivating the Raf/MEK/ERK signaling pathway [17]. All of these data were conducive to the proving of the mechanism and function of miR-429, CrkL and the MEK/ERK signaling pathway in human diseases. In conclusion, our study provides evidence that the down-regulation of miR-429 could alleviate the oxidative stress and inflammation reaction of rats with CHD. Furthermore, down-regulated miR-429 has the ability to suppress the apoptosis of cardiomyocyte, resulting in a protective impact on the myocardium of CHD rats. These findings would contribute to the treatment of CHD. Nevertheless, more efforts are needed to be carried on to further identify the function mechanisms of miR-429 in the progression of CHD. Acknowledgments We would like to acknowledge the reviewers for their helpful comments on this paper. Disclosure statement No potential conflict of interest was reported by the authors. Ethical statement Written informed consents were obtained from all patients prior to the study. The protocols of this study were approved by the Ethic Committee of The second Affiliated Hospital of Anhui Medical University and based on the ethical principles for medical research involving human subjects of the Helsinki Declaration. Animal experiments were strictly in accordance with the Guide to the Management and Use of Laboratory Animals issued by the National Institutes of Health. The protocol of animal experiments was approved by the Institutional Animal Care and Use Committee of The second Affiliated Hospital of Anhui Medical University. Consent for publication Not applicable Availability of data and material Not applicable Authors’ contributions Guarantor of integrity of the entire study: Yang Qin; Study concepts: Li Jingrong; Study design: Zhang Hao; Experimental studies: Zuo Heping; Statistical analysis: Zhang Qinglong; Manuscript editing: Cheng Jinglin ==== Refs References [1] Gaziano TA , Halberg DL , Sands C et al. 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==== Front Cell Cycle Cell Cycle KCCY kccy20 Cell Cycle 1538-4101 1551-4005 Taylor & Francis 31095447 1618120 10.1080/15384101.2019.1618120 Research Paper Hsa_circ_0101432 promotes the development of hepatocellular carcinoma (HCC) by adsorbing miR-1258 and miR-622 H. ZOU ET AL. CELL CYCLE Zou Haibo a Xu Xiangang b Luo Lanyun a Zhang Yu c Luo Le a Yao Yutong a Xiang Guangming a Huang Xiaolun a http://orcid.org/0000-0003-3439-3327 Wang Guan a a Department of Hepatobiliary Surgery, Sichuan Academy of Medical Sciences & Sichuan Provincial People’s Hospital, Chengdu, Sichuan, China b Department of Hepatobiliary Surgery, Guizhou Provincial People’s Hospital, Guiyang, Guizhou, China c Graduate School, Chengdu Medical College, Chengdu, Sichuan, China CONTACT Guan Wang Email [email protected] * Haibo Zou and Xiangang Xu contributed equally to this work. 2019 1 7 2019 18 19 23982413 4 10 2018 8 5 2019 8 5 2019 © 2019 Informa UK Limited, trading as Taylor & Francis Group 2019 Informa UK Limited, trading as Taylor & Francis Group ABSTRACT The present research was major in investigating the regulation association among hsa_circ_0101432 (has_circ_RPPH1), miR-1258, miR-622 and MAPK1 in hepatocellular carcinoma (HCC), and we explored the mechanism underlying pathogenesis of HCC. Microarray analysis was employed to detect hsa_circ_0101432 expression in HCC. Hsa_circ_0101432 was verified as a circRNA by testing divergent primers and RNase R. And qRT-PCR was performed to determine the expression of hsa_circ_0101432, miR-1258, miR-622 and MAPK1 mRNA. Furthermore, miRanda predicted that mRNAs targeted miR-1258 and miR-622. CCK-8 assay, colony formation assay, flow cytometry as well as transwell assay were performed to detect cell viability, proliferation, apoptosis and invasive ability, respectively. Xenograft in nude mice was applied to observe tumor growth in vivo. Up-regulated hsa_circ_0101432 and down-regulated miR-1258 and miR-622 were detected in HCC while Hsa_circ_0101432 enhanced expression of MAPK1 mRNA by targeting miR-1258 and miR-622. Knocking down hsa_circ_0101432 or overexpressing miR-1258 and miR-622 inhibited proliferation and invasive ability of HCC cell and promoted cell apoptosis. Hsa_circ_0101432 was confirmed to promote tumor growth via inhibiting miR-1258 and miR-622 expression and promoting MAPK1 mRNA expression by in vivo experiment. Hsa_circ_0101432 inhibited HCC cell apoptosis, promoted cell proliferation, invasive ability and HCC tumor growth by targeting miR-1258 and miR-622 and upregulating MAPK1 mRNA expression. KEYWORDS Hepatocellular carcinoma Hsa_circ_0101432 MAPK1 ==== Body Introduction Regarded as one of the most malignant and common cancer, hepatocellular carcinoma (HCC) is the fifth leading cancer and the second preeminent cause of cancer-related deaths in the world [1]. Despite the critical factors which play important roles in HCC incidence and development are found, HCC patients have not substantially gotten an enhanced survival rate over the past decades [2]. As HCC is notoriously refractory for standard system therapy, surgery, radiotherapy and chemotherapy still have a deficiency for patients’ diagnosis of advanced stages, while patients of the early stages of HCC can be successfully cured [3].The positron emission tomography (PET) scan is the newest imaging modality used in detecting HCC tumor foci [4]. As both HCC cells and healthy hepatocytes keep high metabolic activity and expend significant amounts of glucose, it brought difficulties in detecting liver tumor foci by PET under physiological conditions [5,6]. Therefore, the identification of the pathogenic mechanisms and novel targets is promptly needed for HCC. Recently, people have paid a lot of attention to circular RNAs (circRNAs), a type of endogenous non-coding RNAs (ncRNAs) that covalently join the 3’ and 5’ ends together and form circular loops [7]. They are important in a variety of biological processes, such as cell development, proliferation, apoptosis, cancer metastasis and invasiveness [8,9]. According to the forecast, they are competitively combined with miRNA binding sites, which could vary miRNA expression. For example, Yu et al. found that circRNA Cdr1as participated in the regulation network of HCC through targeting miR-7 [10]. The development of HCC is a complex process that involves the accumulation of gene regulation alteration at multiple levels, and ceRNAs (competing endogenous RNAs) are identified to be influenced by molecules such as transcriptional factors. Lin et al. performed an integrated analysis of differentially expressed circRNAs, miRNAs and mRNAs in HCC and discovered the circRNA-miRNA-mRNA regulatory network of hsa_circ_0078279, hsa_circ_0007456 and hsa_circ_0004913 in HCC [11]. Fu et al. explored the hsa_circ_0005986/miR-129-5p/NOTCH1 axis to find that hsa_circ_0005986 could affect the cell cycle and cell proliferation of HCC via the G0/G1 phase transition regulation [3]. These researches illustrate why miRNA–circRNA interaction is regarded as one of the best therapeutic directions [12]. Hsa_circ_0101432 belongs to the family of circRNAs and targets miR-1258 and miR-622 simultaneously, but the exact role of hsa_circ_ 0101432 in HCC remains to be confirmed. MicroRNAs (miRNAs), small RNA molecules composed of 19–23 nucleotide acids, are able to regulate their target genes directly by binding with their 3ʹ-untranslated regions [13]. The dysregulated expression of miRNAs has been correlated with many human diseases, including HCC. It was proved that miR-1258 had inhibitory effects on cell growth and proliferation in HCC. MiR-1258 could also suppress the progression of HCC by targeting CDC28 protein kinase regulatory subunit 1B [14]. Furthermore, several evidence indicated that miR-622 was frequently downregulated in gastric cancer [15], pancreatic adenocarcinoma and ampullary adenocarcinoma [16]. Song et al. investigated the abnormal expression and prognostic significance of miR-520a-3p in HCC, and found that miR-520a-3p suppressed the growth and induced apoptosis of HCC cell lines MHHC-97H and Hep3B [17]. Liu et al. indicated that the coordinated expression of EZH2/miR-622/CXCR4 axis might be a prediction of worse prognostic in patients suffered HCC [18]. Thus, the relation of miR-1258 and miR-622 is considered to be an ideal direction of therapy in HCC. Mitogen-Activated Protein Kinase 1 (MAPK1), the downstream signal of MAPK, is a protein kinase that consists of serine-threonine kinases [19]. MAPK1 constitutes an essential signal transduction cascade that is pivotal in cell growth, differentiation, proliferation, apoptosis, and migration [20]. The MAPK signaling pathway includes some key signaling components and phosphorylation events which play a pivotal role in tumorigenesis [21] and MAPK has been reported to change the signaling pathway of human cancer. Zou et al. identified that MAPK1 mutation might be involved in the tumorigenesis of ovarian mixed germ cell tumors [22]. In addition, the report of Kong et al. showed that lncRNA XIST regulated expression of MAPK1 to promote HCC progression [23]. All of these researches indicate that MAPK1 can be a potential therapeutic target to treat HCC. In this study, we probed the expressions of hsa_circ_ 0101432, miR-1258, miR-622 and MAPK1 in HCC tissues by bioinformatics assay. Meanwhile, it was verified that miR-1258 and miR-622 were the common target miRNAs of hsa_circ_0101432 and MAPK1 in vitro. Hsa_circ_0101432 was proved to promote the development of HCC by regulating expression levels of miR-1258, miR-622 and MAPK1. Materials and methods Tissue sample collection Sichuan Academy of Medical Sciences & Sichuan Provincial People’s Hospital provided us with 43 pairs of HCC tissues and matched normal liver tissues, and clinical characteristics of HCC patients are provided in Supplementary Table 1. And all of the patients did not receive any chemotherapy or radiotherapy before surgery. We used a histopathological evaluation to diagnose HCC. Tissue samples were snap frozen in liquid nitrogen and stored at −80°C for further use. To carry on this study, we gained the approval of the Sichuan Academy of Medical Sciences & Sichuan Provincial People’s Hospital. Microarray analysis Gene expression data of GSE78520, GSE94508 and GSE97332 were obtained from the Biotechnology Information (NCBI) Gene Expression Omnibus (GEO). GSE78520 of HCC samples from three donors, including three adjacent normal tissues and three liver cancer tissues. GSE94508 contained 10 human samples derived from HCC tissues and paracancerous tissues. GSE97332 included seven normal hepatic tissues and seven HCC tumor tissues. GPL19978 platform was used to analyze three gene chips. Screening for differentially expressed genes was used in the Limma package, with the screening criteria \|log2(FC)\|>1, P < 0.05. The target miRNAs of hsa_circ_0101432 were predicted by https://circinteractome.nia.nih.gov/. Cell culture Normal liver cell line (HL-7702) and four human HCC cell lines (Huh-7, SK-HEP-1, HepG2 and HLE) were obtained from BeNa Culture Collection (Beijing, China). HL-7702 cells, SK-HEP-1 cells and HLE cells were maintained in RPMI-1640 medium (Sigma, St. Louis, MO, USA) with 10% fetal bovine serum (FBS) (Invitrogen, Carlsbad, CA, USA) while Huh-7 cells and HepG2 cells were cultured in High Glucose Dulbecco’s modified Eagle’s medium (DMEM-H) (Sigma, St. Louis, MO, USA) with 10% FBS. All cells were cultured at 37°C with 5% CO2. Cell transfection Cells in good condition were set on six-well plates (1 × 106). Transfection was performed by following the instructions on Lipofectamine® 2000 (Life Technologies, Gaithersburg, MD, USA). Discard the medium solution, add the prepared transfection mixture to the plate, and then incubated the cells at 37 ℃. It was divided into nine groups of cells: 1. NC group; 2. si-circ-1 group; 3. si-circ-2 group; 4. miR-1258 mimics group; 5. miR-1258 inhibitor group; 6. miR-622 mimics group; 7. miR-622 inhibitor group; 8. si-circ+miR-1258 inhibitor group; 9. si-circ+miR-622 inhibitor group. Sequences of hsa_circ_0101432 siRNAs, miRNAs mimics and inhibitor are shown in Table 1.10.1080/15384101.2019.1618120-T0001 Table 1. Sequence of siRNA, mimics and inhibitor. Name Sequence 5’→3’ hsa_circ_0101432 Si-1 5’- CAGGAGATGCCTGCGTCCTGT −3 Si-2 5’- AGATGCCTGCGTCCTGTCACT −3’ miR-1258 mimics 5’- AGUUAGGAUUAGGUCGUGGAA −3’ inhibitor 5’- UCAAUCCUAAUCCAGCACCUU −3’ miR-1296 mimics 5’- GAGUGGGGCUUCGACCCUAACC −3’ inhibitor 5’- CUCACCCCGAAGCUGGGAUUGG −3’ miR-622 mimics 5’- ACAGUCUGCUGAGGUUGGAGC −3’ inhibitor 5’- UGUCAGACGACUCCAACCUCG −3’ miR-326 mimics 5’- CCUCUGGGCCCUUCCUCCAG −3’ inhibitor 5’- GGAGACCCGGGAAGGAGGUC −3’ QRT-PCR Total RNA was extracted and quantified by TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) as well as NanoDrop 2000 (Thermo Fisher Scientific Inc., USA). RNA was transcribed into cDNA by using the ReverTra Ace qPCR RT Kit (Toyobo, Osaka, Japan). QRT-PCR was conducted by THUNDERBIRD SYBR qPCR Mix (Toyobo, Osaka, Japan). U6 and β-actin were used as internal quality controls. 2−∆∆Ct method was used to determine comparative quantification. The sequences of primer were provided in Table 2. All experiments were duplicated three times.10.1080/15384101.2019.1618120-T0002 Table 2. Primers sequences for qRT-PCR. Name Sequences hsa_circ_0101432 Forward primers: 5’ – AGCTTCGGGGAGGTGAGTT – 3’ Reverse primers: 5’ – TGGCCCTAGTCTCAGACCTT – 3’ miR-1258 Forward primers: 5’ – CTGCGAGTCCCTGGAGTTAG – 3’ Reverse primers: 5’ -CGGTCCCCTAACTACCCATT – 3’ miR-1296 Forward primers: 5’ – ATCCCAGGGAGACAGAGATCGAGG – 3’ Reverse primers: 5’ – AAGCTTGGTGGTGGACTTTTGGTTGT – 3’ miR-622 Forward primers: 5’ -ATCCCAGGGAGACAGAGATCGAGG – 3’ Reverse primers: 5’ -AAGCTTGGTGGTGGACTTTTGGTTGT – 3’ miR-326 Forward primers: 5’ – CGTGTTGCAGATCCAGACCA – 3’ Reverse primers: 5’- GGATAAGCCAAGACGGGCTG – 3’ MAPK1 Forward primers: 5’ – CTTCGGCAGCACATATAC – 3’ Reverse primers: 5’ – GAACGCTTCACGAATTTGC – 3’ GAPDH Forward primers: 5’ – TGGTCACCAGGGCTGCTT – 3’ Reverse primers: 5’ – AGCTTCCCGTTCTCAGCC – 3’ U6 Forward primers: 5’ -CTCGCTTCGGCAGCACA – 3’ Reverse primers: 5’ -AACGCTTCACGAATTTGCGT – 3’ CircRNA confirmatory assay CircRNAs could be amplified by both divergent primers and convergent primers while lncRNAs were only amplified by convergent primers. Hsa_circ_0101432 and hsa_circ_0101432 genomic DNA (gDNA) were amplified by THUNDERBIRD SYBR qPCR Mix (Toyobo, Osaka, Japan) with convergent primers or divergent primers. Total RNA (5 μg) was kept in 3 U/μg of RNase R (Epicentre Biotechnologies, Madison, WI, USA) for 15 min at 37°C. Two RNase R digestion reactions were performed according to previously published methods. To detect cDNA and gDNA PCR products, we use TE buffer (Thermo Scientific, Waltham, MA, USA) in the agarose gel. All experiments were duplicated three times. RNA pull-down assay The Huh-7 cells were transfected in 50 nM biotinylated probes of the miR-1258 and miR-622 for 48 h. Then, cells were washed with ice-cold PBS, and were harvested and incubated in a lysis buffer (Sigma, St. Louis, MO, USA) on ice for 10 min. After that, we centrifugated the lysates. Fifty μL of the sample was aliquoted as input, and the remaining lysates were incubated with M-280 streptavidin magnetic beads (Sigma, St. Louis, MO, USA) coated with RNase-free BSA and yeast tRNA (Sigma, St. Louis, MO, USA). The beads were incubated at 4°C for 3 h and then washed thoroughly with buffer. The bound RNAs were isolated and then qRT-PCR was performed to detect RNA levels. All experiments were duplicated three times. Dual luciferase reporter gene assay Wild-type and mutated-type of MAPK1-3`UTR sequences were inserted into pmirGLO vectors in order to construct pGL3-MAPK1-3`UTR-WT and pGL3-MAPK1-3`UTR-MUT. MiRNA mimics (miR-1258 mimics or miR-622 mimics) and reporters were co-transfected by Lipofectamine® 2000 (Life Technologies, Gaithersburg, MD, USA). After transfection 24–48 h, the luciferase reporter activities were determined by the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, United States). All experiments were duplicated three times. Cell proliferation assay After transfection, Huh-7 and HepG2 cells (1.0×104/well) were seeded in 96-well plates for 0 h, 24 h, 48 h and 72 h, and then 10 μL of CCK-8 solution (Dojindo, Kumamoto, Japan) was joined into each well at the specified time points. The absorbance of each well was detected at a wavelength of 450 nm by Microplate Reader (Bio-Rad, Hercules, CA, USA). All experiments were duplicated three times. Colony formation assay Cells were digested by trypsin and 500 cells were seeded in six-well plates at 37°C for 2 weeks. The supernatant medium changed per 3 days. The cells were mixed with 4% formaldehyde and stained by 0.1% crystal violet for 1 h. The number of colonies which were >50 cells were counted by Image J software. All experiments were duplicated three times. Flow cytometry assay (FCM) Si-circ-1, si-circ-2, miR-1258 and miR-622 mimics and inhibitors or NC oligonucleotides were transfected into Huh-7 cells. After transfection48 h, cells were harvested and washed by using PBS, and then resuspended in binding buffer. Cells were incubated in propidium iodide (PI) and Annexin V-fluresceinisot hiocyanate (FITC) in the dark at room temperature for 15 min. The ratio of cells undergoing apoptosis was calculated by a FACS Calibur (BD Biosciences, San Jose, CA, USA). All experiments were duplicated three times. Transwell assay Huh-7 invasive ability was assayed by using Transwell Matrigel invasive ability Chambers (BD Biosciences, San Jose, CA, USA). After 24 h transfection, the cells were seeded in serum-free DMEM-H on the upper wells of 24-well Transwell Matrigel chambers. DMEM-H medium with 10% FBS was served as chemoattractant filling the lower chamber. The non-invading cells and Metrigel matrix were wiped by cotton swabs. The invasive cells were mixed in methanol, stained with 0.1% crystal violet, and counted at 200 times magnification by an inverted microscope (Thermo Fisher Scientific, Waltham, MA, USA). Each sample from a total of five random fields was calculated. All experiments were duplicated three times. Western blot BCA Kit (Sigma-Aldrich, St. Louis, MO, USA) detected the protein concentration. After separating proteins by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE), the protein was transferred to a polyvinylidene fluoride membrane (PVDF, Millipore, Billerica, MA, USA) and blocked by TBST containing 5% skim milk. Primary antibodies MAPK1 (1:500, ab32081, Abcam, Cambridge, MA, USA) and β-actin (1:500, ab8227, Abcam, Cambridge, MA, USA) were incubated at 4°C overnight, and then the membrane was incubated in the secondary antibody (goat anti-rabbit IgG labeled with horseradish peroxidase, 1:5000, Abcam, MA, USA) for 1 h. The blots were measured through an ECL system (Life Technologies, Gaithersburg, MD, USA), and the densitometric analysis was performed via the Image J software. All experiments were duplicated three times. Xenograft in nude mice BALB/c nude mice were obtained from Sichuan Academy of Medical Sciences & Sichuan Provincial People’s Hospital and assigned to one of five groups randomly: NC, si-circ-1 group, si-circ-2 group, agomir miR-1258 group, and agomir miR-622 group transfected by miR-622 mimics. There were five nude mice per group. One hundred μL (1 × 107 cells/mL) lentivirus-infected Huh-7 cells mixture was subcutaneously injected into the right flank of nude mice. Tumor volume was measured by caliper every 7 days. After 4 weeks, mice were sacrificed to get the tumor xenograft tissues and weighed the tissues. All animal assays were conducted in accordance with the ethical guidelines for laboratory animal use and approved by the Sichuan Academy of Medical Sciences & Sichuan Provincial People’s Hospital. Statistical analysis All of the statistical data were analyzed by GraphPad Prism 6.0 software (GraphPad Software Inc., USA). The results were presented by the mean + SD from three independent experiments. Student t-test and one-way ANOVA were performed to analyze whether the comparison between two or more groups was statistically significant. P < 0.05 was regarded as statistically significant. Results Hsa_Circ_0101432 was up-regulated in HCC tumor tissues Previously, the expression of circRNA was analyzed in GSE78520, GSE94508 and GSE97332. In heat map, CircRNA presented differentially expressed in HCC tissues matched non-tumor tissue, \|log2(FC)\|>1, P < 0.05 (219 differentially expressed circRNAs in GSE78520 (n = 6), 240 differentially expressed circRNAs in GSE94508 (n = 10) and 713 differentially expressed circRNAs in GSE97332 (n = 14)). Ten circRNAs were significantly expressed in all the three chips. (Figure 1a). In 3 chips, 10 circRNAs were up-regulated in HCC tissues and displayed in the heat map compared to matched non-tumor tissues analyzed by circRNAs Arraystar chips (Figure 1b-d). The 10 intersection circRNAs are showed in Supplementary Table 2. Our attention was caught by hsa_circ_0101432, because it was one of the differentially expressed circRNAs that were up-regulated in HCC tumor tissues samples among the three gene chips.10.1080/15384101.2019.1618120-F0001 Figure 1. Hsa-circ_0101432 was up-regulated in hepatocellular carcinoma tumor tissues. (a) 10 circRNAs were significantly expressed in three Chips（GSE78520, GSE94508, GSE97332). (b-d) The heat map showed the upregulated circRNAs in HCC tissues as compared to that in the matched non-tumor tissues analyzed by circRNAs Arraystar Chip. Hsa_circ_0101432 was verified as a circRNA We used convergent primers and divergent primers to amplify hsa_circ_0101432 to further validate the structure of hsa_circ_0101432 (Figure 2a). Since it could be amplified by divergent primers but divergent primers cannot amplify linear DNA theoretically, hsa_circ_0101432 might be a circRNA but not a lncRNA (Figure 2b). By adding RNase R, the luminance of hsa_circ_0101432 was basically the same, whereas linear RNA was degraded (Figure 2c). RNase R could degrade linear RNAs rather than circRNAs (Figure 2d). Hsa_circ_0101432 was a circRNA verified by these results. It was further verified hsa_circ_0101432 was overexpressed in tumor tissues and HCC cell lines compared with non-tumor tissues and normal hepatocyte cell line (Figure 2e-f). Huh-7 and HepG2 cell lines were selected for the following experiment duo to the expressions of hsa_circ_0101432, which were much higher than those of other HCC cell lines (Figure 2f). The expressions of hsa_circ_0101432 in Huh-7 and HepG2 cells were reduced when cells were transfected in hsa_circ_0101432 silence RNAs, si-circ-1 and si-circ-2 (Figure 2g-h).10.1080/15384101.2019.1618120-F0002 Figure 2. The experiments were to verify that hsa_circ_0101432 was circRNA. (a) Both convergent primers and divergent primers were used to amplify circRNA. (b) Divergent primers can amplify the circRNA but cannot amplify the genomic DNA. (c) The luminance of circRNA was basically the same in RNase R+ group and RNase R- group, while linear RNA was degraded by RNase R. (d) Linear RNA can be degraded by RNase R while circRNAs can’t be degraded. (e-f) The qRT-PCR results showed that hsa_circ_0101432 was a significantly high expression in HCC tissues and cell lines compared with the matched non-tumor tissues or normal hepatocyte cell line. (g-h) The hsa_circ_0101432 expression was detected by qRT-PCR in HCC cell lines (Huh-7 and HepG2). Similar results were obtained in three independent experiments. * P < 0.05, compared with NC group. Hsa_circ_0101432 downregulated expressions of miR-622 and miR-1258 in HCC cells The target miRNAs of hsa_circ_0101432 were predicted by bioinformation that targeted miR-1258, miR-326, miR-622 and miR-1296 (Figure 3a). Expressions of miR-1258, miR-326, miR-622 and miR-1296 in HCC tissues and matched non-tumor tissues were detected by qRT-PCR and miR-1258 as well as miR-622 was significantly down-regulated in HCC tissues (Figure 3b). Negative correlation of miR-1258 and hsa_circ_0101432 expression as well as negative correlation of miR-622 and hsa_circ_0101432 expression was presented by spearman’s rank correlation coefficient (Figure 4a-b). To investigate whether the decrease in miR-1258 and miR-622 expression was caused by overexpression of hsa_circ_0101432, we compulsively overexpressed hsa_circ_0101432 in vitro and found the expression of miR-1258 and miR-622 was inhibited (Supplementary Figure 1A-B). Pull down assay verified the target relation between hsa_circ_0101432 and miR-1258 and between hsa_circ_0101432 and miR-622 (Figure 4c). The expressions of miR-1258 and miR-622 in Huh-7 and HepG2 cells were increased when transfected with si-circ-1 and si-circ-2 (Figure 4d-e). In conclusion, hsa_circ_0101432 could bind miR-1258 and miR-622, and inhibit their expression in HCC cells.10.1080/15384101.2019.1618120-F0003 Figure 3. Deregulated miRNAs in HCC patients. (a) Hsa_circ_0101432 shared binding sites with miR-1258, miR-326, miR-622 and miR-1296 by Circular RNA Interactome (https://circinteractome.nia.nih.gov/). (b) MiR-1258 and miR-622 were distinctly downregulated in HCC tissues compared to that in the matched non-tumor tissues. P < 0.05, ns: no significant difference, compared with matched non-tumor tissues. 10.1080/15384101.2019.1618120-F0004 Figure 4. Hsa_circ_0101432 bound miR-1258 and miR-622 in HCC cells. (a) There was a negative correlation between miR-1268 and hsa_circ_0101432 expression level. Spearman’s rank correlation coefficient rho = −0.8206 (P < 0.0001). (b) There was a negative correlation between miR-622 and hsa_circ_0101432 expression level. Spearman’s rank correlation coefficient rho = −0.7768 (P < 0.0001). (c) Biotinylated miRNA pull-down assay was used to confirm the binding between hsa_circ_0101432/miR-1258 and hsa_circ_0101432/miR-622. The bond hsa_circ_0101432 expression level was detected by qRT-PCR. (d-e) MiR-1258 and miR-622 expressions were upregulated by hsa_circ_0101432 inhibition in Huh-7 cells and HepG2 cells. Similar results were obtained in three independent experiments. P < 0.05, compared with the Input group or the NC group. MAPK1 was a direct target both of miR-1258 and miR-622 mRNAs related to HCC and targeted miR-1258 and miR-622 were analyzed by DigSee and miRanda, and then narrowed down to three intersection genes (Figure 5a-b). Dual luciferase reporter assays confirmed that the luciferase activity of the MAPK1-WT was inhibited by miR-1258 or miR-622 mimics, while the luciferase activity of the MAPK1-MUT remained unchanged (Figure 5c-d). The results of biotinylated miRNA pull-down assay indicated that miR-1258 and miR-622 could directly bind to MAPK1 mRNA (Figure 5e). MiR-1258 and miR-622 mimics or inhibitors were transfected into Huh-7 and HepG2 cell lines, respectively. The expression of miR-1258 and miR-622 was promoted by mimics and inhibited by mimics (Figure 6a-b). But it was confirmed that miR-1258 or miR-622 could not regulate hsa_circ_0101432 expression in HCC cells (Supplementary Figure 2A-B). Expression of MAPK1 mRNA was downregulated by miR-1258 and miR-622 mimics but upregulated by inhibitors of miR-1258 and miR-622 (Figure 6c-d). These results revealed that miR-1258 and miR-622 could target MAPK1 and decrease its expression.10.1080/15384101.2019.1618120-F0005 Figure 5. MAPK1 was a direct target both of miR-1258 and miR-622. (a) MRNAs associated with HCC as well as binding both miR-1258 and miR-622, were predicted by DigSee and miRanda. (b) The targeted mRNAs were narrowed down to three using Venny. (c-d) MiR-1258 and miR-622 significantly inhibited the luciferase activity of the MAPK1 wild type 3’-UTR but not that of the mutant. (e) Biotinylated miRNA pull-down assay was used to confirm the binding between miR-1258/MAPK1 and miR-622/MAPK1. The bond MAPK1 expression level was detected by qRT-PCR. Similar results were obtained in three independent experiments. P < 0.05, compared with NC group. 10.1080/15384101.2019.1618120-F0006 Figure 6. MiR-1258 and miR-622could regulated MAPK1 expression. (a) The expressions of miR-1258 were examined through qRT-PCR in Huh-7 and HepG2 cell lines. (b) The expressions of miR-622 were decreased by miR-622 inhibitor and increased by miR-622 mimics. (c-d) MiR-1258 and miR-622 mimics could upregulate MAPK1 expression level in vitro while the miR-1258 and miR-622 inhibitor had the opposite effects. Similar results were obtained in three independent experiments. P < 0.05, compared with NC group. Silencing hsa_circ_0101432 or transfection of miR-1258 and miR-622 suppressed HCC progression in vitro MAPK1 mRNA expression was inhibited when hsa_circ_0101432 was silenced in Huh-7 and HepG2 cells (Figure 7a-b). Furthermore, CCK-8 assay, clone formation assay, flow cytometry assay and transwell assay were conducted to reveal the roles of hsa_circ_0101432, miR-1258 and miR-622 in proliferation, apoptosis and invasive ability of HCC cells. Cell proliferation was inhibited by si-circ-1, si-circ-2, miR-1258 and miR-622 mimics (Figure 7c-d). Clone cells were also reduced through transforming has_circ_0101432 siRNAs, miR-1258 and miR-622 mimics to Huh-7 cells (Figure 7e). Cell apoptosis ratios in si-circ-1 group, si-circ-2 group, miR-1258 mimics group and miR-622 mimics group were remarkably higher than those in NC group, si-circ+miR-1258 inhibitor group and si-circ+miR-622 inhibitor group (Figure 8a). On the contrary, hsa_circ_0101432 siRNAs, miR-1258 mimics or miR-622 mimics inhibited cell invasive ability (Figure 8b). It could be deduced that knockdown expression of hsa_circ_0101432 or overexpression of miR-1258 and miR-622 could inhibit cells proliferation, invasive ability and promote cell apoptosis of HCC.10.1080/15384101.2019.1618120-F0007 Figure 7. Hsa_circ_0101432 inhibition, miR-1258 and miR-622 mimics suppressed HCC cell proliferation and colony cloning. (a-b) Hsa_circ_0101432 was silenced by its siRNAs in Huh-7 and HepG2 cells. The mRNA expression level of MAPK1 was measured by qRT-PCR. (c-d) Hsa_circ_0101432 knockdown and miR-1258, miR-622 overexpression inhibited proliferation. (e) The number of clone cells was reduced through transforming si-circRNAs, miR-1258 mimics and miR-622 mimics to Huh-7 cells. Similar results were obtained in three independent experiments. P < 0.05, compared with NC group. 10.1080/15384101.2019.1618120-F0008 Figure 8. Hsa_circ_0101432 inhibition, miR-1258 and miR-622 mimics promoted cell apoptosis and suppressed cell invasion. (a) The apoptosis ratios of Huh-7 cells were measured by FCM assay. (b) The invasion of Huh-7 cells was measured by transwell assay. Similar results were obtained in three independent experiments. P < 0.05, compared with the NC group. The reduction of hsa_circ_0101432 inhibited tumor growth in vivo After injecting Huh-7 cells stably transfected by negative control, si-circ, miR-1258 mimics and miR-622 mimics for 4 weeks, the nude mice were sacrificed to harvest tumor tissues (Figure 9a). The tumor weight was lighter and tumor volume got smaller in si-circ-1 group, si-circ-2 group, agomir miR-1258 group and agomir miR-622 group than those in the NC group (Figure 9a-b). siRNAs of hsa_circ_0101432 inhibited hsa_circ_0101432 expression while miR-1258 and miR-622 agomirs had no effect on hsa_circ_0101432 expression (Figure 9c). In the tumor tissues of si-circ-1 group, si-circ-2 group, agomir miR-1258 group and agomir miR-622 group, expressions of MAPK1 mRNA were reduced but those of miR-1258 and miR-622 were increased (Figure 9d-f). Western blot also verified that protein expression of MAPK1 was inhibited by hsa_circ_0101432 siRNAs, miR-1258 and miR-622 mimics (Figure 9f). To sum up, HCC tumor growth could be repressed through silencing hsa_circ_0101432.10.1080/15384101.2019.1618120-F0009 Figure 9. Hsa_circ_0101432 overexpression promoted tumor growth in vivo. (a) The nude mice were sacrificed after four weeks and there were the solid tumors and weight of these tumor tissues. (b) Tumor volume of nude mice was measured every 7 days. (c-f) The qRT-PCR was done to detect hsa_circ_0101432, miR-1258, miR-622 and MAPK1 mRNA expression levels. (f) MAPK1 protein expression level was measured by Western Blot. *P < 0.05, compared with NC group. Discussion In this study, has_circ_0101432 and its target miRNAs were selected by the dysregulated microarray analysis and common database, and at the same time, miR-1258 as well as miR-622 was screened out for further study. MAPK1 was selected as the research gene in HCC by STRING and Venny 2.1.0. Has_circ_0101432 and MAPK1 mRNA were confirmed to be overexpressed in HCC cell lines and tissue samples by qRT-PCR. In vitro, the results demonstrated that has_circ_0101432 not only promoted cell proliferation and invasion ability of HCC, but also suppressed its apoptosis. Furthermore, it was also verified in vivo that has_circ_0101432 promoted the process of HCC by adsorbing miR-1258 and miR-622. In the present study, hsa_circ_0101432 was overexpressed. What is more, it accelerated cell proliferation, colony formation and cell invasive ability while inhibited cell apoptosis by targeting miR-1258 and miR-622 in cell lines and tissue samples of HCC. Although hsa_circ_0101432 was barely studied in the past, growing evidence clarified the functions of different circRNAs in HCC [24]. Consistently with our study, Li et al. confirmed that hsa_circ_0005075 was overexpressed in tissue samples and cell lines of HCC, which increased numbers of proliferative, migration and invasive ability of SMMC-7721 cells through inhibiting miR-431 [25]. In the study of Jiang et al., expression of hsa_circ_0000673 was remarkably high in HCC tissues. Knockout hsa_circ_0000673 could significantly inhibit cells proliferation and invasive ability of HCC and tumor growth, which was confirmed by the result of in vivo [26]. On the contrary, downregulation of some circRNAs predicted a poor prognosis. Zhang et al. demonstrated that hsa_circ_0001649 suppressed cells proliferation, migration and invasive capability of HCC whereas it promoted the apoptosis of HCC cells [27]. Moreover, cell experiment results indicated that knocking down the miR-1258 and miR-622 would lead to the enhancement of proliferation, colony formation and invasiveness in HCC cells and the decline of apoptosis ratio via upregulation of MAPK1. Other studies received the same relationship between the miRNA and MAPK1 mRNA. MiR-22 affected cancers of the bladder epithelial and snails by inhibiting MAPK1/Slug/vimentin feedback loop-mesenchymal transition [28]. Hu et al. shed light on the role of miR-585 in targeting MAPK1 and inhibiting gastric cancer cell proliferation and migration [29]. Furthermore, Chang et al. applied a series of assays to witness that miR-143 might suppress the proliferation, migration and invasive ability of Endometrial cancer (EC) cells while promote the EC cells apoptosis by decreasing MAPK1 [30]. MAPK1 was regarded as a tumor promoter because of its role in tumorigenesis [19]. In study of Zou et al., MAPK1 mutation was associated with the tumorigenesis of ovarian mixed germ cell tumors [22]. In addition, downregulation of MAPK1 by miR-378 displayed a remarkable reduction in prostate cancer tumor growth [31]. As for HCC, Kong et al. elucidated that miR-194-5p targeted and suppressed the expression of MAPK1 to inhibit tumor progression [23]. These researches provided a mighty reference for the role of our study of MAPK1 in HCC. However, some limitations were inevitable in our research. For example, in the current study, we just investigated the functions of hsa_circ_0101432, miR-1258 and miR-622 in HCC, while the signaling pathway related MAPK1 in HCC should be taken into account, which can expand the information and understanding of molecule mechanisms. Because HCC was known to be resistant to conventional therapies [32], influence of si-circ_0101432, miR-1258 and miR-622 is still needed to recheck. In summary, our finding acknowledged the overexpression of hsa_circ_0101432 in HCC. Additionally, hsa_circ_0101432 enhanced the expression of MAPK1 through sponging miR-1258 and miR-622, eventually promoted HCC progression. Experiments in vitro indicated that silencing hsa_circ_0101432 or transfection of miR-1258 and miR-622 mimics suppressed cell proliferation, colony cloning as well as invasive ability of HCC and promoted cell apoptosis, while in vivo experiments demonstrated that the reduction of hsa_circ_0101432 inhibited HCC tumor growth. Our results may provide us with new diagnostic and therapeutic targets of HCC in clinical practice. Disclosure statement No potential conflict of interest was reported by the authors. Ethics approval This article does not contain any studies with human participants or animals performed by any of the authors. Supplementary material: Supplemental data for this article can be accessed here. 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==== Front Cell Cycle Cell Cycle KCCY kccy20 Cell Cycle 1538-4101 1551-4005 Taylor & Francis 31537148 1667707 10.1080/15384101.2019.1667707 Research Paper LncRNA UCA1 affects epithelial-mesenchymal transition, invasion, migration and apoptosis of nasopharyngeal carcinoma cells R. HAN ET AL. CELL CYCLE Han Ri a Chen Shunjin b Wang Jianqi c Zhao Yunteng a Li Gang a a Department of Otolaryngology-Head & Neck Surgery, Nanfang Hospital, Southern Medical University, Guangzhou, PR. China b Department of Otolaryngology, Dongguan People’s Hospital, Dongguan, PR. China c Department of Otolaryngology, The Third Affiliated Hospital of Southern Medical University, Guangzhou, PR. China CONTACT Gang Li [email protected] 2019 19 9 2019 18 21 30443053 9 8 2019 3 9 2019 6 9 2019 © 2019 Informa UK Limited, trading as Taylor & Francis Group 2019 Informa UK Limited, trading as Taylor & Francis Group ABSTRACT Objective: In this study, long non-coding RNA urothelial carcinoma associated 1 (lncRNA UCA1) in nasopharyngeal carcinoma (NPC) and its effect on the malignant phenotype of NPC cells was investigated. Methods: Initially, the expression of UCA1 in NPC tissues and cells was detected. NPC cell line that with highest expression of UCA1 was selected for subsequent cell function test. A series of experiments were used to detect proliferation, colony formation, cell cycle distribution, apoptosis, invasion and migration of NPC cells with the interference of UCA1 expression. Western blot analysis was carried out to detect the expression of E-cadherin and vimentin for verifying the effect of UCA1 on epithelial mesenchymal transition (EMT). Results: The expression of UCA1 was found to be upregulated in NPC tissues and cells. The expression of UCA1 in stage Ⅲ + IV of NPC tissues and in patients with lymph node metastasis was significantly higher than that in patients at stage Ⅰ + Ⅱ and in patients without lymph node metastasis. Inhibition of UCA1 repressed proliferation, EMT, colony formation, invasion and migration while stimulating apoptosis of NPC cells. Conclusion: Our study suggests that UCA1 expression was overexpressed in NPC. Additionally, UCA1 suppression could inhibit proliferation, EMT, invasion and migration, and promote apoptosis of NPC cells. KEYWORDS Nasopharyngeal carcinoma UCA1 Colony formation Natural Science Foundation of Guangdong Province10.13039/5011000034532015A030313237 Social Science and Technology Development Project of Dongguan201750715001465 National Natural Science Foundation81472534 This work was supported by the Natural Science Foundation of Guangdong Province [2015A030313237]; Social Science and Technology Development Project of Dongguan [201750715001465]; National Natural Science Foundation [81472534]. ==== Body Introduction Nasopharyngeal carcinoma (NPC) is known as one of the most frequent malignant head and neck tumors, which arises in the surface epithelium of nasopharynx [1–3]. According to the statistics, high incidences of NPC are found in Southeast Asia and southern China [4], particularly in the Cantonese region of Guangzhou, which contributing to serious healthcare problems [5]. Referring to the World Health Organization (WHO) classification in 1991, NPC is grouped into two main histological subtypes: keratinizing squamous cell carcinoma (KSCC) and non-KSCC [6]. NPC is a multifactorial disease that is caused by complicated etiological factors, such as genetic predisposition, Epstein-Barr virus (EBV) infection as well as other environmental risk factors [7]. The treatment with radiotherapy and chemotherapy is suggested to control the primary tumor temporarily, while most of patients present radioresistance and chemoresistance, which remains the major obstacles for patient’s survival of NPC [8,9]. Despite great advances in the treatment of NPC, no obvious improvements have been found in the overall survival because of local or regional failure and recurrence [10]. Therefore, it is of great importance to identify more accurate predictive biomarkers and seek for the molecular mechanisms of NPC to further understand NPC cell biology. Long non-coding RNAs (lncRNAs) are considered as new regulators of different biological functions, which playing an essential role in both oncogenesis and tumor progression [11,12]. A growing number of evidence have suggested that lncRNAs are of great importance in various biological processes, such as X chromosome inactivation, regulation of gene expression, post-transcriptional modification, chromatin remodeling as well as translational control [13–17]. Urothelial carcinoma-associated 1 (UCA1) is a member of lncRNA family with three exons, which encodes a 1.4 kb isoform and a 2.2 kb isoform [18]. As previously reported, UCA1 is remarkably upregulated in numerous cancers, such as hepatocellular carcinoma, colorectal cancer, gastric cancer, endometrial cancer and medulloblastoma, indicating that UCA1 could function as a vital oncogene in human cancers [19–23]. In view of this, we could speculate that lncRNA UCA1 may exert its function in the progression and metastasis of cancers, while the role of UCA1 in NPC and its underlying mechanism remains undiscovered. Therefore, the purpose of this study is to figure out the expression of lncRNA UCA1 in NPC and its effect on the malignant phenotype of NPC cells. Materials and methods Ethical statement This study was approved by the ethics committee of Nanfang Hospital, Southern Medical University. All the subjects signed the informed consent. Study subjects A total of 68 patients with NPC (40 males and 28 females, with an average age of 45.3 ± 11.5 years) diagnosed and treated in Nanfang Hospital, Southern Medical University from October 2014 to October 2016 were selected for experiment. The NPC tissues and adjacent normal tissues (over 5 cm away from the tumor margin) were selected from NPC patients after surgical resection. The inclusion criteria were as follows: patients were diagnosed with NPC by histology or cytology; patients had not receive preoperative radiotherapy, chemotherapy and other adjuvant treatments. The corresponding exclusion criteria were: patients had distant metastasis or with other malignant tumors. The clinicopathological features of the patients were collected. Cell selection and culture NPC cell lines CNE1, CNE2, HONE1 and C666-1 were obtained from Shanghai Zishi Biotechnology Co., Ltd (Shanghai, China), and cultured in an incubator at 37°C with 5% CO2 in RPMI 1640 medium containing 10% fetal bovine serum (FBS, Gibco, Grand Island, NY, USA). Normal nasopharyngeal epithelial cell line NP69 was purchased from Shanghai Xcess Biotechnology Co., Ltd. (Shanghai, China) and cultured in an incubator at 37°C with 5% CO2 in KMSF medium (Gibco, Grand Island, NY, USA). The solution was changed every 2–3 days. When the cell confluence was between 80% and 90%, the passage culture began. The expression of UCA1 in all cells was detected by quantitative reverse transcription polymerase chain reaction (qRT-PCR), and the cells with the greatest difference from NP69 cells were selected for cell function test. Cell grouping and treatment NPC in logarithmic growth stage was assigned into three groups, namely the blank group and the negative control (NC) group (cells transfected with empty plasmid) and the UCA1 siRNA group (cells transfected with UCA1 siRNA plasmid). The siRNA sequences for UCA1 were si-UCA1-1: 5ʹ-AGUAUGUUGUUUGUUGUUAGA-3ʹ (Sense) and 5ʹ-UAACAACAAACAACAUACUUU-3ʹ (Antisense), si-UCA1-2: 5ʹ-UUAAUCCAGGAGACAAAGATT-3ʹ (Sense) and 5ʹ-UCUUUGUCUCCUGGAUUAATT-3ʹ (Antisense) and si-UCA1-3: 5ʹ-GCACCUUGUUAGCUACAUAAA-3ʹ (Sense) and 5ʹ-UAUGUAGCUAACAAGGUGCCA-3ʹ (Antisense), and the sequences for NC were: 5ʹ-UUCUCCGAACGUGUCACGUTT −3ʹ (Sense) and 5ʹ-ACGUGACACGUUCGGAGAATT-3ʹ (Antisense). The empty plasmid, si-UCA1-1, si-UCA1-2 and si-UCA1-3 plasmids were purchased from Shanghai GenePharma Co., Ltd (Shanghai, China). According to the instructions of Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA), the cells after transfection were cultured in an incubator for 48 hours to detect the expression of UCA1. qRT-PCR The one-step method of Trizol (Invitrogen, Carlsbad, CA, USA) was used to extract the total RNA in cells and tissues, and the high-quality RNA was confirmed by ultraviolet (UV) analysis and the detection of formaldehyde denaturation electrophoresis. cDNA was obtained by avian myeloblastosis virus (AMV) reverse transcriptase (ThermoFisher Scientific, Massachusetts, USA) after the acquirement of l μg RNA. Design and synthesis of PCR primer was designed and synthesized by Invitrogen, Carlsbad, CA, USA (Table 1). Glyceraldehyde phosphate dehydrogenase (GAPDH) was used as an internal control (U6 as an internal control of the nuclear RNA). The PCR amplification conditions were as follows: pre-denaturation at 94°C for 5 min, a total of 40 cycles of denaturation at 94°C for 40 s, annealing at 60°C for 1 min, extension at 72°C for 1 min and finally, extension at 72°C for 10 min. The product was verified by agarose gel electrophoresis. The threshold cycle (Ct) value of each reaction tube was obtained by manually selecting the threshold at the lowest point of parallel rise of each logarithmic expansion curve. 2−ΔΔCt method [24] was used to analyze the ratio relation of target gene expression between the experimental group and the control group. The formula is as follows: ΔΔCt = [Ct (target gene) – Ct (internal control gene)] the experimental group – [Ct (target gene) – Ct (internal control gene)] the control group. The experiment was repeated for 3 times to obtain the average value.10.1080/15384101.2019.1667707-T0001 Table 1. Primer sequence. Gene Sequence UCA1 F: 5ʹ-CATCGCGACCCTACATTAAAGCTAAT −3’ R: 5ʹ- GCTTCAAGTGTGACCAGGGACT −3’ GAPDH F: 5ʹ- TGGGTGTGAACCATGAGAAG-3’ R: 5ʹ- GTGTCGCTGTTGAAGTCAGA-3’ Note: UCA1, urothelial carcinoma associated 1; GAPDH, glyceraldehyde phosphate dehydrogenase. Western blot analysis The proteins from cells in each group were extracted and the protein concentrations were determined according to the instructions of the bicinchoninic acid (BCA) assay (Wuhan Boster Biological Technology LTD, Wuhan, China). The extracted protein was added to the sample buffer and then boiled at 95°C for 10 min, with each well for 30 μg protein. Following 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (Wuhan Boster Biological Technology LTD, Wuhan, China), protein samples were transferred to a nitrocellulose (NC) membrane using the wet transfer method, with the electrophoretic voltage from 80 v to 120 v, the trarsmembrane voltage of 100 mv and the time for 45–70 min. Subsequently, the protein samples were transferred to polyvinylidene fluoride (PVDF) membrane and blocked with 5% bovine serum albumin (BSA). Afterward, the membranes were added with the primary antibodies of E-cadherin (1: 1000; ab15148), Vimentin (1: 1000; ab137321) and β-actin (1: 3000; ab227387) (all from Abcam, Cambridge, MA, USA) and incubated at 4°C overnight. The membranes were rinsed with Tris-buffered saline and Tween 20 (TBST) for 3 times, each time for 5 min, and the corresponding secondary antibodies (Shanghai Miao Tong Biotechnology Company, Shanghai, China) were incubated at room temperature for 1 h to wash the membranes for 3 times, each time for 5 min, and an electrogenerated chemiluminescence (ECL) solution was used for developing. β-actin was regarded as an internal control. Gel Doc EZ formatter (Bio-rad, California, USA) was used for developing. The gray value analysis of target band was analyzed by Image J software (National Institutes of Health, Bethesda, Maryland, USA). The experiment was repeated for three time to obtain the average value. Cell counting kit-8 (CCK-8) assay The cell suspensions of each group were diluted with a certain concentration and then inoculated into 96-well plates at the density of 1 × 103 cells/100 μL/per well. Each group was set for 15 parallel wells. The cells were classified into 5 groups according to the culture time of 0 h, 24 h, 48 h, 72 h and 96 h, and each group was set 3 multiple wells. CCK-8 solution (Sigma, St. Louis, MO, USA) was added to the cell-free medium as a blank control, and the culture plate was cultured at 37°C and 5% CO2. At each time point, 10 μL CCK-8 solution was added to the corresponding well and incubated in an incubator for 4 hours. The optical density (OD) value of each well was measured at the wavelength of 450 nm by a microplate reader (ThermoFisher Scientific, Massachusetts, USA). Colony formation assay The detached cells were fully dispersed and inoculated with 200 cells into 6-well plates, and the culture plate was gently shaking so that the cells were dispersed evenly and cultured for 2 ~ 3 weeks. When the cell colony was visible to the naked eye, the culture was terminated, the culture solution was abandoned, and washed with the cells with phosphate buffer saline (PBS) and fixed with 4% paraformaldehyde for 30 min. After that, the cells were washed with PBS for 3 times, stained with Giemsa application solution (Beyotime Biotechnology, Shanghai, China) for 60 min, then washed out slowly by water, and dried in air. The number of cell colonies were counted under a microscope. Flow cytometry The detached cells in each group were collected and centrifuged at 1000 rpm for 5 min, then the supernatant was abandoned. Subsequently, the cells were suspended and washed with PBS to make the cell concentration adjusted to 1 × 106 cells/mL, thus the single cell suspension was prepared. The single cell suspension was centrifuged for 5 min, and the supernatant was removed. The volume fraction of about 70% ethanol (500 μL) was added to each group, and then fixed for 2 hours at 4°C until overnight. The fixative solution was discarded, and 1 mL PBS was supplemented to further eluate fixation solution, centrifuged at 2000 rpm for 3min, with the supernatant removed. Afterward, 100 μL RNase A (Sigma, St. Louis, MO, USA) was added to the cells at 37°C for 30 min, then 400 μL PI (Sigma, St. Louis, MO, USA) was added to the cells and mixed at 4°C for 30 min devoid of light. The red fluorescence at the excitation wavelength of 488 nm was recorded. The flow cytometer (BD, New Jersey, USA, USA) was used for cell cycle distribution detection. After the cells in logarithmic growth phase were detached, the suspension cells were mixed, centrifuged to collect the cells, with the supernatant discarded. The cells were washed with 4°C pre-cooled PBS for 5 min, and this step was repeated for two times. The cell concentration was adjusted to 106 cells/mL, and 200 μL of the cells were washed with 1 mL pre-cooled PBS for twice and then centrifuged. The cells were suspended in 100 μL binding buffer and added with 2 μL Annexin-V-FITC (20 μg/mL, Sigma, St. Louis, MO, USA), gently mixed, and placed on the ice for 15 min. Next, the cells were turned to the flow detection tube, added with 300 μL PBS, and also added with 1 μLPI (50 μg/mL, Sigma, St. Louis, MO, USA) to each sample before going on the machine, and the detection was lasted for 30 min. The criteria for determine the results were as follows: Annexin V was regarded as horizontal axis, and PI as longitudinal axis; left upper quadrant as mechanically injured cells, right upper quadrant as late apoptotic cells or necrotic cells, left lower quadrant as negative normal cells and right lower quadrant as early apoptotic cells. Transwell assay After 48 hours of transfection, the cells of each group were detached, and each Transwell chamber (Corning, Lowell, MA, USA) was spread 1:8 Matrigel (80 μL) and seeded with 1 × 105 cells. Subsequently, the cells were added with 100 μL serum-free RPMI 1640 medium, added with complete culture medium in the basolateral chamber and incubated for 24 hours. After that, the cells in the apical chamber were erased with cotton swabs, fixed with 4% paraformaldehyde for 15 min, and stained with crystal violet staining solution for 10 min. Under a microscope, 5 visual fields were selected for photographing and counting. Scratch test At the back of the 6-well plate, the marker pen was used to draw a uniform horizontal line against the ruler about every 0.8 cm or so, crossing the well. Each well passed through at least 5 lines. When about 5 × 105 cells were added into each well, the cell confluence reached 100%. The next day, a 10 μL gun head was perpendicular to the back of the horizontal line against the ruler scratch, and the gun head was vertical and could not be tilted. After scratching, the cells were gently washed with PBS for 3 times, then gently adhered to the wall and added with PBS. The cells were washed and removed, then added to the culture medium and cultured in a CO2 incubator at 37°C. At the time point of 0 h and 24 h, the sampling was performed so as to take pictures under an inverted microscope. The wound healing area was calculated with National instrument Vision Assistant 8.6 software (Texas, Austin, USA). Cell migration rate = wound healing area/initial scratch wound area × 100. The experiment was repeated three times to obtain the average value. Statistical analysis The data were analyzed by SPSS21.0 software (SPSS, Inc, Chicago, IL, USA). The data were normally distributed by Kolmogorov-Smirnov test. The results were expressed as mean ± standard deviation. The comparison between the two groups was performed by t test. One-way analysis of variance (ANOVA) was used in comparison among multiple groups. After ANOVA analysis, the Fisher’s least significant difference t test (LSD-t) was utilized for pairwise comparison. All tests were bilateral, and the significant difference was defined as P < 0.05. Results Lncrna UCA1 is highly expressed in NPC tissues and NPC cells The expression of UCA1 in NPC tissues and their adjacent normal tissues was detected by qRT-PCR. The results showed that the expression of UCA1 in NPC tissues was significantly higher than that in adjacent normal tissues (P < 0.01; Figure 1a).10.1080/15384101.2019.1667707-F0001 Figure 1. Expression level of UCA1 in nasopharyngeal carcinoma tissues and nasopharyngeal carcinoma cells. Note: A. qRT-PCR was used to detect the expression of UCA1 in nasopharyngeal carcinoma tissues and adjacent normal tissues; t test was used to analyze the data; N = 68; , P < 0.01 vs. adjacent normal tissues; B. The expression of UCA1 in nasopharyngeal carcinoma cells and normal nasopharyngeal epithelial cells was detected by qRT-PCR; , P < 0.01 vs. NP69 cells; C. The expression level of UCA1 in CNE2 cells was detected by qRT-PCR; *, P < 0.01 vs. the control group; One-way ANOVA was used in comparison among multiple groups. After ANOVA analysis, the LSD-t was utilized for pairwise comparison; the experiment was independently repeated for three times. The expression level of UCA1 in CNE1, CNE2, HONE1 and C666-1 cells was significantly higher than that in normal nasopharyngeal epithelial cells NP69 (all P < 0.01), and the UCA1 expression level in CNE2 cells was the highest, so CNE2 cells were selected to perform functional tests (Figure 1b). The results of qRT-PCR indicated that in CNE2 cells, the expression of UCA1 in the cells of the si-UCA1-1, si-UCA1-2 and si-UCA1-3 groups was significantly lower than that in the blank group and the NC group (all P < 0.01; Figure 1c), suggesting CNE2 cells with low expression of UCA1 were successfully constructed. Among them, si-UCA1-3 was superior to si-UCA1-1 and si-UCA1-2, so si-UCA1-3 was selected for subsequent experiments, which was named as UCA1 siRNA. Expression of UCA1 is related to clinical stage and lymph node metastasis of NPC The relationship between the expression of UCA1 and the clinicopathological features of NPC was analyzed. The results demonstrated that the expression of UCA1 in patients with stage III-IV in NPC tissues was significantly higher than that in stage I-II(P < 0.05), and the expression of UCA1 in NPC tissues with lymph node metastasis was significantly higher than that in patients without lymph node metastasis (P < 0.05). There was no significant correlation between expression of UCA1 with the gender and T stage of NPC patients (both P > 0.05; Table 2).10.1080/15384101.2019.1667707-T0002 Table 2. Relationship between expression of UCA1 and clinicopathological characteristics of nasopharyngeal carcinoma. Clinicopathological characteristic Case UCA1 expression t/F P Age (years) 1.00 0.323 ≤ 50 43 4.39 ± 0.34 > 50 25 4.44 ± 0.29 Gender 1.67 0.100 Male 40 4.46 ± 0.32 Female 28 4.33 ± 0.31 T stage 1.29 0.202 T1 + T2 35 4.36 ± 0.31 T3 + T4 33 4.46 ± 0.33 Clinical stage 2.44 0.018 Ⅰ + Ⅱ 24 4.29 ± 0.32 Ⅲ + IV 44 4.48 ± 0.30 Lymph node metastasis 2.92 0.005 With 41 4.50 ± 0.30 Without 27 4.28 ± 0.31 Note: The date were analyzed by the unpaired t test. Inhibition of UCA1 inhibits proliferation and colony formation of NPC cells The results of MTT assay showed that there was no significant difference in proliferation of NPC cells in the NC group compared with the blank group (P > 0.05), but the proliferation rate of the UCA1 siRNA group was significantly slower than that of the NC group, and the proliferation rate was decreased significantly (P < 0.05; Figure 2a).10.1080/15384101.2019.1667707-F0002 Figure 2. Effect of down-regulation of UCA1 on the proliferation and colony formation of nasopharyngeal carcinoma cells. Note: A. MTT assay for the proliferation of CNE2 cells in each group; B. Detection of colony number of CNE2 cells in each group by clone forming experiment; , P < 0.01 vs. the blank group; One-way ANOVA was used in comparison among multiple groups. After ANOVA analysis, the LSD-t was utilized for pairwise comparison; the experiment was independently repeated for three times. Colony formation assay was used to detect the change of cell colony formation ability in each group. The results suggested that there was no significant difference in cell colony number between the blank and the NC groups (P > 0.05), but the colony number of the UCA1 siRNA group was significantly lower than that of the NC group (P < 0.05; Figure 2b). It suggested that down-regulation of UCA1 could inhibit the proliferation and colony formation of NPC cells. Inhibition of UCA1 inhibits cell cycle progression and promotes apoptosis of NPC cells The cell cycle distribution of each group was detected by flow cytometry. The results showed that there was no significant difference in the proportion of G0/G1, S and G2/M cells between the blank and the NC group (P > 0.05). Compared with the blank group and the NC group, the cells in G0/G1 phase in the UCA1 siRNA group were significantly increased, and the number of cells in S phase and G2/M phase decreased significantly (all P < 0.05; Figure 3a). Based on which, we could conclude that down-regulation of UCA1 expression can inhibit cell cycle progression and arrested cells at G0/G1 phase in NPC cells.10.1080/15384101.2019.1667707-F0003 Figure 3. Effect of down-regulation of UCA1 on cell cycle distribution and apoptosis of nasopharyngeal carcinoma cells. Note: A. Flow cytometry was used to detect the cell cycle distribution of CNE2 in each group; B. Detection of apoptosis rate of CNE2 cells in each group by flow cytometry; , P < 0.01 vs. the blank group; One-way ANOVA was used in comparison among multiple groups. After ANOVA analysis, the LSD-t was utilized for pairwise comparison; the experiment was independently repeated for three times. The results of flow cytometry showed that there was no significant difference in apoptosis rate between the blank and the NC groups (P > 0.05), but the apoptosis rate of the UCA1 siRNA group was significantly higher than that of the blank group and NC group (both P < 0.05; Figure 3b). These results suggest that inhibiting the expression of UCA 1 can promote the apoptosis of NPC cells. Inhibition of UCA1 inhibits epithelial-mesenchymal transition (EMT), invasion and migration of NPC cells The expressions of E-cadherin and vimentin were detected by western blot analysis. The results revealed that compared with the blank group, the expression of E-cadherin and Vimentin were not significantly changed in the NC group (both P > 0.05), the expression of E-cadherin increased significantly and the expression of Vimentin decreased significantly in the UCA1 siRNA group (both P < 0.05; Figure 4a).10.1080/15384101.2019.1667707-F0004 Figure 4. Effect of down-regulation of UCA1 on epithelial-mesenchymal transition, invasion and migration in nasopharyngeal carcinoma cells. Note: A. The expression level of epithelial interstitial marker protein in CNE2 cells in each group was detected by western blot analysis; B. The invasion number of CNE2 cells in each group was detected by Transwell assay (× 400); C. Detection of migration rate of CNE2 cells in each group by scratch test; , P < 0.01 vs. the blank group; One-way ANOVA was used in comparison among multiple groups. After ANOVA analysis, the LSD-t was utilized for pairwise comparison; the experiment was independently repeated for three times. The results of Transwell assay showed that there was no significant difference in the number of cell invasion in the NC group compared with the blank group (P > 0.05), and the number of cell invasion in the UCA1 siRNA group was significantly decreased in contrast to the blank group and the NC group (both P < 0.05; Figure 4b). The results of scratch test indicated that compared with the blank group and the NC group, the migration ability of cells in the UCA1 siRNA group decreased significantly and the migration rate was also decreased significantly (both P < 0.05; Figure 4c). These results suggest that inhibition of UCA1 can inhibit EMT, invasion and migration of NPC cells. Discussion Emerging data strongly suggests lncRNAs in the basic regulation of protein-coding genes, which at both the transcriptional and the posttranscriptional levels, are central to normal development and oncogenesis [25–28]. Thus, differential expression of lncRNAs may contribute to cancer diagnosis and prognosis as well as select potential therapeutics. More and more evidence has been recently revealed the function of UCA1 as possessing the oncogenic roles in tumorigenesis [2,29,30]. Therefore, we tried to understand the role of lncRNA UCA1 in the occurrence and development of NPC. The results of this study showed that down-regulation of UCA1 could inhibit EMT, invasion and migration, and promote apoptosis of NPC cells, implying that UCA1 functions as an oncogene in NPC, which might be a potential biological target of NPC therapy. In the present study, we determined the expression of UCA1 in NPC tissues and their adjacent normal tissues by qRT-PCR, and the results showed that the expression of UCA1 in NPC tissues was significantly higher than that in adjacent normal tissues. Additionally, we also found that the expression level of UCA1 in CNE1, CNE2, HONE1 and C666-1 cells was significantly higher than that in normal nasopharyngeal epithelial cells NP69. In accordance with the results in our study, Zheng et al. [31] and Gao et al. [32] also proposed that UCA1 was upregulated in both gastric cancer tissues and cell lines in contrast to that in normal control tissues. Besides, the expression of UCA1 in non-small cell lung carcinoma tissues and oral squamous cell carcinoma tissues and cells was significantly higher than that in pairmatched adjacent nontumourous tissues and cells [33,34]. Taken together, these findings supported our previous hypothesis that UCA1 might function as an oncogene in the progression of NPC. Subsequently, the relationship between the expression of UCA1 and the clinicopathological features of NPC was analyzed, and the corresponding results revealed that the expression of UCA1 was associated with clinical stage and lymph node metastasis. A previous study suggested that UCA1 expression was related to TNM stage and lymph node metastases of gastric cancer [35,36]. Additionally, the UCA1 expression was found to be closely related to TNM stage and tumor differentiation, and the overexpression of UCA1 implying poor prognosis [12]. Based on which, UCA1 is therefore regarded as a potential oncogene that could play a pivotal regulatory part in the clinical progression of NPC. In accordance with our results, Luo et al. found that downregulation of UCA1 was observed to be negatively related to cell invasion ability and inhibited EMT of bladder cancer cells [37]. Gain and loss of function analysis showed that siRNA-UCA1 inhibited invasion and migration and promote apoptosis of NPC cells. Notably, some articles have demonstrated that the depletion of lncRNA UCA1 can attenuate the cell migration and invasion ability of bladder cancer, esophageal squamous cell carcinoma and ovarian cancer [38–40], suggesting that lncRNA UCA1 could act as a vital regulator of migration and invasion of cells. EMT is a well-characterized process, which could facilitate both the invasion and metastatic dissemination of cancers [41,42]. Therefore, we further discussed whether lncRNA UCA1 could control EMT of NPC cells. The obtained results revealed that the expression of E-cadherin increased significantly and the expression of Vimentin decreased significantly in the UCA1 siRNA group. These data have shown that UCA1 may control cell invasion and migration by promoting EMT in NPC cells In conclusion, this present study demonstrates the functional significance of UCA1 expression in tumorigenesis of NPC, and the results of our study indicated that UCA1 expression was upregulated in NPC tissues and cell lines. Besides, the upregulated UCA1 was related to the clinical stage and lymph node metastases of NPC. Furthermore, the poor expression of UCA1 underlined an inhibitory role in the inhibition of EMT, invasion and migration, and promote apoptosis of NPC cells. Thus, UCA1 acts as a biomarker in clinical application and holds great promise as a new diagnostic and prognostic marker as well as a therapeutic target for NPC. However, further studies which concentrated on the specific mechanisms, such as signaling pathway, of UCA1 on NPC cells were performed to verify our results. Acknowledgments We would like to acknowledge the reviewers for their helpful comments on this paper. This study was supported by National Natural Science Foundation (No: 81472534), Natural Science Foundation of Guangdong Province (No: 2015A030313237), Social Science and Technology Development Project of Dongguan (201750715001465). Disclosure statement No potential conflict of interest was reported by the authors. ==== Refs References [1] Zeng Z , Huang H , Zhang W , et al Nasopharyngeal carcinoma: advances in genomics and molecular genetics. Sci China Life Sci. 2011;54 (10 ):966–975.22038010 [2] Han Y , Yang Y-N , Yuan -H-H , et al UCA1, a long non-coding RNA up-regulated in colorectal cancer influences cell proliferation, apoptosis and cell cycle distribution. Pathology. 2014;46 (5 ):396–401.24977734 [3] Chua MLK , Wee JTS , Hui EP , et al Nasopharyngeal carcinoma. 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Sci Signal. 2014;7 (344 ):re8.25249658	31537148	PMC6791705	NO-CC CODE	2022-03-24 23:15:08	yes	Cell Cycle. 2019 Sep 19; 18(21):304417-3053
==== Front Cell Cycle Cell Cycle KCCY kccy20 Cell Cycle 1538-4101 1551-4005 Taylor & Francis 31564203 1669388 10.1080/15384101.2019.1669388 Research Paper MicroRNA-129-5p alleviates nerve injury and inflammatory response of Alzheimer’s disease via downregulating SOX6 Z. ZENG ET AL. CELL CYCLE Zeng Zhilei a* Liu Yajun b* Zheng Wei a Liu Liubin b Yin Honglei b Zhang Simiao a Bai Hongying a Hua Linlin a Wang Shanshan b Wang Zhen b Li Xiaodong a Xiao Jianhao a Yuan Qian a Wang Yunliang ab a Department of Neurology, The Second Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, China b Department of Neurology, The 960th PLA hospital, Zibo, shandong, China CONTACT Yunliang Wang [email protected] * They are coauthor 2019 29 9 2019 18 22 30953110 25 7 2019 4 9 2019 6 9 2019 © 2019 Informa UK Limited, trading as Taylor & Francis Group 2019 Informa UK Limited, trading as Taylor & Francis Group ABSTRACT There is growing evidence of the position of microRNAs (miRs) in Alzheimer’s disease (AD), thus our objective was to discuss the impact of miR-129-5p regulating nerve injury and inflammatory response in AD rats by modulating SOX6 expression. The AD rat model was established by injecting Aβ25-35 into the brain. The pathological changes, ultrastructure, number of neurons, cell degeneration and apoptosis of hippocampal tissue were observed in vivo. MiR-129-5p, SOX6, IL-1β, TNF-α, Bcl-2 and Bax expression in serum and hippocampal tissues were detected by ELISA, RT-qPCR or western blot analysis. The successfully modeled hippocampal neuronal cells of AD were transfected with miR-129-5p mimic, SOX6-siRNA or their controls to figure out their roles in proliferation, apoptosis and inflammatory reaction in vitro. Low expression of SOX6 and high expression of miR-129-5p in vivo of rats would shorten the escape latent period and increase the times of crossing platforms, alleviate the pathological injury, inhibit neuronal apoptosis and reduce the inflammatory reaction. Up-regulation of miR-129-5p and down-regulation of SOX6 promoted proliferation, suppressed apoptosis and degraded the inflammatory reaction of neuronal cells in vitro. Up-regulation of SOX6 reversed the expression of miR-129-5p to reduce the damage and inflammatory response of the cell model of AD. Our study presents that up-regulation of miR-129-5p or down-regulation of SOX6 can reduce nerve injury and inflammatory response in rats with AD. Thus, miR-129-5p may be a potential candidate for the treatment of AD. KEYWORDS MicroRNA-129-5p SOX6 Interleukin-1β Henan provincial Commission of Health and Family Planning Science and Technology Innovative Talents “51282” ProjectPrepare WeiKe (2016)32) This work was supported by Henan provincial Commission of Health and Family Planning Science and Technology Innovative Talents “51282” Project [Grant no. Prepare WeiKe (2016)32]. ==== Body Introduction Alzheimer disease (AD) is an emblematical neurodegenerative disease with progressive and destructive characteristics [1]. In 2015, almost 47 million people in the world were affected by AD, and it is estimated to be about 55 million by 2030 and 131 million by 2050, with the largest expected growth in low-and middle-income countries [2]. The clinical characteristic of AD is from intermittent memory matters to slow decline of overall cognitive function, making the patient in the end-stage of AD bedridden and dependent on guardianship, dying within an average of 9 years after diagnosis [3]. The etiology and pathogenesis of AD have not been definitely explained [1], and it is a multi-factor disease caused by genetic (70%) and environment (30%) factors [4]. A variety of scales developed to evaluate the function of patients with AD have been included in clinical trials and observational studies to evaluate the effects of treatment or intervention [2]. The acetylcholinesterase inhibitors and memantine are the only drugs approved for AD’s treatment so far, which provide only symptomatic improvement [5], while there is no treatment that can be used to stop or reverse the underlying pathology of identified disease [2]. MicroRNAs (miRNAs) is a class of non-coding RNA with small molecules characteristics (19–25 nucleotides), single-strand and extensive expression noncoding RNA [6]. MiRNAs are considered to negatively modulate gene expression via binding to 3ʹ untranslated regions (UTRs) through translational restraint and destabilization of mRNAs [2]. A study has reported the changes of miRNA expression in cerebrospinal fluid in young patients with AD [2]. Another study also revealed that miR-129-5p can regulate the expression of fragile X mental retardation 1 protein (FMRP), thus ensuring the normal positioning of cerebral cortex neurons [2]. It was proved that up-regulated miR-129-5p could restrain the revascularization of induced intracerebral hemorrhage rats [2]. Sry-related high-mobility box (SOX) gene family is a transcription factor coding gene which possess highly conserved HMG-box sequence [7]. SOX6 is a multifaceted transcription factor participated in terminal differentiation of many cell types in vertebrates [2]. It was reported that SOX transcription factors play a key role in modulating nervous system of embryonic and adult, containing maintenance of the multipotency, renewal and cell fate of neural trunk/progenitor cells [8]. SOX6 gene serves pivotal roles in the nervous system for creating neuronal diversity. Specifically, there existed expression of this gene in neuron progenitor cells in the dorsal telencephalon and induced it to differentiate into interneurons [2]. Thus, the objective of this study was to examine the impact of miR-129-5p/SOX6 axis regulating nerve injury and inflammatory response in AD rats. Materials and methods Ethics statement All animal experiments were in line with the Guide for the Care and Use of Laboratory Animal of the National Institutes of Health. The protocol was permitted by the Committee on the Ethics of Animal Experiments of The Second Affiliated Hospital of Zhengzhou University. Study subjects Ninety specific pathogen free (SPF) male Sprague-Dawley (SD) rats (Shanghai SLAC Laboratory Animal Co., Ltd., Shanghai, China) aging 8 w, weighting between 160 and 200 g, were housed in the SPF experimental animal center. The environment was set at 24 ± 5°C with normal circadian rhythm of water and food intake. Ten neonatal SD rats (Shanghai SLAC Laboratory Animal Co., Ltd., Shanghai, China) aging 1–3 d, were housed in the SPF experimental animal center. The environment was set at 20 – 23°C and a humidity of 45 – 50%, with normal circadian rhythm of water and food intake. Preparation of rat models of AD After the SD rat was anesthetized with 3% pentobarbital sodium (50 mg/kg, Sigma-Aldrich, St. Louis, MO, USA), the head of the rat was fixed, the skull was exposed through operation, the anterior fontanel was the starting point and the puncture point was positioned behind 3–4 mm of the anterior fontanel with 2 mm right transverse incision. Skull was opened with an electric drill prior to the incubated Aβ25-35 (1 μL, 10 μg, Sigma-Aldrich, St. Louis, MO, USA) was injected vertically into the CA1 area of bilateral hippocampus, with pin retracted after 5 min. The control group was injected with saline in the same way. The wound was sutured and partially coated with gentamicin. The postoperative rats had the right of freely moving and eating. Animal grouping Seventy successful modeled SD rats were assigned into 7 groups, with 10 rats in each group: AD group (induced by Aβ), agomir-negative control (NC) group (50 μL of miR-129-5p agonist NC (1 nmol/50 μL, Guangzhou RiboBio Co., Ltd., Guangdong, China) was given by the lateral ventricle under stereotaxis 24 h after successful induction of AD), agomir-miR-129-5p group (50 μL of miR-129-5p agonist (1 nmol/50 μL, Guangzhou RiboBio Co., Ltd., Guangdong, China) was given by the lateral ventricle under stereotaxis 24 h after successful induction of AD), siRNA-NC group (NC of 15 μL downregulated SOX6 vector (Shanghai GenePharma Co. Ltd., Shanghai, China) was given by the lateral ventricle under stereotaxis 24 h after successful induction of AD), SOX6-siRNA group (15 μL downregulated SOX6 vector (Shanghai GenePharma Co. Ltd., Shanghai, China) was given by the lateral ventricle under stereotaxis 24 h after successful induction of AD), agomir-miR-129-5p + overexpressed (OE)-NC group (50 μL of miR-129-5p agonist and 4 μL overexpressed SOX6 vector NC (GeneCopoeia, Guangzhou, China) was given by the lateral ventricle under stereotaxis 24 h after successful induction of AD), agomir-miR-129-5p + OE-SOX6 group (50 μL of miR-129-5p agonist and 4 μL over-expressed SOX6 vector (GeneCopoeia, Guangzhou, China) was given by the lateral ventricle under stereotaxis 24 h after successful induction of AD). Meanwhile, the normal group (only saline was injected into the abdominal cavity, 10 rats) was set as a control. The Morris water maze test was carried out after 2 w of AD, and the blood of the femoral artery was taken prior to the rats of each group were euthanized and hippocampal tissue was taken for correlation detection. Morris water maze test The latency was set at 90 s. If the rat found and stayed on the platform for 5 s during the latent period, the actual latency was recorded. In the place navigation test, a hidden platform was placed in the barrel target quadrant. One of the four quadrants of the barrel plane was selected as the water entry point of the rat, and the rat was gently placed into the water along the bucket wall facing the experimenter. The swimming track of the rat was recorded simultaneously via software. In case the rat could not find the platform, it would be guided to the platform and stayed on 15 s. Each rat was trained in one quadrant a day, resting for 10 min after each quadrant was completed, and trained continuously for 6 days at a fixed time. The latency of each quadrant was recorded in each rat. In the event of the indoor temperature was too low, each rat will dry the water quickly after completing a round of the experiment and pay attention to keeping warm. After 6 d of training, probe trial testing was carried out after 1 day. Removing the platform from the barrel, and the rest of the conditions were the same as the place navigation test. One quadrant (avoiding the target quadrant) was placed into the rat, and the residence time in the target quadrant, the number of times the rat crossed the original platform position and the movement track were recorded during the 90 s [9]. Hematoxylin-eosin (HE) staining The specimens were fixed by 10% formaldehyde, and sliced into 4-μm paraffin-embedded continuous sections. First, the baked tissue slices were sequentially dewaxed by xylene I and II for 10 min. Second, the dewaxed tissue slices were sequentially immersed in the absolute ethyl alcohol I and II, 95%, 80%, 70% alcohol for 2 min, respectively. Third, it was stained with hematoxylin for 3 min, and rinsed with tap water for 3 min. Fourth, color separated for 2 min by 1% hydrochloric acid, slices was soaked in 50%, 70%, 80% alcohol for 2 min in turn, immersed in eosin for 5 s, and washed under tap water for 3 min. Fifth, the slices were sequentially penetrated into 95% of alcohol, absolute ethyl alcohol I and absolute ethyl alcohol II for 3 min, respectively. Finally, the slices were, respectively, penetrated for 5 min with xylene I and the xylene II, blocked with the neutral gum and the microscopic examination was carried out. Electron microscopic observation Hippocampal tissues were fastened in 40 g/L glutaraldehyde for 1 h, rinsed with 0.1 mol/L phosphate buffer saline (PBS, pH 7.4) for 3 times, 5 min per time. The tissues were fixed by osmium tetroxide (10 g/L) for 1.5 h, and rinsed with 0.1 mol/L PBS (pH 7.4) 3 times. The tissues were dehydrated with gradient ethanol, immersed into the mixture of acetone and Epon812 for 3 h, and embedded by Epon812, then polymerized at 60°C for 48 h. The sections were stained with uranyl acetate (40 g/L) for 20 min and lead nitrate (27 g/L) for 20 min, and observed under an electron microscope (Thermo Fisher Scientific, Massachusetts, USA). Nissl staining Hippocampal tissue sections were immersed in xylene, 100%, 95%, 80%, 70% alcohol for 2 min, respectively, prior to stained with 1% toluidine blue (Beyotime Biotechnology Co., Shanghai, China) at 56°C for 40 min. Next, the sections were rinsed with running water for 8 min, and dehydrated in 70%, 80%, 95% and 100% alcohol for 2 min in turn, then cleared with xylene I and II for 3 min, respectively, and sealed with neutral gum. Finally, Nissl’s positive cells were observed under a microscope and counted. Fluoro-Jade C (FJC) staining Hippocampal tissues slices were immersed into 1% NaOH-80% ethanol mixture liquor for 5 min, transferred to 70% ethanol for 2 min, then immersed in distilled water for 2 min. Next, the slices were immersed in 0.06% potassium permanganate solution for 10 min, transferred to distilled water for 2-min washing. FJC staining solution (0.0001%, containing 0.1% acetic acid) (AmyJet Scientific Inc, Wuhan, Hubei, China) was evenly added to the treated brain slices for 10 min, and the reacted sections were rinsed in distilled water for 3 times, 1 min each time. The sections were dried and cleared with xylene (1 min, blocked with neutral gum). Under an Olympus BX60 fluorescence microscope (Olympus, Tokyo, Japan), blue filter (excitation wavelength 450–490 nm) was adopted to collect the image. TdT-mediated dUTP-biotin nick end-labeling (TUNEL) assay The paraffin-embedded slices were dehydrated by routine dewaxing, which were hatched with pepsin (0.25–0.5% HCl solution) for 25 min. Then, the sections were mixed with 50 μL TUNEL reaction mixed solution (Roche, Basel, Switzerland), incubated in a 37°C wet box for 3 times. Next, the sections were added with 50 μL conversion agent-peroxidase (Roche, Basel, Switzerland) and hatched in wet box for 30 min. The sections were added with diaminobenzidine reagent to observe whether it was colored or not via a microscope, and the coloration was stopped through adding water. The sections were put into the hematoxylin for 2 min, dipped in 95% ethanol I – II, immersed in anhydrous ethanol I-II 3–5 min, and xylene I-II 3–5 min, respectively. Subsequently, the sections were blocked with neutral gum, and observed under a light microscope. Hoechst 33,258 staining The paraffin sections were bathed with citrate buffer at 97°C for 15 min after xylene and gradient alcohol dewaxing. Hoechst 33,258 staining solution (Beyotime Biotechnology Co., Shanghai, China) was added and stained for 5 min. After the staining solution was removed, a drop of anti-quenching sealing liquid was dripped, and then covered with a clean cover glass. The sections were then captured by a fluorescence microscopy (excitation wavelength of 350 nm, emission wavelength of 460 nm). After Hoechst 33,258 staining, the apoptotic nuclei were condensed and bright blue. The number of positive cells and total number of cells in the same area were determined by IPP 6.0 software (Media Cybernetics, Maryland, USA), and the cell apoptosis rate was expressed by number of positive cells/total number of cells × 100%. Enzyme-linked immunosorbent assay (ELISA) After anesthesia, the blood of thigh artery was taken, the serum samples were amassed by centrifugation, and the hippocampal tissue was taken out. The hippocampal tissue was ground into homogenate, and the supernatant was obtained by centrifugation. The cells were gathered and fostered to be supernatant fluid and then packed in aseptic Eppendorf (EP) tube. In the light of IL-1β and TNF-α ELISA kit (RayBiotech, Norcross, GA, USA), 8 standard products were prepared, the eighth well was set as blank control group. Standard products and samples (100 μL) were appended to 96-well plates, and incubated for 2 h. Cleaning solution (300 μL) was acceded, then the liquid in the well was removed after mixing. Primary antibody (100 μL) was added to each well and incubated for 1 h. After cleaning, second antibody (100 μL) was mixed into each well and put on the shaking bed for 45 min. Next, tissues were hatched with color reagent (100 μL) avoiding light for 30 min. Lastly, terminating liquid (50 μL) was mixed into each well to stop the reaction. The absorbance and concentration were gauged and the standard curve of each well was drawn immediately. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) Total RNA in tissue specimens and cells was abstracted through Trizol extraction Kit (Invitrogen, Carlsbad, California, USA). Primers were devised and compounded by Takara (Dalian, China) (Table 1). Then, RNA was reverse transcribed into cDNA referring to the protocols of PrimeScript RT kit (Takara, Dalian, China). The reaction solution was utilized for fluorescence quantitative PCR, with reference to the instructions of SYBR® Premix Ex TaqTM II kit. The fluorescence quantitative PCR was performed in ABI PRISM® 7300 system. U6 was an internal parameter of miR-129-5p, and glyceraldehyde phosphate dehydrogenase (GAPDH) was internal parameters of SOX6, IL-1β, TNF-α, Bcl-2, and Bax. The relative transcriptional levels of target genes were computed by 2−△△Ct method [2].10.1080/15384101.2019.1669388-T0001 Table 1. Primer sequence. Gene Sequence (5ʹ→3ʹ) miR-129-5p F: 5ʹ-CUUUUUGCGGUCUGGGCUUGC-3’ R: 5ʹ-AAGCCCAGACCGCAAAAAGUU-3’ U6 F: 5ʹ- CTCGCTTCGGCAGCACA- 3’ R: 5ʹ- AACGCTTCACGAATTTGCGT- 3’ SOX6 F: 5ʹ- CCCCTCTGAACATGGTGGTGGC- 3’ R: 5ʹ-TGAGACTGCCCCTGCCGAGT- 3’ IL-1β F: 5ʹ-GACTTCACCATGGAACCCGT- 3’ R: 5ʹ-GGAGACTGCCCATTCTCGAC- 3’ TNF-α F: 5ʹ-TTACAGGAAGTCCCTCACCCTC- 3’ R: 5ʹ- CCCAGAGCCACAATTCCCTT- 3’ Bcl-2 F: 5ʹ-ACTTCTCTCGTCGCTACCGTCG- 3’ R: 5ʹ- CCCTGAAGAGTTCCTCCACCACC- 3’ Bax F: 5ʹ- TGGGCTGGACACTGGACTTC- 3’ R: 5ʹ- CTTCCAGATGGTGAGTGAGGC- 3’ GAPDH F: 5ʹ-TCTCCCTCACAATTTCCATCCC- 3’ R: 5ʹ-TTTTTGTGGGTGCAGCGAAC-3’ Note: F, forward; R, reverse; miR-129-5p, microRNA-129-5p; IL-1β, Interleukin-1β; TNF-α, Tumor necrosis factor-α; GAPDH, glyceraldehyde phosphate dehydrogenase. Western blot analysis Total proteins were abstracted from hippocampal tissues and cells. The protein concentration of each sample was confirmed and adjusted by the deionized water to ensure that the sample size was consistent. Sodium dodecyl sulfate separation gel and spacer gel (10%) were prepared. The sample was mixed with sample loading buffer and boiled at 100°C for 5 min. After centrifugation, the same amount of sample was added to carry out electrophoretic separation, and the protein on the gel was transferred to nitrocellulose membrane. The nitrocellulose membrane was blocked with skimmed milk powder (5%) at 4°C for overnight. The cells were hatched with primary antibody against SOX6 (1:1000), IL-1β, TNF-α (1:1000, Abcam Inc., Cambridge, UK), Bcl-2 and Bax (1:500, Proteintech, Chicago, USA) overnight. And then incubated with IgG (1:1000, Boster Biological Technology Co. Ltd., Wuhan, Hubei, China) secondary antibody labeled with horseradish peroxide at 37°C for l h. The membrane was immersed in enhanced chemiluminescence reaction solution (Pierce, Rockford, IL, USA) for 1 min. Using GAPDH as internal reference, protein marker was purchased from Piercenet (# 84,785). Protein imprinting images were analyzed by ImageJ2x software (National Institutes of Health (NIH), Maryland, USA). Culture and molding of neuronal cells The Sprague-Dawley (SD) rats born within 24 h were treated with 75% ethanol, and the rat was euthanized by neck dislocation and placed in the D-Hank’s solution (Procell Life technology co., Ltd., Wuhan, Hubei, China). The hippocampus was isolated in passivation, the meninges and blood vessels were removed and cut, and then 1.25 g/L trypsin was added and detached in water bath at 37°C for 20–25 min. The upper layer of the trypsin was removed, 4 mL of the culture medium was added to terminate the detachment, the cells were gently triturated with a flame-polished straw for about 20 times, and centrifuged for 5 min at 1000 r/min. After centrifugation, a proper amount of culture solution was mingled with the precipitation prior to triturating into the single-cell suspension and filtered by 200-mesh cell screen. Counted by trypan blue staining, the final concentration of the diluted cells was 1 × 108 L−1. The cell suspension was planted in the L-polylysine-coated 96-well plates, five parallel wells were arranged in each group, cultured in a saturated humidity at 37°C with 5% CO2. The culture medium was renewed in the full amount after 24 h, and the culture medium was exchanged with half amount of liquid to maintenance medium every 3 days. The growth of cells during culture was observed regularly under an inverted phase contrast microscope (Olympus, Tokyo, Japan). On the 9th day, the cells were identified by SP immunocytochemical staining, and neure was labeled with neurofilament protein monoclonal antibody (1:200, Sigma-Aldrich Chemical Company, Missouri USA). The hippocampal neurons cultured in vitro on the 9th day were sucked off the culture medium, and the cells were treated with Aβ to construct AD cell model [10]. Grouping The cells were divided into 8 groups: control group (normal rat hippocampal neuron cell); AD group (AD rat hippocampal neuron cell); mimic NC group (AD rat hippocampal neuron cells transfected with mimic-NC); miR-129-5p mimic group (AD rat hippocampal neuron cells transfected with miR-129-5p mimic, GeneCopoeia Co., Ltd. Guangzhou, China); siRNA-NC group (AD rat hippocampal neuron cells transfected with SOX6-siRNA vector NC); SOX6-siRNA group (AD rat hippocampal neuron cells transfected with SOX6-siRNA vector, GenePharma Ltd. Company, Shanghai, China); miR-129-5p mimic + OE-NC group (AD rat hippocampal neuron cells transfected with miR-129-5p mimic and overexpression of SOX6 vector NC); miR-129-5p mimic + OE-SOX6 group (AD rat hippocampal neuron cells transfected with miR-129-5p mimic and overexpression SOX6 vector, GeneCopoeia Co., Ltd. Guangzhou, China). The cells were inoculated in six-well plate before 24 h with transfection. When the confluence reached about 50%, neuronal cells were transiently transfected under the mediation of lipofectamine 2000 (Invitrogen, Carlsbad, California, USA), transfected for 6 h, and collected for subsequent experiments after cultured for 48 h. 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay Hippocampal neuron cells in logarithmic growth were detached with 0.25% trypsin and made single-cell suspension to counted. The cell concentration was adjusted to 5 × 103 cell/mL and seeded in 96-well plates with 100 μL/well. There were 6 parallel wells in each group, and a blank control without cells was set up. After cultured in a 5% CO2 incubator for 48 h, 5 μg/mL MTT solution (20 μL, Sigma-Aldrich Chemical Company, Missouri, USA) was added to per well, and hatched for 4 h. After discarding the supernatant, dimethyl sulfoxide (200 μL/well, Sigma-Aldrich Chemical Company, Missouri, USA) was added and oscillated for 10 min, and lastly, the optical density (OD490) was read by a microplate reader (BioRad company, California, USA). Hoechst 33,342 staining The cells of each group were gathered, and incubated with the RPMI-1640 medium (2 mL) containing 2% fetal bovine serum (FBS) (Gibco, Carlsbad, California, USA), and then supplanted with 5 μg/mL Hoechst 33,342 staining solution (Beijing Biolab Technology Co., Ltd., Beijing, China) at 37°C for 90 min in the dark room and added with 1 mL of RPMI-1640 medium containing 2% FBS. Fluorescence microscope was used to observe and shoot. Flow cytometry Annexin V-APC/propidium iodide (PI) double-staining method for detecting cell apoptosis: cells in each group were amassed and the medium was discarded. Cells were suspended with 250 μL of binding buffer in the light of the apoptosis detection kit (Annexin V-APC Apoptosis Detection Kit) of BD company (Becton, Dickinson and Company, NJ, USA). And 5 μL of Annexin V-APC and 5 μL of PI were mixed and then hatched for 15 min avoiding light. Flow cytometer (Beckman Colter Life Sciences, Brea, CA, USA) was used for cell apoptosis analysis. Dual luciferase reporter gene assay The targeting relationship between miR-129-5p and SOX6 and the binding site between miR-129-5p and SOX6 3ʹuntranslated region (UTR) were forecasted by bioinformatics software http://www.targetscan.org. The 3ʹUTR fragment of SOX6 gene was amplified by PCR and cloned into pmirGLO vector to construct the recombinant luciferase reporter plasmid of wild type plasmid (SOX6-WT) and mutant type plasmid (SOX6-MUT). SOX6-WT and SOX6-MUT plasmids were extracted according to the steps of the purchased plasmid extraction kit (Promega, Madison, Wisconsin, USA). The logarithmic cells were inoculated into 96-well plates. When the cell confluence was about 70%, Lipofectamine 2000 was utilized for transfection. SOX6-WT and SOX6-MUT were mixed with mimic NC and miR-129-5p mimic (Shanghai GenePharma Co. Ltd., Shanghai, China) respectively, and then co-transfected to neuronal cells. The cells were amassed and lysed after transfected 48 h, and luciferase activity was detected by luciferase detection kit (BioVision, San Francisco, CA, USA) and Glomax 20/20 luminometer (Promega, Madison, Wisconsin, USA). Statistical analysis All data were analyzed by SPSS 21.0 (IBM-SPSS Inc., Chicago, IL, USA) software. The measurement data were expressed as mean ± standard deviation. Comparisons among multiple groups were assessed by one-way analysis of variance (ANOVA) and the pairwise comparison between groups after ANOVA were analyzed by Tukey’s post hoc test. P value <0.05 was indicative of statistically significant difference. Results Low expression of SOX6 and high expression of miR-129-5p shorten the escape latent period and increase the times of crossing platforms in rats with AD It was demonstrated by place navigation test (Figure 1(a)) that compared to the normal group, the average escape latency of the rats in the AD group was prolonged (P < 0.05). In relation to the agomir-NC group and siRNA-NC group, the average escape latency of the agomir-miR-129-5p group and the SOX6-siRNA group was shortened (both P < 0.05). In contrast with the agomir-miR-129-5p + OE-NC group, the average escape latency of the agomir-miR-129-5p + OE-SOX6 group was prolonged (P < 0.05).10.1080/15384101.2019.1669388-F0001 Figure 1. The escape latent period would shorten and the times of crossing platforms increase when SOX6 was poorly expressed and miR-129-5p was highly expressed in vivo of AD rats. (a) Average escape latency period in each group of rats. (b) Times of crossing platforms for rats in each group. * P < 0.05 vs. the normal group. + P < 0.05 vs. the agomir-NC group. # P < 0.05 vs. the siRNA-NC group. & P < 0.05 vs. the agomir-miR-129-5p + OE-NC group. N = 10. Measurement data were depicted as mean ± standard deviation. Comparison among multiple groups were assessed by one-way ANOVA and the comparisons between groups after ANOVA were analyzed by Tukey’s post hoc test. The results of probe trial testing presented that the times of crossing platform in the AD group was lower than that in the normal group (P < 0.05). In relation to the agomir-NC group and the siRNA-NC group, the times of crossing platform in the agomir-miR-129-5p group and the SOX6-siRNA group was raised (both P < 0.05). By comparison with the agomir-miR-129-5p + OE-NC group, the times of crossing the platform in the agomir-miR-129-5p + OE-SOX6 group was reduced (P < 0.05) (Figure 1(b)). Poor expression of SOX6 and high expression of miR-129-5p alleviate the pathological injury in hippocampal tissues The results of HE staining presented that compared to the normal group, the hippocampal neurons in the AD group were solidified and atrophied, the whole cell was deeply stained and the cell was necrotic. And the cell necrosis of hippocampal neurons in the agomir-miR-129-5p group and the SOX6-siRNA group were decreased in relation to the agomir-NC group and the siRNA-NC group. By comparison with the agomir-miR-129-5p + OE-NC group, the intercellular structure of hippocampal neurons in the agomir-miR-129-5p + OE-SOX6 group was loose and the cell necrosis was ascended (Figure 2(a)).10.1080/15384101.2019.1669388-F0002 Figure 2. Downregulation of SOX6 and upregulation of miR-129-5p in hippocampal tissues will alleviate the pathological injury. (a) Observation of neuron staining in each group of rats (400 ×). (b) The results of the ultrastructure of the neurons in each group of rats (500 nm). C&D: Comparison of neuronal cell injury in each group (400 ×). * P < 0.05 vs. the normal group. + P < 0.05 vs. the agomir-NC group. # P < 0.05 vs. the siRNA-NC group. & P < 0.05 vs. the agomir-miR-129-5p + OE-NC group. N = 10. Measurement data were depicted as mean ± standard deviation. Comparison among multiple groups were assessed by one-way ANOVA and the comparisons between groups after ANOVA were analyzed by Tukey’s post hoc test. Electron microscope observation suggested that the hippocampal neurons in the normal group has a regular morphology, a uniform distribution of chromatin, a clear structure of endoplasmic reticulum and endoplasmic reticulum, and an abundant ribosome. The nucleus of hippocampal neurons in the AD group was irregular, the heterochromatin increased and aggregated, the perinuclear space increased obviously, and the mitochondria showed swelling or vacuole. Compared to the agomir-NC group and the siRNA-NC group, the ultrastructure damage of hippocampal neurons in the agomir-miR-129-5p group and the SOX6-siRNA group was alleviated, most nuclear chromatin was evenly distributed and the organelle structure was clear. In relation to the agomir-miR-129-5p + OE-NC group, the nucleus of hippocampal neurons in the agomir-miR-129-5p + OE-SOX6 group was irregular, the heterochromatin increased and accumulated, the perinuclear space increased and the mitochondria swelled or vacuolated (Figure 2(b)). Nissl staining findings reported that in contrast to the normal group, the number of neuronal cells in the AD group decreased (P < 0.05). The number of neuronal cells in the agomir-miR-129-5p group and the SOX6-siRNA group was higher than the agomir-NC group and the siRNA-NC group (both P < 0.05). By comparison with the agomir-miR-129-5p + OE-NC group, the number of neuronal cells in the agomir-miR-129-5p + OE-SOX6 group degraded (P < 0.05) (Figure 2(c-d)). Overexpression of miR-129-5p and poor expression of SOX6 inhibit neuronal apoptosis in hippocampal tissues No FJC positive cells were found in the normal group, FJC positive cells with bright yellow green fluorescence could be seen clearly in FJC-stained brain sections in the AD group. FJC positive cells in the agomir-miR-129-5p group and the SOX6-siRNA group reduced in contrast with the agomir-NC group and the siRNA-NC group. And FJC positive cells in the agomir-miR-129-5p + OE-SOX6 group enhanced by comparison with the agomir-miR-129-5p + OE-NC group (Figure 3(a)).10.1080/15384101.2019.1669388-F0003 Figure 3. Neuronal apoptosis would be suppressed by upregulated miR-129-5p and downregulated SOX6. (a) FJC-positive denatured neuron in rats (400 ×). (b) Observation results of neuronal apoptosis by TUNEL staining (400 ×). (c) Observation of Hoechst 33,258 staining in rat hippocampal tissue (400 ×). (d) Apoptosis rate of hippocampal neurons in rats. (e) Detection of Bax and Bcl-2 expression by RT-qPCR. F&G: Detection of Bax and Bcl-2 expression by western blot analysis, 1–8 indicated normal group, AD group, agomir-NC group, agomir-miR-129-5p group, siRNA-NC group, SOX6-siRNA group, agomir-miR-129-5p + OE-NC group, and agomir-miR-129-5p + OE-SOX6 group, respectively. * P < 0.05 vs. the normal group. + P < 0.05 vs. the agomir-NC group. # P < 0.05 vs. the siRNA-NC group. & P < 0.05 vs. the agomir-miR-129-5p + OE-NC group. N = 10. Measurement data were depicted as mean ± standard deviation. Comparison among multiple groups were assessed by one-way ANOVA and the comparisons between groups after ANOVA were analyzed by Tukey’s post hoc test. The results of TUNEL staining presented that in hippocampal tissues, those with dark brown nucleus were positive. The amount of TUNEL positive cells in the normal group was less than that in the AD group, in the agomir-miR-129-5p group and the SOX6-siRNA group was less than that in the agomir-NC group and the siRNA-NC group. And the number of TUNEL positive cells in the agomir-miR-129-5p + OE-SOX6 group was more than that in the agomir-miR-129-5p + OE-NC group (Figure 3(b)). After Hoechst 33,258 staining, apoptotic nucleus condensed and appeared bright blue in hippocampal tissues. The apoptosis rate of the normal group was lower than that of the AD group (P < 0.05). The apoptosis rate of the agomir-miR-129-5p group and the SOX6-siRNA group was less than that of the agomir-NC group and the siRNA-NC group (both P < 0.05). The apoptosis rate of the agomir-miR-129-5p + OE-SOX6 group was higher than that of the agomir-miR-129-5p + OE-NC group (P < 0.05) (Figure 3(c-d)). The results of RT-qPCR and western blot analysis displayed that the expression of Bcl-2 degraded, and Bax enhanced in the AD group compared to the normal group (both P < 0.05). In contrast with the agomir-NC group and the siRNA-NC group, Bcl-2 expression raised while Bax expression depressed in the agomir-miR-129-5p group and the SOX6-siRNA group (all P < 0.05). In relation to the agomir-miR-129-5p + OE-NC group, Bcl-2 expression degraded and Bax expression elevated in the agomir-miR-129-5p + OE-SOX6 group (both P < 0.05) (Figure 3(e-g)). Upregulated miR-129-5p and down-regulated SOX6 reduce the inflammatory reaction in hippocampal tissues ELISA demonstrated that the levels of IL-1β and TNF-α in serum and hippocampal tissues of the AD group were higher than those of the normal group (all P < 0.05), and the levels of IL-1β and TNF-α in serum and hippocampal tissues of the agomir-miR-129-5p group and the SOX6-siRNA group were lower than those of the agomir-NC group and the siRNA-NC group (all P < 0.05). In relation to the agomir-miR-129-5p + OE-NC group, the levels of IL-1β and TNF-α in serum and hippocampal tissue of the agomir-miR-129-5p + OE-SOX6 group were raised (all P < 0.05) (Figure 4(a)).10.1080/15384101.2019.1669388-F0004 Figure 4. Overexpression of miR-129-5p and down-regulation of SOX6 depress the inflammatory reaction in hippocampal tissues. (a) Detection of the expression of IL-1β and TNF-α in the serum and hippocampal tissues of each group by ELISA. (b) Expression of miR-129-5p, SOX6, IL-1β, and TNF-α by RT-qPCR. C&D: SOX6, IL-1β and TNF-α protein expression tested by western blot analysis. 1–8 indicate normal group, AD group, agomir-NC group, agomir-miR-129-5p group, siRNA-NC group, SOX6-siRNA group, agomir-miR-129-5p + OE-NC group, and agomir-miR-129-5p + OE-SOX6 group, respectively. * P < 0.05 vs. the normal group. + P < 0.05 vs. the agomir-NC group. # P < 0.05 vs. the siRNA-NC group. & P < 0.05 vs. the agomir-miR-129-5p + OE-NC group. N = 10. Measurement data were depicted as mean ± standard deviation. Comparison among multiple groups were assessed by one-way ANOVA and the comparisons between groups after ANOVA were analyzed by Tukey’s post hoc test. RT-qPCR and western blot analysis revealed that compared to the normal group, miR-129-5p expression was decreased, and the expression of SOX6 and the inflammatory factor IL-1β and TNF-α in the AD group were heightened (all P < 0.05). By comparison with the agomir-NC group and the siRNA-NC group, the expression SOX6 and the inflammatory factor IL-1β and TNF-α in the agomir-miR-129-5p group and the SOX6-siRNA group was reduced (all P < 0.05). In contrast with the agomir-miR-129-5p + OE-NC group, the expression SOX6 and the inflammatory factor IL-1β and TNF-α in the agomir-miR-129-5p + OE-SOX6 group was elevated (all P < 0.05) (Figure 4(b-d)). Low expression of SOX6 and overexpression mir-129-5p promote proliferation of neuronal cells The growth of the rat hippocampal neurons cultured in vitro was as follows: after cultured for 3 h, some of the neuronal cells had attached to the wall, and a few cells had grown 1–2 tiny protrusions, and the majority of the adherent cells were in the shape of an ellipse. After 6 h, the number of the outgrowth protrusions increased gradually, small number of the protrusions were up to 10 μm. After cultured for 24 h, the cells were adhered to the wall completely, most of the cells grow protrusions, and the protrusions were prolonged correspondingly. After 7 d culture, the protrusions of the cells extended further to form a dense network, mainly multipolar neurons with full cell bodies, most of them were fusiform, conical, halo around, and strong stereosensory, rich in cytoplasm, large in nucleus and visible in nucleolus (Figure 5(a)).10.1080/15384101.2019.1669388-F0005 Figure 5. Poor expression of SOX6 and high expression of miR-129-5p advance proliferation of neuronal cells. (a) Morphological observation of the hippocampus neurons in rat (200 ×). (b) Expression of NF in hippocampal neurons of rats (400 ×). (c) Comparison of the proliferation ability of each group of cells tested by MTT assay. * P < 0.05 vs. the control group. + P < 0.05 vs. the mimic NC group. # P < 0.05 vs. the siRNA-NC group. & P < 0.05 vs. the miR-129-5p mimic + OE-NC group. Measurement data were depicted as mean ± standard deviation. Comparison among multiple groups were assessed by one-way ANOVA and the comparisons between groups after ANOVA were analyzed by Tukey’s post hoc test. The experiment repeated three times. Identification of rat hippocampal neurons cultured in vitro: the cultured cells were chemically stained with anti-NF immune cells. The positive cells observed by the inverted microscope were about 90%, the cytoplasm was brown, and the cytoplasm of the NC cells was hyacinthine (Figure 5(b)). The proliferation of neuronal cells verified by MTT assay. In the AD group, the proliferation was lower than that of the control group (P < 0.05). Compared to the mimic NC group and the siRNA-NC group, the proliferation of the neuronal cells of the miR-129-5p mimic group and the SOX6-siRNA group was raised (both P < 0.05). By comparison with the miR-129-5p mimic + OE-NC group, the proliferation of the neuronal cells in the miR-129-5p mimic + OE-SOX6 group was reduced (P < 0.05) (Figure 5(c)). Upregulation of miR-129-5p and down-regulation of SOX6 suppress apoptosis of neuronal cells The hippocampal neurons in the control group showed a uniform dispersion of blue fluorescence, indicating that there were a large number of living cells. In the AD group, neuron nucleus were concentrated or broken, showing dense or dispersed particle bulk fluorescence, indicating a large number of apoptotic cells. In the miR-129-5p mimic group and the SOX6-siRNA group, there was less particulate blue fluorescence than the mimic NC group and the siRNA-NC group, the apoptotic cells were less than the mimic NC group and the siRNA-NC group. In the miR-129-5p mimic + OE-SOX6 group, there were more particulate blue fluorescence, the apoptotic cells were more than the miR-129-5p mimic + OE-NC group (Figure 6(a)).10.1080/15384101.2019.1669388-F0006 Figure 6. Apoptosis of neuronal cells would be restrained by upregulation of miR-129-5p and down-regulation of SOX6. (a) Detection of apoptosis of neuronal cells by Hoechst 33,342 staining. (b) Apoptosis of neuronal cells detect by flow cytometry. (c) Comparison of cell apoptosis rate in each group. (d) Bcl-2 and Bax mRNA expression by RT-qPCR. E: Bcl-2 and Bax protein expression determined by Western blot analysis. 1–8 indicate control group, AD group, mimic NC group, miR-129-5p mimic group, siRNA-NC group, SOX6-siRNA group, miR-129-5p mimic + OE-NC group, and miR-129-5p mimic + OE-SOX6 group, respectively. * P < 0.05 vs. the control group. + P < 0.05 vs. the mimic NC group. # P < 0.05 vs. the siRNA-NC group. & P < 0.05 vs. the miR-129-5p mimic + OE-NC group. Measurement data were depicted as mean ± standard deviation. Comparison among multiple groups were assessed by one-way ANOVA and the comparisons between groups after ANOVA were analyzed by Tukey’s post hoc test. The experiment repeated three times. The results of AnnexinV-APC/PI double staining reported that the apoptosis rate of neuronal cells in the AD group was higher than that in the control group (P < 0.05), and that in the miR-129-5p mimic group and the SOX6-siRNA group was lower than that in the mimic NC group and the siRNA-NC group (P < 0.05). In relation to the miR-129-5p mimic + OE-NC group, the apoptosis rate in the miR-129-5p mimic + OE-SOX6 group was raised (P < 0.05) (Figure 6(b-c)). RT-qPCR and western blot analysis revealed that compared to the control group, the expression of Bcl-2 degraded, and Bax enhanced in the AD group (both P < 0.05). In contrast with the mimic NC group and the siRNA-NC group, Bcl-2 expression raised while Bax expression depressed in the miR-129-5p mimic group and the SOX6-siRNA group (both P < 0.05). In relation to the miR-129-5p mimic + OE-NC group, Bcl-2 expression degraded and Bax expression elevated in the miR-129-5p mimic + OE-SOX6 group (both P < 0.05) (Figure 6(d-f)). SOX6 is the target gene of miR-129-5p, and upregulated miR-129-5p and downregulated SOX6 degrade the inflammatory reaction of neuronal cells The SOX6 gene sequence had a specific binding region with the miR-129-5p sequence and the SOX6 was the target gene of the miR-129-5p (Figure 7(a)) which tested by the on-line software analysis. Luciferase activity assay revealed that (Figure 7(b)) compared to the NC group, miR-129-5p mimic decreased the luciferase activity of Wt-miR-129-5p/SOX6 plasmid (P < 0.05), while had no effect on the luciferase activity of Mut-miR-129-5p/SOX6 (P > 0.05). Thus, miR-129-5p could specifically bind to SOX6 gene.10.1080/15384101.2019.1669388-F0007 Figure 7. Overexpression of miR-129-5p and poor expression of SOX6 reduce the inflammatory reaction of neuronal cells and SOX6 is the target gene of miR-129-5p. (a) Prediction of binding sites of miR-129-5p on SOX6 3ʹUTR. (b) Luciferase activity detection for verifying the relationship between miR-129-5p on SOX6. (c) Detection of IL-1β and TNF-α expression in the supernatant of each group by ELISA. (d) Expression of miR-129-5p, SOX6, IL-1β, and TNF-α tested by RT-qPCR. E: SOX6, IL-1β and TNF-α protein expression determined by western blot analysis. 1–8 indicate control group, AD group, mimic NC group, miR-129-5p mimic group, siRNA-NC group, SOX6-siRNA group, miR-129-5p mimic + OE-NC group, and miR-129-5p mimic + OE-SOX6 group, respectively. * P < 0.05 vs. the control group. + P < 0.05 vs. the mimic NC group. # P < 0.05 vs. the siRNA-NC group. & P < 0.05 vs. the miR-129-5p mimic + OE-NC group. Measurement data were depicted as mean ± standard deviation. Comparison among multiple groups were assessed by one-way ANOVA and the comparisons between groups after ANOVA were analyzed by Tukey’s post hoc test. The experiment repeated three times. The results of ELISA presented that the levels of IL-1β and TNF-α in the supernatant of the AD group were higher than those in the control group (both P < 0.05), and the levels of IL-1β and TNF-α in the miR-129-5p mimic group and the SOX6-siRNA group were lower than those in the mimic NC group and the siRNA-NC group (both P < 0.05). In contrast with the miR-129-5p mimic + OE-NC group, the levels of IL-1β and TNF-α in the miR-129-5p mimic + OE-SOX6 group were elevated (both P < 0.05) (Figure 7(c)). RT-qPCR and western blot analysis displayed that compared to the control group, the expression of SOX6 and the inflammatory factor IL-1β and TNF-α in the AD group were heightened (all P < 0.05). By comparison with the mimic NC group and the siRNA-NC group, the expression SOX6 and the inflammatory factor IL-1β and TNF-α in the miR-129-5p mimic group and the SOX6-siRNA group was reduced (all P < 0.05). In contrast with the miR-129-5p mimic + OE-NC group, the expression of SOX6 and the inflammatory factor IL-1β and TNF-α in the miR-129-5p mimic + OE-SOX6 group was enhanced (all P < 0.05) (Figure 7(d-f)). Discussion AD is one of the most common neurodegenerative and multifactorial diseases in the world [1], showing grave global health and economic challenges [2]. A previous study has proved that some miRNAs, such as miR-132 and miR-124, are thought to be candidates for regulating the process of AD [2]. However, no evidence has presented the association between miR-129-5p and AD. Moreover, it was reported that SOX transcription factors exert an enormous function on regulating neurogenesis in embryonic and adult nervous system [8]. As the related mechanisms of miR-129-5p in AD remains to be excavated, the objective of our study was to investigate the impact of the miR-129-5p regulating nerve injury and inflammatory response in AD rats via regulating SOX6. In this study, low expression of miR-129-5p and high expression of SOX6 was found in hippocampal tissues of AD rats. Consistent with our study, a study reported that the level of miR-129-5p in prostate cancer was lower than that in normal prostate cancer [6]. Another study has presented that the expression level of serum miR-129-5p in patients with liver cancer was degraded [11]. The above evidence suggests the suppressive role of miR-129-5p in diseases. It is presented that SOX6 mRNA expression was positively correlated with copy number variation and elevated in skeletal muscle cell differentiation [2]. The expression of SOX6 in AD needs further verification. Our study also presented that SOX6 was the target gene of miR-129-5p. Similarly, there existed a relationship between other miRNAs with SOX6. For example, a previous study has proved that miR-202 directly targets SOX6 [12]. Another study has presented miR-96 can boost cell invasion, migration, and proliferation of hepatocellular carcinoma by targeting SOX6 [2]. Furthermore, it was displayed that miR-671 directly targets tumor suppressor SOX6 in the 3ʹUTR to restrain its expression [2]. In addition, it was revealed that up-regulating miR-129-5p descended the apoptosis and ascended the proliferation of neuronal cells of AD rats and can alleviate the neuronal injury and inflammatory response in AD rats. It has been suggested previously that the migration and invasion of the cells were suppressed when the expression of miR-129-5p in Hep2 cells was up-regulated [2]. Another study has verified that intrathecal injection of miR-129-5p mimic reduced the levels of toll-like receptor 3, high-mobility group box-1, TNF-α and IL-1β [2]. Moreover, it was suggested up-regulation of miR-129-5p can efficiently inhibits proliferation and induces apoptosis of ovarian cancer cells [13]. The study also showed that down-regulating SOX6 descended the apoptosis and ascended the proliferation of neuronal cells of AD rats as well as alleviate the neuronal injury and inflammatory response in AD rats. It is revealed that silencing SOX6 eliminated the promotive effect of low expression of miR-765 on the apoptosis and proliferation of multiple myeloma cells [14]. Other study also proved that the knockdown of SOX6 completely saved the phenotype of Trbp-mutant, while the overexpression of SOX6 shows TrbpcKO phenotype [2]. Furthermore, it has been suggested in this present study that up-regulation of SOX6 can reverse the expression of miR-129-5p to reduce the damage and inflammatory response of the cell model of AD. A study proved that SOX6 partially reversed the effects of miR-499-5p on up-regulating Bcl-2 level and down-regulating the expression of Bax and caspase-3 [7]. It has been suggested that up-regulation of SOX6 can reverse the anti-apoptotic and proliferation effects of miR-499 [2]. Also, it was found that overexpression of SOX6 can reverse dendritic development and neuronal differentiation mediated by miR-135a-5p [15]. Moreover, Xie et al. have found that the ectopic expression of SOX6 can reverse the growth-promoting characteristics of the miR-155 [2]. All these studies revealed the functions of miR-129-5p and SOX6 in AD remain to be elucidated. In conclusion, our study provides evidence that upregulation of miR-129-5p or down-regulation of SOX6 can reduce nerve injury and inflammatory response in rats with AD. This paper provides a new idea for further study the pathogenesis of AD. We expect to find more association of miR-129-5p/SOX6 axis with patients with AD by this way to offer a more scientific basis for clinical decision-making. Acknowledgments We would like to acknowledge the reviewers for their helpful comments on this paper. Availability of data and material Not applicable Authors’ contributions Guarantor of integrity of the entire study: Zhilei Zeng, Yajun Liu study design: Wei Zheng, Liubin Liu, Honglei Yin, Simiao Zhang, Hongying Bai, Linlin Hua experimental studies: Shanshan Wang, Zhen Wang, Xiaodong Li, Jianhao Xiao, Qian Yuan manuscript editing: Yunliang Wang Consent for publication Not applicable Disclosure statement No potential conflict of interest was reported by the authors. Ethical statement This study was approved and supervised by the animal ethics committee of The Second Affiliated Hospital of Zhengzhou University. The treatment of animals in all experiments conforms to the ethical standards of experimental animals. ==== Refs References [1] Chen L , Guo X , Li Z , et al Relationship between long non-coding RNAs and Alzheimer’s disease: a systematic review. Pathol Res Pract. 2019;215 (1 ):12–20.30470438 [2] Sevigny J , Chiao P , Bussière T, et al. The antibody aducanumab reduces Aβ plaques in Alzheimer's disease[j]. Nature. 2016;537 (7618 ):50–56. [3] Citron M. Alzheimer’s disease: strategies for disease modification. Nat Rev Drug Discov. 2010;9 (5 ):387–398.20431570 [4] Dorszewska J , Prendecki M , Oczkowska A , et al Molecular basis of familial and sporadic Alzheimer’s disease. Curr Alzheimer Res. 2016;13 (9 ):952–963.26971934 [5] Anand R , Gill KD , Mahdi AA. Therapeutics of Alzheimer’s disease: past, present and future. Neuropharmacology. 2014;76 Pt A :27–50.23891641 [6] Gao G , Xiu D , Yang B , et al miR-129-5p inhibits prostate cancer proliferation via targeting ETV1. 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==== Front Open Access Maced J Med SciOpen Access Maced J Med SciOpen Access Macedonian Journal of Medical Sciences1857-9655Republic of Macedonia ID Design 2012/DOOEL Skopje OAMJMS-7-307310.3889/oamjms.2019.776Research ArticleA Black Hole at the Center of Earth Plays the Role of the Biggest System of Telecommunication for Connecting DNAs, Dark DNAs and Molecules of Water on 4+N- Dimensional Manifold Fioranelli Massimo 1Sepehri Alireza 1Roccia Maria Grazia 1Linda Cota 1Rossi Chiara 1Vojvodic Petar 2Lotti Jacopo 1Barygina Victoria 3Vojvodic Aleksandra 4Wollina Uwe 5Tirant Michael 6Thuong Nguyen Van 7Lotti Torello 81 Department of Nuclear Physics, Sub-nuclear and Radiation, G. Marconi University, Rome, Italy2 Clinic for Psychiatric Disorders “Dr. Laza Lazarevic”, Belgrade, Serbia3 Department of Biomedical Experimental and Clinical Sciences, University of Florence, Florence, Italy4 Department of Dermatology and Venereology, Military Medical Academy, Belgrade, Serbia5 Department of Dermatology and Allergology, Städtisches Klinikum Dresden, Dresden, Germany6 G. Marconi University, Rome, Italy7 Vietnam National Hospital of Dermatology and Venereology, Hanoi, Vietnam8 Department of Dermatology, University of G. Marconi, Rome, Italy Correspondence: Massimo Fioranelli. Department of Nuclear Physics, Sub-nuclear and Radiation, G. Marconi University, Rome, Italy. E-mail: [email protected] 9 2019 30 8 2019 7 18 3073 3080 16 6 2019 04 7 2019 05 7 2019 Copyright: © 2019 Massimo Fioranelli, Alireza Sepehri, Maria Grazia Roccia, Cota Linda, Chiara Rossi, Petar Vojvodic, Jacopo Lotti, Victoria Barygina, Aleksandra Vojvodic, Uwe Wollina, Michael Tirant, Nguyen Van Thuong, Torello Lotti.2019This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License (CC BY-NC 4.0)Recently, some scientists from NASA have claimed that there may be a black hole like structure at the centre of the earth. We show that the existence of life on the earth may be a reason that this black hole like object is a black brane that has been formed from biological materials like DNA. Size of this DNA black brane is 109 times longer than the size of the earth’s core and compacted interior it. By compacting this long object, a curved space-time emerges, and some properties of black holes emerge. This structure is the main cause of the emergence of the large temperature of the core, magnetic field around the earth and gravitational field for moving around the sun. Also, this structure produces some waves which act like topoisomerase in biology and read the information on DNAs. However, on the four-dimensional manifold, DNAs are contracted at least four times around various axis’s and waves of earth couldn’t read their information. While, by adding extra dimensions on 4 +n-dimensional manifold, the separation distance between particles increases and all of the information could be recovered by waves. For this reason, each DNA has two parts which one can be seen on the four-dimensional universe, and another one has existed in extra dimensions, and only it’s e_ects is observed. This dark part of DNA called as a dark DNA in an extra dimension. These dark DNAs not only exchange information with DNAs but also are connected with some of the molecules of water and helps them to store information and have memory. Thus, the earth is the biggest system of telecommunication which connects DNAs, dark DNAs and molecules of water. BlackholeTelecommunicationEarthDark DNAWaves ==== Body Introduction Black holes and their related subjects are of main puzzles in science which many scientists work on them [1]. Physics of these objects is approximately known. However, they are lost, and many cosmological detectors and telescopes try to find them. On the other hand, the concept of a black hole isn’t limited to cosmology, and in some high energy colliders, some objects may emerge that change space-time [2]. These objects are known as Tev black holes or mini black holes that are formed from concentrating of a large amount of energy in a small place at large hadron collider [3], [4], [5]. Thus, some types of black hole-like structures could be observed on earth. Newly, some scientists who worked in NASA claimed that there is a black hole at the centre of the earth which is the main cause of the high temperature of the core and magnetic field around the earth [6]. This idea may change some old beliefs about the formation of the earth and solar system. In this model, the earth is a planet that has been formed around a black hole and has properties of that object in its core. In this paper, we show that this idea could have more results and help us to explore the origin of life. One of the main questions in science is about the relation between the inner core of the earth, life, water and waves. Each of these subjects has itself puzzles centre, and all of them together make a more main puzzle. For example, our knowledge about the inner core of the earth isn’t complete, and only limited information has been obtained from the waves of the earthquake [7], [8], [9], [10], [11]. These considerations have shown that there is equatorial anisotropy in the inner part of the Earths inner core [7], [8]. Also, scientists have estimated the temperature of the inner core from the melting temperature of impure iron at the pressure which iron is under at the boundary of the inner core (about 330 GPa) [9]. From these considerations, they have estimated its temperature as between 5,400 K (5,100 C; 9,300 F) and 5,700 K (5,400 C; 9,800 F). However, in 2013, some others have obtained experimentally a substantially higher temperature for the melting point of iron, 6230 [10], [11]. The reason for this high temperature is unclear. However, some scientists believe that some nuclear interactions occur interior of the core that is the main cause of high temperature [12], [13], [14]. In parallel, the origin of life on the earth and its relation with evolutions of the core is unclear. Also, there are some puzzles about controlling life by some special dark genes which couldn’t be observed and detected by present devices. These genes have been discovered by Hargreaves and his colleagues. They have encountered a dark part of DNA when sequencing the genome of the sand rat (Psammomys obesus), a species of gerbil that lives in deserts. In particular, they wanted to study the gerbil’s genes related to the production of insulin, to understand why this animal is particularly susceptible to type 2 diabetes. But when they looked for a gene called Pdx1 that controls the secretion of insulin, they found it was missing, as were 87 other genes surrounding it. Some of these missing genes, including Pdx1, are essential and without them, an animal cannot survive. The first clue was that, in several of the sand rats body tissues, they found the chemical products that the instructions from the missing genes would create. This would only be possible if the genes were present somewhere in the genome, indicating that they weren’t missing but just hidden [15]. Now, the question arises that how we can communicate with these special dark DNAs which their effects can be seen; however, they are themselves lost. Another puzzle in science is the ability of molecules of water for exchanging information with DNAs and storing their information [16], [17], [18]. The chemical structure of water (H2O) is very simple and has no ability to store information. Thus, how these molecules communicate with other molecules and DNAs. Also, it seems that there is a relation between molecules of water, earth and DNA. Because the water of rain has a better effect on the plants and their growth. To respond to all of these questions, we should design a model which explains the relationship between earth, water and life. To this aim, we can use ideas of scientists for the existence of a black hole at the centre of the earth. This black hole may be constructed from a DNA black brane with 109 times longer than the core of the earth which is compacted interior of the core. The number of excited states of this object is similar to the number of microstates of a black hole. However, its material is similar to the material of a DNA. This structure produces a temperature around 6000 K which is in agreement with the predicted temperature of the core. Also, this structure is the main cause of the emergence of the magnetic field around the earth and gravitational waves for moving around the sun. We show that DNA black brane of the earth is the biggest system of telecommunications which exchange waves with all DNAs and molecules of water. Also, we introduce a new type of DNAs called dark DNAs on the eleven-dimensional manifold. In fact, on the four-dimensional manifold, DNAs are contracted at least four times around various axes and waves of earth couldn’t read their information. However, by adding extra dimensions, the separation distance between particles increases and all of the information could be recovered by waves. For this reason, each DNA has two parts which one can be seen on the four-dimensional universe, and another one has existed in extra dimensions, and only it’s effects can be observed. This extra dark part of DNA called as a dark DNA in an extra dimension. Waves of the earth’s DNA connect DNAs on four-dimensional universe and dark DNAs in extra dimensions and act like topoisomerases in biology. These waves are different for males and females and also different from linear waves which radiate by electronic devices. On the other hand, experiments show that radiated waves of the earth interact with molecules of water and store information in their memory. The memory of water is in contradiction to its chemical structure (H2O). Thus, there should be extra dark DNAs in related to the molecules of water that help them in storing information and exchanging waves with DNAs of earth. This means that molecules of water could have gender like DNAs. On the other hand, the earth could emit some special waves to molecules of water and extract DNA black brane from extra dimensions. This could be the origin of life on earth. Thus, earth, water and DNA form the best system of telecommunication which controls all evolutions of life. The outline of the paper is as follows. In section II, we show that a DNA black brane interior of the core may be the cause of the emergence of the magnetic field, gravitational waves and high temperature. In section III, we show that the core of the earth, DNA, waves and molecules of water create the biggest system of telecommunication. The emergence of Magnetic Field, Gravitational Wave and High Temperature of Earth’s Core by a DNA Black Brane In this section, we will show that all DNAs of creatures are imaged on its core and produce a DNA black brane in it (See Figure 1). This structure has around 109 times longer than the core of the earth and is compacted interior of the core. We will show that this structure is the main cause of the gravitational field, magnetic field and high temperature of the core. Figure 1 Induced DNA black brane interior of the core by imaging all DNAs on its meta First, we calculate Hamiltonian of one DNA and then, we generalise it to a DNA black brane. Each DNA is formed from hexagonal and pentagonal molecules (See Figure 2). Figure 2 Each DNA is formed from joining hexagonal and pentagonal molecules [19] Also, each hexagonal and pentagonal molecule is formed from six or -ve strings (See Figure 3). Thus, we will use the action of strings for them [20], [21]; Figure 3 Each hexagonal molecule is formed from joining six strings The topology of DNA has a direct effect on its radiation [19], Figure 4 Each DNA and it’s hexagonal and pentagonal manifolds are coiled several times around the axis without background axes. Here, t is time, r is the radius of the page of DNA and θ is the angle of rotation. The action of this DNA can be given by: where gMN is the background metric, θM(σa)’s are scalar fields which are produced by pairing electrons (ϕ = ψup ψdown), N is a number of exchanged photons between DNAs, σa’s are the DNA coordinates, a, b = 0, 1, … 3 are world-volume indices of DNA and M, N = 0, 1 …, are a number of paired electrons. Also, G is the nonlinear field, and A is the photon which exchanges between DNAs. Using the metric in equation (1), we can write below relations between coordinates of bulk and a DNA [20], [21]: Using the above relations, for this DNA in at space time, the action is given by [20], [21]: For this action, it has been asserted that momentum density is given by [20], [21]: where ‘denotes the derivative respect to the field (F). On the other hand, it has been asserted that there is a relation between momentum density and σ [20], [21]: Using equations (5 and 6) and assuming (z’ << G(F)) and also following method in [20], [21], [22], we can obtain: 3.3 Above equation shows that Hamiltonian of bases depends on their shape, the separation distance between atoms and angles. Each DNA is coiled several times, and thus, its hexagonal and pentagonal manifolds are coiled several times around various axes (See Figure 4). Coiling around the axes leads to the motion of electrons in various directions and emergence of the magnetic field. Thus, radiation of DNAs depend on their topology, and for this reason, radiation of chromosomes of males is different respect to radiations of chromosomes of females. For coiled hexagonal and pentagonal manifold, we obtain: which are rounded around axes, using Hamiltonians in (9 and 10), we can obtain below Hamiltonian: where is the angle between two atoms of respect to the center of hexagonal and pentagonal manifolds, and ϕ is the angle between a hexagonal and a pentagonal manifold. Consequently, Hamiltonian of a DNA can be obtained as: This Hamiltonian depends on the separation distance and the angle between atoms. Topology of DNA has a direct e_ect on its energy, and for example, the energy of DNAs in males and females are different. By using some special values for angles between atoms, we can obtain the known energy of the earth of the above Hamiltonian: Above equation shows that DNA black brane interior of the core can produce expected gravitational energy. Thus, this theory is in agreement with known laws of physics. Now, we can assert that the magnetic of earth can be obtained by summing over exchanged electromagnetic fields between atoms: where n is the number of magnetic fields between elements of DNA black brane. Above equation shows that the magnetic field of the earth can be obtained by summing over magnetic fields of pentagonal and heptagonal manifolds. This magnetic field depends on the topology of DNA, and for example, for a DNA with the gender of male, the magnetic field is different respect to the DNA with the gender of female. Now, we want to obtain the temperature of the core. To this aim, we put a number of microstates of all pentagonal and hexagonal manifolds equal to the number of microstate of DNA black brane. To calculate a number of microstates, we use a normal thermal distribution which is used for Bose-Einstein correlation in statistics: This equation shows that the temperature of DNA black brane depends on the Hamiltonian of DNA black brane, Hamiltonian of hexagonal-pentagonal molecules and temperatures of each manifold. Assuming that temperatures of hexagonal and pentagonal manifolds be constant and around the temperature of the room and using equations (12, 13 and 15), we can obtain dependency of temperature in terms of size of the core and plot it in Figure 5. Figure 5 Temperature of DNA black brane in terms of its size It is clear that for a DNA black brane with the size around 109 times longer than the core of the earth, the temperature will be around 6000 K. This temperature is in agreement with predicted temperature for the core. It is concluded that a DNA black brane with 109 meters which is compacted interior of core, can produce all known physics of the earth. Formation of a System of Telecommunication by Waves, DNAs, Dark DNAs and Molecules of Water Now, we consider the process of communications between earth, DNAs and molecules of water. In Figure 6, we show that a system like a DNA may be coiled in four dimensions. However, by adding extra strings in extra dimensions, it will be open, and its topology will be transferred to a circle. Figure 6 Topology of a system by adding some extra strings in extra dimensions change and shrinks to a circle These extra strings may be related to waves or dark DNAs in extra dimensions. We can write below relation between waves, DNAs and dark DNAs in extra dimensions: where NCircle is the number of microstates for a circle which is produced by joining dark. DNA, waves and DNA. Also, NDNA is the number of microstates for DNA, NDark-DNA is the number of microstates for dark DNA, and Nwave is the number of microstates for waves. This equation shows that waves connect DNAs in four dimensions to dark DNAs in extra dimensions and deform their topology, open their coilings and transfer them to circles (See Figure 7). Figure 7 Dark DNA in extra dimensions Thus, a number of microstates of waves depend on the number of microstates of DNAs and dark DNAs in extra dimensions. We can write below relation for the interaction of molecules of water with dark DNAs and waves: Figure 8 The relation between molecules of water and dark DNAs in extra dimensions Above equation shows that there is a big system of telecommunication which is formed by the core of the earth, DNAs, waves, molecules of water and dark DNAs in an extra dimension. This big system of telecommunication controls all evolutions of life on the earth. Also, the shape of DNAs has a direct effect on their number of microstates and consequently is in relation to a number of microstates of waves, molecules of water and dark DNAs in an extra dimension. Some Results Of The Existence Of The Biggest System Of Telecommunication We can write below results from our model and calculations: 1. Molecules of water are in related to dark DNAs in extra dimensions. On the other hand, dark DNAs have gender like normal DNAs. Thus, molecules of water can have some properties like gender, and each molecule of water with the gender of the male can attract by DNAs with the gender of female and reversely, each molecule of water with the gender of a female can attract with molecules of water with the gender of male (See Figures 9 and 10). Figure 9 Molecules of water is related to dark DNAs with the gender of female Equation (19, 20 and 21) give this possibility that the number of microstates of molecules of water has a relation with a number of microstates of DNAs and dark DNAs. This means that by radiating some waves to molecules of water, we can increase the number of microstates of molecules of water and obtain below relation: Figure 10 Molecules of water is related to dark DNAs with the gender of male 2. Above equation shows that by radiating some waves to water, we can extract properties of DNAs. This result is in good agreement with predictions of Montagnier and his colleagues (see Figure 11). Figure 11 Emergence of life by waves of the earth in pure water 3. DNA black brane interior of core, DNAs on the earth, dark DNAs in extra dimensions, waves and molecules of water form the best system of telecommunication (See figure 12). Figure 12 The biggest system of telecommunication from DNA black brane interior of core, DNAs on the earth, dark DNAs in extra dimensions and molecules of water Conclusions In this paper, we have shown that the earth’s core is the biggest system of telecommunication which exchanges waves with all DNAs and molecules of water. Imaging of DNAs on the interior of the metal of the core produces a DNA black brane with around 109 times longer than the core of the earth which is compacted and creates a structure similar to a black hole or black brane. We have shown that this DNA black brane is the main cause of high temperature of core and magnetic of earth. Also, this structure produces gravitational fields of earth and leads to the motion of the earth around the sun. We have argued that DNA black brane of earth exchange some non-linear waves with DNAs and recover their information. The shape of these waves depends on the topology of DNAs and are different for DNAs of males and females. Each DNA is compacted several times around various axes and reading it’s information is hard. However, by adding extra dimensions to four dimensions of the universe, the separation distance between elements of DNAs increases and waves of earth could recover their information. Thus, each DNA has an extra dark part in extra dimension which we call them dark DNAs. These extra parts couldn’t be observed, however, their effects can be seen. DNA black brane of the earth’s core exchange waves with both dark and light parts of DNA and connect them. These waves are different for males and females and play the role of topoisomerases in biology. On the other hand, our calculations and experiments show that these waves interact with molecules of water. However, the chemical structure of water (H2O) is very simple and cant store any information. This means that there are some extra dark DNAs on the 4+n-dimensional manifold which are related to molecules of water and play the role of memory for it. These dark DNAs have gender like other DNAs and give properties of gender to molecules of water. On the other hand, DNA black brane of the earth could emit some special waves to molecules of water and extract dark DNAs from extra dimensions. This means that the origin of life could be a system of telecommunication which is formed by DNA black brane interior of the earth, dark DNAs, waves and molecules of water. Funding: This research did not receive any financial support Competing Interests: The authors have declared that no competing interests exist ==== Refs 1 Hawking SW Ellis GF The large scale structure of space-time Cambridge university press 1973 https://doi.org/10.1017/CBO9780511524646 2 Hawking SW Virtual black holes Physical Review D 1996 53 6 3099 3107 https://doi.org/10.1103/PhysRevD.53.3099 PMid:10020307 3 Dimopoulos S Landsberg G Black holes at the large hadron collider Physical Review Letters 2001 87 16 161602 https://doi.org/10.1103/PhysRevLett.87.161602 PMid:11690198 11690198 4 Giddings SB Thomas S High energy colliders as black hole factories:The end of short-distance physics Physical Review D 2002 65 5 056010 https://doi.org/10.1103/PhysRevD.65.056010 5 CMS Collaboration JHEP 2018 11 042 arxiv: 1805.06013 6 Riofrio L Scientist Claims Theres a Black Hole in Center of the Earth 2019 5 3 https://mysteriousuniverse.org/2019/05/scientist-claims-theres-a-black-hole-in-center-of-the-earth 7 Wang T Song X Xia HH Equatorial anisotropy in the inner part of Earth's inner core from autocorrelation of earthquake coda Nature Geoscience 2015 8 3 224 https://doi.org/10.1038/ngeo2354 8 Tkalcic H Pham TS New Constraints on the Earth's Core From Global Correlation Wavefield In EGU General Assembly Conference Abstracts 2018 20 5834 9 Souriau A Souriau M Ellipticity and density at the inner core boundary from subcritical PKiKP and PcP data Geophysical Journal International 1989 98 1 39 54 https://doi.org/10.1111/j.1365-246X.1989.tb05512.x 10 Alfe D Gillan MJ Price GD Temperature and composition of the Earth's core Contemporary Physics 2007 48 2 63 80 https://doi.org/10.1080/00107510701529653 11 Anzellini S Dewaele A Mezouar M Loubeyre P Morard G Melting of iron at Earth's inner core boundary based on fast X-ray diffraction Science 2013 340 6131 464 6 https://doi.org/10.1126/science.1233514 PMid:23620049 23620049 12 Fukuhara M Possible generation of heat from nuclear fusion in Earth's inner core Scientific reports 2016 6 37740 https://doi.org/10.1038/srep37740 PMid:27876860 PMCid:PMC5120317 27876860 13 de Meijer RJ van Westrenen WS Afr J Sci (in the press) 14 Schuiling RD Dye S.T Is there a Nuclear Reactor at the Center of the Earth? 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==== Front Open Access Maced J Med SciOpen Access Maced J Med SciOpen Access Macedonian Journal of Medical Sciences1857-9655Republic of Macedonia ID Design 2012/DOOEL Skopje OAMJMS-7-308510.3889/oamjms.2019.777Review ArticleRecovery of Brain in Chick Embryos by Growing Second Heart and Brain Fioranelli Massimo 1Sepehri Alireza 1Roccia Maria Grazia 1Linda Cota 1Rossi Chiara 1Dawodo Amos 1Vojvodic Petar 2Lotti Jacopo 1Barygina Victoria 3Vojvodic Aleksandra 4Wollina Uwe 5Tirant Michael 6Thuong Nguyen Van 7Lotti Torello 81 Department of Nuclear Physics, Sub-nuclear and Radiation, G. Marconi University, Rome, Italy2 Clinic for Psychiatric Disorders “Dr. Laza Lazarevic”, Belgrade, Serbia3 Department of Biomedical Experimental and Clinical Sciences, University of Florence, Florence, Italy4 Department of Dermatology and Venereology, Military Medical Academy, Belgrade, Serbia5 Department of Dermatology and Allergology, Städtisches Klinikum Dresden, Dresden, Germany6 G. Marconi University, Rome, Italy7 Vietnam National Hospital of Dermatology and Venereology, Hanoi, Vietnam8 Department of Dermatology, University of G. Marconi, Rome, Italy Correspondence: Massimo Fioranelli. Department of Nuclear Physics, Sub-nuclear and Radiation, G. Marconi University, Rome, Italy. E-mail: [email protected] 9 2019 30 8 2019 7 18 3085 3089 23 6 2019 06 7 2019 07 7 2019 Copyright: © 2019 Massimo Fioranelli, Alireza Sepehri, Maria Grazia Roccia, Cota Linda, Chiara Rossi, Amos Dawodo, Petar Vojvodic, Jacopo Lotti, Victoria Barygina, Aleksandra Vojvodic, Uwe Wollina, Michael Tirant, Nguyen Van Thuong, Torello Lotti.2019This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License (CC BY-NC 4.0)To recover chick embryos damaged the brain, two methods are presented. In both of them, somatic cells of an embryo introduced into an egg cell and an embryo have emerged. In one method, injured a part of the brain in the head of an embryo is replaced with a healthy part of the brain. In the second method, the heart of brain embryo dead is transplanted with the embryo heart. In this mechanism, new blood cells are emerged in the bone marrow and transmit information of transplantation to subventricular zone (SVZ) of the brain through the circulatory system. Then, SVZ produces new neural stem cells by a subsequent dividing into neurons. These neurons produce new neural circuits within the brain and recover the injured brain. To examine the model, two hearts of two embryos are connected, and their effects on neural circuits are observed. ChickEmbryoBrainRecovery ==== Body Introduction Several years ago, some investigators proved the existence a little brain on the heart which acts like a real brain in the head [1]. This ‘little brain’ on the heart is comprised of spatially distributed sensory (afferent), interconnecting (local circuit) and motor (adrenergic and cholinergic efferent) neurones that communicate with others in intrathoracic extracardiac ganglia, all under the tonic influence of central neuronal command and circulating catecholamines. Neurones residing from the level of the heart to the insular cortex form temporally dependent reflexes that control overlapping, spatially determined cardiac indices [2]. Until now, fewer discussions have been done on this subject. For example, some researchers have argued that cardiac function is under the control of the autonomic nervous system, composed by the parasympathetic and sympathetic divisions, which are finely tuned at different hierarchical levels. They have shown that while a complex regulation occurs in the central nervous system involving the insular cortex, the amygdala and the hypothalamus, a local cardiac regulation also takes place within the heart, driven by an intracardiac nervous system. This complex system consists of a network of ganglionic plexuses and interconnecting ganglions and axons [3]. Now, the question arises that what happens for this little brain during heart transplantation? Recent investigations show that patients who gave hearts from donors, obtain some characteristics of them. One of them was Sylvia who declared that soon after her operation, she felt like drinking beer, something she hadn’t particularly been fond of before. Later, she observed an uncontrollable urge to eat chicken nuggets and found herself drawn to visiting the popular chicken restaurant chain, et al., [4]. This means that the little brain could be transformed from one body to another during heart transplantation. On the other hand, the existence of the little brain on the heart could help to head transplantation and make it possible. In fact, during cutting heads, this little brain plays the main role in decisions and does all activities of a real brain. Until now, some scientists have reported the head transplantation in animals. For example, in 1908, some scientists have tried to graft the head of one dog on an intact second dog; the grafted head showed some reflexes early on but deteriorated quickly, and the animal was killed after a few hours [5]. There were few animal experiments on head transplantation for many years after this [6], [7]. In 2016 some investigators published a review of attempted as well as possible neuroprotection strategies that they said should be researched for potential use in a head transplantation procedure; they discussed various protocols for connecting the vasculature, the use of various levels of hypothermia, the use of blood substitutes, and the possibility of using hydrogen sulfide as a neuroprotective agent [8]. Besides these considerations, one of the interesting claims in head transplantation has been made by Sergio Canavero. He published a protocol and said would make human head transplantation possible [9]. This transplantation is possible if there be a second brain for doing activities of the brain in the absence of the head. This second brain could be a little brain in the heart. On the other hand, using cells transplantation may save some brain-damaged patients. However, the new head or brain should be formed from cells of the patient his / her self. This may be possible through the reprogramming of cells. During reprogramming, cells can convert to induced pluripotent stems and then these cells produce new specialised cells [9], [10], [11], [12]. In one of the methods, an oocyte can reprogram an adult nucleus into an embryonic state after somatic cell nuclear transfer, so that a new organism can be developed from such cell [13]. In some other methods, some factors (Oct4, Sox2, Klf4, and c-Myc) are used to generate induced pluripotent stem cells (iPSCs) [14]. Using these methods, we can produce new neural stem cells that could produce new neurons and form new neural circuits within the injured or dead brain. In this paper, we propose two methods for recovering chick embryos dead brains. In both methods, we injected a cell of the patient into an egg cell (for women, we use their egg cell) and put it in a uterus. After a period of time, neural networks and blood circulatory systems are produced. For a dead brain, we could transplant initial circuits of the initial brain with circuits of the second brain. For an injured brain, we can transplant the initial heart with the second one. This causes the formation of new blood cells in bone marrows and new neural stem cells in the subventricular zone (SVZ) of the brain. New neurons which are emerged in this process, produce new neural circuits and cure injured brain. The outline of this paper is as follows: In section II, we make a review of connections between the neural network and the circulatory system. In section III, we propose two methods for recovering injured brains. In section IV, we test one of the methods on chick embryos. A review of connections between nervous and circulatory systems Previously, it has been shown that there is a little brain in the heart that can control some activities of the body [1], [2]. In Figure 1, the location and distribution of intrinsic cardiac ganglia are shown [15]. A ganglion is a nerve cell cluster or a group of nerve cell bodies located in the autonomic nervous system and sensory system, mostly outside the central nervous system except for certain nuclei [16]. This system is a bridge between the nervous system and neural network. The origin of these systems is genetic circuits of initial DNAs. These genetic circuits act as the receiver or sender of radio eaves [17]. Figure 1 Distributions of neurons in a heart [1], [15] To transfer information of these genetic circuits, two types of circuits have emerged. First circuits are built of neurons and form a neural network within the brain. Second circuits are formed from vessels of blood cells and form the circulatory system. There are some connections between the nervous system and the heart which are known as cardiac ganglia. Also, there are some connections between the circulatory system and the neural network in the brain (see Figure 2). Figure 2 Connections between neural and circulatory systems Two methods for recovering injured and dead brains To recover injured or dead brains, we should replace some hurt circuits with healthy ones. To produce these circuits, we can use of reprogramming. The best way for reprogramming is by removing the nucleus of an egg cell and replacing it by the nucleus of a body cell of a patient. If we put this system under normal conditions like conditions of a uterus, this cell divides into more cells, and an embryo emerges. This embryo has a brain and a heart which are like the initial brain and heart (See Figure 3). Figure 3 Formation of second heart and brain by introducing a somatic cell into an egg cell During the formation of the brain in an embryo, first, neural plate and neural tube are emerged [18] (See Figure 4) which can be transplanted with the nervous system of the related patient and pass other stages inside his/her body. Figure 4 Stages of developments of a brain in a chick embryo [18] For some brain-dead patients, some parts of initial brain should be replaced by normal and healthy parts of the second brain (See Figure 5). Figure 5 Formation and Transplantation of normal part of the second brain into the injured part of the first brain Maybe this question arises that what is the fate of memory and personality during this replacement. We can hope that some information is exchanged between circuits of brain and heart, and thus, the initial heart has a copy of memory in its neural system. After replacing neural circuits of the dead brain with new ones, this memory can be transformed into the brain. For patients who their SVZ part of the brain isn’t hurt and is healthy, one can transplant initial heart with the heart of related embryo. In these conditions, some new blood cells have emerged in bone marrows. These new cells reach the SVZ and communicate with neural stem cells. Consequently, some new neurons have emerged which produce neural circuits and recover brain (See Figure 6). Figure 6 Emergence of new neural circuits after transplantation of second heart into an initial one Testing the model for chick embryos To observe the effects of transplantation of two hearts on neural systems, we can use of chick embryos. First, we incubate fertilised eggs for 58 h. Then, we break them and pour them in a tube or vessel of a shell-less culture system. In this system, similar to [19], we apply a 450 ml polystyrene plastic cup as the pod for the culture vessel. We also make a 1-1.5 cm diameter hole in the side of the cup approximately 2 cm from the bottom and plug the hole with a cotton pledget as a filter. We insert a 2 mm diameter plastic tube through the space between the pledget and the hole to provide an oxygen supply. We add an aqueous solution (40 ml) of benzalkonium chloride to the cup. We form a polymethylpentene film into a concave shape, carefully avoiding wrinkles and installed as an artificial culture vessel in the pod. Finally, we place a polystyrene plastic cover on top of the culture vessel [19]. In one of the vessels, we put normal embryo, and in another, we try to connect two embryos from their hearts. We put two types of vessels in an incubator (see Figure 7). Figure 7 Transplantation of two embryos We connect two systems to the scope and measure related currents. We observe that there is a significant difference between radiated waves of neurons within a normal vessel and vessel, including two connected embryos (See Figure 8). This shows that transplantation of two hearts has a direct effect on the formation of neural circuits. Figure 8 Comparing currents which are emerged by the neural system of a chick embryo (blue colour) with currents of two interacting embryos (red colour) Conclusion In this paper, we have shown that there is a direct relation between the neural network and the blood circulatory system. Both of them are emerged to transfer information of initial genes in an initial stem cell. In fact, each gene acts as the receiver or sender of waves and produce two types of circuits, one related to the neural circuit and another related to the blood circuit. These circuits exchange information with each other through some connections. These connections are some neurons within the heart and some vessels with the head. This may help us to introduce some methods for recovering dead and injured brains. In these methods, we inject a cell of a patient into a bare egg cell and put this system in a uterus. After some time, two new neural and circulatory systems emerge. Then, we have two ways. In one way, we can transplant injured parts of the initial brain with some neural circuits of the second brain. In the second way, we can transplant the initial heart of a patient with a second heart of embryo. In these conditions, bone marrows produce new stem blood cells and cause to produce new blood cells. These blood cells move along the circulatory system and reach to SVZ part of brain. Then, SVZ produce some new neurons related to the second heart and create new neural circuits. These circuits are replaced with ruined circuits and recover dead brain. We have tested the model in chick embryos and shown that transplantation has a direct effect on neural circuits. Funding: This research did not receive any financial support Competing Interests: The authors have declared that no competing interests exist ==== Refs 1 Armour JA The little brain on the heart Cleve Clin J Med 2007 74 1 S48 51 https://doi.org/10.3949/ccjm.74.Suppl_1.S48 PMid:17455544 17455544 2 Armour JA Potential clinical relevance of the 'little brain'on the mammalian heart Exp Physiol 2015 100 4 348 353 25833107 3 Campos ID Pinto V Sousa N Pereira VH A brain within the heart:A review on the intracardiac nervous system Journal of molecular and cellular cardiology 2018 119 1 9 https://doi.org/10.1016/j.yjmcc.2018.04.005 PMid:29653111 29653111 4 Pearsall Paul The Heart's Code:Tapping the wisdom and power of our heart energy 1999 New York Broadway Books 5 Lamba N Holsgrove D Broekman ML The history of head transplantation:a review Acta Neurochirurgica 2016 158 12 2239 2247 https://doi.org/10.1007/s00701-016-2984-0 PMid:27738901 PMCid:PMC5116034 27738901 6 Furr A Hardy MA Barret JP Barker JH Surgical, ethical, and psychosocial considerations in human head transplantation International journal of Surgery 2017 41 190 5 https://doi.org/10.1016/j.ijsu.2017.01.077 PMid:28110028 PMCid:PMC5490488 28110028 7 Čartolovni A Spagnolo AG Ethical considerations regarding head transplantation Surgical Neurology International 2015 6 1 103 https://doi.org/10.4103/2152-7806.158785 PMid:26110084 PMCid:PMC4476134 8 Ren X Orlova EV Maevsky EI Bonicalzi V Canavero S Brain protection during cephalosomatic anastomosis Surgery 2016 160 1 5 10 https://doi.org/10.1016/j.surg.2016.01.026 PMid:27143608 27143608 9 Canavero S HEAVEN:The head anastomosis venture Project outline for the first human head transplantation with spinal linkage (GEMINI) Surgical Neurology International 2013 4 1 S335 42 https://doi.org/10.4103/2152-7806.113444 PMid:24244881 PMCid:PMC3821155 24244881 10 Gurdon JB The developmental capacity of nuclei taken from intestinal epithelium cells of feeding tadpoles Journal of Embryology and Experimental Morphology 1962 10 622 40 13951335 11 Takahashi K Yamanaka S Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors cell 2006 126 4 663 76 https://doi.org/10.1016/j.cell.2006.07.024 PMid:16904174 16904174 12 Baker M Adult cells reprogrammed to pluripotency, without tumors Nature Reports Stem Cells 2007 https://doi.org/10.1038/stemcells.2007.124 13 Hochedlinger K Jaenisch R Nuclear reprogramming and pluripotency Nature 2006 441 7097 1061 7 https://doi.org/10.1038/naturne04955 PMid:16810240 16810240 14 Paull D Sevilla A Zhou H Hahn AK Kim H Napolitano C Tsankov A Shang L Krumholz K Jagadeesan P Woodard CM Automated, high-throughput derivation, characterization and differentiation of induced pluripotent stem cells Nature methods 2015 12 9 885 92 https://doi.org/10.1038/nmeth.3507 PMid:26237226 26237226 15 https://www.heartmath.org/our-heart-brain/ 16 Brodal P The Central Nervous System Oxford University Press In the CNS, such a group is called a nucleus and in the peripheral nervous system (PNS), a ganglion 2010 5 17 Sepehri A A mathematical model for DNA Int J Geom Methods Mod Phys 2017 14 1750152 https://doi.org/10.1142/S02198≍7501523 18 Estomih Mtui Gregory Gruener (2006) Clinical Neuroanatomy and Neuroscience 2006 1 Philadelphia Saunders 19 Tahara Y Obara K A Novel Shell-less Culture System for Chick Embryos Using a Plastic Film as Culture Vessels Journal of Poultry Science 2014 51 3 307 312 https://doi.org/10.2141/jpsa.0130043 20 Sepehri A Fioranelli M Roccia MG Shoorvazi S The role of entropic penalties of circular DNA assembly in spectroscopy and imaging J Theor Appl Phys 2019 https://doi.org/10.1007/s40094-019-0321-8	31850128	PMC6910787	NO-CC CODE	2021-01-06 05:12:17	yes	Open Access Maced J Med Sci. 2019 Aug 30; 7(18):3085-3089
==== Front Open Access Maced J Med SciOpen Access Maced J Med SciOpen Access Macedonian Journal of Medical Sciences1857-9655Republic of Macedonia ID Design 2012/DOOEL Skopje OAMJMS-7-299110.3889/oamjms.2019.778Research ArticleNew System Delivering Microwaves Energy for Inducing Subcutaneous Fat Reduction: In - Vivo Histological and Ultrastructural Evidence Zerbinati Nicola 1d’Este Edoardo 2Cornaglia Antonia Icaro 3Riva Federica 3Farina Aurora 3Calligaro Alberto 3Gallo Giovanni 4Perrotta Emanuele Rosario 5Protasoni Marina 1Bonan Paolo 6Vojvodic Aleksandra 7Fioranelli Massimo 8Thuong Nguyen Van 9Lotti Torello 10Tirant Michael 11Vojvodic Petar 121 Department of Medicine and Surgery School of Medicine, University of Insubria (Varese), Italy2 Centro Medico Polispecialistico, Pavia, Italy3 Department of Public Health, Experimental and Forensic Medicine, Unit of Histology and Embryology, University of Pavia, Pavia, Italy4 Laser Unit of Medi-Este-Laser Center, Canicattì, Italy5 Department of General Surgery and Surgical Medical Specialties, Hospital Cannizzaro, Catania, Italy6 Laser Cutaneous Cosmetic & Plastic Surgery Unit, Villa Donatello Clinic, Florence, Italy7 Department of Dermatology and Venereology, Military Medical Academy, Belgrade, Serbia8 Department of Nuclear Physics, Sub-nuclear and Radiation, G. Marconi University, Rome, Italy9 Director of National Hospital of Dermatology and Venereology Vietnam, Head of Dermatology and Venereology Faculty, Hanoi Medical University, Hanoi, Vietnam10 Department of Dermatology, University Guglielmo Marconi, Rome, Italy11 University Guglielmo Marconi, Rome, Italy12 Clinic for Psychiatric Disorders “Dr. Laza Lazarevic”, Belgrade, Serbia Correspondence: Massimo Fioranelli. Department of Nuclear Physics, Sub-nuclear and Radiation, G. Marconi University, Rome, Italy. E-mail: [email protected] 9 2019 30 8 2019 7 18 2991 2997 12 6 2019 04 7 2019 05 7 2019 Copyright: © 2019 Nicola Zerbinati, Edoardo d’Este, Antonia Icaro Cornaglia, Federica Riva, Aurora Farina, Alberto Calligaro, Giovanni Gallo, Emanuele Rosario Perrotta, Marina Protasoni, Paolo Bonan, Aleksandra Vojvodic, Massimo Fioranelli, Nguyen Van Thuong, Torello Lotti, Michael Tirant, Petar Vojvodic.2019This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License (CC BY-NC 4.0)BACKGROUND: Recently, it has been developed a new technology for the reduction of subcutaneous adipose tissue through a non-invasive treatment by microwaves. The main objective of the present study is to demonstrate the feasibility of utilising a non-invasive, localised microwaves (MW) device to induce thermal modifications into subcutaneous adipose tissue only by a controlled electromagnetic field that heats up fat preferentially. This device is provided with a special handpiece appropriately cooled, directly contacting the cutaneous surface of the body, which provides a calibrated energy transfer by microwaves. AIM: In this paper, microscopic and ultrastructural modifications of subcutaneous adipose tissue induced by microwaves irradiation are evaluated. METHODS: Our experimental plan was designed for collecting biopsy samples, for each skin region treated with a single irradiation session, 1) before treatment (control), 2) immediately after treatment, 3) after 6 hrs, 4) after 1 month, 5) after 2 months. Bioptic samples from each step were processed for light microscopy and transmission electron microscopy. At the same time, each region where biopsies were collected was subjected to ultrasound examination. Recorded images permitted to evaluate the thickness of different layers as epidermis, dermis, hypodermis, connective fasciae, until to muscle layer, and related modifications induced by treatment. RESULTS: In every biopsy collected at different time-steps, epidermis and superficial dermis appeared not modified compared to control. Differently, already in the short-term biopsies, in the deep dermis and superficial hypodermis, fibrillar connective tissue appeared modified, showing reduction and fragmentation of interlobular collagen septa. The most important adipose tissue modifications were detectable following 1 month from treatment, with a significant reduction of subcutaneous fat, participating both the lysis of many adipocytes and the related phagocytic action of monocytes/macrophages on residuals of compromised structures of adipocytes. In the samples collected two months following treatment, the remnants of adipose tissue appeared normal, and macrophages were completely absent. CONCLUSIONS: Ultrasound, microscopic and ultrastructural evidence are supporting significant effectiveness of the new device treatment in the reduction of subcutaneous fat. In this paper, the possible mechanisms involved in the activation of the monocytes/macrophages system responsible for the removal of adipocytes residuals have also been discussed. MicrowavesSubcutaneousFatFat reduction ==== Body Introduction The trafficking of fatty acids into and out adipocytes is a physiological mechanism regulated by a complex series of proteins and enzymes and is under control by a variety of hormonal and metabolic factors (Thompson 2011) [1]. Many Authors have presented theoretical and experimental papers using methods and devices with as objective the reduction of adipose tissue. Particular interesting a recent paper (Asan 2017) [2] in which it has been considered the properties of some tissues related to the absorption of microwaves energy in different models using equivalent phantom and ex-vivo measurements. The most of experimental evaluations available in the literature, related to fat reduction, have been performed in vitro conditions, which only in few cases could represent what and how happens in vivo, in the complex dynamics inside tissues and organs in the individuals. In this paper, we present morphological observations on the subcutaneous adipose tissue following microwaves irradiation of the skin. A controlled electromagnetic field @2,45 GHz, perpendicularly applied to the skin, is selectively absorbed by the subdermal fat layer thanks to the dielectric properties of the different tissues (epidermis, dermis, fat) crossed by the applied EMF. The high-controlled emission of the EMF can establish a perfect coupling only in the presence of a fat layer, due to the absorption characteristics of this tissue at the established frequency. In the present in-vivo study on an animal model, 7 minutes thermal exposure to 50°C, histological and ultrastructural evidences show conservation without damage to the epidermis and dermis layers, while the subdermal fat is modified through a series of molecular processes stimulating a massive delivery of fat droplets and the beginning of “auto-adipolysis”. Material and Methods A mathematical model was used to correlate the frequency of the electromagnetic field and the dielectric properties of the tissues to induce localised hyperthermia only in the subcutaneous fat layer. The non-invasive pseudo-transcutaneous electromagnetic field (Coolwaves™ by Onda, DEKA, Florence, Italy) was applied to thermally induce adipocytes’ damage. During the treatment, applying a transmitting handpiece on the surface of the skin, the electromagnetic field energy was delivered into the subcutaneous adipose tissue (SAT) of a Vietnamese pig (this study on the animal model was carried out in full compliance with international guidelines for safety and compliance with their use) with a 50.000 J total dose delivered in 7 minutes over a cutaneous area 15 x 15 cm2. The handpiece used was maintained at a temperature of 5°C through a specific cooling system. The experimental plan was designed on the basis of a single irradiation session, and for each skin region treated, biopsy samples were collected and processed for light and electron microscopy, owing to the following steps: T0 (before treatment, as control), T1 (immediately after treatment), T2 (6h following treatment), T3 (1 month following treatment) and T4 (2 months following treatment). Temperature monitoring was performed superficially by the thermometric infrared camera vision system and internally, in the subdermal fat layer (at 7mm depth), by sterile thermometric glass fibre. The epidermal temperature in the treated area was always observed in a safety-rage of 15-25°C, while at 4 to 7 mm in depth was of 50°C (Figure 1A and 1B). Figure 1 A and B are showing the tip of the temperature probe (arrow) by ultrasound A) and the temperature measured inside the hypodermal layer B) respectively; C) and D) correlation of ultrasound image C) and biopsy sample D) tissue layers; The different layers are clearly identifiable: Epidermis and Dermis (E + D), Subcutis (Hypodermal Adipose Tissue), Fascia, Muscle In each time step, biopsy samples were collected, immediately immersed in the fixative solutions and processed both for light microscopy and transmission electron microscopy. Light microscopy Biopsy samples were immediately immersed in a 4% paraformaldehyde/sodium phosphate buffer solution for 24 hrs and then processed (dehydration, paraffin embedding and sectioning) for light microscopy. Sections were stained with Haematoxylin and Eosin, and other sections were stained with Picrosirius Red for collagen staining. Electron microscopy Fixation was performed by immersion of biopsy samples in a 2.5% glutaraldehyde (EM grade)-2% paraformaldehyde in 0.1M sodium cacodylate buffer solution (pH 7.3) for 6 hours at 4°C. After washing in the same buffer, samples were post-fixed for 2 h in osmium tetroxide 1.33% in 0.1 M s-collidine buffer, dehydrated in a graded series of ethanol (30%, 50%, 70%, 80%, 95%, 100%), propylene oxide and finally embedded in epoxy resin Epon 812. Semithin (0.2 µm) and ultrathin (40-60 nm) sections were obtained at the ultramicrotome Reichert Ultracut S provided with a diamond knife. Semithin sections were stained with Toluidine blue and ultrathin sections, previously collected on 200 µm mesh copper grids, were counterstained with lead citrate and uranyl acetate. A Zeiss EM 902 transmission electron microscope, operating at 80 kV with an objective aperture of 30 / 60 µm, was used for direct observation. Electron micrographs were recorded on Kodak 4489 Electron Image film and finally digitised with an Epson Perfection V750 Pro scanner at 1200 dpi. Results The description of our observations will follow the time sequence of samples collection, using the most suitable presentation of our findings to better understand the functional dynamics. Before sample collection, it has been used ultrasound imaging to define the thickness of the different structural layers, particularly the depth and extension of the subcutaneous tissue as the target of the microwave energy transfer (Figure 1C) and compare it with biopsy sample (Figure 1D). At time T0, defining the control samples collected before the treatment, microscopic morphology of adipocytes appeared normal, spherical in shape, with the whole volume of cells appearing filled with a homogeneous content, surrounded by a very thin layer of peripheral cytoplasm, where an elongated and flattened nucleus was well identifiable (Figure 2A). All around adipocytes and between them, a loose connective tissue was appreciable, with some cells (fibroblasts) and an extracellular matrix constituted by collagen fibrils relatively dispersed inside a faint ground substance (Figure 2A). Figure 2 T0 (Control); A) Semithin section at the light microscope from epoxy resin embedding, stained with toluidine blue. Adipocytes show peripheral nucleus (N) with dispersed chromatin, a very thin peripheral layer of cytoplasm, and a homogeneous lipidic content. Around them, elongated fibroblasts and bundles of collagen fibres are also observable; B) Ultrastructure of the very thin peripheral cytoplasmic of an adipocyte and inside it two elongated mitochondria were observable. A continuous basal lamina close to the plasma membrane and collagen microfibrils in the surrounding connective tissue is also visible. The homogeneous fat is constituting the inner content of the adipocyte Small blood vessels were also identifiable. At the electron microscope (Figure 2B), the very thin peripheral cytoplasm appeared to contain dispersed organelles, as mitochondria, rare profiles of the endoplasmic reticulum and small vesicles involved realistically in the physiological transport of materials (lipids included), responsible of the correct homeostatic processes across the cytoplasm to and from the interstitial connective tissue. At time T1 (immediately the following treatment), small single or multiple vesicles were visible at the light microscope in the peripheral cytoplasm of adipocytes (Figure 3A). At time T2 (6 hrs after treatment), many vesicles containing material very similar to the big lipidic content of the adipocyte were detectable inside adipocyte peripheral cytoplasm. In ultrathin sections observed at the electron microscope, the whole peripheral cytoplasm was completely occupied by vesicles containing a relatively electron transparent material. This feature appears particularly evident in the oblique sections of the inner surface of the peripheral cytoplasm as a holey structure (Figure 3B). In a relatively thick section at the electron microscope, we observed blebbing structures at the surface of adipocytes (Figure 3C) projecting part of their lipidic content towards the interstitial connective tissue. Figure 3 T1, T2, T3; A) T1 (immediately the following treatment): Semithin section at the light microscope from epoxy resin embedding, stained with toluidine blue. Inside the thin peripheral cytoplasm of adipocytes, some vesicles with the content similar to the big lipid content of cells are visible; B) T2 (6 hrs following treatment): at the electron microscope the peripheral cytoplasm of adipocytes shows numerous vesicles occupying the whole thickness of the cytoplasm. In the oblique sections, the internal cytoplasmic surface appears fully of vesicles; C) T3 (6 hrs following treatment): the high magnification at the electron microscope represents the superficial blebbing as a direct extrusion of a lipid droplet (asterisk) through peripheral cytoplasm from the inside of an adipocyte (left) to the outside, in the interstitial connective tissue (ICT). Arrows mark the thin peripheral cytoplasm of the adipocyte Further, some cytological details of adipocytes involved in these mechanisms, have been observed at the electron microscope, as mitochondrial swelling with few disorganised cristae and interruptions of the inner membrane, interruptions of the plasma membrane, dilations of the endoplasmic reticulum (data not shown). The whole of these features suggests adipolysis via necrotic processes. At time T3 (1 month after treatment), observing transverse sections of the wall of adipocytes, numerous vesicles were evident inside an electron-dense cytoplasm. Some of them appeared as invaginations of the inner side of the peripheral cytoplasm with a content continuous with the big lipidic sphere of the adipocyte; others appeared as openings towards the interstitial connective tissue. These features are suggesting a mechanism of endo-exo-cytosis of lipids from the inside of the adipocyte to the outside towards the interstitial connective tissue. At time T3, in some areas of the biopsies an inflammatory infiltrate was detectable. This infiltrates appeared constituted by numerous cells penetrating the interstitial tissue, singularly or grouped encircling single adipocytes (Figure 4A). Figure 4 T3 (1 month following treatment); A) Light microscopy of a “Crown-Like-Structure”, constituted by a crown of single monocytes, joined between them by thin cytoplasmic processes, distributed all around an adipocyte. These monocytes appear to contain in the cytoplasm small droplets similar to the content of the adipocyte. Realistically these droplets are the result of phagocytosis due to macrophage activity; B) The smaller adipocyte appears completely surrounded by a multinucleated structure originated by the fusion of single monocytes forming a single syncytial macrophage. The small dimensions of the adipocyte are realistically due to stimulated phagocytosis of the fat of necrotic adipocyte. Semithin sections from epoxy resin embedding, toluidine blue-stained; C) In the high left part of the electron micrographs (red arrow), a portion of the big lipidic content of the adipocyte is directly contacting the interstitial tissue. The remnant part of this content is directly contacting the plasma membrane of a macrophage (small black arrows), which has already internalised in his cytoplasm numerous lipid droplets. The lack of the peripheral cytoplasm of the adipocyte demonstrates that this adipocyte has completed the process of cellular death. Furtherly, the basal membrane, far from the adipocyte, appears highly disorganised inside interstitial tissue (asterisks); N: macrophage nucleus The infiltrate is constituted mainly by monocytes distributing free in the interstitial connective tissue or close contact with adipocytes, covering their external surface. Single monocytes appeared provided with cytoplasmic processes projecting free in the interstitial connective tissue or directly contacting free vesicles released by adipocytes. The first contact of monocytes with lipids released by necrotic adipocytes is realistically suggested by a direct contact representing the result of the “find me” action of the extracellular lipid structures towards monocytes and the starting of the mechanism “eat me” stimulating the phagocytic action of macrophages. The following step in the mechanism of adipolysis observed at the same time T3 (after 1 month from treatment) is represented by the formation of Crown-Like-Structures as the most typical feature. CLS are constituted by single monocytes closely related one with the other forming a pluricellular structure directly encircling adipocytes (Figure 4A). Inside these cells, small droplets appear very similar to the adipocyte content, suggesting the starting of a phagocytosis mechanism. In a following step of the mechanism suggested by images, single monocytes forming the “crown” around adipocyte, fuse their cytoplasm forming a unique big cytoplasm with many nuclei inside (syncytium) all around adipocytes (Figure 4B), realistically for a more effective action of phagocytosis of the content of these cells and the cytoplasmic residuals of the same necrotic adipocytes. Remark in Figure 4B the small dimensions of the represented adipocyte, due to the action of fat removal by macrophages. At the electron microscope, a single monocyte of the crown appears closely contacting the surface of the adipocyte (Figure 4C). The surface of this cell appears expanded in numerous and complex superficial projections towards both the interstitium and the surface of adipocyte, constituting a sort of labyrinth of spaces realistically ready to endocytosis. Inside this cell, some small droplets of phagocytosis are detectable (Figure 7C). Adipocyte peripheral cytoplasm appears highly fragmented or completely absent, permitting a direct exposure of the adipocyte content to the interstitium and direct contact between the plasma membrane of macrophage and the big lipid droplet of the same adipocyte. Furtherly, remark the absence of the basal membrane at the surface of the necrotic adipocyte. Disorganised residuals of this structure are recognisable in the interstitium externally to the macrophage (Figure 4C). The interstitium of the adipose tissue is constituted by a loose connective tissue containing finely distributed collagen fibres and a highly permeable ground substance formed by glycosaminoglycans, proteoglycans and multi adhesive glycoproteins. The interstitial connective tissue is also provided with cells, like fibroblasts, mesenchymal cells capable of differentiation, migrating cells, endothelial cells forming the inner lining of blood and lymphatic vessels. Highly extended lymphatic vessels have been detected even at the light microscope, as a delicate structure with a very fine endothelial wall expanded between adipocytes. Inside lymphatic vessels, free macrophages containing lipid droplets. Realistically, following phagocytosis, macrophages migrate inside connective tissue, then pass through the thin endothelial wall of lymphatics and finally they are transported with the lymph to lymph nodes where the terminal lysis of the phagocytised material is performed (data not shown). At time T4 (two months after treatment) the normal structure of the adipose tissue appear restored, as shown in semithin sections from epoxy resin embedding (Figure 5A). Normal features of adipocytes morphology two months after treatment are also confirmed at the electron microscope (Figure 5B). Figure 5 T4 (2 months following treatment); A) Semithin section at the light microscope from epoxy resin embedding, toluidine blue-stained. Adipocytes appear as T0 (control), without any vesicle in the thin peripheral cytoplasm and a very thin interstitial tissue between them. No cell infiltrate detectable; B) At the electron microscope, the peripheral cytoplasm of two adjacent adipocytes, with the interstitial connective tissue (ICT) between them, shows normal features, as in the control samples. Abnormal modifications of the plasma membrane or cytoplasmic structures are never detectable. The very small vesicles observable in the peripheral cytoplasm, as in control, are related to physiological trafficking of molecules across peripheral cytoplasm of adipocytes The effectiveness of the treatment in terms of fat reduction has been demonstrated by ultrasound measurements of thickness from the surface of epidermis to the muscle layer, significantly reduced due to the fat reduction (Table 1). Table 1 Comparative Table of Thickness from the epidermal surface to the muscle (excluded) Time Table Thickness Abdomen A (right) Thickness Abdomen B (left) T0 Control 2.7 cm 3.1 cm T1 Immediately post treatment 2.6 cm 3.0 cm T2 6 hrs after treatment 2.38 cm 2.59 cm T3 1 Month after treatment 1.38 cm 1.3 cm T4 2 Months after treatment 1.31 cm 1.4 cm Discussion Controlled hyperthermia-and maybe other effects not directly evaluable due to the treatment (among them the resonance of some molecular species-both structural and enzymatic-with the frequency of irradiation) determines in the adipose tissue (with particular effect on hypertrophic adipocytes), severe adipolysis with necrotic-like features which in turn stimulate an immune response with the active participation of monocytes and macrophages. Realistically, it could result in adipocytes a response of functional surcharge in the transporting mechanisms through membranes of the peripheral cytoplasm. In adipocytes, some key-structures for the life of cells appear involved, as the plasma membrane, the outer and inner membranes of mitochondria, and the membranes of the endoplasmic reticulum. The plasma membrane is essential for maintaining the intracellular microenvironment and for the whole of the finely controlled transmembrane transport processes which guarantee the correct ionic and molecular gradients and the related homeostatic equilibrium with the extracellular environment. Mitochondria are the cytoplasmic organelles responsible for oxidative phosphorylation, providing adenosine triphosphate (ATP) for most energetic cellular processes. Endoplasmic reticulum (particularly the rough e. r.) is the site of protein synthesis in the polyribosomes attached to the membranes of the organelle, controlling at the same time the 3D conformation of proteins form which specific biological functions depend. Concerning mitochondria, it is possible to consider that the increased catabolism induced by irradiation, in the mitochondria of hypertrophic adipocytes would start a condition of oxidative stress with production of Reactive Species of Oxygen and free radicals (Giordano 2013) [3], promoting an excess of delivery of fatty acids. The excess of transport of different molecular species through lipid droplets well identifiable morphologically at the electron microscope (Kranedonk 2014) [4]-fatty acids, adipokines and pro-inflammatory molecules – to the outside of the cell towards the interstitial connective tissue (Giordano 2013) [3], overtake the normal physiologic homeostatic flow through the very thin peripheral cytoplasm of adipocytes (Gao 2017) [5]. As a consequence of these structural and functional events, “necrosis-like” modifications are initiated, similarly to what happens in experimental obesity demonstrated in mouse and humans (Cinti 2005) [6]. Through a chemotactic mechanism mediated by receptors specifically called “find me”, microparticles delivered by adipocytes stimulate the recruitment of cells, both resident and coming from the blood, specifically monocytes and macrophages (Eguchi 2015) [7]. To all that, it follows the starting of phagocytosis through molecular triggering signals (“eat me” signals) of phosphatidylserine (a glycerophospholipid) delivered by adipocytes (Krahling 1999, Engin 2017) [8], [9]. They are also released pro-inflammatory mediators, as (TNF)α, nitric oxide synthase, interleukin (IL)-6 and (IL)-1β. The most of macrophages arrange in close relationship with adipocytes which have started the necrotic process, constituting characteristic “Crown Like Structures” (Cinti 2005) [6]. In these structures, activated monocytes and macrophages, starting with single contacts, arrange constituting extended multinuclear syncytia, as demonstrated in our observations. The function of these structures is the removal of the excess of free fatty acids and lipid droplets free in the interstitial tissue, together with the residuals of necrotic adipocytes. The big amount of small insoluble lipid droplets released from adipocytes constitute an important cytotoxic source of cholesterol and free fatty acids, which can damage other adipocytes (Unger 2002) [10]. Because of that, the removal of these molecular species from the interstitial tissue by macrophages represents a defence mechanism to restore the physiological homeostatic equilibrium. To all that it is added the release of further other molecules chemoattractant for macrophages, also stimulating the recruitment of blood monocytes through diapedesis from vessels with synergistic action of resident macrophages (Curat 2004) [11]. The excess of free fatty acids, via “Toll-like receptors” (Nguyen 2007) [12], stimulate the immune- system of the interstitial tissue, represented by resident cells (subpopulation of macrophages also identified as “dendritic cells”). These cells, similarly to dendritic cells and Langerhans cells in the epidermis, perform patrolling functions in the interstitium and favour the recall of other monocytes from the blood. In these cells, phagocytic functions are stimulated and optimised through the aggregation of single monocytes forming multinucleated phagocyting macrophages which arrange all around the most damaged adipocytes. In this process are also involved the lipases in the adipocytes (Fujimoto 2011) [13], as enzymes acting on constitutive triglycerides inside adipocytes, with the delivery of glycerol and fatty acids. In this way, these molecules are delivered by adipocytes which already started a necrotic process, to which significantly contribute Reactive Oxygen Species (Ventura 2004, Green 2014) [14], [15]. These molecular species aggravate the cytotoxic stress, with rupture of cells membranes (plasma membrane, mitochondria and endoplasmic reticulum membranes), with free delivery of both cell fragments (or molecular components no more contained inside membranous compartments) and enzymes into the interstitial tissue. Here, through phagocytosis, macrophages remove these materials from interstitium and after that, migrate inside lymphatic vessels in order to complete terminal lysis of phagocytized material from involved adipocytes Funding: This research did not receive any financial support Competing Interests: The authors have declared that no competing interests exist ==== Refs 1 Thompson BR Lobo S Bernlohr DA Fatty acid flux in adipocytes;the in's and out's of fat cell lipid trafficking Mol Cell Endocrinol 2010 318 1-2 24 33 https://doi.org/10.1016/j.mce.2009.08.015 PMid:19720110 PMCid:PMC2826553 19720110 2 Asan NB Noreland D Hassan E Redzwan Mohd Shah S Rydberg A Blokhuis TJ Carlsson PO Voigt T Augustine R Intra-body microwave communication through adipose tissue Healthc Technol Lett 2017 4 4 115 121 https://doi.org/10.1049/htl.2016.0104 PMid:28868147 PMCid:PMC5569712 28868147 3 Giordano A Murano I Mondini E Perugini J Smorlesi A Severi I Barazzoni R Scherer PE Cinti S Obese adipocytes show ultrastructural features of stressed cells and die of pyroptosis J Lipid Res 2013 54 9 2423 36 https://doi.org/10.1194/jlr.M038638 PMid:23836106 PMCid:PMC3735940 23836106 4 Kranendonk ME Visseren FL van Balkom BW Nolte-'t Hoen EN van Herwaarden JA de Jager W Schipper HS Brenkman AB Verhaar MC Wauben MH Kalkhoven E Human adipocyte extracellular vesicles in reciprocal signaling between adipocytes and macrophages Obesity (Silver Spring) 2014 22 5 1296 308 https://doi.org/10.1002/oby.20679 PMid:24339422 24339422 5 Gao X Salomon C Freeman DJ Extracellular vesicles from Adipose tissue - A potential Role in Obesity and type 2 Diabetes?Front Endocrinol 2017 8 202 https://doi.org/10.3389/fendo.2017.00202 PMid:28868048 PMCid:PMC5563356 6 Cinti S Mitchell G Barbatelli G Murano I Ceresi E Faloia E Wang S Fortier M Greenberg AS Obin MS Adipocyte death defines macrophage localization and function in adipose tissue of obese mice and humans J Lipid Res 2005 46 11 2347 2355 https://doi.org/10.1194/jlr.M500294-JLR200 PMid:16150820 16150820 7 Eguchi A Mulya A Lazic M Radhakrishnan D Berk MP Povero D Gornicka A Feldstein AE Microparticles release by adipocytes act as “find-me“signals to promote macrophage migration PLoS One 2015 10 4 e0123110 https://doi.org/10.1371/journal.pone.0123110 PMid:25849214 PMCid:PMC4388837 25849214 8 Krahling S Callahan MK Williamson P Schlegel RA 1999 Exposure of phosphatidylserine is a general feature in the phagocytosis of apoptotic lymphocytes by macrophages. Cell Death Differ 1999 6 2 183 9 https://doi.org/10.1038/sj.cdd.4400473 PMid:10200565 10200565 9 Engin AB Adipocyte-Macrophage Cross-Talk in Obesity Adv Exp Med Biol 2017 960 327 343 https://doi.org/10.1007/978-3-319-48382-59514 PMid:28585206 28585206 10 Unger R Lipotoxic diseases Annu Rev Med 2002 53 319 336 https://doi.org/10.1146/annurev.med.53.082901.104057 PMid:11818477 11818477 11 Curat C Miranville A Saengenes C Diehl M Tonus C Busse R Boulcumie A From blood monocytes to adipose tissue-resident macrophages:induction of diapedesis by human mature adipocytes Diabetes 2004 53 1285 1292 https://doi.org/10.2337/diabetes.53.5.1285 PMid:15111498 15111498 12 Nguyen MA Favelyukis S Nguyen AK Reichart D Scott PA Jenn A Liu-Bryan R Glass CK Neels JG Olefsky JM A subpopulation of macrophages infiltrates hypertrophic adipose tissue and is activated by free fatty acids via Toll-like receptors 2 and 4 and JNK-dependent pathways Journal of Biological Chemistry 2007 282 48 35279 92 https://doi.org/10.1074/jbc.M706762200 PMid:17916553 17916553 13 Fujimoto T1 Parton RG Not just fat:the structure and function of the lipid droplet Cold Spring Harb Perspect Biol 2011 3 3 a004838 https://doi.org/10.1101/cshperspect.a004838 PMid:21421923 PMCid:PMC3039932 21421923 14 Ventura J Cogswell P Flavell R Baldwin AJ Davis R JNK potentiates TNF-stimulated necrosis by increasing the production of cytotoxic reactive oxygen species Genes Dev 2004 8 2905 2915 https://doi.org/10.1101/gad.1223004 PMid:15545623 PMCid:PMC534651 15 Green DR Galluzzi L Kroemer G Cell biology Metabolic control of cell death Science 2014 345 6203 1250256 https://doi.org/10.1126/science.1250256 PMid:25237106 PMCid:PMC4219413 25237106	31850107	PMC6910790	NO-CC CODE	2021-01-06 05:12:18	yes	Open Access Maced J Med Sci. 2019 Aug 30; 7(18):2991-2997
==== Front Open Access Maced J Med SciOpen Access Maced J Med SciOpen Access Macedonian Journal of Medical Sciences1857-9655Republic of Macedonia ID Design 2012/DOOEL Skopje OAMJMS-7-309610.3889/oamjms.2019.767Research ArticleDNA Waves and Their Applications in Biology Fioranelli Massimo 1Sepehri Alireza 1Roccia Maria Grazia 1Rossi Chiara 1Lotti Jacopo 1Vojvodic Petar 2Barygina Victoria 3Vojvodic Aleksandra 4Vlaskovic-Jovicevic Tatjana 2Peric-Hajzler Zorica 5Matovic Dusica 5Vojvodic Jovana 2Dimitrijevic Sanja 6Sijan Goran 7Wollina Uwe 8Tirant Michael 9Thuong Nguyen Van 10Lotti Torello 111 Department of Nuclear Physics, Sub-nuclear and Radiation, G. Marconi University, Rome, Italy2 Clinic for Psychiatric Disorders “Dr. Laza Lazarevic”, Belgrade, Serbia3 Department of Biomedical Experimental and Clinical Sciences, University of Florence, Florence, Italy4 Department of Dermatology and Venereology, Military Medical Academy, Belgrade, Serbia5 Military Medical Academy, Belgrade, Serbia6 Department of Gynecology, Military Medical Academy, Belgrade, Serbia7 Clinic for Plastic Surgery and Burns, Military Medical Academy, Belgrade, Serbia8 Department of Dermatology and Allergology, Städtisches Klinikum Dresden, Dresden, Germany9 G. Marconi University, Rome, Italy10 Vietnam National Hospital of Dermatology and Venereology, Hanoi, Vietnam11 Department of Dermatology, University of G. Marconi, Rome, Italy Correspondence: Massimo Fioranelli. Department of Nuclear Physics, Sub-nuclear and Radiation, G. Marconi University, Rome, Italy. E-mail: [email protected] 9 2019 11 9 2019 7 18 3096 3100 02 9 2019 14 9 2019 15 9 2019 Copyright: © 2019 Massimo Fioranelli, Alireza Sepehri, Maria Grazia Roccia, Chiara Rossi, Jacopo Lotti, Petar Vojvodic, Victoria Barygina, Aleksandra Vojvodic, Tatjana Vlaskovic-Jovicevic, Zorica Peric-Hajzler, Dusica Matovic, Jovana Vojvodic, Sanja Dimitrijevic, Goran Sijan, Uwe Wollina, Michael Tirant, Nguyen Van Thuong, Torello Lotti.2019This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License (CC BY-NC 4.0)AIM: In this research, we show that DNA waves have many applications in biology. DNA is formed by the joining of quantum particles like electrons and charged atoms. DNA has different motions during transcription, translation, and replication, in which the charged particles move, accelerate, and emit waves. Thus, DNA could emit quantum waves. METHODS: Two methods are proposed to observe the effect of DNA waves. The first proposed method measures DNA waves emitted by bacteria suspended in the milk. The vessel of milk is placed in the interior of an inductor. One side of the vessel is connected to a generator and another side to a scope. By sending a current to the inductor, an input electromagnetic field is produced. Bacteria interact with the input field, change it and produce new output signals. Using the scope, the output signals are observed and compared with the input signals. The number of DNA waves produced also depends on temperature. RESULTS: At lower temperatures, bacterial replication is less, and fewer DNA waves are produced. Conversely, more bacteria are generated at higher temperatures, and more DNA waves are produced. The second proposed method acquires and images of DNA signals of chick embryos. In this method, a circuit is constructed that consists of a graphene or metal tube, generator, inductor, scope, DNA in the interior of eggs and DNA exterior to the eggs. Magnetic waves pass the interior and exterior DNA as well as the graphene. The DNA is excited and the exciting interior/exterior DNA exchanges waves. Some of these waves interact with electrons in the graphene tube, and a current is generated. Properties of the chick embryo DNA can be explored by analysing changes in the waves that emerge from the eggs. CONCLUSION: It is concluded that DNA waves could be used extensively in imaging and provide for us the exact information about evolutions of DNAs interior of biological systems. Quantum BiologyChick EmbryoDNA WaveBacterial DNA ==== Body Introduction Quantum biology is a field of science that explores the applications of quantum mechanics in biology. Erwin Schrödinger first coined the term “quantum mechanics” in biology and proposed the idea of an “aperiodic crystal” that contains genetic information in its configuration of covalent chemical bonds [1]. Also, he suggested that mutations could be explained by “quantum leaps”. The term “quantum biology” was coined by Per-Olov Löwdin for this new field of science, when he introduced proton tunnelling as another mechanism for DNA mutation [2]. Quantum biology has many applications in the evolution and continuity of life. One application is to propose a model for DNA mutation. This mutation is, in fact, an error in the DNA code, which occurs during the copying of a DNA strand during cell reproduction. A DNA mutation model has been proposed in which a nucleotide may change its form through the process of quantum tunnelling. The changed nucleotide will lose its ability to pair with its original base pair, which will change the structure and order of the DNA strand [3]. This DNA mutation may be produced by exposure to ultraviolet rays and other types of radiation [4]. Another application of quantum biology in biological systems is to explain the mechanisms for vision and the involved scientific process of phototransduction. In this process, a photon is absorbed by a chromophore in a light receptor, which causes photoisomerisation. This change in structure induces a change in the structure of the photoreceptor and the resulting signal transduction pathways that lead to a visual signal [5]. This process is very rapid (< 200 femtoseconds) and has a high yield. Models have been proposed in which quantum effects shape the ground state and excited state potentials to achieve the visual signal [6]. Yet another application of quantum biology involves magnetoreception, in which animals can navigate using the inclination of the Earth’s magnetic field [7]. This biological event can be described by the entangled radical pair mechanism in quantum mechanics [8], [9]. Other biological events, such as photosynthesis [10], [11] and enzymatic activity, have been described through the quantum field theory [12], [13]. In addition to these applications, some observations can only be explained by quantum biology. For example, Montagnier and his collaborators argued about the capacity of some bacterial DNA sequences to emit very low-frequency electromagnetic waves when extensively diluted in an aqueous fluid. The authors discussed that the genomic DNA of most pathogenic bacteria includes sequences that can create such signals [14]. Another study by the same group described the experimental conditions under which electromagnetic waves of low frequency can be emitted by dilute aqueous solutions of some bacterial and viral DNAs. Also, the authors observed this transduction process in living human cells exposed to electromagnetic wave irradiation and suggested a quantum field theory analysis of the phenomenon [15]. Given this importance of quantum biology in biological systems, its origin is important to consider. We have approached this issue by considering the structure of DNA. We demonstrate the involvement of quantum charged particles that join together. Due to the motion of these objects, their charged particles create electrical currents and emit electromagnetic waves. We suggest some mechanisms for applying quantum waves in imaging of DNA packages like viruses, bacteria, and embryonic cells. The outline of the paper is as follows. In section II, we show that DNA is constructed from quantum particles and radiates quantum waves. In section III, we propose methods for detecting the signals of DNA inside the virus and bacteria. In section IV, we describe the use of quantum waves in imaging. DNA quantum waves In this section, we propose several reasons (1-5) why DNA could radiate waves. 1. Each DNA is formed from a base pairing between A (Adenine) and T (Thymine), and between C (Cytosine) and G (Guanine). A and G are constructed from hexagonal and pentagonal manifolds. T and C are hexagonal [16], [17]. Each of these manifolds is constructed from charged atoms like nitrogen and carbon, and electrons. The electrical charges of each base differ from the others. Consequently, the A-T and C-G base pairs form two types of electrical moments (Figure 1). Figure 1 Each base in DNA is constructed from electrical moments Moreover, different DNAs have different activities that cause the motion-related electrical charges and moments. For example, during transcription and translation, some regions of the genetic information on DNA are copied to form RNAs and proteins, which interact with DNA and lead to the motion of the DNA. According to the laws of physics, the motion of electrical charges produces a magnetic field and results in the emission of electromagnetic waves. Thus, each DNA can radiate various types of waves depending on the nature of its interaction with biological material like DNA and RNA (Figure 2). Figure 2 During transcription and translation, electrical pairs become separated, and some waves emerge 2. During cell division, the DNA in each cell replicates so that the two daughter cells have the same genetic information as the parent cell [18]. In this process, the two strands of the original DNA double helix separate and each strand’s complementary DNA sequence is recreated as catalysed by DNA polymerase. In this mechanism, charged pairs are separated and then joined to each other. Consequently, the motions of these charged particles produce electromagnetic waves (Figure 3). Figure 3 During replication, electrical pairs become separated, and electromagnetic waves emerge 3. The DNA structure is very similar to a solenoid or coil. Consequently, the motion of electrons the structure produces magnetic fields (Figure 4). Figure 4 The structure of DNA is very similar to a coil 4. Each part of DNA acts similar to an electronic device. For example, hexagonal and pentagonal molecules store waves and energy and act as a capacitor. Coiled regions of DNA produce a solenoid. The collective circuits produce a system similar to a radio wave receiver or transmitter (Figure 5). Figure 5 Each part of DNA acts similar to an electronic device [19] 5. Some waves act like topoisomerases and unwind DNA to allow reading of the information. Topoisomerases are enzymes that participate in the rewinding or unwinding of DNA. The winding problem of DNA arises due to the intertwined nature of its double-helical structure. Topoisomerases act on the topology of DNA [20]. Similar to these enzymes, some waves participate in the unwinding of DNA. These waves are coupled to the structure of DNA and produce topologically simple structures. This causes the exchange of information between DNAs (Figure 6). Figure 6 Topoisomerase-like wave couples to the wound structure of DNA and make it unwind topologically A method for detecting waves of DNA packages like bacteria To observe DNA waves, it is best to use biological versions of packaged DNA; virus and bacteria are suitable. When not packaged, DNA cannot undergo normal actions like replication and will not produce waves. For this method, bacteria and the viruses that infect them can be contained in a vessel that houses a fluid, such as milk, which can be used by the bacteria for growth. Also, since bacteria replicate autonomously, but virus do not (bacteriophage require bacteria to replicate), we need to bacterial packages. In this experiment, we didn’t use the chemical medium and use of natural material like milk to show communication between DNAs and effects of DNA waves in a natural medium. In this procedure, a vessel of milk containing bacteria and virus were placed in an inductor. One side of the vessel was connected to a generator and the other side to a scope (Figure 7). Figure 7 Detection of signals of DNA packages (bacteria) in a vessel of milk in an external magnetic field A current is supplied to the inductor to produce a magnetic field. The bacteria and virus suspended in the milk interact with the magnetic field, alter it, and produce a new type of output electromagnetic field. The entire system can be placed in an incubator to observe the types of interactions between bacteria, viruses and magnetic field changes at different temperatures. The signals obtained from bacteria growing at various temperatures are displayed in Figure 8. With time, the number of DNA packages (i.e., bacteria) in the milk increases, and more waves are emitted. The pattern depends on temperature. For example, at 5°C, fewer bacteria are produced, and fewer waves are detected, while more bacteria (and hence more waves) are produced at higher temperatures. Figure 8 The growth signals of bacteria in milk in terms of time for 40°C (blue), 38°C (red), and 5°C (grey) Use of DNA waves in imaging The concepts of quantum biology and DNA waves can be exploited for imaging. For example, information about the properties of DNAs of chick embryos residing inside eggs might be obtained by analysing the waves exchanged between the DNA inside the egg with the DNA exterior to the egg. To this aim, we build a circuit from a graphene or metal tube, generator, inductor, scope, DNA in the interior of eggs, and DNA exterior to the eggs. Magnetic waves pass through the interior / exterior DNA, and the graphene. The DNAs are excited and exchange waves. Some of these waves interact with the electrons in the graphene tube, which generates a current. The changes that occur in these waves when exiting the eggs permit the analysis of the properties of the chick embryo DNA (Figure 9). Figure 9 A circuit for using exchanged DNA waves between cells interior and exterior of egg in imaging To obtain the exterior DNA, a culture system devoid of the shell was used for chick embryos. Similar to Tahara and Obara (2014) [19] and Sepehri et al., (2019) [20], [21], [22], a 450 ml polystyrene plastic cup was used as the pod for the culture vessel. A whole 1 to 1.5 cm in diameter was made at the side of the cup approximately 2 cm from the bottom, and a cotton pledget was installed in the hole as a filter. Then, a 2 mm diameter plastic tube was positioned in the space between the pledget and the hole to provide oxygen that was necessary for bacterial growth. A concave polymethyl pentene film was placed in the pod as an artificial culture vessel. A polystyrene plastic cover was put on top of the culture vessel. The vessel containing broken fertilised eggs was put in an incubator, and the shell-less cultures were incubated at 38°C and rotated 120° clockwise twice a day. After 54 h, initial cells of chick embryos were evident (Figure 10). Figure 10 Incubation of shell-less cultures of chick embryos to observe the growth of cells We explored whether the DNA waves that were generated could be used in imaging and to determine the gender of chick embryos. In Figure 11, the current value could distinguish males (red) and females (blue). The signal type differed between males and females. This is because that topology of some chromosomes in cells of males is different from the chromosomes of females. The motions of charged particles and electrons depend on the topology of the chromosomes and type of coiling, winding, and packing of DNA in them. Thus, by changing the shape of chromosomes and the DNA topology, ways and degrees of freedom of electrons change. Changing the motions of electrons changes their radiated waves, which explains the difference in the signals of males and females. Figure 11 Comparison of signals of male (red) and female (blue) In conclusion, in this research, we have shown that DNA waves play major roles in the evolution of biological systems. We propose two models for imaging by using concepts of quantum biology and DNA waves. The models are useful in charting bacterial growth and in distinguishing the gender of chick embryos. Funding: This research did not receive any financial support Competing Interests: The authors have declared that no competing interests exist ==== Refs 1 Erwin Schrödinger 1967 96 New York Cambridge University Press (Originally published in 1944.) 2 Lowdin PO Quantum genetics and the aperiodic solid Some aspects on the Biological problems of heredity, mutations, aging and tumours in view of the quantum theory of the DNA molecule Advances in Quantum Chemistry 1965 2 213 360 https://doi.org/10.1016/S0065-3276(08)60076-3 3 Trixler F Quantum Tunnelling to the Origin and Evolution of Life Current Organic Chemistry 2013 17 16 1758 1770 https://doi.org/10.2174/13852728113179990083 PMid:24039543 PMCid:PMC3768233 24039543 4 Yu SL Lee SK Ultraviolet radiation:DNA damage, repair, and human disorders Molecular- Cellular Toxicology 2017 13 1 21 28 https://doi.org/10.1007/s13273-017-0002-0 5 Johnson PJ Farag MH Halpin A Morizumi T Prokhorenko VI Knoester J Jansen TL Ernst OP Miller RD The primary photochemistry of vision occurs at the molecular speed limit J Phys Chem B 2017 121 16 4040 7 https://doi.org/10.1021/acs.jpcb.7b02329 PMid:28358485 28358485 6 Schoenlein RW Peteanu LA Mathies RA Shank CV The first step in vision:femtosecond isomerization of rhodopsin Science 1991 254 5030 412 5 https://doi.org/10.1126/science.1925597 PMid:1925597 1925597 7 Hore PJ Mouritsen H The Radical-Pair Mechanism of Magnetoreception Annual Review of Biophysics 2016 45 1 299 344 https://doi.org/10.1146/annurev-biophys-032116-094545 PMid:27216936 8 Schulten K Swenberg CE Weller A A biomagnetic sensory mechanism based on magnetic field modulated coherent electron spin motion Zeitschrift für Physikalische Chemie 1978 111 1 1 5 https://doi.org/10.1524/zpch.1978.111.1.001 9 Kominis IK The radical-pair mechanism as a paradigm for the emerging science of quantum biology Mod Phys Lett B 2015 29 1 1530013 https://doi.org/10.1142/S0217984915300136 10 Lee H Cheng YC Fleming GR Quantum coherence accelerating photosynthetic energy transfer 2009 Springer, Berlin, Heidelberg In Ultrafast Phenomena XVI 607 609 https://doi.org/10.1007/978-3-540-95946-5_197 11 Fujihashi Y Fleming GR Ishizaki A Impact of environmentally induced fluctuations on quantum mechanically mixed electronic and vibrational pigment states in photosynthetic energy transfer and 2D electronic spectra J Chem Phys 2015 142 21 212403 https://doi.org/10.1063/1.4914302 PMid:26049423 26049423 12 Nagel ZD Klinman JP Tunneling and Dynamics in Enzymatic Hydride Transfer ChemInform 2006 37 43 https://doi.org/10.1002/chin.200643274 13 Nagel ZD Klinman JP Tunneling and Dynamics in Enzymatic Hydride Transfer Chemical Reviews 2006 106 8 3095 3118 https://doi.org/10.1021/cr050301x PMid:16895320 16895320 14 Montagnier L Aissa J Ferris S Montagnier JL Lavalléee C Electromagnetic signals are produced by aqueous nanostructures derived from bacterial DNA sequences Interdiscip Sci Comput Life Sci 2009 1 2 81 90 https://doi.org/10.1007/s12539-009-0036-7 PMid:20640822 15 Montagnier L Del Giudice E Aïssa J Lavallee C Motschwiller S Capolupo A Polcari A Romano P Tedeschi A Vitiello G Transduction of DNA information through water and electromagnetic waves Electromagnetic biology and medicine 2015 34 2 106 12 https://doi.org/10.3109/15368378.2015.1036072 PMid:26098521 26098521 16 Alberts B Johnson A Lewis J Raff M Roberts K Walter P Molecular Biology of the Cell (6th ed.) 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==== Front Open Access Maced J Med SciOpen Access Maced J Med SciOpen Access Macedonian Journal of Medical Sciences1857-9655Republic of Macedonia ID Design 2012/DOOEL Skopje OAMJMS-7-312110.3889/oamjms.2019.774Research ArticleA Mathematical Model for the Signal of Death and Emergence of Mind Out of Brain in Izhikevich Neuron Model Fioranelli Massimo 1Sepehri Alireza 1Roccia Maria Grazia 1Rossi Chiara 1Lotti Jacopo 1Barygina Victoria 2Vojvodic Petar 3Vojvodic Aleksandra 4Vlaskovic-Jovicevic Tatjana 3Vojvodic Jovana 3Dimitrijevic Sanja 5Peric-Hajzler Zorica 6Matovic Dusica 6Sijan Goran 7Wollina Uwe 8Tirant Michael 9Thuong Nguyen Van 10Lotti Torello 111 Department of Nuclear Physics, Sub-nuclear and Radiation, G. Marconi University, Rome, Italy2 Department of Biomedical Experimental and Clinical Sciences, University of Florence, Florence, Italy3 Clinic for Psychiatric Disorders “Dr. Laza Lazarevic”, Belgrade, Serbia4 Department of Dermatology and Venereology, Military Medical Academy, Belgrade, Serbia5 Department of Gynecology, Military Medical Academy, Belgrade, Serbia6 Military Medical Academy, Belgrade, Serbia7 Clinic for Plastic Surgery and Burns, Military Medical Academy, Belgrade, Serbia8 Department of Dermatology and Allergology, Städtisches Klinikum Dresden, Dresden, Germany9 University G. Marconi, Rome, Italy10 Vietnam National Hospital of Dermatology and Venereology, Hanoi, Vietnam11 Department of Dermatology, University of G. Marconi, Rome, Italy Correspondence: Massimo Fioranelli. Department of Nuclear Physics, Sub-nuclear and Radiation, G. Marconi University, Rome, Italy. E-mail: [email protected] 9 2019 11 9 2019 7 18 3121 3126 03 7 2019 14 8 2019 15 8 2019 Copyright: © 2019 Massimo Fioranelli, Alireza Sepehri, Maria Grazia Roccia, Chiara Rossi, Jacopo Lotti, Victoria Barygina, Petar Vojvodic, Aleksandra Vojvodic, Tatjana Vlaskovic-Jovicevic, Jovana Vojvodic, Sanja Dimitrijevic, Zorica Peric-Hajzler, Dusica Matovic, Goran Sijan, Uwe Wollina, Michael Tirant, Nguyen Van Thuong, Torello Lotti.2019This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License (CC BY-NC 4.0)AIM: In this paper, using a mathematical model, we will show that for special exchanged photons, the Hamiltonian of a collection of neurons tends to a constant number and all activities is stopped. These photons could be called as the dead photons. To this aim, we use concepts of Bio-BIon in Izhikevich Neuron model. METHODS: In a neuron, there is a page of Dendrite, a page of axon’s terminals and a tube of Schwann cells, axon and Myelin Sheath that connects them. These two pages and tube form a Bio-Bion. In a Bio-Bion, exchanging photons and some charged particles between terminals of dendrite and terminals of axon leads to the oscillation of neurons and transferring information. This Bion produces the Hamiltonian, wave equation and action potential of Izhikevich Neuron model. Also, this Bion determines the type of dependency of parameters of Izhikevich model on temperature and frequency and obtains the exact shape of membrane capacitance, resting membrane potential and instantaneous threshold potential. RESULTS: Under some conditions, waves of neurons in this BIon join to each other and potential shrinks to a delta function. Consequently, total Hamiltonian of the system tends to a constant number and system of neuron act like a dead system. Finally, this model indicates that all neurons have the ability to produce similar waves and signals like waves of the mind. CONCLUSION: Generalizing this to biology, we can claim that neurons out of the brain can produce signals of minding and imaging and thus mind isn’t confined to the brain. IzhikevichNeuronBio-BionAction Potential ==== Body Introduction Recently, Izhikevich has proposed a neuronal dynamical model which is a simple model that faithfully reproduces all the neurocomputational dynamical features of the neuron [1]. This model is obtained by reducing some Biological aspects of Hodgkin-Huxley (HH) neuron using bifurcation methods [2]. Until now, many discussions have been done on this subject. For example, in one research, authors have focused herein on the Izhikevich neuron model and compared the characteristics of Chaotic resonance in the chaotic states arising through the period-doubling or tangent bifurcation routes. They have found that the signal response in Chaotic resonance had a unimodal maximum concerning the stability of chaotic orbits in the tested chaotic states [3]. In another research, authors have presented a Multiplier less Noisy Izhikevich Neuron (MNIN) model, which was used for digital implementation of Biological neural networks in large scale. Simulation results have shown that the MNIN model reproduces the same operations of the original noisy Izhikevich neuron [4]. In another paper, authors have performed numerical simulations of synaptically coupled Izhikevich networks under the effect of general non-Gaussian Lvy noise. Firing dynamics of an all-to-all coupled Izhikevich network and two excitatory coupled Izhikevich networks with differing adaptation properties have been studied in response to applied Lvy noise [5]. And in one of newest works, authors have considered the effect of synaptic interaction (electrical and chemical) as well as structural connectivity on synchronisation transition in network models of Izhikevich neurons which spike regularly with beta rhythms. They have found a wide range of behaviour including continuous transition, explosive transition, as well as lack of global order [6]. In this paper, we show that in an Izhikevich Neuron model, neurons join to each other and build a stable system. In some conditions, exchanged photons between neurons join to each other produce a constant Hamiltonian. This leads to a stop in transferring information and the death of the system. To this aim, we show that a neuron has a structure similar to Bio-BIons. These Bio-BIons are formed from a page of Dendrite, a page of axon’s terminals and a tube of Schwann cells, axon and Myelin Sheath that connects them. Previously, it has been shown that exchanged photons between sheets of a graphene system could produce a nano-Bion [7]. The same Bion can emerge along neurons. Hamiltonian, action potential and wave equation in this Bio-Bion is very similar to an action potential and wave equation in Izhikevich Neuron model. We also use the concepts in [8] and propose a new temperature model for oscillating neurons. For some temperatures and rotating velocity, total potential tends to delta function and system is dead. We can save the system by removing some neurons. For example, in a body, we can remove neurons of the brain and prevention of death. In these conditions, neurons of the spinal cord do tasks of brain-like minding. Figure 1 Similarity between the structure of a neuron and a Bio-BIon The outline of the paper is as follows. In section II, we will construct c in a Bio-Bion. In section III, we will consider the dependency of Izhikevich parameters on temperature and frequency. In section IV, we will consider the origin of waves of death in the Izhikevich model. In section V, we will discuss the results of experiments on birds. We also show that birds without a brain can continue minding. The last section is devoted to conclusions. Constructing the Izhikevich Neuron Model in Bio-Bion The Izhikevich Neuron model which reproduce spiking and bursting behaviour of many known types of neurons are described by the pair of the differential equation: where t is time, C is membrane capacitor, V is membrane potential, Vr is the resting membrane potential, V is the instantaneous threshold potential, U is the recovery variable, S is stimulus (synaptic: excitatory or inhibitory, external, noise) and a, b, D are some constants. To consider the rotating neuron, we specialise to an embedding of the neuron world volume in Minkowski space-time with metric [9]; Without background fluxes. Here, t is time, r is the radius of the page of Dendrite and θ is the angle of rotation. When neurons oscillate with frequency ω, a rotating velocity emerges, and this velocity produces an acceleration (a). We can write: In this case, the relation between the world volume coordinates of the rotating neuron (τ, σ) and the coordinates of Minkowski space-time (t, r) are [9]; Where T is the temperature of the BIon and T0 is the critical temperature. However, this relation is questionable. Based on this relation, the superconductivity phenomena depend on the system velocity!! You can move a system with special velocities to reduce its temperature to the less than of its critical temperature, and then the system shows superconductivity by itself!! It means that a physical phenomenon (superconductivity) depends on the system velocity, a result in direct conflict with the relativity law claiming that the physical laws are independent of the observer velocity. This relativistic relation for temperature is not a true relation, and in fact, the temperature’s relation depends on the thermocouple apparatus used. A true thermocouple rejects this definition of temperature (For example, see [8], [10], [11]). Thus, to obtain a true relation between temperature and acceleration, we use concepts of BIon: Previously, thermo-dynamical parameters have been obtained in [12]: using relation (8) in relation (7), we can obtain an explicit form of temperature in an accelerating neuron: Above equations show the explicit relation between temperatures and acceleration in a neuron. However, to obtain the relation between temperature and rotating velocity, we should take a derivation of the above equations, put and obtain the below relation: where T0 is the temperature of non-rotating neuron and ω is the frequency. Also, ω0 is the upper limit frequency of neurons. Substituting equation (10) in equations (5), we obtain: Above metrics correspond to thermal rotating neurons. These metrics depend on the temperature and rotating velocity of neurons. To obtain the spectrum of the rotating neuron, we should obtain the action. To this aim, we will use of the concept of BIon model for in [7]. For flat space-time, the action of a neuron is [9]: where gMN is the background metric, XM (σa)’s are scalar fields, N is number of exchanged photons between Dendrite and axon, σ a’s are the neuron coordinates, a, b = 0, 1, …, 3 are world-volume indices of rotating neuron and M, N = 0, 1, …, 10 are neuron dimensional spacetime indices. Also, G is the nonlinear field [9], and A is the photon which exchanges between Dendrite and axon. With the metric of equation (11), the action (12) should be re-written as: Using the method in ref [9], we can obtain the Hamiltonian from equation (13) for neuron: By substituting equation (15) in equation (14), we obtain: where N is the number of terminals of Dendrite and axon. Above equation shows that Hamiltonian of an oscillating neuron depends on the frequency and temperature. By increasing temperature, more photons are exchanged between neurons and energy of neurons increases. Also, by increasing the number of exchanged photons, frequency of system increases and Hamiltonian grows. Also, the above Hamiltonian depends on parameters of Izhikevich neuron model. The dependency of Izhikevich Parameters on Temperature and Frequency in Bio- Bion In this section, we will obtain the exact dependency of parameters of Izhikevich neuron model on temperature and frequency. To this aim, we extract the wave equation from 10 equation (20): Above equation indicates that all parameters of Izhikevich Neuron model could be produced in a Bio-BIon. Also, the exact form of these parameters and type of their dependency on frequency and temperature could be determined in a Bio-BIon. Figure 2 shows the Membrane potential of Izhikevich Neuron model, which is produced in a Bio-BIon. This shape is very the same with results in [1], [2]. Neuron acts 13 like a Bio-BIon and transmits photons, Sodium and other charged particles which carry information. Figure 2 Membrane potential of Izhikevich Neuron model in a Bio-BIon Signal of Death in Izhikevich Neuron Model In this section, we will show that neurons can join to each other and produce a stable system. In these conditions, Hamiltonian of the system tends to a constant number, and no information is transferred. First, we rewrite equation (20) as: Some of the neurons oscillate reverse to some others and emit some photons with opposite momentums. We can sum over Hamiltonians of all neurons: Above equation shows that Hamiltonian of the neuronic system may be a constant number. In these conditions, this system is strongly stable, and there isn’t any equation of state. This means that information couldn’t be transformed and thus, the system has been dead. Birds Without Brain In Izhikevich Neuron Model: Emergence Of Mind Out Of Brain Until now, we have considered some conditions that Hamiltonian of the total system tends to one. To prevent this state, we can remove some neurons of the system. For example, in a system which includes brain and spinal cord, we can remove neurons of the brain. As a result, equation (21) can be re-written as: Above equation shows that Hamiltonian of the spinal cord depends on the Hamiltonian of the brain. Thus, after cutting the brain, the spinal cord could do some activities of brain-like minding. Discussion In this research, we have shown that neurons in the Izhikevich Neuron model may join to each other, and the total Hamiltonian of the system tends to a constant number. For some exchanged waves, transferring of information between neurons is stopped, and the system of neuron acts as a dead system. These waves are known as the waves of death. Also, we have considered the 15 origins of these waves and Izhikevich Neuron model in a Bio-Bion system. This system was constructed from a page of Dendrite, a page of axon’s terminals and a tube of Schwann cells, axon and Myelin Sheath that connects them. Evolutions of parameters of Izhikevich Neuron model like membrane capacitance, resting membrane potential and instantaneous threshold potential depend on temperature and frequency of Bio-Bion. Our calculations in Izhikevich Neuron model show that before death, a signal is emerged in the brain and suggest to all parts of bod to stop their activities. If we remove this signal, other parts of the body could continue to their activities. To show this in experiments, we cut the brain of some birds suddenly and observe that their other parts of bodies continue to activity for a long time and we hope that control their life for more times. Also, until now, scientists believed that by cutting the brain, the mind is disappeared. However, our calculations show that Hamiltonian of the spinal cord depends on the Hamiltonian of the brain. Thus, after cutting the brain, the spinal cord could do some activities of brain-like minding. Also, our experiments show that birds without a brain can determine the best way to escape or passing barriers. This means that other neurons out of the brain have also a role in imaging. Funding: This research did not receive any financial support Competing Interests: The authors have declared that no competing interests exist ==== Refs 1 Izhikevich EM Simple model of spiking neurons IEEE Transactions on neural networks 2003 14 6 1569 72 https://doi.org/10.1109/TNN.2003.820440 PMid:18244602 18244602 2 Izhikevich EM Dynamical systems in neuroscience MIT press 2007 https://doi.org/10.7551/mitpress/2526.001.0001 PMCid:PMC14∌9 3 Nobukawa S Nishimura H Yamanishi T Chaotic resonance in typical routes to chaos in the Izhikevich neuron model Scientific reports 2017 7 1 1331 https://doi.org/10.1038/s41598-017-01511-y PMid:28465524 PMCid:PMC5430992 28465524 4 Haghiri S Zahedi A Naderi A Ahmadi A Multiplierless implementation of noisy Izhikevich neuron with low-cost digital design IEEE transactions on biomedical circuits and systems 2018 12 6 1422 30 https://doi.org/10.1109/TBCAS.2018.286∪ PMid:30188839 30188839 5 Vinaya M Ignatius RP Effect of Lévy noise on the networks of Izhikevich neurons Nonlinear Dynamics 2018 94 2 1133 50 https://doi.org/10.1007/s11071-018-4414-8 6 Montakhab A Khoshkhou M Beta-rhythm oscillations and synchronization transition in network models of Izhikevich neurons:effect of topology and synaptic type Frontiers in Computational Neuroscience 2018 12 59 https://doi.org/10.3389/fncom.2018.00059 PMid:30154708 PMCid:PMC6103382 30154708 7 Sepehri A The nano-BIon in nanostructure Physics Letters A 2016 380 16 1401 7 https://doi.org/10.1016/j.physleta.2016.02.026 8 Montakhab A Ghodrat M Barati M Statistical thermodynamics of a two-dimensional relativistic gas Physical Review E 2009 79 3 031124 https://doi.org/10.1103/PhysRevE.79.031124 PMid:19391919 9 Sepehri A Shoorvazi S Ghaforyan H The European Physical Journal Plus 2018 133 280 https://doi.org/10.1140/epjp/i2018-12127-6 10 Ghodrat M Montakhab A Time parametrization and stationary distributions in a relativistic gas Physical Review E 2010 82 1 011110 https://doi.org/10.1103/PhysRevE.82.011110 PMid:20866568 11 Farías C Pinto VA Moya PS What is the temperature of a moving body?Scientific reports 2017 7 1 17657 https://doi.org/10.1038/s41598-017-17526-4 PMid:29247189 PMCid:PMC5732238 12 Beesham A Sepehri A Emergence and expansion of cosmic space in an accelerating BIon The European Physical Journal C 2018 78 11 968 https://doi.org/10.1140/epjc/s10052-018-6463-z	31850137	PMC6910805	NO-CC CODE	2021-01-06 05:12:18	yes	Open Access Maced J Med Sci. 2019 Sep 11; 7(18):3121-3126

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