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Next generation sequencing analysis of gastric cancer identifies the leukemia inhibitory factor receptor (LIFR) as a driving factor in gastric cancer progression and as a predictor of poor prognosis Gastric cancer (GC) is the third cause of cancer-related-death worldwide. Nevertheless, because GC screening programs are not cost-effective, most patients receive diagnosis in the advanced stages, when surgical options are limited because the presence of diffuse disease. Peritoneal dissemination occurs in approximately one third of patients with GC and is a strong predictor of poor outcome. Despite the clinical relevance, biological and molecular mechanisms underlying the formation of peritoneal metastasis in GC remain poorly defined. To investigate this point, we conducted a high-throughput sequencing of transcriptome expression in paired samples of normal and neoplastic gastric mucosa in 31 GC patients with or without peritoneal carcinomatosis. The RNAseq analysis led to the discovery of a group of highly upregulated or downregulated genes that were differentially modulated in patients with peritoneal disease in comparison to GC patients without peritoneal involvement. Among these genes the leukemia inhibitory factor receptor (LIFR) and the one cut domain family member (ONECUT)2 were the only two genes that predicted survival at univariate statistical analysis. Because LIFR was the highest regulated gene we have further assessed whether this receptor plays a mechanistic role in GC dissemination. For this purpose, we have first assessed the expression of LIF, a member of IL-6 cytokine family, and LIFR in GC cell lines. Our results demonstrate that exposure of MKN45 cells to LIF, promoted a concentration-dependent proliferation and epithelial-mesenchymal transition (EMT) as shown by modulation of E-cadherin/vimentin gene expression along with JAK and STAT 3 phosphorylation and acquisition of a migratory phenotype. These features were reversed by in vitro treatment with a LIFR antagonist. Together, these data provide support to the notion that development of LIF/LIFR inhibitors might have a role in the treatment of GC. | cancer biology |
A QuantCrit investigation of society's educational debts due to racism, sexism, and classism in biology student learning We investigated the intersectional relationships between racism, sexism, and classism in inequities in student conceptual knowledge in introductory biology courses using a quantitative critical framework. Using Bayesian hierarchical linear models, we examined students conceptual knowledge as measured by the Introductory Molecular and Cell Biology Assessment. The data came from the LASSO database and included 6,547 students from 87 introductory courses at 11 institutions. The model indicated that students with marginalized identities by race, gender, and class tended to start with lower scores than continuing-generation, White men. We conceptualized these differences as educational debts society owed these students due to racism, sexism, and classism. Instruction added to these educational debts for most marginalized groups, with the largest increases for students with multiple marginalized identities. After instruction, society owed Black and Hispanic, first-generation women an educational debt equal to 60-80% of the average learning in the courses. These courses almost all (85/87) used collaborative learning and half (45/87) supported instruction with learning assistants. While research shows collaborative learning better serves students than lecture-based instruction, these results indicate it does not repay educational debts due to racism, sexism, and classism. | scientific communication and education |
A unique epigenomic landscape defines CD8+ tissue-resident memory T cells Memory T cells provide rapid and long-term protection against infection and tumors. The memory CD8+ T cell repertoire contains phenotypically and transcriptionally heterogeneous subsets with specialized functions and recirculation patterns. While these T cell populations have been well characterized in terms of differentiation potential and function, the epigenetic changes underlying memory T cell fate determination and tissue-residency remain largely unexplored. Here, we examined the single-cell chromatin landscape of CD8+ T cells over the course of acute viral infection. We reveal an early bifurcation of memory precursors displaying distinct chromatin accessibility and define epigenetic trajectories that lead to a circulating (TCIRC) or tissue-resident memory T (TRM) cell fate. While TRM cells displayed a conserved epigenetic signature across organs, we demonstrate that these cells exhibit tissue-specific signatures and identify transcription factors that regulate TRM cell populations in a site-specific manner. Moreover, we demonstrate that TRM cells and exhausted T (TEX) cells are distinct epigenetic lineages that are distinguishable early in their differentiation. Together, these findings show that TRM cell development is accompanied by dynamic alterations in chromatin accessibility that direct a unique transcriptional program resulting in a tissue-adapted and functionally distinct T cell state.
Graphical Abstract
O_FIG O_LINKSMALLFIG WIDTH=192 HEIGHT=200 SRC="FIGDIR/small/490680v1_ufig1.gif" ALT="Figure 1">
View larger version (56K):
org.highwire.dtl.DTLVardef@10a866aorg.highwire.dtl.DTLVardef@1febe9corg.highwire.dtl.DTLVardef@1941dd2org.highwire.dtl.DTLVardef@131dc58_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIscATAC atlas reveals the epigenetic variance of memory CD8+ T cell subsets over the course of acute infection
C_LIO_LIEarly bifurcation of memory precursors leads to circulating versus tissue-resident cell fates
C_LIO_LIIntegrating transcriptional and epigenetic analyses identified organ-specific TRM cell regulators including HIC1 and BACH2
C_LIO_LIEpigenetic distinction of TRM cells and TEX cell subsets
C_LI | immunology |
Identification of viral dose and administration time in simulated phage therapy occurrences The rise in multidrug-resistant bacteria has sprung a renewed interest in applying phages as antibacterial, a procedure Western practitioners eventually abandoned due to several downfalls, including poor understanding of the dynamics between phages and bacteria. A successful phage therapy needs to account for the loss of infective virions and the multiplication of the hosts. The parameters critical inoculation size (VF) and failure threshold time (TF) have been introduced to assure that the viral dose (v{phi}) and administration time (t{phi}) would lead to an effective treatment. The problem with the definition of VF and TF is that they are non-linear equations with two unknowns; thus, their solution is cumbersome and not unique. The current study used machine learning in the form of a decision tree algorithm to determine ranges for the viral dose and administration times required to achieve an effective phage therapy. Within these ranges, a Pareto optimal solution of a multi-criterial optimization problem (MCOP) provides values leading to effective treatment. The algorithm was tested on a series of microbial consortia that described allochthonous invasions (the outgrowing of a species at high cell density by another species initially present at low concentration) to inhibit the growth of the invading species. The present study also introduced the concept of mediated phage therapy, where targeting a booster bacteria might decrease the virulence of a pathogen immune to phagial infection. The results demonstrated that the MCOP could provide pairs of v{phi} and t{phi} that could effectively wipe out the bacterial target from the considered micro-environment. In summary, the present work introduced a novel method for investigating the phage/bacteria interaction that could help increase the effectiveness of phage therapy.
Author summaryPhage therapy is a treatment that can help fight infections with bacteria resistant to antibiotics. However, several phage therapy application have failed, possibly because phages were administered at the wrong time or in insufficient amounts. The present study implemented a machine learning protocol to correctly calculate the administration time and viral load to obtain effective phage therapy. Four simulated microbial consortia, including one case where the pathogen was not directly a phages host, were employed to prove the procedures concept. The results demonstrated that the procedure is suitable to help the microbiologists to instantiate an effective phage therapy and clear infections. | microbiology |
Molecular insights into the effect of alkanediols on FUS liquid-liquid phase separation Numerous cell biology studies have used high concentrations of 1,6-hexanediol to dissolve membraneless organelles and disordered protein biomolecular condensates. Yet, little is known about how alkanediols effect liquid-liquid phase separation (LLPS), and why certain alkanediol isomers are more effective. Here, we evaluate the effect of various alkanediols on the archetypal phase separating protein FUS. Low-complexity domain and full-length FUS LLPS is decreased varyingly, while LLPS of FUS RGG-RNA condensates is even enhanced by some alkanediols. NMR experiments show that all diols act similarly, correlating atomistic changes with LLPS-preventing effects. Furthermore, we find no evidence for specific residue interactions - the largest perturbations are seen at backbone and glutamine side-chain hydrogen bonding sites, not hydrophobic/aromatic residues. Furthermore, 1,6 hexanediol favors formation of protein-solvent hydrogen bonds and increases FUS local motions. These findings show how alkanediols affect water-disordered protein interactions, underscoring the difficulty in using alkanediol-derivatives to target dissolution of specific membraneless organelles. | biochemistry |
Skull and scalp segmentation in neonatal cerebral MRI using subject-specific probability models This study presents a new approach for the segmentation of cranial bones in magnetic resonance images (MRIs) acquired from neonates in the age range of 39 to 42 weeks, gestational age. This approach uses subject specific probability maps of the skull and scalp, which are created from atlas computed tomography (CT) images taken retrospectively from neonates in the same age range. Our method uses also a subject specific probability map of cerebrospinal fluid (CSF), which is constructed from retrospective atlas MRIs. To construct skull, scalp and CSF probability maps, a subject specific bimodal MR-CT neonatal head template is created (using atlas MR and CT images), and employed. In the next step, the subject specific probability maps are fed to expectation maximization algorithm in conjunction with Markov random field method implemented in FSL software to segment skull and scalp from the input MR image. The results of the proposed method were evaluated through various experiments. First, we computed the similarity between frontal and occipital sutures (reconstructed from segmented cranial bones) and the ground truth (created manually by an expert radiologist). For this purpose, modified versions of Dice similarity index (DSI) were adopted and used. Second, the size of anterior fontanel was compared to its normal size as reported for the neonates in the same age range. Third, the thickness of cranial bones was computed and compared to its normal values as reported for healthy neonates. Finally, a retrospective data including MRI and CT images was used which have been acquired from the same neonate within a short time interval. After aligning the two images, the similarity between cranial bones of the MR and CT image was compared using DSI and modified Hausdorff distance. The results of these experiments demonstrated the success of our segmentation method. | bioengineering |
Systematic benchmarking of all-in-one microbial SNP calling pipelines Clinical and public health microbiology is increasingly utilising whole genome sequencing (WGS) technology and this has lead to the development of a myriad of analysis tools and bioinformatics pipelines. Single nucleotide polymorphism (SNP) analysis is an approach used for strain characterisation and determining isolate relatedness. However, in order to ensure the development of robust methodologies suitable for clinical application of this technology, accurate, reproducible, traceable and benchmarked analysis pipelines are necessary. To date, the approach to benchmarking of these has been largely ad-hoc with new pipelines benchmarked on their own datasets with limited comparisons to previously published pipelines.
In this study, Snpdragon, a fast and accurate SNP calling pipeline is introduced. Written in Nextflow, Snpdragon is capable of handling small to very large and incrementally growing datasets. Snpdragon is benchmarked using previously published datasets against six other all-in-one microbial SNP calling pipelines, Lyveset, Lyveset2, Snippy, SPANDx, BactSNP and Nesoni. The effect of dataset choice on performance measures is demonstrated to highlight some of the issues associated with the current available benchmarking approaches.
The establishment of an agreed upon gold-standard benchmarking process for microbial variant analysis is becoming increasingly important to aid in its robust application, improve transparency of pipeline performance under different settings and direct future improvements and development.
Snpdragon is available at https://github.com/FordeGenomics/SNPdragon.
Impact statementWhole-genome sequencing has become increasingly popular in infectious disease diagnostics and surveillance. The resolution provided by single nucleotide polymorphism (SNP) analyses provides the highest level of insight into strain characteristics and relatedness. Numerous approaches to SNP analysis have been developed but with no established gold-standard benchmarking approach, choice of bioinformatics pipeline tends to come down to laboratory or researcher preference. To support the clinical application of this technology, accurate, transparent, auditable, reproducible and benchmarked pipelines are necessary. Therefore, Snpdragon has been developed in Nextflow to allow transparency, auditability and reproducibility and has been benchmarked against six other all-in-one pipelines using a number of previously published benchmarking datasets. The variability of performance measures across different datasets is shown and illustrates the need for a robust, fair and uniform approach to benchmarking.
Data SummaryO_LIPreviously sequenced reads for Escherichia coli O25b:H4-ST131 strain EC958 are available in BioProject PRJNA362676. BioSample accession numbers for the three benchmarking isolates are:
O_LIEC958: SAMN06245884
C_LIO_LIMS6573: SAMN06245879
C_LIO_LIMS6574: SAMN06245880
C_LI
C_LIO_LIAccession numbers for reference genomes against the E. coli O25b:H4-ST131 strain EC958 benchmark are detailed in table 2.
C_LIO_LISimulated benchmarking data previously described by Yoshimura et al. is available at http://platanus.bio.titech.ac.jp/bactsnp (1).
C_LIO_LISimulated datasets previously described by Bush et al. is available at http://dx.doi.org/10.5287/bodleian:AmNXrjYN8 (2).
C_LIO_LIReal sequencing benchmarking datasets previously described by Bush et al. are available at http://dx.doi.org/10.5287/bodleian:nrmv8k5r8 (2).
C_LI
O_TBL View this table:
org.highwire.dtl.DTLVardef@3cb198org.highwire.dtl.DTLVardef@af9421org.highwire.dtl.DTLVardef@1261244org.highwire.dtl.DTLVardef@793b42org.highwire.dtl.DTLVardef@1217387_HPS_FORMAT_FIGEXP M_TBL O_FLOATNOTable 2.C_FLOATNO O_TABLECAPTIONReference genomes used in the EC958 benchmarking dataset and the percent identity against the three included samples.
C_TABLECAPTION C_TBL | bioinformatics |
Automated systematic evaluation of cryo-EM specimens with SmartScope Propelled by improvements in hardware for data collection and processing, single particle cryo-electron microscopy has rapidly gained relevance in structural biology. Yet, finding the conditions to stabilize a macromolecular target for imaging remains the most critical barrier to determining its structure. Attaining the optimal specimen requires the evaluation of multiple grids in a microscope as conditions are varied. While automation has significantly increased the speed of data collection, optimization is still carried out manually. This laborious process which is highly dependent on subjective assessments, inefficient and prone to error, often determines the success of a project. Here, we present SmartScope, the first framework to streamline, standardize, and automate specimen evaluation in cryo-electron microscopy. SmartScope employs deep-learning-based object detection to identify and classify features suitable for imaging, allowing it to perform thorough specimen screening in a fully automated manner. A web interface provides remote control over the automated operation of the microscope in real time and access to images and annotation tools. Manual annotations can be used to re-train the feature recognition models, leading to improvements in performance. Our automated tool for systematic evaluation of specimens streamlines structure determination and lowers the barrier of adoption for cryo-electron microscopy. | biophysics |
An interactive mass spectrometry atlas of histone posttranslational modifications in T-cell acute leukemia The holistic nature of omics studies makes them ideally suited to generate hypotheses on health and disease. Sequencing-based genomics and mass spectrometry (MS)-based proteomics are linked through epigenetic regulation mechanisms. However, epigenomics is currently mainly focused on DNA methylation status using sequencing technologies, while studying histone posttranslational modifications (hPTMs) using MS is lagging, partly because reuse of raw data is impractical. Yet, targeting hPTMs using epidrugs is an established promising research avenue in cancer treatment. Therefore, we here present the most comprehensive MS-based preprocessed hPTM atlas to date, including 21 T-cell acute lymphoblastic leukemia (T-ALL) cell lines. We present the data in an intuitive and browsable single licensed Progenesis QIP project and provide all essential quality metrics, allowing users to assess the quality of the data, edit individual peptides, try novel annotation algorithms and export both peptide and protein data for downstream analyses, exemplified by the PeptidoformViz tool. This data resource sets the stage for generalizing MS-based histone analysis and provides the first reusable histone dataset for epidrug development. | cancer biology |
M2-polarized macrophages control LSC fate by enhancing stemness, homing, immune evasion and metabolic reprogramming While it is increasingly becoming clear that cancers are a symbiosis of diverse cell types and tumor clones, the tumor microenvironment (TME) in acute myeloid leukemias (AML) remains poorly understood. Here, we uncover the functional and prognostic relevance of an M2-polarized macrophage compartment. Intra bone marrow co-injection of M2d-macrophages together with leukemic blasts that fail to engraft on their own now induce fatal leukemia in mice. Even a short-term two-day in vitro exposure to M2d macrophages can "train" leukemic blasts after which cells are protected against phagocytosis, display increased mitochondrial metabolism and improved in vivo homing, resulting in full-blown leukemia. Single-cell RNAseq analysis of AML associated macrophages revealed metabolic-related pathways such as Fatty Acid Oxidation and NAD+ generation as therapeutical targetable vulnerabilities. Our study provides insight into the mechanisms by which the immune landscape contributes to aggressive leukemia development and provides alternatives for effective targeting strategies. | cancer biology |
Nav1.1 in mammalian sensory neurons is required for normal motor behaviors AO_SCPLOWBSTRACTC_SCPLOWThe mammalian voltage-gated sodium channel (NaV), NaV1.1, has been well-studied in the central nervous system; conversely, its contribution to peripheral sensory neuron function is more enigmatic. Here, we report a new role for peripherally expressed NaV1.1 in murine motor behaviors. RNAscope analysis found 100% of proprioceptors express NaV1.1 transcript, consistent with in vitro patch clamp recordings showing this channel is required for repetitive firing in proprioceptors. Notably, genetic deletion of NaV1.1 in all sensory neurons caused profound motor coordination deficits in homozygous conditional knockout animals of both sexes, a phenotype similar to conditional Piezo2-knockout animals. Movement deficits were also observed in heterozygotes, demonstrating that NaV1.1 haploinsufficiency in sensory neurons leads to motor deficiencies. This behavioral phenotype was not due to reduced proprioceptor numbers or abnormal muscle spindle formation; however, we observed decreased proprioceptor innervation of motor neurons in the spinal cord in conditional knockouts, indicating loss of NaV1.1 in sensory neurons alters spinal cord circuitry. Ex vivo muscle afferent recordings also support the notion that loss of NaV1.1 leads to aberrant proprioceptor function. Collectively, these data provide the first evidence that NaV1.1 in mammalian sensory neurons is essential for motor coordination. Importantly, human patients harboring NaV1.1 loss-of-function mutations often present with motor delays and ataxia. Thus, our data suggest sensory neuron dysfunction may contribute to the clinical manifestations and co-morbidities of neurological disorders in which NaV1.1 function is compromised. | neuroscience |
Probabilistic Tracking U-fiber on the Superficial White Matter Surface The short association fibers or U-fibers connect two neighboring gyri and travel in the superficial white matter (SWM) beneath the cortical layer. These U-fibers are essential for the understanding of neurodevelopment and neurodegeneration. However, the complex structures and the high curvature of the U-fibers lead to erroneous streamlines reconstruction of the traditional tractography since the volume-based tractography cannot use the biological characteristic of U-fibers that they tightly beneath the cortical layer. In this work, we proposed a surface-based framework for probabilistic tracking of the U-fibers on the triangular mesh of the SWM. We develop a novel approach to project the fiber orientation distributions (FODs) data onto the tangent space of the SWM surface. With the projected FODs, an advanced probabilistic tracking technique, which regularizes the streamlines based on the intrinsic geometry of the surface, is developed to reconstruct the highly bent U-fibers on the SWM surface. In the experiment, we demonstrate our method based on the high-resolution diffusion imaging data from the Human Connectome Project (HCP). We quantitatively compare the proposed method with state-of-the-art volume-based tractography from MRTrix and another surface-based tractography on the U-fibers of the central sulcus. Moreover, we show the reconstructed U-fibers on the parietal lobe and frontal lobe. The results show that our method outperforms the other two methods and successfully reconstructs the U-fibers on the cortical regions with high intersubject variability. | neuroscience |
Bacterial association with metals enables in vivo tracking of microbiota using magnetic resonance imaging Bacteria constitute a significant part of the biomass of the human microbiota, but their interactions are complex and difficult to replicate outside the host. Exploiting the superior resolution of magnetic resonance imaging (MRI) to examine signal parameters of selected human isolates may allow tracking of their dispersion throughout the body. We investigated longitudinal and transverse MRI relaxation rates and found significant differences between several bacterial strains. Common commensal strains of lactobacilli display notably high MRI relaxation rates, partially explained by outstanding cellular manganese content, while other species contain more iron than manganese. Lactobacillus crispatus show particularly high values, 4-fold greater than any other species; over 10-fold greater signal than relevant tissue background; and a linear relationship between relaxation rate and fraction of live cells. Different bacterial strains have detectable, repeatable MRI relaxation rates that in future may enable tracking of their persistence in the human body for enhanced molecular imaging.
IMPORTANCE
To understand how spatial and temporal distribution of microbiota impact human health, dynamic tools for monitoring microbiota landscapes inside the host are needed. Particularly when considering the complexity of the gastrointestinal tract and the microbiota that dwell within, tools for monitoring deep segments of the gut non-invasively are required. Medical imaging provides solutions that enable the study of microorganisms in their preferred niche regardless of health status. To bootstrap this technology, we investigated the magnetic resonance imaging (MRI) properties of bacterial isolates and showed that outstanding signal detection is an inherent property of several strains. Among these, we showed that bacteria relying on manganese metabolism have an MRI characteristic that is distinct from mammalian cells. Our findings will lead to direct and safe imaging of bacteria; influence how we monitor both infection and gut health; and help direct the use of antibiotics to curtail the growing threat of antibiotic resistance. | microbiology |
Acinetobacter baumannii and cefiderocol between cidality and adaptability Among the bacterial species included in the ESKAPE group, Acinetobacter baumannii is of great interest due to its intrinsic and acquired resistance to many antibiotic classes and its ability to infect different body districts. Cefiderocol is a novel cephalosporin active against Gram-negative bacteria with promising efficacy on A. baumannii infections, but some studies have reported therapeutic failures even in the presence of susceptible A. baumannii strains.
This study aims to investigate the interactions between cefiderocol and ten A. baumannii strains with different susceptibility profiles to this drug. We confirmed diverse susceptibility profiles in the strains, with resistance values close to the EUCAST-proposed breakpoints. MBC/MIC (minimal bactericidal concentration/minimal inhibitory concentration) ratios, demonstrated bactericidal activity of the drug; on the other hand, bacterial regrowth was evident after exposition to cefiderocol, as were changes in the shape of colonies and bacterial cells. A switch to a non-susceptible phenotype in the presence of high cefiderocol concentrations was found as adaptation mechanisms implemented by these A. baumannii strains to overcome the cidal activity of this antibiotic, also confirmed by the presence of heteroresistant, unstable subpopulations. As our isolates harbored numerous {beta}-lactamase genes, {beta}-lactamase inhibitors showed the ability to restore the antimicrobial activity of cefiderocol regardless of the different non-susceptibility levels of the tested strains.
These in vitro results, can sustain the concept of using combination therapy to eliminate drug-adapted subpopulations and regain full cefiderocol activity in this difficult-to-treat species. | microbiology |
Polyimidazolium protects against an invasive clinical isolate of Salmonella Typhimurium Frequent outbreaks of Salmonella Typhimurium infection in both the animal and human population with potential for zoonotic transmission pose a significant threat to the public health sector. The rapid emergence and spread of more invasive multidrug-resistant clinical isolates of Salmonella further highlight the need for the development of new drugs with effective broad-spectrum bactericidal activities. Synthesis and evaluation of main-chain cationic polyimidazolium 1 (PIM1) against several gram-positive and gram-negative bacteria have previously demonstrated the efficacy profile of PIM1. The present study focuses on antibacterial and anti-biofilm activities of PIM1 against Salmonella both in vitro and in ovo setting. In vitro, PIM1 exhibited bactericidal activity against all tested three strains of Salmonella at a low dosage of 8 g/ml. Anti-biofilm activity of PIM1 was evident with complete inhibition for the initial attachment of biofilms at 16 g/ml and degradation of pre-formed biofilms in a dose-dependent manner. During the host cell infection process, PIM1 reduces extracellular bacterial adhesion and invasion rates to limit the establishment of infection. Once intracellular, the drug-resistant strain was tolerant and protected from PIM1 treatment. In a chicken egg infection model, PIM1 exhibited therapeutic activity for both Salmonella strains with stationary-phase and exponential-phase inocula. Moreover, PIM1 showed a remarkable efficacy against the stationary phase inocula of drug-resistant Salmonella by eliminating the bacteria burden in >50% of infected chicken egg embryos. Collectively, PIM1 has demonstrated its potential as a drug candidate for treatment of Salmonella infections, as well as a solution to tackle egg contamination issues on poultry farms. | microbiology |
Serine Deamination as a New Acid Resistance Mechanism in Escherichia coli Escherichia coli associates with humans early in life and can occupy several body niches either as a commensal in the gut and vagina, or as a pathogen in the urinary tract. As such, E. coli has an arsenal of acid response mechanisms that allow it to withstand the different levels of acid stress encountered within and outside the host. Here, we report the discovery of an additional acid response mechanism that involves the deamination of L-serine to pyruvate by the conserved L-serine deaminases SdaA and SdaB. L-serine is the first amino acid to be imported in E. coli during growth in laboratory media, as the culture senesces. However, there remains a lack in knowledge as to why L-serine is preferred and how it is utilized. We show that in acidified media, L-serine is brought into the cell via the SdaC transporter and deletion of both SdaA and SdaB renders E. coli susceptible to acid stress, with a phenotype similar to other acid stress deletion mutants. We also show that the pyruvate produced by L-serine de-amination activates the pyruvate sensor BtsS, which in concert with the non-cognate response regulator YpdB upregulates the putative transporter YhjX, similar to what has been reported for this system during transition of E. coli to stationary phase. Based on these observations, we propose that L-serine deamination constitutes another acid response mechanism in E. coli that may function to protect E. coli as it transitions to stationary phase of growth.
IMPORTANCEThe observation that L-serine uptake occurs as an E. coli culture senesces is well-established, yet the benefit E. coli garners from this uptake remains unclear. Here, we report a novel acid resistance mechanism, where L-serine is deaminated to pyruvate and ammonia, promoting acid tolerance in E. coli. This study is important as it provides evidence of the use of L-serine as an acid response strategy, not previously reported for E. coli. | microbiology |
Development of recombinant monoclonal antibodies targeting conserved VlsE epitopes in Lyme disease pathogens VlsE (variable major protein-like sequence, expressed) is an outer surface protein of the Lyme disease pathogen (Borreliella species) and a key diagnostic biomarker of Lyme disease. However, the high sequence variability of VlsE poses a challenge to the development of consistent VlsE-based diagnostics and therapeutics. In addition, the standard diagnostic protocols detect immunoglobins elicited by the Lyme pathogen, not the presence of pathogen or its derived antigens. Here we describe the development of recombinant monoclonal antibodies (rMAbs) that bind specifically to conserved epitopes on VlsE. We first quantified amino-acid sequence variability encoded by the vls genes from thirteen B. burgdorferi genomes by evolutionary analyses. We showed broad inconsistencies of the sequence phylogeny with the genome phylogeny, indicating rapid gene duplications, losses, and recombination at the vls locus. To identify conserved epitopes, we synthesized peptides representing five long conserved invariant regions (IRs) on VlsE. We tested the antigenicity of these five IR peptides using sera from three mammalian host species including human patients, the natural reservoir white-footed mouse (Peromyscus leucopus), and VlsE-immunized New Zealand rabbits (Oryctolagus cuniculus). The IR4 and IR6 peptides emerged as the most antigenic and reacted strongly with both the human and rabbit sera, while all IR peptides reacted poorly with sera from natural hosts. Four rMAbs binding specifically to the IR4 and IR6 peptides were identified, cloned, and purified. Given their specific recognition of the conserved epitopes on VlsE, these IR-specific rMAbs are promising diagnostic and theragnostic agents for direct detection of Lyme disease pathogens regardless of strain heterogeneity. | microbiology |
Condensation of the fusion focus by the intrinsically disordered region of the formin Fus1 is essential for cell-cell fusion Spatial accumulation of secretory vesicles underlies various cellular processes, such as neurotransmitter release at neuronal synapses [1], hyphal steering in filamentous fungi [2, 3], and local cell wall digestion preceding the fusion of yeast gametes [4]. Secretory vesicles transported on actin filaments by myosin V motors form clusters that serve as pool for local content release. During fission yeast Schizosaccharomyces pombe gamete fusion, the actin fusion focus assembled by the formin Fus1 concentrates secretory vesicles carrying cell wall digestive enzymes [5-7]. Focus position and coalescence are controlled by local signalling and actin-binding proteins to prevent inappropriate cell wall digestion that would cause lysis [6, 8-10], but the mechanisms of focusing have been elusive. Here, we show that the regulatory N-terminus of Fus1 contains an intrinsically disordered region (IDR) that mediates Fus1 condensation in vivo and forms dense assemblies that exclude other macromolecules. Fus1 lacking its IDR fails to condense in a tight focus and causes cell lysis during attempted cell fusion. Remarkably, replacement of Fus1 IDR with a heterologous low-complexity region that forms liquid condensates fully restores Fus1 condensation and function. By contrast, replacement of Fus1 IDR with a domain that forms more stable oligomers restores condensation but poorly supports cell fusion, suggesting that condensation is tuned to yield a structure selectively permeable for secretory vesicles. We propose that condensation of actin structures by an intrinsically disordered region may be a general mechanism for actin network organisation and the selective local concentration of secretory vesicles. | cell biology |
Incorporating uniparental markers and demographic information in kinship analysis Knowledge of kinship relations between members of wild populations is of great importance in ecological and conservation genetic studies. The bi-parentally inherited autosomal markers has been the Golden Standard in kinship analysis. However, analysis of kin relationship can be challenging in wild populations. The uni-parentally inherited markers and population demographic information can be helpful for identifying false-positive in kinship analysis. Here we showed how incorporating uniparental genetic and demographic information can improve the correct classification rate of kinship analyses by reanalyzing data of a recent study published in Science Advances. The application of next generation high-throughput sequencing to address fundamental ecological questions is of immense benefit to the field of molecular ecology, which could also generate uniparentally inherited organelle genomes together with nuclear data. We strongly recommended that uniparental genetic markers and demographic information be seriously considered in kinship analyses of wild populations. | ecology |
Depth normalization for single-cell genomics count data Single-cell genomics analysis requires normalization of feature counts that stabilizes variance while accounting for variable cell sequencing depth. We discuss some of the trade-offs present with current widely used methods, and analyze their performance on 526 single-cell RNA-seq datasets. The results lead us to recommend proportional fitting prior to log transformation followed by an additional proportional fitting. | bioinformatics |
The dependence of EGFR oligomerization on environment and structure: A camera-based N&B study Number and Brightness analysis (N&B) is a fluorescence spectroscopy technique to quantify protein oligomerization. Accurate results, however, rely on a good knowledge of non-fluorescent states of the fluorescent labels, especially of fluorescent proteins (FP), which are widely used in biology. FPs have been characterized for confocal but not camera-based N&B, which allows in principle faster measurements over larger areas. Here, we calibrate camera-based N&B implemented on a total internal reflection fluorescence (TIRF) microscope for various fluorescent proteins by determining their propensity to be fluorescent. We then apply camera-based N&B in live CHO-K1 cells to determine the oligomerization state of the epidermal growth factor receptor (EGFR), a transmembrane receptor tyrosine kinase that is a crucial regulator of cell proliferation and survival with implications in many cancers. EGFR oligomerization in resting cells and its regulation by the plasma membrane microenvironment is still under debate. Therefore, we investigate the effects of extrinsic factors, including membrane organization, cytoskeletal structure, and ligand stimulation, and intrinsic factors, including mutations in various EGFR domains, on the receptors oligomerization. Our results demonstrate that EGFR oligomerization increases with removal of cholesterol or sphingolipids, or the disruption of GM3-EGFR interactions, indicating raft association. However, oligomerization was not significantly influenced by the cytoskeleton. Mutations in either I706/V948 residues or E685/E687/E690 residues in the kinase and juxtamembrane domains, respectively, led to a decrease in oligomerization, indicating their necessity for EGFR dimerization. Finally, EGFR phosphorylation is oligomerization-dependent involving the extracellular domain (550-580 residues). Coupled with biochemical investigations, camera-based N&B indicates that EGFR oligomerization and phosphorylation is the outcome of several molecular interactions involving the lipid content and structure of the cell membrane and multiple residues in the kinase, juxtamembrane, and extracellular domains.
STATEMENT OF SIGNIFICANCENumber and brightness (N&B) analysis is a powerful tool to determine protein association but is mostly conducted in confocal microscopes. This work determines the brightness and fluorescence probability of a range of fluorescent proteins for camera-based N&B on a total internal reflection microscope, demonstrating that with proper calibration different fluorescent proteins provide the same answers on oligomerization within the margins of error. This camera-based approach allows measuring N&B values across whole cell basal membranes up to an area of ~1,000 m2 simultaneously. N&B is then used in combination with biochemical assays to investigate the oligomerization and activation of the epidermal growth factor receptor (EGFR), a prototypical receptor tyrosine kinase with importance in cell signalling, division and survival and implicated in various cancers. The results indicate that EGFR oligomerization and activation is governed by an interplay between membrane structure and composition and key amino acid residues of EGFR that span the extracellular to the intracellular domains. | biophysics |
SWEET13 transport of sucrose, but not gibberellin, restores male fertility in Arabidopsis sweet13;14 SWEET sucrose transporters play important roles in the allocation of sucrose in plants. Some SWEETs were shown to also mediate transport of the plant growth regulator gibberellin (GA). The close physiological relationship between sucrose and GA raised the questions of if there is a functional connection, and whether one or both of the substrates are physiologically relevant. To dissect these two activities, molecular dynamics were used to map the binding sites of sucrose and GA in the pore of SWEET13 and predicted binding interactions that might be selective for sucrose or GA. Transport assays confirmed these predictions. In transport assays, the N76Q mutant had 7x higher relative GA3 activity, and the S142N mutant only transported sucrose. The impaired pollen viability and germination in sweet13;14 double mutants were complemented by the sucrose-selective SWEET13S142N but not by the SWEET13N76Q mutant, indicating that sucrose is the physiologically relevant substrate and that GA transport capacity is dispensable in the context of male fertility. Therefore, GA supplementation to counter male sterility may act indirectly via stimulating sucrose supply in male sterile mutants. These findings are also relevant in the context of the role of SWEETs in pathogen susceptibility. | plant biology |
Transcriptional fluctuations govern the serum dependent cell cycle duration heterogeneities in Mammalian cells Mammalian cells exhibit a high degree of intercellular variability in cell cycle period and phase durations. However, the factors orchestrating the cell cycle duration heterogeneities remain unclear. Herein, by combining cell cycle network-based mathematical models with live single-cell imaging studies under varied serum conditions, we demonstrate that fluctuating transcription rates of cell cycle regulatory genes across cell lineages and during cell cycle progression in mammalian cells majorly govern the robust correlation patterns of cell cycle period and phase durations among sister, cousin, and mother-daughter lineage pairs. However, for the overall cellular population, alteration in serum level modulates the fluctuation and correlation patterns of cell cycle period and phase durations in a correlated manner. These heterogeneities at the population level can be finetuned under limited serum conditions by perturbing the cell cycle network using a p38-signaling inhibitor without affecting the robust lineage level correlations. Overall, our approach identifies transcriptional fluctuations as the key controlling factor for the cell cycle duration heterogeneities, and predicts ways to reduce cell-to-cell variabilities by perturbing the cell cycle network regulations.
Significance statementIn malignant tumors, cells display a diverse pattern in cell division time. This cell-to-cell variability in cell cycle duration had been observed even under culture conditions for various mammalian cells. Here we used live-cell imaging studies to monitor FUCCI-HeLa cells and quantified the cell cycle period and time spent in different phases under varied serum conditions. We proposed a set of stochastic cell cycle network-based mathematical models to investigate the live-cell imaging data and unraveled that the transcription rate variation across cell lineages and during cell cycle phases explains every aspect of the cell cycle duration variabilities. Our models identified how different deterministic effects and stochastic fluctuations control these variabilities and predicted ways to alter these cell cycle duration variabilities. | systems biology |
Obesity medication lorcaserin requires brainstem GLP-1 neurons to reduce food intake in mice Overweight and obesity are rapidly becoming the "new normal" in developed countries, which promotes a widespread negative impact on human health. Amongst recently developed obesity medications are the serotonin 2C receptor (5-HT2CR) agonist lorcaserin and glucagon-like peptide-1 receptor (GLP-1R) agonists, but the brain circuits employed by these medications to produce their therapeutic effects remain to be fully defined. 5-HT2CRs and GLP-1Rs are widely distributed in the brain, including in the key homeostatic region the nucleus of the solitary tract (NTS) where GLP-1 is produced by preproglucagon (PPGNTS) neurons. PPGNTS cells were profiled using histochemistry and single nucleus RNA sequencing (Nuc-Seq) of mouse brainstem. Transcriptomic analyses revealed 5-HT2CR expression was widespread in PPGNTS clusters. Demonstrating the functional significance of this co-expression, lorcaserin required PPGNTS to reduce food intake. Analysis of second order neurons revealed that local GLP1-R neurons within the NTS are necessary for 5-HT2CRNTS food intake suppression. In contrast, GLP-1RNTS were not required for GLP-1R agonist liraglutide and exendin-4s short term feeding reduction, suggesting scope for lorcaserin and GLP1-R agonist combination therapy. In support of this, lorcaserin+liraglutide and lorcaserin+exendin-4 produced greater reductions in food intake when administered in combination as compared to monotherapies. These data provide insight into the therapeutic mechanisms of lorcaserin and identify a combination strategy to improve the therapeutic profile of lorcaserin and GLP1-R agonists. | neuroscience |
The LC3B FRET biosensor monitors the modes of action of ATG4B during autophagy in living cells Although several mechanisms of autophagy have been dissected in the last decade, following this pathway in real time remains challenging. Among the early events leading to autophagy activation, the ATG4B protease primes the key autophagy player LC3B. Given the lack of reporters to follow this event in living cells, we developed a Forsters Resonance Energy Transfer (FRET) biosensor responding to the priming of LC3B by ATG4B. The biosensor was generated by flanking LC3B within a pH-resistant donor-acceptor FRET pair, Aquamarine/tdLanYFP. We here showed that the biosensor has a dual readout. First, FRET indicates the priming of LC3B by ATG4B and the resolution of the FRET image allows to characterize the spatial heterogeneity of the priming activity. Second, quantifying the number of Aquamarine-LC3B puncta determines the degree of autophagy activation. We then showed that there are small pools of unprimed LC3B upon ATG4B downregulation, and that the priming of the biosensor is completely abolished in ATG4B knockout cells. The lack of priming can be rescued with the wild-type ATG4B or with the partially active W142A mutant, but not with the catalytically dead C74S mutant. Last, we screened for commercially-available ATG4B inhibitors, and we illustrated their differential mode of action by implementing a spatially-resolved, broad-to-sensitive analysis pipeline combining FRET and the quantification of autophagic puncta. Therefore, the LC3B FRET biosensor paves the way for a highly-quantitative monitoring of the ATG4B activity in living cells and in real time, with unprecedented spatiotemporal resolution. | cell biology |
Urine-derived exosomes from individuals with IPF carry pro-fibrotic cargo. BackgroundMicroRNAs (miRNA) and other components contained in extracellular vesicles may reflect the presence of a disease. Lung tissue, sputum and sera of individuals with idiopathic pulmonary fibrosis (IPF) show alterations in miRNA expression. We designed this study to test whether urine and/or tissue derived exosomal miRNAs from individuals with IPF carry cargo that can promote fibrosis.
MethodsExosomes were isolated from urine (U-IPFexo), lung tissue myofibroblasts (MF-IPFexo), serum from individuals with IPF (n=16) and age/sex-matched controls without lung disease (n=10). We analyzed microRNA expression of isolated exosomes and their in vivo bio-distribution. We investigated the effect on ex vivo skin wound healing and in in vivo mouse lung models.
ResultsU-IPFexo or MF-IPFexo expressed miR let-7d, miR-29a-5p, miR 181b-3p and miR-199a-3p consistent with previous reports of miRNA expression obtained from lung tissue/sera from patients with IPF. In vivo bio-distribution experiments detected bioluminescent exosomes in the lung of normal C57Bl6 mice within 5 minutes after intravenous infusion, followed by distribution to other organs irrespective of exosome source. Exosomes labeled with gold nanoparticles and imaged by transmission electron microscopy were visualized in alveolar epithelial type I and type II cells. Treatment of human and mouse lung punches obtained from control, non-fibrotic lungs with either U-IPFexo or MF-IPFexo produced a fibrotic phenotype. A fibrotic phenotype was also induced in a human ex vivo skin model and in in vivo lung models.
ConclusionsOur results provide evidence of a systemic feature of IPF whereby exosomes contain pro-fibrotic miRNAs when obtained from a fibrotic source and interfere with response to tissue injury as measured in skin and lung models.
FundingThis work was supported in part by Lester and Sue Smith Foundation and The Samrick Family Foundation and NIH grants R21 AG060338 (SE and MKG), U01 DK119085 (IP, RS, MTC). | cell biology |
Depth and evenness of sequence coverage are associated with assembly quality, genome structure, and choice of sequencing platform in archived plastid genomes In plastid genomes, the depth and evenness of sequence coverage are considered important indicators for assembly quality. However, the precise manifestations that sequencing depth and evenness can have in the assembly of these genomes, as well as any differences across individual genome sections, have yet to be evaluated. This investigation aims to identify the impact that sequencing depth and evenness can have on the assembly of plastid genomes and how both metrics are related to plastid genome structure. Specifically, we assess if sequencing evenness and reduced sequencing depth have significant correlations with, or significant differences among, individual genome sections, assembly quality metrics, the sequencing platforms employed, and the software tools used for genome assembly. To that end, we retrieve published plastid genomes as well as their sequence reads and genome metadata from public databases, measure sequencing depth and evenness across their sequences, and test several hypotheses on genome assembly and structure through non-parametric statistical tests. The results of our analyses show significant differences in sequencing depth across the four structural partitions as well as between the coding and non-coding sections of the plastid genomes, a significant correlation between sequencing evenness and the number of ambiguous nucleotides per genome, and significant differences in sequencing evenness between various sequencing platforms. Based on these results, we conclude that the observed differences and correlations are not a product of chance alone but possibly genuine manifestations of sequencing depth and evenness during the assembly of these genomes. | genomics |
Plasmid overlap and evolution between Enterobacterales isolates from bloodstream infections and non-human compartments Horizontal gene transfer in Enterobacterales allows mobile genetic elements to move between strains, species, and genera. In particular, the movement of plasmids is a known route for rapid dissemination of antimicrobial resistance (AMR) genes. However, it is difficult to establish to what extent plasmids are shared between Enterobacterales causing human infections and those from non-human sources, such as livestock or the environment. While some previous studies have found only limited evidence for genetic overlap, these have often been limited in size, restricted to drug-resistant isolates, and have used fragmented genome assemblies. Here, we report a collection of geographically and temporally restricted isolates from human bloodstream infections (BSI), environmental soils, livestock (cattle, pigs, poultry, sheep), wastewater (influent, effluent), and rivers. Isolates were all collected between 2008-2018 from sampling sites <60km apart. The combined dataset contains 1,458 complete Enterobacterales genomes, including 3,697 circularised plasmids of which one third were unclassifiable. We find eight groups of near-identical plasmids seen in both human BSI and non-human isolates, of which two are conjugative F-type plasmids carrying AMR genes. We cluster plasmids based on alignment-free distances and find that 73/247 (30%) plasmid clusters contain plasmids from both human BSI and non-human isolates. Pangenome-style analyses of the 69 most prevalent clusters (1,832/3,697 plasmids) reveals sets of shared core genes alongside accessory gene repertoires. Core-gene phylogenies suggest an intertwined ecology where well-conserved putative plasmid backbones carry diverse accessory functions, potentially linked to niche adaptation. Furthermore, we show that closely related human and non-human plasmids are frequently found across distantly related bacterial hosts. Our findings underline the importance of diverse sampling in One Health approaches for AMR management. | genomics |
Reinforcement waves as a mechanism for discontinuous learning Problem-solving and reasoning involve mental exploration and navigation in sparse relational spaces. A physical analogue is spatial navigation in structured environments such as a network of burrows. Recent experiments with mice navigating a labyrinth show a sharp discontinuity during learning, corresponding to a distinct moment of sudden insight when mice figure out long, direct paths to the goal. This discontinuity is seemingly at odds with reinforcement learning (RL), which involves a gradual build-up of a value signal during learning. Here, we show that biologically-plausible RL rules combined with persistent exploration generically exhibit discontinuous learning. In structured environments, positive feedback from learning generates a traveling reinforcement wave. The discontinuity occurs when the wave reaches the starting point. Task difficulty and the learning rule alter its profile and speed, which are determined by the nonlinear dynamics between the nose and bulk of the wave. Predictions explain existing data and motivate specific experiments to isolate the phenomenon. Additionally, we characterize the exact learning dynamics of various RL rules for a complex sequential task. | animal behavior and cognition |
Crystal structure and biochemical analysis suggest that YjoB ATPase is a substrate-specific molecular chaperone AAA+ ATPases are ubiquitous proteins associated with most cellular processes, including DNA unwinding and protein unfolding. Their functional and structural properties are typically determined by domains and motifs added to the conserved ATPases domain. Currently, the molecular function and structure of many ATPases remain elusive. Here, we report the crystal structure and biochemical analyses of YjoB, a Bacillus subtilis AAA+ protein. The crystal structure revealed that the YjoB hexamer forms a bucket hat-shaped structure with a porous chamber. Biochemical analyses showed that YjoB prevents the aggregation of vegetative catalase KatA and gluconeogenesis-specific glyceraldehyde-3 phosphate dehydrogenase GapB, but not citrate synthase, a conventional substrate. Structural and biochemical analyses further showed that the internal chamber of YjoB is necessary for the chaperone activity. Our results suggest that YjoB, conserved in the class Bacilli, is a molecular chaperone acting in the starvation/stationary phases of B. subtilis growth. | biochemistry |
Nucleation and stability of branched versus linear Arp2/3-generated actin filaments Activation of the Arp2/3 complex by VCA-domain-bearing NPFs results in the formation of daughter actin filaments branching off the sides of pre-existing mother filaments. Alternatively, when stimulated by SPIN90, Arp2/3 directly nucleates linear actin filaments. Uncovering the similarities and differences of these two activation mechanisms is fundamental to understanding the regulation and function of Arp2/3. Analysis of individual filaments reveals that the catalytic VCA domain of WASP, N-WASP and WASH, accelerate the Arp2/3-mediated nucleation of linear filaments by SPIN90, in addition to their known branch-promoting activity. Unexpectedly, these VCA domains also destabilize existing branches, as well as SPIN90-Arp2/3 at filament pointed ends. Furthermore, cortactin and GMF, which respectively stabilize and destabilize Arp2/3 at branch junctions, have a similar impact on SPIN90-activated Arp2/3. However, unlike branch junctions, SPIN90-Arp2/3 at the pointed end of linear filaments is not destabilized by piconewton forces, and does not become less stable with time. It thus appears that linear and branched Arp2/3-generated filaments respond similarly to regulatory proteins, albeit with quantitative differences, and that they differ greatly in their responses to aging and to mechanical stress. These results indicate that SPIN90- and VCA-activated Arp2/3 complexes adopt similar yet non-identical conformations, and that their turnover in cells may be regulated differently. | biochemistry |
Mechanism of threonine ADP-ribosylation of F-actin by a Tc toxin Tc toxins deliver toxic enzymes into host cells by a unique injection mechanism. One of these enzymes is TccC3, an ADP-ribosyltransferase from Photorhabdus luminescens. Once TccC3 is translocated into the target cell, the enzyme ADP-ribosylates actin, resulting in clustering of the actin cytoskeleton and ultimately cell death. Here, we combine biochemistry, solution and solid-state NMR spectroscopy and cryo-EM to show in atomic detail how TccC3 modifies actin. We find that the ADP-ribosyltransferase does not bind to G-actin but interacts with two consecutive actin subunits of F-actin. The binding of TccC3 to F-actin occurs via an induced-fit mechanism that facilitates access of NAD+ to the nucleotide binding pocket. The following nucleophilic substitution reaction results in the transfer of ADP-ribose to threonine-148 of F-actin. We demonstrate that this site-specific modification of F-actin prevents its interaction with depolymerization factors, such as cofilin, which impairs actin network turnover and leads to steady actin polymerization. Our findings reveal in atomic detail a new mechanism of action of a bacterial toxin through specific targeting and modification of F-actin. | biochemistry |
ReCSAI Recursive compressed sensing artificial intelligence for confocal lifetime localization microscopy Localization-based super-resolution microscopy resolves macromolecular structures down to a few nanometers by computationally reconstructing fluorescent emitter coordinates from diffraction-limited spots. The most commonly used algorithms are based on fitting parametric models of the point spread function (PSF) to a measured photon distribution. These algorithms make assumptions about the symmetry of the PSF and thus, do not work well with irregular, non-linear PSFs that occur for example in confocal lifetime imaging, where a laser is scanned across the sample. An alternative method for reconstructing sparse emitter sets from noisy, diffraction-limited images is compressed sensing, but due to its high computational cost it has not yet been widely adopted. Deep neural network fitters have recently emerged as a new competitive method for localization microscopy. They can learn to fit arbitrary PSFs, but require extensive simulated training data and do not generalize well. A method to efficiently fit the irregular PSFs from confocal lifetime localization microscopy combining the advantages of deep learning and compressed sensing would greatly improve the acquisition speed and throughput of this method.
Here we introduce ReCSAI, a compressed sensing neural network to reconstruct for confocal dSTORM, together with a simulation tool to generate training data. We implemented and compared different artificial network architectures, aiming to combine the advantages of compressed sensing and deep learning. We found that a U-Net with a recursive structure inspired by iterative compressed sensing showed the best results on realistic simulated datasets with noise, as well as on real experimentally measured confocal lifetime scanning data. Adding a trainable wavelet denoising layer as prior step further improved the reconstruction quality.
Our deep learning approach can reach a similar reconstruction accuracy for confocal dSTORM as frame binning with traditional fitting without requiring the acquisiton of multiple frames. In addition, our work provide generic insights on the reconstruction of sparse measurements from noisy experimental data by combining compressed sensing and deep learning. We provide the trained networks, the code for network training and inference as well as the simulation tool as python code and Jupyter notebooks for easy reproducibility. | bioinformatics |
Biophysical characterization of calcium-binding and modulatory-domain dynamics in a pentameric ligand-gated ion channel Pentameric ligand-gated ion channels (pLGICs) perform electrochemical signal transduction in organisms ranging from bacteria to humans. Among the prokaryotic pLGICs there is architectural diversity involving N-terminal domains (NTDs) not found in eukaryotic relatives, exemplified by the calcium-sensitive channel (DeCLIC) from a Desulfofustis deltaproteobacterium, which has an NTD in addition to the canonical pLGIC structure. Here we have characterized the structure and dynamics of DeCLIC through cryo-electron microscopy (cryo-EM), small-angle neutron scattering (SANS), and molecular dynamics (MD) simulations. In the presence and absence of calcium, cryo-EM yielded structures with alternative conformations of the calcium binding site. SANS profiles further revealed conformational diversity at room temperature beyond that observed in static structures, shown through MD to be largely attributable to rigid body motions of the NTD relative to the protein core, with expanded and asymmetric conformations improving the fit of the SANS data. This work reveals the range of motion available to the DeCLIC NTD and calcium binding site, expanding the conformational landscape of the pLGIC family. Further, these findings demonstrate the power of combining low-resolution scattering, high-resolution structural, and MD-simulation data to elucidate interfacial interactions that are highly conserved in the pLGIC family. | biophysics |
Barley powdery mildew effector CSEP0162 targets multivesicular body-associated MON1 important for immunity Encasements formed around haustoria and biotrophic hyphae as well as hypersensitive reaction (HR) cell death are essential plant immune responses to filamentous pathogens. Here we study a possible reason why these responses are absent in susceptible barley attacked by the powdery mildew fungus. We find that the effector CSEP0162 from this pathogen targets plant MON1, important for fusion of multivesicular bodies to their target membranes. Over-expression of CSEP0162 and silencing of barley MON1 both inhibit encasement formation. We find that the Arabidopsis ecotype No-0 has partial resistance to powdery mildew, and that this is dependent on MON1. Surprisingly, we find the MON1-dependent resistance in No-0 not only include an effective encasement response, but also HR. Similarly, silencing of MON1 in barley also blocked Mla3-mediated HR-based powdery mildew resistance. These data indicate that MON1 is a vital plant immunity component, and we speculate that the barley powdery mildew fungus introduces the effector CSEP0162 to target MON1 and reduce encasement formation and HR.
HighlightMON1 is essential for MVB fusion to plasma membrane. We find that MON1 also is important for immunity, and that it is targeted by the barley powdery mildew effector CSEP0162. | plant biology |
Compartment-driven imprinting of intestinal CD4 (regulatory) T cells in inflammatory bowel disease and homeostasis ObjectiveThe mucosal immune system is implicated in the etiology and progression of inflammatory bowel diseases. The lamina propria and epithelium of the gut mucosa constitute two separate compartments, containing distinct T cell populations. Human CD4 T cell programming and regulation of lamina propria and epithelium CD4 T cells, especially during inflammation, remains incompletely understood.
DesignWe performed imaging mass cytometry, flow cytometry, bulk and single-cell RNA-sequencing to profile ileal lamina propria and intraepithelial CD4 T cells (CD4CD8, regulatory T cells (Tregs), CD69- and CD69high Trm T cells) in controls and Crohns disease (CD) patients (paired non-inflamed and inflamed).
ResultsInflammation results in alterations of the CD4 T cell population with a pronounced increase in Tregs and migrating/infiltrating cells. On a transcriptional level, inflammation within the epithelium induced T cell activation, increased IFN{gamma} responses and effector Treg differentiation. Conversely, few transcriptional changes within the lamina propria were observed. Key regulators including the chromatin remodelers ARID4B and SATB1 were found to drive compartment-specific transcriptional programming of CD4 T(reg) cells.
ConclusionInflammation in CD patients primarily induces changes within the epithelium and not the lamina propria. Additionally, there is compartment-specific CD4 T cell imprinting, driven by shared regulators, upon translocation from the lamina propria to the epithelium. The main consequence of epithelial translocation, irrespective of inflammation, seems to be an overall dampening of broad (pro-inflammatory) responses and tight regulation of lifespan. These data suggest differential regulation of the lamina propria and epithelium, with a specific regulatory role in the inflamed epithelium. | immunology |
Suppression of tumor/host intrinsic CMTM6 drives anti-tumor cytotoxicity in a PD-L1 independent manner CKLF-like MARVEL transmembrane domain-containing 6 (CMTM6) has been identified as a regulator of membranal programmed death ligand 1 (PD-L1) stability and a factor associated with malignancy progression, but the effects and mechanisms of CMTM6 on tumor growth, as well as its potential for therapy, are still largely unknown. Here, we show that tumor CMTM6 increased with progression in both clinical patients and mice. Ablation of CMTM6 resulted in significant retardation of human and murine tumor growth dependent on T-lymphocyte immunity. Tumor CMTM6 suppression broke resistance to immune checkpoint inhibitors and remodeled the tumor immune microenvironment, as specific antitumor cytotoxicity was enhanced and contributed primarily to tumor inhibition. Further, without the PD-1/PD-L1 axis, CMTM6 suppression still significantly dampened tumor growth dependent on cytotoxic cells. Notably, we identified that CMTM6 was widely expressed on immune cells. T-cell CMTM6 increased with sustained immune activation and intratumoral immune exhaustion and affected the T-cell-intrinsic PD-L1 levels. Host CMTM6 knockout significantly restrained tumor growth dependent on CD8+ T-cells, and similarly, not entirely dependent on PD-L1. Thus, we developed and evaluated the antitumor efficacy of CMTM6-targeting adeno-associated virus (AAV), which effectively mobilized antitumor immunity and could be combined with various antitumor drugs. Our findings reveal that both tumor and host CMTM6 are deeply involved in tumor immunity with or without the PD-1/PD-L1 axis and that gene therapy targeting CMTM6 is a promising strategy for cancer immunotherapy.
One Sentence SummaryEven in the absence of the PD-1/PD-L1 axis, tumor or host CMTM6 deficiency can mediate cytotoxicity-dependent anti-tumor immune responses, allowing CMTM6 to be a novel target for scAAV-mediated oncoimmunology gene therapy and combination treatment. | immunology |
Orai3 and Orai1 are essential for CRAC channel function and metabolic reprogramming in B cells The essential role of store-operated Ca2+ entry (SOCE) through Ca2+ release-activated Ca2+ (CRAC) channels in T cells is well established. In contrast, the contribution of individual Orai isoforms to SOCE and their downstream signaling functions in B cells are poorly understood. Here, we demonstrate changes in expression of Orai isoforms in response to B cell activation. We show that Orai3 and Orai1 are essential components of native CRAC channels in B cells and are critical for primary B cell proliferation and survival. The combined loss of Orai1 and Orai3 strongly impairs SOCE, nuclear factor for activated T cells (NFAT) activation, mitochondrial respiration, glycolysis, and the metabolic reprogramming of B cells in response to antigenic stimulation. Our results clarify the molecular composition and cellular functions of SOCE in B lymphocytes. | immunology |
Understanding the role of c-di-AMP signaling in determining antibiotic tolerance in Mycobacterium smegmatis: generation of resistant mutants and regrowth of persisters In this study, we probe the role of secondary messenger c-di-AMP in drug tolerance, which includes both persister and resistant mutant characterization of Mycobacterium smegmatis. Specifically, with the use of c-di-AMP null and overproducing mutants, we showed how c-di-AMP plays a significant role in resistance mutagenesis against antibiotics with different mechanisms of action. We elucidated the specific molecular mechanism linking the elevated intracellular c-di-AMP level and high mutant generation and highlighted the significance of non-homology-based DNA repair. Further investigation enabled us to identify the unique mutational landscape of target and non-target mutation categories linked to intracellular c-di-AMP levels. Overall fitness cost of unique target mutations was estimated in different strain backgrounds, and then we showed the critical role of c-di-AMP in driving epistatic interactions between resistance genes, resulting in the evolution of multi-drug tolerance. Finally, we identified the role of c-di-AMP in persister cells regrowth and mutant enrichment upon cessation of antibiotic treatment. | microbiology |
A new microbiological weapon against lepidopteran pests Nowadays researchers provide more and more evidence that it is necessary to develop an ecologically friendly approach to pest control. This is reflected in a sharp increase in the value of the biological insecticide market in recent decades. In our study, we found a virus strain belonging to the genus Cypovirus (Reoviridae); the strain was isolated from Dendrolimus sibiricus: that possesses attractive features as a candidate for mass production of biological agents for lepidopteran-pest control. We describe morphological, molecular, and ecological features of the new Cypovirus strain. This strain was found to be highly virulent to D. sibiricus (half-lethal dose is 68 occlusion bodies per second-instar larva) and to have a relatively wide host range (infects representatives of five families of Lepidoptera: Erebidae, Sphingidae, Pieridae, Noctuidae, and Lasiocampidae). The virus strain showed a strong interaction with a nontoxic adjuvant (optical brightener), which decreased the lethal dose for both main and alternative hosts, decreased lethal time, and may expand the host range. Moreover, we demonstrated that the insecticidal features were preserved after passaging through the most economically suitable host. By providing strong arguments for possible usefulness of this strain in pest control, we call on virologists, pest control specialists and molecular biologists to give more attention to the Cypovirus genus, which may lead to new insights in the field of pest control research and may provide significant advantages to compare with baculoviruses and Bacillus thuringiensis products which are nowadays main source of bioinsecticides.
Significance statementWithin this article we are describing unique set of features of newly discovered cypovirus strain which possess by significant premises for modern biological insecticides requirements: high potency, universality, true regulating effect, flexible production (possibility to choose host species for production), interaction with enhancing adjuvants, ecologically friendly. Basing on genome alignment we suggest that increasing of host range of new strain is the sequence of evolutionary event which was occurred after coinfection of different CPV species within same host. This finding open new perspective to consider CPVs as perspective agent of biocontrol products. | microbiology |
Metalation calculators for E. coli strain JM109 (DE3): Aerobic, anaerobic and hydrogen peroxide exposed cells cultured in LB media Three web-based calculators, and three analogous spreadsheets, have been generated that predict in vivo metal occupancies of proteins based on known metal affinities. The calculations exploit estimates of the availabilities of the labile buffered pools of different metals inside a cell. Here, metal availabilities have been estimated for a strain of E. coli that is commonly used in molecular biology and biochemistry research, for example in the production of recombinant proteins. Metal availabilities have been examined for cells grown in LB medium aerobically, anaerobically and in response to H2O2 by monitoring the abundance of a selected set of metal-responsive transcripts by qPCR. The selected genes are regulated by DNA-binding metal sensors that have been thermodynamically characterised in related bacterial cells enabling gene expression to be read-out as a function of intracellular metal availabilities expressed as free energies for forming metal complexes. The calculators compare these values with the free energies for forming complexes with the protein of interest, derived from metal affinities, to estimate how effectively the protein can compete with exchangeable binding sites in the intracellular milieu. The calculators then inter-compete the different metals, limiting total occupancy of the site to a maximum stoichiometry of 1, to output percentage occupancies with each metal. In addition to making these new and conditional calculators available, an original purpose of this article was to provide a tutorial which discusses constraints of this approach and presents ways in which such calculators might be exploited in basic and applied research, and in next-generation manufacturing. | cell biology |
Fluorescence fluctuation based super resolution microscopy, basic concepts for an easy start. Due to the wave nature of light, optical microscopy has a lower-bound lateral resolution limit of about half of the wavelength of the detected light, i.e., within the range of 200 to 300 nm. The Fluorescence Fluctuation based Super Resolution Microscopy (FF-SRM) encompases a collection of image analysis techniques which rely on the statistical processing of temporal variations of fluorescence to reduce the uncertainty about the fluorophore positions within a sample, hence, bringing spatial resolution down to several tens of nm. The FF-SRM is known to be suitable for live-cell imaging due to its compatibility with most fluorescent probes and lower instrumental and experimental requirements, which are mostly camera-based epifluorescence instruments. Each FF-SRM approach has strengths and weaknesses, which depend directly on the underlying statistical principles through which enhanced spatial resolution is achieved. In this review, the basic concepts and principles behind a range of FF-SRM methods published to date are revisited. Their operational parameters are explained and guidance for its selection is provided. | bioinformatics |
A glutamine-based single α-helix scaffold to target globular proteins The binding of intrinsically disordered proteins to globular ones often requires the folding of motifs into [a]-helices. These interactions offer opportunities for therapeutic intervention but their modulation with small molecules is challenging because they bury large surfaces. Linear peptides that display the residues that are key for binding can be targeted to globular proteins when they form stable helices, which in most cases requires their chemical modification. Here we present rules to design peptides that fold into single [a]-helices by instead concatenating glutamine side chain to main chain hydrogen bonds recently discovered in polyglutamine helices. The resulting peptides are uncharged, contain only natural amino acids, and their sequences can be optimized to interact with specific targets. Our results provide design rules to obtain single [a]-helices for a wide range of applications in protein engineering and drug design. | biophysics |
Nanoscale prognosis of colorectal cancer metastasis from AFM image processing of histological sections Early ascertainment of metastatic tumour phases is crucial to improve cancer survival, formulate an accurate prognostic report of disease advancement and, most important, quantify the metastatic progression and malignancy state of primary cancer cells with a universal numerical indexing system. This work proposes an early improvement of cancer detection with 97 nm spatial resolution by indexing the metastatic cancer phases from the analysis of atomic force microscopy images of human colorectal cancer histological sections. The procedure applies variograms of residuals of Gaussian filtering and theta statistics of colorectal cancer tissue image settings. The methodology elucidates the early metastatic progression at the nanoscale level by setting metastatic indexes and critical thresholds from relatively large histological sections and categorising the malignancy state of a few suspicious cells not identified with optical image analysis. In addition, we sought to detect early tiny morphological differentiations indicating potential cell transition from epithelial cell phenotypes of low to high metastatic potential. The metastatic differentiation, also identified by higher moments of variograms, sets different hierarchical levels for the metastatic progression dynamic, potentially impacting therapeutic cancer protocols. | cancer biology |
Sinking a giant: quantitative macroevolutionary comparative methods debunk qualitative assumptions Myhrvold et al.1 suggest that our inference of subaqueous foraging among spinosaurids2 is undermined by selective bone sampling, inadequate statistical procedures, and use of inaccurate ecological categorizations. Myhrvold et al.1 ignore major details of our analyses and results, and instead choose to portray our inferences as if they were based on qualitative interpretations of our plots, without providing additional analyses to support their claims. In this manuscript, we thoroughly discuss all the concerns exposed by Myhrvold et al.1. Additional analyses based on our original datasets2 and novel data presented by Myhrvold et al.1 do not change our original interpretations: while the spinosaurid dinosaurs Spinosaurus and Baryonyx are recovered as subaqueous foragers, Suchomimus is inferred as a non-diving animal. | paleontology |
Density-Dependent Color Scanning Electron Microscopy (DDC-SEM) for calcified tissue and pathological calcification. Scanning electron microscopy (SEM) is widely used for materials characterization. It has also been successfully applied to the imaging of biological samples, providing invaluable insights into the topography, morphology and composition of biological structures, including pathological minerals, in diseases affecting cardiovascular, kidney and ocular tissues. Here we provide a comprehensive and detailed guide on how to use colored SEM to aid the visualization and characterization of pathological calcification, and identify the effects of different sample preparation protocols for the visualisation of these minerals. | pathology |
Piezo buffers mechanical stress via modulation of intracellular Ca2+ handling in the Drosophila heart Throughout its lifetime the heart is buffeted continuously by dynamic mechanical forces resulting from contraction of the heart muscle itself and fluctuations in haemodynamic load and pressure. These forces are in flux on a beat-by-beat basis, resulting from changes in posture, physical activity or emotional state, and over longer timescales due to altered physiology (e.g. pregnancy) or as a consequence of ageing or disease (e.g. hypertension). It has been known for over a century of the hearts ability to sense differences in haemodynamic load and adjust contractile force accordingly1-4. These adaptive behaviours are important for cardiovascular homeostasis, but the mechanism(s) underpinning them are incompletely understood. Here we present evidence that the mechanically-activated ion channel, Piezo, is an important component of the Drosophila hearts ability to adapt to mechanical force. We find Piezo is a sarcoplasmic reticulum (SR)-resident channel and is part of a mechanism that regulates Ca2+ handling in cardiomyocytes in response to mechanical stress. Our data support a simple model in which Drosophila Piezo transduces mechanical force such as stretch into a Ca2+ signal, originating from the SR, that modulates cardiomyocyte contraction. We show that Piezo mutant hearts fail to buffer mechanical stress, have altered Ca2+ handling, become prone to arrhythmias and undergo pathological remodelling. | physiology |
Allocation of visuospatial attention indexes evidence accumulation for reach decisions Visuospatial attention is a prerequisite for the performance of visually guided movements: Perceptual discrimination is regularly enhanced at target locations prior to movement initiation. It is known that this attentional prioritization evolves over the time of movement preparation; however, it is not clear whether this build-up simply reflects a time requirement of attention formation or whether, instead, attention build-up reflects the emergence of the movement decision. To address this question, we combined behavioral experiments, psychophysics, and computational decision-making models to characterize the time course of attention build-up during motor preparation. Participants (n = 46, 29 female) executed center-out reaches to one of two potential target locations and reported the identity of a visual discrimination target that occurred concurrently at one of various time-points during movement preparation and execution. Visual discrimination increased simultaneously at the two potential target locations but was modulated by the experiment-wide probability that a given location would become the final goal. Attention increased further for the location that was then designated as the final goal location, with a time course closely related to movement initiation. A sequential sampling model of decision-making predicted key temporal characteristics of attentional allocation. Together, these findings provide evidence that visuospatial attentional prioritization during motor preparation does not simply reflect that a spatial location has been selected as movement goal, but rather indexes the time-extended, cumulative decision that leads to selection, hence constituting a link between perceptual and motor aspects of sensorimotor decisions.
Significance statementWhen humans perform a goal-directed movement such as a reach, attention shifts towards the goal location already before movement initiation, indicating that motor goal selection relies on the use of attention. Here, we demonstrate that key temporal aspects of visuospatial attention are predicted by a well-known computational model of decision-making. These findings suggest that visual attention signals much more than simply that a motor goal has been selected: instead, the time-course of emergent, visuospatial attention reflects the time-extended, cumulative decision that leads to goal selection, offering a window onto the tight link of perceptual and motor aspects in sensorimotor decision-making. | neuroscience |
Conserved population dynamics in the cerebro-cerebellar system between waking and sleep Despite the importance of the cerebellum for motor learning, and the recognised role of sleep in motor memory consolidation, surprisingly little is known about activity in the sleeping cerebro-cerebellar system. Here we used wireless recording from M1 and the cerebellum in monkeys to examine the relationship between patterns of single-unit spiking activity observed during waking behaviour and in natural sleep. Across the population of recorded units, we observed similarities in the timing of firing relative to local field potential features associated with both movements during waking and up-states during sleep. We also observed a consistent pattern of asymmetry in pair-wise cross-correlograms, indicative of preserved sequential firing in both wake and sleep at low frequencies. Despite the overall similarity in population dynamics between wake and sleep, there was a global change in the timing of cerebellar activity relative to motor cortex, from contemporaneous in the awake state, to motor cortex preceding the cerebellum in sleep. We speculate that similar population dynamics in waking and sleep may imply that cerebellar internal models are activated in both states, but that their output is decoupled from motor cortex in sleep. Nevertheless, spindle frequency coherence between the cerebellum and motor cortex may provide a mechanism for cerebellar computations to influence sleep-dependent learning processes in the cortex.
Significance statementIt is well known that sleep can lead to improved motor performance. One possibility is that synaptic changes during sleep result from off-line repetitions of neuronal activity patterns in brain areas responsible for the control of movement. In this study we show for the first time that neuronal patterns in the cerebro-cerebellar system are conserved during both movements and sleep up-states, albeit with a shift in the relative timing between areas. Additionally, we show the presence of simultaneous M1-cerebellar spike coherence at spindle frequencies associated with up-state replay and postulate that this is a mechanism whereby a cerebellar internal models can shape plasticity in neocortical circuits during sleep. | neuroscience |
Subthalamic beta bursts correlate with dopamine-dependent motor symptoms in 106 Parkinson's patients BackgroundPathologically increased beta power has been described as a biomarker for Parkinsons disease (PD) and related to prolonged bursts of subthalamic beta synchronization.
MethodsHere, we investigate the association between subthalamic beta dynamics and motor impairment in a cohort of 106 Parkinsons patients in the ON- and OFF-medication state, suing two different methods of beta burst determination.
ResultsWe report a frequency-specific correlation of low beta power and burst duration with motor impairment OFF dopaminergic medication. Furthermore, reduction of power and burst duration correlated significantly with symptom alleviation through dopaminergic medication. Importantly, qualitatively similar results were yielded with two different methods of beta burst definition.
ConclusionsOur findings validate the robustness of previous results on pathological changes in subcortical oscillations both in the frequency- as well as in the time-domain in the largest cohort of PD patients to date with important implications for next-generation adaptive deep brain stimulation control algorithms. | neuroscience |
A novel mouse model of postpartum depression and the neurobiological effects of fast-acting antidepressant treatments postpartum depression (PPD) is a severe psychiatric disorder that affects up to 15% of mothers and impairs mother-infant bonding with devastating consequences on the child development and the mother health. Several studies indicate a possible dysregulation of glutamatergic and GABAergic signalling in the corticolimbic system, as well as a downregulation of the allopregnanolone levels in serum of PPD patients. Although brexanolone, an allopregnanolone-based treatment, has recently emerged as fundamental PPD treatment, there is scarce evidence on its neurobiological action mechanism. Moreover, ketamine appears to be a promising antidepressant treatment preventing PPD, nevertheless whether it might be a more effective than allopregnanolone for some patients remain unknown. Therefore, the present study is aimed to evaluate the depressive-like phenotype of postpartum females undergoing maternal separation with early weaning (MSEW) protocol, as well as to compare the effectiveness of ketamine and allopregnanolone treatments. MSEW dams show increased despair-like behaviour, anhedonia and disrupted maternal behaviour. Moreover, lower allopregnanolone serum levels, reduction of vesicular transporters for GABA (VGAT) and glutamate (VGLUT1) in the infralimbic cortex, as well as decreased hippocampal cellular proliferation are found in MSEW females. As for the antidepressant treatments, both drugs prevent despair-like behaviour, whereas only ketamine reverts anhedonia present in MSEW females. In addition, both treatments induce pro-neurogenic effects in the dorsal hippocampus but only allopregnanolone increases the VGAT and VGLUT1, without altering the excitatory/inhibitory ratio. Altogether, we propose a new mice model that recapitulates the core symptomatology and alterations in glutamatergic and GABAergic systems shown in PPD patients, which allows us to investigate the therapeutic mechanisms of allopregnanolone and ketamine. | neuroscience |
Endogenous retroviruses promote prion-like spreading of proteopathic seeds Endogenous retroviruses, remnants of viral germline infections, make up a substantial proportion of the mammalian genome. While usually epigenetically silenced, retroelements can become upregulated in neurodegenerative diseases associated with protein aggregation, such as amyotrophic lateral sclerosis and tauopathies. Here we demonstrate that spontaneous upregulation of endogenous retrovirus gene expression drastically affects the dissemination of protein aggregates between murine cells in culture. Viral glycoprotein Env mediates membrane association between donor and recipient cells and promotes the intercellular transfer of protein aggregates packaged into extracellular vesicles. Proteopathic seed spreading can be inhibited by neutralizing antibodies targeting Env as well as drugs inhibiting viral protein processing. Importantly, we show that also overexpression of a human endogenous retrovirus Env elevates intercellular spreading of pathological Tau. Our data highlight the potential influence of endogenous retroviral proteins on protein misfolding diseases and suggest that antiviral drugs could represent promising candidates for inhibiting protein aggregate spreading. | neuroscience |
Unraveling the hidden diversity of endogenous bornavirus-like elements derived from ancient orthobornaviral X and P genes in mammalian genomes Endogenous bornavirus-like elements (EBLs) are heritable sequences derived from bornaviruses in vertebrate genomes, which are strongly suggested to originate from transcripts of ancient bornaviruses. EBLs have been detected by sequence similarity searches, such as tBLASTn, and thus EBLs derived from small and/or rapidly evolving viral genes, such as viral X and P genes, are difficult to be detected. Indeed, no EBLs derived from X and P genes of orthobornaviruses were detected. Thus, although previous studies comprehensively analyzed the presence of EBLs in vertebrate genomes, there may still be undetectable EBLs. In this study, we developed a novel strategy to detect such "hidden" EBLs by focusing on the nature of readthrough transcription of orthobornavirus. We showed a series of evidence supporting that there are EBLs derived from orthobornaviral X and P genes in mammalian genomes. Therefore, this study contributes to a deeper understanding of the evolution of ancient viruses and of virus-host relationships. Further, our data also suggest that endogenous viral elements detected thus far are just the tip of the iceberg, and thus further studies are required to understand ancient viruses more accurately. | microbiology |
Vertical gradients in physiological function and resource allocation of white spruce diverge at the northern- and southern-most range extremes Light availability drives vertical canopy gradients in photosynthetic functioning and carbon (C) balance, yet patterns of variability in these gradients remain unclear. We measured light availability, photosynthetic CO2 and light response curves, foliar C, nitrogen (N) and pigment concentrations, and the photochemical reflectance index (PRI) on upper and lower canopy needles of white spruce trees (Picea glauca) at the northern and southern extremes of the species range. We combined our photosynthetic data with previously published respiratory data to compare and contrast canopy C balance between latitudinal extremes. We found steep canopy gradients in irradiance, photosynthesis, and leaf traits at the southern range limit, but clear convergence across canopy positions at the northern range limit. Thus, unlike many tree species from tropical to mid-latitude forests, high latitude trees do not require vertical gradients of metabolic activity to optimize photosynthetic C gain. Consequently, accounting for self-shading is less critical for predicting gross primary productivity at northern relative to southern latitudes. Northern trees also had a significantly smaller net positive C balance than southern trees suggesting that, regardless of canopy position, low photosynthetic rates coupled with high respiratory costs may ultimately constrain the northern range limit of this widely distributed boreal species.
SUMMARY STATEMENTCanopy gradients in photosynthetic capacity of white spruce diminish at high compared to low latitudes. Low carbon balance in high latitude trees may determine the extent of northern treeline. | ecology |
Phasing of de novo mutations using a scaled-up multiple amplicon long-read sequencing approach De novo mutations (DNMs) play an important role in severe genetic disorders that reduce fitness. To better understand the role of DNMs in disease, it is important to determine the parent-of-origin and timing of the mutational events that give rise to the mutations, especially in sex-specific developmental disorders such as male infertility. However, currently available short-read sequencing approaches are not ideally suited for phasing as this requires long continuous DNA strands that span both the DNM and one or more informative SNPs. To overcome these challenges, we optimised and implemented a multiplexed long-read sequencing approach using the Oxford Nanopore technologies MinION platform. We specifically focused on improving target amplification, integrating long-read sequenced data with high-quality short-read sequence data, and developing an anchored phasing computational method. This approach was able to handle the inherent phasing challenges that arise from long-range target amplification and the normal accumulation of sequencing error associated with long-read sequencing. In total, 77 out of 109 DNMs (71%) were successfully phased and parent-of-origin identified. The majority of phased DNMs were prezygotic (90%), the accuracy of which is highlighted by the average mutant allele frequency of 49.6% and a standard error margin of 0.84%. This study demonstrates the benefits of using an integrated short-read and long-read sequencing approach for large-scale DNM phasing. | genomics |
Quantification of hypoxic regions distant from occlusions in cerebral penetrating arteriole trees The microvasculature plays a key role in oxygen transport in the mammalian brain. Despite the close coupling between cerebral vascular geometry and local oxygen demand, recent experiments have reported that microvascular occlusions can lead to unexpected distant tissue hypoxia and infarction. To better understand the spatial correlation between the hypoxic regions and the occlusion sites, we used both in vivo experiments and in silico simulations to investigate the effects of occlusions in cerebral penetrating arteriole trees on tissue hypoxia. In a rat model of microembolisation, 25 {micro}m microspheres were injected through the carotid artery to occlude penetrating arterioles. In representative models of human cortical columns, the penetrating arterioles were occluded by simulating the transport of microspheres of the same size and the oxygen transport was simulated using a Greens function method. The locations of microspheres and hypoxic regions were segmented, and two novel distance analyses were implemented to study their spatial correlation. The distant hypoxic regions were found to be present in both experiments and simulations, and mainly due to the hypoperfusion in the region downstream of the occlusion site. Furthermore, a reasonable agreement for the spatial correlation between hypoxic regions and occlusion sites is shown between experiments and simulations, which indicates the good applicability of in silico models in understanding the response of cerebral blood flow and oxygen transport to microemboli.
Author summaryThe brain function depends on the continuous oxygen supply through the bloodstream inside the microvasculature. Occlusions in the microvascular network will disturb the oxygen delivery in the brain and result in hypoxic tissues that can lead to infarction and cognitive dysfunction. To aid in understanding the formation of hypoxic tissues caused by micro-occlusions in the penetrating arteriole trees, we use rodent experiments and simulations of human vascular networks to study the spatial correlations between the hypoxic regions and the occlusion locations. Our results suggest that hypoxic regions can form distally from the occlusion site, which agrees with the previous observations in the rat brain. These distant hypoxic regions are primarily due to the lack of blood flow in the brain tissues downstream of the occlusion. Moreover, a reasonable agreement of the spatial relationship is found between the experiments and the simulations, which indicates the applicability of in silico models to study the effects of microemboli on the brain tissue. | bioengineering |
SimBu: Bias-aware simulation of bulk RNA-seq data with variable cell type composition MotivationAs complex tissues are typically composed of various cell types, deconvolution tools have been developed to computationally infer their cellular composition from bulk RNA sequencing (RNA-seq) data. To comprehensively assess deconvolution performance, gold-standard datasets are indispensable. Gold-standard, experimental techniques like flow cytometry or immunohistochemistry are resource-intensive and cannot be systematically applied to the numerous cell types and tissues profiled with high-throughput transcriptomics. The simulation of pseudo-bulk data, generated by aggregating single-cell RNA-seq (scRNA-seq) expression profiles in pre-defined proportions, offers a scalable and cost-effective alternative. This makes it feasible to create in silico gold standards that allow fine-grained control of cell-type fractions not conceivable in an experimental setup. However, at present, no simulation software for generating pseudo-bulk RNA-seq data exists.
ResultsWe developed SimBu, an R package capable of simulating pseudo-bulk samples based on various simulation scenarios, designed to test specific features of deconvolution methods. A unique feature of SimBu is the modelling of cell-type-specific mRNA bias using experimentally-derived or data-driven scaling factors. Here, we show that SimBu can generate realistic pseudo-bulk data, recapitulating the biological and statistical features of real RNA-seq data. Finally, we illustrate the impact of mRNA bias on the evaluation of deconvolution tools and provide recommendations for the selection of suitable methods for estimating mRNA content.
ConclusionSimBu is a user-friendly and flexible tool for simulating realistic pseudo-bulk RNA-seq datasets serving as in silico gold-standard for assessing cell-type deconvolution methods.
AvailabilitySimBu is freely available at https://github.com/omnideconv/SimBu as an R package under the GPL-3 license.
Contactalex.dietrich@tum.de and markus.list@tum.de
Supplementary informationSupplementary data are available at Bioinformatics online. | bioinformatics |
Multiple instance learning to predict immune checkpoint blockade efficacy using neoantigen candidates A successful response to immune checkpoint blockade treatment (ICB) depends on the functional re-invigoration of neoantigen-specific T cells and their anti-tumoral activity. Previous studies showed that the patients neoantigen candidate load is an imperfect predictor of the response to ICB. Further studies provided evidence that the overall response to ICB is also affected by the qualitative properties of a few or even single candidates, limiting the predictive power based on candidate quantity alone.
To our knowledge, this is the first study to predict the response to ICB therapy based on qualitative neoantigen candidate profiles in the context of the mutation type, using a multiple instance learning approach. Multiple instance learning is a special branch of machine learning which classifies labelled bags that are formed by a set of unlabeled instances. The multiple instance learning approach performed systematically better than random guessing and was independent of the neoantigen candidate load. Qualitative modeling performed better in comparison to the quantitative approach, in particular for modelling low-abundant fusion genes. Our findings suggest that multiple instance learning is an appropriate method to predict immunotherapy efficacy based on qualitative neoantigen candidate profiles without relying on direct T-cell response information and provide a foundation for future developments in the field. | bioinformatics |
Spatio-temporal diversity of dietary preferences and stress sensibilities of early and middle Miocene Rhinocerotidae from Eurasia: impact of climate changes Major climatic and ecological changes are documented in terrestrial ecosystems during the Miocene epoch. The Rhinocerotidae are a very interesting clade to investigate the impact of these changes on ecology, as they are abundant and diverse in the fossil record throughout the Miocene. Here, we explored the spatio-temporal evolution of rhinocerotids paleoecology during the early and middle Miocene in Europe and Pakistan. We studied the dental texture microwear (proxy for diet) and enamel hypoplasia (stress indicator) of 19 species belonging to four sub-tribes and an unnamed clade of Rhinocerotidae, and coming from nine Eurasian localities ranging from MN2 to MN7/8. Our results suggest a clear niche partitioning based on diet at Kumbi 4 (MN2, Pakistan), Sansan (MN6, France), and Villefranche dAstarac (MN7/8, France), while dietary overlap and subtle variations are discussed for Beon 1 (MN4, France) and Gra[c]anica (MN5/6, Bosnia-Herzegovina). All rhinocerotids studied were browsers or mixed-feeders, and none had a grazing nor frugivore diet. Regarding hypoplasia, the prevalence was moderate (~ 10%) to high (> 20 %) at all localities but Kumbi 4 (~ 6 %), and documented quite well the local conditions. Sansan and Devinska Nova Ves (MN6, Slovakia), both dated to the MN6 (i.e., by the middle Miocene Climatic Transition, ca. 13.9 Mya), had moderate hypoplasia prevalence. Besides locality, species and tooth locus were also important factors of variation for the prevalence of hypoplasia. The very large hippo-like Brachypotherium brachypus was one of the most affected species at all concerned localities (but Sansan), while early-diverging elasmotheriines were very little affected. | paleontology |
A new device for continuous non-invasive measurements of leaf water content using NIR-transmission allowing dynamic tracking of water budgets Leaf water content (LWC) permanently fluctuates under variable transpiration rate and sap flow and influences e.g. stomatal responses and osmotic adjustment of plant cells. Continuous recordings of LWC are therefore central for the investigation of the regulatory networks stabilizing leaf hydration. Available measurement methods, however, either influence local hydration, interfere with the local leaf micro-environment or cannot easily be combined with other techniques. To overcome these limitations a non-invasive sensor was developed which uses light transmission in the NIR range for precise continuous recordings of LWC. For LWC measurements the transmission ratio of two NIR wavelengths was recorded using a leaf-specific calibration. Pulsed measurement beams enabled measurements under ambient light conditions. The contact-free sensor allows miniaturization and can be integrated into many different experimental settings. Example measurements of LWC during disturbances and recoveries of leaf water balance show the high precision and temporal resolution of the LWC sensor and demonstrate possible method combinations. Simultaneous measurements of LWC and transpiration allows to calculate petiole influx informing about the dynamic leaf water balance. With simultaneous measurements of stomatal apertures the relevant stomatal and hydraulic processes are covered, allowing insights into dynamic properties of the involved positive and negative feed-back loops. | plant biology |
Heart Rate Variability Covaries with Amygdala Functional Connectivity During Voluntary Emotion Regulation The Neurovisceral Integration Model posits that shared neural networks support the effective regulation of emotions and heart rate, with heart rate variability (HRV) serving as an objective, peripheral index of prefrontal inhibitory control. Prior neuroimaging studies have predominantly examined both HRV and associated neural functional connectivity at rest, as opposed to contexts that require active emotion regulation. The present study sought to extend upon previous resting-state functional connectivity findings, examining HRV and corresponding amygdala functional connectivity during a cognitive reappraisal task. Seventy adults (52 old and 18 young adults, 18-84 years, 51% male) received instructions to cognitively reappraise negative and neutral affective images during functional MRI scanning. HRV measures were derived from a finger pulse signal throughout the scan. During the task, young adults exhibited a significant inverse association between HRV and amygdala-medial prefrontal cortex (mPFC) functional connectivity, in which higher HRV was correlated with weaker amygdala-mPFC coupling, whereas old adults displayed a slight positive, albeit non-significant correlation. Furthermore, voxelwise whole-brain functional connectivity analyses showed that higher HRV was linked to weaker right amygdala-posterior cingulate cortex connectivity across old and young adults, and in old adults, higher HRV positively correlated with stronger right amygdala - right ventrolateral prefrontal cortex connectivity. Collectively, these findings highlight the importance of assessing HRV and neural functional connectivity during active regulatory contexts to further identify neural concomitants of HRV and adaptive emotion regulation. | neuroscience |
Single neurons and networks in the claustrum integrate input from widespread cortical sources The claustrum is highly interconnected with many structures in the brain, but the organizing principles governing its vast connectivity have yet to be fully explored. We investigated the defining characteristics and activity of single claustrum neurons, the nature of their relationship with the cortex, and their connectivity within the claustrum itself to gain a comprehensive view of claustral circuits. We show that the claustrum is composed of excitatory and inhibitory cell types that are connected through intraclaustral excitatory synapses, especially between neurons with disparate projection targets. Investigations of corticoclaustral innervation demonstrated that axons from the cortex localize to distinct dorsoventral modules depending on their region of origin. In vitro dual-color optogenetic mapping experiments revealed that individual claustrum neurons integrated inputs from more than one cortical region in a cell type- and projection target-specific manner. Integration in claustrum neurons was observed to be particularly common between areas of the frontal cortex and less so for sensory, motor, and association cortices. Finally, activity in claustrum axons in midline cortical areas recorded with in vivo two-photon calcium imaging showed responses to multimodal sensory stimuli. Our findings shed light on the organizing principles of claustrum circuits, demonstrating that individual claustrum neurons integrate cortical inputs and redistribute this information back to cortex after performing output target- and cell type-dependent local computations. | neuroscience |
The vectoring competence of the mite Varroa destructor for Deformed wing virus of honey bees is highly dynamic and affects survival of the mite. The ectoparasitic mite, Varroa destructor and the viruses it vectors, including types A and B of Deformed wing virus (DWV), pose a major threat to honey bees, Apis mellifera. Analysis of 256 mites collected from the same set of colonies on five occasions from May to October 2021 showed that less than a half of them, 39.8% (95% confidence interval (CI): 34.0 - 46.0%), were able to induce an overt-level DWV infection with more than 109 viral genomes per bee in the pupa after 6 days of feeding, with both DWV-A and DWV-B being vectored at similar rates. To investigate the effect of the phoretic stage on the mites ability to vector DWV, the mites from two field collection events were divided into two groups, one of which was tested immediately for their infectiveness, and the other was kept with adult worker bees in cages for 12 days prior to testing their infectiveness. We found that while 39.2 % (95% CI: 30.0 - 49.1%) of the immediately tested mites induced overt-level DWV infections, 12-day phoretic passage significantly increased the infectiousness of mites to 89.8% (95% CI: 79.2 - 95.6%). It is likely that Varroa mites that survive brood interruptions in field colonies are increasingly infectious. We found that mite lifespan was significantly affected by the type of DWV it transmitted to pupae. The mites, which induced overt DWV-B but not DWV-A infection had an average lifespan of 15.5 days (95% CI: 11.8 - 19.2 days), which was significantly shorter than those of the mites which induced overt DWV-A but not DWV-B infection, with an average lifespan of 24.3 days (95% CI: 20.2 - 28.5), or the mites which did not induce high levels of DWV-A or DWV-B, with an average survival of 21.2 days (95% CI: 19.0 - 23.5 days). The mites which transmitted high levels of both DWV-A and DWV-B had an intermediate average survival of 20.5 days (95% CI: 15.1 - 25.9 days). The negative impact of DWV-B on mite survival could be a consequence of the ability of DWV-B, but not DWV-A to replicate in Varroa mites. | microbiology |
ACE2 nanoparticles prevent cell entry of SARS-CoV-2 We have now been in the grip of the COVID-19 pandemic for over two years with devastating consequences. The continual evolution of the virus has challenged the efficacy of many vaccines and treatment options based on immunotherapies are compromised by this viral escape. One treatment strategy that averts viral escape is the use of constructs based on its entry receptor Angiotensin-Converting Enzyme 2 (ACE2) acting as decoys. Here, we combined full-length human ACE2 with viral vectors commonly used for gene therapy to form nanoparticles that present ACE2 on their surface analogous to human cells. Using cell-based assays and direct, multiscale imaging including cryogenic cellular tomography, we show that these ACE2 nanoparticles are highly efficient in preventing entry of SARS-CoV-2, the virus causing COVID-19, in model cell systems as well as human respiratory tract ex-vivo cultures. Thus, ACE2 nanoparticles have high potential as the next generation therapeutics for addressing the immediate needs of the current pandemic and possible future outbreaks. | cell biology |
Intracellular connections between basal bodies promote the coordinated behavior of motile cilia Hydrodynamic flow produced by multi-ciliated cells is critical for fluid circulation and cell motility. Hundreds of cilia beat with metachronal synchrony for fluid flow. Cilia-driven fluid flow produces extracellular hydrodynamic forces that cause neighboring cilia to beat in a synchronized manner. However, hydrodynamic coupling between neighboring cilia is not the sole mechanism that drives cilia synchrony. Cilia are nucleated by basal bodies (BBs) that link to each other and to the cells cortex via BB-associated appendages. The intracellular BB and cortical network is hypothesized to synchronize ciliary beating by transmitting cilia coordination cues. The extent of intracellular ciliary connections and the nature of these stimuli remain unclear. Moreover, how BB connections influence the dynamics of individual cilia has not been established. We show by FIB-SEM imaging that cilia are coupled both longitudinally and laterally in the ciliate Tetrahymena thermophila by the underlying BB and cortical cytoskeletal network. To visualize the behavior of individual cilia in live, immobilized Tetrahymena cells, we developed Delivered Iron Particle Ubiety Live Light-(DIPULL) microscopy. Quantitative and computer analyses of ciliary dynamics reveal that BB connections control ciliary waveform and coordinate ciliary beating. Loss of BB connections reduces cilia-dependent fluid flow forces.
SummarySoh et al investigate whether intracellular connections between basal bodies control ciliary behavior in multi-ciliated cells. Using a Tetrahymena live cell immobilization technique to quantify ciliary dynamics, they show that inter-BB connections are required for effective ciliary waveform and coordinated ciliary beating that promotes fluid flow. | cell biology |
A Saccharomyces cerevisiae Model for Overexpression of Ntg1 a Base Excision DNA Repair Protein Reveals Novel Genetic Interactions The Base Excision Repair (BER) pathway repairs oxidative DNA damage, a common and detrimental form of damage to the genome. Although biochemical steps in BER have been well define, little is understood about how the pathway is regulated. Such regulation is critical, as cells must respond rapidly to DNA damage while avoiding aberrant activation of repair proteins that can produce DNA damage as intermediates in the repair pathway. Indeed, overexpression of the human BER protein, NTHL1, a DNA N-glycosylase, can cause genomic instability and early cellular hallmarks of cancer. We developed a Saccharomyces cerevisiae model to explore how overexpression of NTHL1 may impair cellular function. Overexpression of Ntg1, the budding yeast orthologue of NTHL1, impairs cell growth. To dissect mechanisms underlying this growth defect, we overexpressed either wild-type Ntg1 or a catalytically inactive variant of Ntg1 (ntg1catdead). Consistent with results obtained for NTHL1, both variants of Ntg1 impair cell growth, but only the wild-type protein causes accumulation of double-strand breaks and chromosome loss. We screened a panel of DNA repair mutants for resistance/sensitivity to overexpression of wild-type Ntg1 or ntg1catdead. This analysis identified several cellular pathways that protect cells from Ntg1-induced damage, providing insight into interplay between DNA repair pathways. Finally, we identified a link to sumoylation and probed how this post-translational modification could contribute to regulation of Ntg1 function. This study describes a budding yeast system to understand how cells regulate and respond to dysregulation of the BER pathway.
Take AwayO_LIOverexpression of a base excision DNA repair protein impairs cell growth
C_LIO_LIOverexpression of a base excision DNA repair protein can cause DNA damage
C_LIO_LIMultiple mechanisms cause DNA damage from overexpression of a repair protein
C_LIO_LIDNA repair pathways functionally interact to protect cells from DNA damage
C_LIO_LIPrecise regulation of the activity of DNA repair proteins is critical
C_LI | genetics |
Insulin Receptor Loss Impairs Mammary Tumorigenesis in Mice Breast cancer (BC) prognosis and outcome are adversely affected by increased body weight, obesity and the obesity-associated type 2 diabetes. Hyperinsulinemia, which is commonly seen as part of metabolic reprograming in the obese state, has been associated with higher risk of death and recurrence in BC. Up to 80% of breast cancers overexpress the insulin receptor (INSR) de novo, which correlates with worse prognosis. To directly probe the role of insulin signaling in mammary tumorigenesis in the mouse, we generated the MMTV-driven polyoma middle T (PyMT) and ErbB2/Her2 BC models, respectively, with coordinate mammary epithelium-restricted deletion of the INSR. In both models, deletion of either one or both copies of the INSR in the mammary gland led to a marked delay in tumor onset and burden, including in mice fed to mimic conditions of human obesity. Longitudinal monitoring of generated mouse models revealed that tumor initiation, rather than progression and metastasis, were impacted by INSR deletion. The similarity of phenotypes elicited by the deletion of one or both copies of INSR raises the possibility that there is a dose-dependent threshold for the contribution of INSR to mammary tumorigenesis. | cancer biology |
Knockout of mitogen-activated protein kinase 3 causes barley root resistance against Fusarium graminearum. The roles of mitogen-activated protein kinases (MAPKs) in plant-fungal pathogenic interactions are less understood in crops. Here, microscopic, phenotyping, proteomic and biochemical analyses revealed that independent TALEN-based knockout lines of Hordeum vulgare MITOGEN-ACTIVATED PROTEIN KINASE 3 (HvMPK3 KO) were resistant against Fusarium graminearum infection. When co-cultured with roots of the HvMPK3 KO lines, F. graminearum hyphae were excluded to the extracellular space, the growth pattern of hyphae was considerably deregulated, mycelia development was less efficient and number of appressoria and their penetration potential were significantly reduced. Intracellular penetration of hyphae was preceded by the massive production of reactive oxygen species (ROS) in attacked cells of the wild type, but it was mitigated in the HvMPK3 KO lines. Suppression of ROS production in these lines coincided with the elevated abundances of catalase and ascorbate peroxidase. Moreover, differential proteomic analysis revealed downregulation of defense-related proteins in wild type, and the upregulation of peroxidases, lipid transfer proteins, and cysteine proteases in HvMPK3 KO lines after 24h of F. graminearum inoculation. Consistently with proteomic analysis, microscopic observations showed an enhanced suberin accumulation in roots of HvMPK3 KO lines, most likely contributing to the arrested infection by F. graminearum. These results suggest that TALEN-based knockout of HvMPK3 leads to the barley root resistance against Fusarium root rot. | plant biology |
Modelling the within-host spread of SARS-CoV-2 infection, and the subsequent immune response, using a hybrid, multiscale, individual-based model. Part I: Macrophages. Individual responses to SARS-CoV-2 infection vary significantly, ranging from mild courses of infection that do not require hospitalisation to the development of disease which not only requires hospitalisation but can be fatal. Whilst many immunological studies have revealed fundamental insights into SARS-CoV-2 infection and COVID-19, mathematical and computational modelling can offer an additional perspective and enhance understanding. The majority of mathematical models for the within-host spread of SARS-CoV-2 infection are ordinary differential equations, which neglect spatial variation. In this article, we present a hybrid, multiscale, individual-based model to study the within-host spread of SARS-CoV-2 infection. The model incorporates epithelial cells (each containing a dynamical model for viral entry and replication), macrophages and a subset of cytokines. We investigate the role of increasing initial viral deposition, increasing delay in type I interferon secretion from epithelial cells (as well as the magnitude of secretion), increasing macrophage virus internalisation rate and macrophage activation, on the spread of infection. | systems biology |
Antigenicity is preserved with fixative solutions used in human gross anatomy: A mice brain immunohistochemistry study. BackgroundHistology remains the gold standard to assess human brain biology, so ex vivo studies using tissue from brain banks are standard practice in neuroscientific research. However, a larger number of specimens could be obtained from gross anatomy laboratories. These specimens are fixed with solutions appropriate for dissections, but whether they also preserve brain tissue antigenicity is unclear. Therefore, we perfused mice brains with solutions used for human body preservation to assess and compare the tissue quality and antigenicity of the main cell populations.
Methods28 C57BL/6J mice were perfused with: 4% formaldehyde (FAS, N=9), salt-saturated solution (SSS, N=9), and alcohol-solution (AS, N=10). The brains were cut into 40m sections for antigenicity analysis and were assessed by immunohistochemistry of four antigens: neuronal nuclei (NeuN), glial fibrillary acidic protein (GFAP-astrocytes), ionized calcium binding adaptor molecule1 (Iba1-microglia), and myelin proteolipid protein (PLP). We compared the fixatives according to multiple variables: perfusion quality, ease of manipulation, tissue quality, immunohistochemistry quality, and antigenicity preservation.
ResultsThe perfusion quality was better using FAS and worse using AS. The manipulation was very poor in SSS brains. FAS and AS fixed brains showed higher tissue and immunohistochemistry quality than the SSS brains. All antigens were readily observed in every specimen, regardless of the fixative solution.
ConclusionSolutions designed to preserve specimens for human gross anatomy dissections also preserve tissue antigenicity in different brain cells. This offers opportunities for the use of human brains fixed in gross anatomy laboratories to assess normal or pathological conditions.
SIGNIFICANCE STATEMENTNeuroscientists currently obtain tissue samples from brain banks. Alternatively, a much larger amount of tissue may be obtained from bodies donated to gross anatomy laboratories. However, they are preserved with different fixative solutions that are used for dissection purposes. We ignore if these solutions also preserve antigenicity of the main cell populations, essential in neuroscientific research. This work is the first to show that two solutions currently used in human gross anatomy laboratories preserve sufficient histological quality, in addition to preserve antigenicity of the main cell populations of the mice brain. This work opens the door to the use of human brain tissue obtained from anatomy laboratories in neuroscientific research. | neuroscience |
Immunological findings in a group of individuals who were non-responders to standard two-dose SARS-CoV-2 vaccines Coronavirus disease (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was declared a pandemic. The virus has infected more than 505 million people and caused more than 6 million deaths. However, data on non-responders to SARS-CoV-2 vaccines in the general population are limited. The objective of the study is to comprehensively compare the immunological characteristics of non-responders to SARS-CoV-2 vaccines in the 18-59 years with that in the 60 years and older using internationally recognized cutoff values. Participants included 627 individuals who received physical examinations and volunteered to participate in COVID-19 vaccination from the general population. The main outcome was an effective seroconversion characterized by anti-SARS-CoV-2 spike IgG level of at least 4-fold increase from baseline. Profiling of naive immune cells was analyzed prior to vaccination to demonstrate baseline immunity. Outcomes of effective seroconversion in the 18-59 years with that in the 60 years and older were compared. The quantitative level of the anti-spike IgG was significantly lower in the 60 years and older and in men among the 18-59 years. There were 7.5% of non-responders among the 18-59 years and 11.7% of non-responders in the 60 years and older using the 4-fold increase parameter. The effective seroconversion rate was significantly related to the level of certain immune cells before vaccination, such as CD4 cells, CD8 cells and B cells and the age. An individual with a titer of anti-SARS-CoV-2 spike IgG that is below 50 BAU/mL might be considered a non-responder between 14-90 days after the last vaccine dose. Booster vaccination or additional protective measures should be recommended for non-responders as soon as possible to reduce disease severity and mortality. | immunology |
ORF6 protein of SARS-CoV-2 inhibits TRIM25 mediated RIG-I ubiquitination to mitigate type I IFN induction. Evasion and antagonism of host cellular immunity upon SARS-CoV-2 infection confers a profound replication advantage on the virus and contributes to COVID-19 pathogenesis. We explored the ability of different SARS-CoV-2 proteins to antagonize the host innate immune system and found that the ORF6 protein mitigated type-I IFN (interferon) induction and downstream IFN signaling. Our findings also corroborated previous reports that ORF6 blocks the nuclear import of IRF3 and STAT1 to inhibit IFN induction and signaling. Here we show that ORF6 directly interacts with RIG-I and blocks downstream type-I IFN induction and signaling by inhibiting K-63 linked ubiquitination of RIG-I by the E3 Ligase TRIM25. This involves ORF6-mediated targeting of TRIM25 for degradation, also observed during SARS-CoV-2 infection. The type-I IFN antagonistic activity of ORF6 was mapped to its C-terminal cytoplasmic tail, specifically to amino acid residues 52-61. Overall, we provide new insights into how the SARS-CoV-2 ORF6 protein inhibits type I-IFN induction and signaling through distinct mechanisms. | microbiology |
Reaction Conditions Promoting the Specific Detection of SARS-CoV-2 NendoU Enzymatic Activity Methods that enable rapid detection of SARS-CoV-2 provide valuable tools for detecting and controlling Covid-19 outbreaks and also facilitate more effective treatment of infected individuals. The predominant approaches developed use PCR to detect viral nucleic acids or immunoassays to detect viral proteins. Each approach has distinct advantages and disadvantages, but alternatives that do not share the same limitations could enable substantial improvements in outbreak detection and management. For instance, methods that have comparable sensitivity to PCR, but that are not prone to the false-positive results that stem from the tendency of PCR to detect molecular degradation products could improve accurate identification of infected individuals. An alternative approach with potential to achieve this entails harnessing the unique enzymatic properties of SARS-CoV-2 enzymes to generate SARS-Cov-2-specific signals that indicate the presence of the virus. This route benefits from the high sensitivity provided by enzymatic signal amplification and also the fact that signal is generated only by intact viral enzymes, not degradation products. Here, we demonstrate enzymatic reaction conditions that enable the preferential detection of NendoU of SARS-CoV-2, versus several of its orthologues, with a fluorogenic oligonucleotide substrate. These compositions provide a possible technical foundation for a novel approach for detecting SARS-CoV-2 that has distinct advantages from current approaches. | microbiology |
GDF15 and ACE2 stratify COVID19 patients according to severity while ACE2 mutations increase infection susceptibility. Coronavirus disease 19 (COVID-19) is a persistent global pandemic with a very heterogeneous disease presentation ranging from a mild disease to dismal prognosis. Early detection of sensitivity and severity of COVID-19 is essential for the development of new treatments. In the present study, we measured the levels of circulating growth differentiation factor 15 (GDF15) and angiotensin-converting enzyme 2 (ACE2) in plasma of severity-stratified COVID-19 patients and healthy control patients and characterized the in vitro effects and cohort frequency of ACE2 SNPs. Our results show that while circulating GDF15 and ACE2 stratify COVID-19 patients according to disease severity, ACE2 missense SNPs constitute a risk factor linked to infection susceptibility. | molecular biology |
Notch signaling functions in non-canonical juxtacrine manner in platelets to amplify thrombogenicity BackgroundNotch signaling is an evolutionarily conserved pathway that dictates cell fate decisions in mammalian cells including megakaryocytes. Existence of functional Notch signaling in enucleate platelets that are generated as cytoplasmic buds from megakaryocytes still remains elusive.
MethodsPlatelets were isolated from human blood by differential centrifugation under informed consent. Expression of transcripts as well as peptides of Notch1 and DLL-4 in platelets was studied by employing RT-qPCR, Western analysis and flow cytometry. Platelet activation responses that include aggregation, secretion of granule contents and platelet-leucocyte interaction were analyzed by Borns aggregometry, flow cytometry, Western analysis and lumi- aggregometry. Shedding of extracellular vesicles from platelets was documented with Nanoparticle Tracking Analyzer. Platelet adhesion and thrombus growth on immobilized matrix was quantified by employing microfluidics platform. Intracellular free calcium in Fura-2-loaded platelets was monitored from ratiometric fluorescence spectrophotometry. Coagulation parameters in whole blood were studied by thromboelastography. Ferric chloride-induced mesenteric arteriolar thrombosis in murine model was imaged by intravital microscopy.
ResultsHere we demonstrate significant expression of Notch1 and its ligand, the Delta-like ligand (DLL)- 4, as well as their respective transcripts, in human platelets. Synthesis and surface translocation of Notch1 and DLL-4 were upregulated when cells were challenged with physiological agonists like thrombin. DLL-4, in turn, instigated neighbouring platelets to switch to activated phenotype, associated with cleavage of Notch receptor and generation of its intracellular domain (NICD). DLL-4-mediated pro-thrombotic attributes were averted by pharmacological inhibition of {gamma}-secretase and phosphatidylinositol 3-kinase. Inhibition of Notch signaling, too, restrained agonist-induced platelet activation, and significantly impaired arterial thrombosis in mice, suggestive of synergism between thrombin- and DLL-4-mediated pathways. Strikingly, prevention of DLL-4-Notch1 interaction by a blocking antibody abolished platelet aggregation and extracellular vesicle shedding induced by thrombin.
ConclusionsOur study presents compelling evidence in support of non-canonical Notch signaling that propagates in juxtacrine manner within platelet aggregates and synergizes with physiological agonists to generate occlusive intramural thrombi. Thus, targeting Notch signaling can be investigated as a potential anti-platelet/anti-thrombotic therapeutic approach.
FundingThis research was supported by J. C. Bose National Fellowship (JCB/2017/000029) and grants received by D. Dash from the Indian Council of Medical Research (ICMR) under CAR (71/4/2018-BMS/CAR), Department of Biotechnology (DBT) (BT/PR-20645/BRB/10/1541/2016) and Science and Engineering Research Board (SERB) (EMR/2015/000583), Government of India. S.N. Chaurasia is a recipient of financial assistance from the ICMR. M. Ekhlak is a recipient of CSIR-SRF and V. Singh is a recipient of UGC-SRF. D. Dash acknowledges assistance from the Humboldt Foundation, Germany. Funders have no role in the design, analysis and reporting of the study. | cell biology |
Allelic gene polymorphisms suspected to diversify the individual early metabolic response upon influenza H3N2 and SARS-CoV-2 infections In this brief report, we point to virus infection time-dependent transcript levels of polymorphic ASMTL genes in human nasal epithelial cells from seven cell origins. Our observations encourage focused top-down and hypothesis-driven studies in support of more efficient allelic genotyping to identify targets for early resilience prediction. | cell biology |
Interrogating the Precancerous Evolution of Pathway Dysfunction in Lung Squamous Cell Carcinoma Using XTABLE Lung squamous cell carcinoma (LUSC) is a type of lung cancer with a dismal prognosis that lacks adequate therapies and actionable targets. This disease is characterized by a sequence of low and high-grade preinvasive stages with increasing probability of malignant progression. Increasing our knowledge about the biology of these premalignant lesions (PMLs) is necessary to design new methods of early detection and prevention, and to identify the molecular processes that are key for malignant progression. To facilitate this research, we have designed XTABLE, an open-source application that integrates the most extensive transcriptomic databases of PMLs published so far. With this tool, users can stratify samples using multiple parameters and interrogate PML biology in multiple manners, such as two and multiple group comparisons, interrogation of genes of interests and transcriptional signatures. Using XTABLE, we have carried out a comparative study of the potential role of chromosomal instability scores as biomarkers of PML progression and mapped the onset of the most relevant LUSC pathways to the sequence of LUSC developmental stages. XTABLE will critically facilitate new research for the identification of early detection biomarkers and acquire a better understanding of the LUSC precancerous stages. | bioinformatics |
Insulin receptors in pancreatic acinar cells contribute to KrasG12D-driven cancer initiation in the context of diet-induced obesity The rising incidence of pancreatic cancer is largely driven by increased prevalence of obesity and type 2 diabetes (T2D). Hyperinsulinemia is a cardinal feature of obesity and T2D, and is associated with increased cancer incidence and mortality. Genetic reduction of insulin production suppresses formation of pancreatic intraepithelial neoplasia (PanIN) pre-cancerous lesions in mice with mutant Kras. However, it remained unclear whether hyperinsulinemia exerts its tumorigenic effects directly on the cells that give rise to PanINs or indirectly on the tumor microenvironment. Here, we tested whether diet-induced hyperinsulinemia contributes to pancreatic cancer directly through insulin receptor (Insr) signaling in KrasG12D-expressing pancreatic acinar cells. Loss of insulin receptors in wild-type or KrasG12D-expressing acinar cells did not significantly influence fasting glucose or insulin. Strikingly, mice lacking Insr in KrasG12D-expressing acinar cells and their progeny had a significant reduction in PanIN plus tumor area in males (2.7-fold) and females (5.3-fold) compared to control mice. Mechanistically, proteome analyses implicated a reduction in digestive enzymes among altered protein networks in mice protected from PanINs, and together with phospho-proteome analysis, linked the spliceosome, ribosome, and secretory pathway to insulin signaling in context of pancreatic cancer initiation. Collectively, these data demonstrate that insulin receptor signaling in acinar cells promotes PanIN initiation in the context of obesity. | cancer biology |
Heterologous expression of a lycophyte protein enhances angiosperm seedling vigor Seedling vigor is a key agronomic trait that determines juvenile plant performance. Angiosperm seeds develop inside fruits and are connected to the mother plant through vascular tissues. Their formation requires plant-specific genes, such as BREVIS RADIX (BRX) in Arabidopsis thaliana roots. BRX family proteins are found throughout the angiosperms but also occur in non-vascular bryophytes and non-seed lycophytes. They consist of four conserved domains, including the tandem "BRX-domains". We found that bryophyte or lycophyte BRX homologs can only partially substitute for Arabidopsis BRX (AtBRX) because they miss key features in the linker between the BRX-domains. Intriguingly however, expression of a BRX homolog from the lycophyte Selaginella moellendorffii (SmBRX) in A. thaliana wildtype background confers robustly enhanced root growth vigor that persists throughout the life cycle. This effect can be traced back to a substantial increase in seed and embryo size, and can be reproduced with a modified, "SmBRX-like" variant of AtBRX. Our results thus suggest that BRX variants could serve as biotechnological tools to boost seedling vigor and shed light on the activity of ancient, non-angiosperm BRX family proteins. | plant biology |
Expansion of information in scientific research papers Presenting information in papers allows readers to see the evidence for the research claims. The amount of information presented to readers is increasing in high impact factor scientific journals. The aim of the present study was to determine whether there was a similar expansion in the amount of information presented to readers in subject-specific journals. We examined 878 research papers that were published in the journals Biology of Reproduction and Reproduction during the first six months of 1989, 1999, 2009, and 2019. Although there were few differences between the journals, we found that between 1989 and 2019 the number of figures increased 1.5-fold, the number of figure panels increased 3.6-fold, and the number of display items increased 5.6-fold. Amongst the display items, the number of images per paper increased 10-fold, and the number of graphs per paper increased 3.7-fold. The median paper in 1989 was 8 pages long, contained 6 tables and/or figures, with 1 image and 4 graphs. In 2019 the median paper was 12 pages long, contained 7 tables and/or figures, with 13 images and 15 graphs. This expansion of information in subject-specific journals implies that authors, reviewers, and editors need to help readers digest complex biological messages without causing information overload.
Lay summaryWe are living in an age of science and information. The amount of information presented in research papers has increased over time in the top science journals. Our research examined whether there has been a similar expansion in information in two influential subject-specific journals. We counted how much information was presented in 878 research papers across a 30-year period in the journals Biology of Reproduction and Reproduction. There were few differences between the two journals. But there was a striking increase in the information presented to readers in 2019 compared with 1989. The typical paper in 1989 was 8 pages long and contained 1 picture and 4 graphs. In 2019 the typical paper was 12 pages long and contained 13 pictures and 15 graphs. This expansion of information means that subject-specific journals must balance the presentation of complex biological messages with the risk of causing information overload. | scientific communication and education |
SARS-CoV-2 evolution and patient immunological history shape the breadth and potency of antibody-mediated immunity Since the emergence of SARS-CoV-2, humans have been exposed to distinct SARS-CoV-2 antigens, either by infection with different variants, and/or vaccination. Population immunity is thus highly heterogeneous, but the impact of such heterogeneity on the effectiveness and breadth of the antibody-mediated response is unclear. We measured antibody-mediated neutralisation responses against SARS-CoV-2Wuhan, SARS-CoV-2, SARS-CoV-2{delta} and SARS-CoV-2o pseudoviruses using sera from patients with distinct immunological histories, including naive, vaccinated, infected with SARS-CoV-2Wuhan, SARS-CoV-2 or SARS-CoV-2{delta}, and vaccinated/infected individuals. We show that the breadth and potency of the antibody-mediated response is influenced by the number, the variant, and the nature (infection or vaccination) of exposures, and that individuals with mixed immunity acquired by vaccination and natural exposure exhibit the broadest and most potent responses. Our results suggest that the interplay between host immunity and SARS-CoV-2 evolution will shape the antigenicity and subsequent transmission dynamics of SARS-CoV-2, with important implications for future vaccine design.
Author SummaryNeutralising antibodies provide protection against viruses and are generated because of vaccination or prior infections. The main target of anti-SARS-CoV-2 neutralising antibodies is a protein called Spike, which decorates the viral particle and mediates viral entry into cells. As SARS-CoV-2 evolves, mutations accumulate in the spike protein, allowing the virus to escape antibody-mediated immunity and decreasing vaccine effectiveness. Multiple SARS-CoV-2 variants have appeared since the start of the COVID-19 pandemic, causing various waves of infection through the population and infecting-in some cases-people that had been previously infected or vaccinated. Since the antibody response is highly specific, individuals infected with different variants are likely to have different repertoires of neutralising antibodies. We studied the breadth and potency of the antibody-mediated response against different SARS-CoV-2 variants using sera from vaccinated people as well as from people infected with different variants. We show that potency of the antibody response against different SARS-CoV-2 variants depends on the particular variant that infected each person, the exposure type (infection or vaccination) and the number and order of exposures. Our study provides insight into the interplay between virus evolution and immunity, as well as important information for the development of better vaccination strategies. | microbiology |
DNA methylation Dependent Restriction of Tyrosine Hydroxylase Contributes to Pancreatic β-cell Heterogeneity The molecular and functional heterogeneity of pancreatic {beta}-cells is well recognized. Pancreatic islets harbor a small subset of {beta}-cells that co-express Tyrosine Hydroxylase (TH), an enzyme involved in synthesis of catecholamines that repress insulin secretion. Restriction of this sub-population within islets is essential for appropriate insulin secretion. However, the distinguishing characteristics of this subpopulation and the mechanisms that restrict TH expression in {beta}-cells are not known. Here, we define the specific molecular and metabolic characteristics of the TH+ {beta}-cells and show that TH expression in {beta}-cells is restricted by DNA methylation patterning during {beta}-cell lineage specification. Ablation of de novo DNA methyltransferase Dnmt3a in the pancreatic- and endocrine-progenitor lineages results in a dramatic increase in the proportion of TH+ {beta}-cells, while {beta}-cell specific ablation of Dnmt3a has no effect on this sub-population. We demonstrate that maintenance of Th promoter DNA methylation patterns is essential for its continued restriction in postnatal {beta}-cells, and that loss of DNA methylation dysregulates TH expression in {beta}-cells in response to chronic overnutrition, contributing to impairment of {beta}-cell identity. These data highlight the essential requirement of DNA methylation patterning in regulating endocrine cell fates, and reveal a novel role of DNA methylation in {beta}-cell heterogeneity. | developmental biology |
Draft genome of six Cuban Anolis lizards and insights into genetic changes during the diversification The detection of various type of genomic variants and their accumulation processes during species diversification and adaptive radiation is important for understanding the molecular and genetic basis of evolution. Anolis lizards in the West Indies are good models for studying the mechanism of the evolution because of the repeated evolution of their morphology and the ecology. In this study, we performed de novo genome assembly of six Cuban Anolis lizards with different ecomorphs and thermal habitats (Anolis isolepis, Anolis allisoni, Anolis porcatus, Anolis allogus, Anolis homolechis, and Anolis sagrei). As a result, we obtained six novel draft genomes with relatively long and high gene completeness, with scaffold N50 ranging from 5.56-39.79 Mb, and vertebrate Benchmarking Universal Single-Copy Orthologs completeness ranging from 77.5% to 86.9%. Subsequently, we performed comparative analysis of genomic contents including those of mainland Anolis lizards to estimate genetic variations that had emerged and accumulated during the diversification of Anolis lizards. Comparing the repeat element compositions and repeat landscapes revealed differences in the accumulation process between Cuban trunk-crown and trunk-ground species, LTR accumulation observed only in A. carolinensis, and separate expansions of several families of LINE in each of Cuban trunk-ground species. The analysis of duplicated genes suggested that the proportional difference of duplicated gene number among Cuban Anolis lizards may be associated to the difference of their habitat range. Furthermore, Pairwise Sequentially Markovian Coalescent analysis proposed that the effective population sizes of each species might have been affected by Cubas geohistory. Hence, these six novel draft genome assemblies and detected genetic variations can be a springboard for the further genetic elucidation of the Anolis lizards diversification.
SignificanceAnolis lizard in the West Indies is excellent model for studying the mechanisms of speciation and adaptive evolution. Still, due to a lack of genome assemblies, genetic variations and accumulation process of them involved in the diversification remain largely unexplored. In this study, we reported the novel genome assemblies of six Cuban Anolis lizards and analyzed evolution of genome contents. From comparative genomic analysis and inferences of genetic variation accumulation process, we detected species- and lineage-specific transposon accumulation processes and gene copy number evolution, considered to be associated with the adaptation to their habitats. Additionally, we estimated past effective population sizes and the results suggested its relationship to Cubas geohistory. | genomics |
Mechanism of the cadherin-catenin F-actin catch bond interaction Mechanotransduction at cell-cell adhesions is crucial for the structural integrity, organization, and morphogenesis of epithelia. At cell-cell junctions, ternary E-cadherin/{beta}-catenin/E-catenin complexes sense and transmit mechanical load by binding to F-actin. The interaction with F-actin, described as a two-state catch bond, is weak in solution but is strengthened by applied force due to force-dependent transitions between weak and strong actin-binding states. Here, we provide direct evidence from optical trapping experiments that the catch bond property principally resides in the E-catenin actin-binding domain (ABD). Consistent with our previously proposed model, deletion of the first helix of the five-helix ABD bundle enables stable interactions with F-actin under minimal load that are well-described by a single-state slip bond, even when E-catenin is complexed with {beta}-catenin and E-cadherin. Our data argue for a conserved catch bond mechanism for adhesion proteins with structurally similar ABDs. We also demonstrate that a stably bound ABD strengthens load-dependent binding interactions between a neighboring complex and F-actin, but the presence of the other E-catenin domains weakens this effect. These results provide mechanistic insight to the cooperative binding of the cadherin-catenin complex to F-actin, which regulate dynamic cytoskeletal linkages in epithelial tissues. | biophysics |
Ongoing Replication Stress Response and New Clonal T Cell Development Discriminate Between Liver and Lung Recurrence Sites and Patient Outcomes in Pancreatic Ductal Adenocarcinoma Background and AimsMetastatic pancreatic adenocarcinoma (mPDAC) is lethal, yet a subset of patients who have metastatic disease that spreads only to the lung have better outcomes. We identified unique transcriptomic and immune features that distinguish patients who develop metastases in the liver (liver cohort) versus those with lung-avid but liver-averse mPDAC (lung cohort).
MethodsWe used clinical data from the Oregon Pancreas Tissue Registry to identify PDAC patients with liver and/or lung metastases. Gene expression and genomic alteration data from 290 PDAC tumors were used to identify features unique to patients from the liver and lung cohorts. In parallel, T cell receptor sequencing data from 289 patients were used to identify immune features unique to patients in the lung cohort.
ResultsLung cohort patients had better survival outcomes than liver cohort patients. Primary tumors from patients in the liver cohort expressed a novel gene signature associated with ongoing replication stress (RS) response predictive of poor patient outcome independent from known subtypes. In contrast, patients with tumors lacking the RS response signature survived longer, especially if their tumors had alterations in DNA damage repair genes. A subset of patients in the lung cohort demonstrated new T cell clonal development in their primary and metastatic tumors leading to diverse peripheral blood TCR repertoires.
ConclusionLiver-avid metastatic PDAC is associated with an ongoing RS response, whereas tumors lacking the RS response with ongoing T cell clonal responses may have unique vulnerabilities allowing long-term survival in patients with lung-avid, liver-averse metastatic PDAC. | cancer biology |
Chromosomal position of ribosomal protein genes impacts long term evolution of Vibrio cholerae. It is unclear how gene order within the chromosome influences bacterial evolution. The genomic location of genes encoding the flow of genetic information is biased towards the replication origin (oriC) in fast-growing bacteria. To study the role of chromosomal location on cell physiology we relocated the S10-spec- locus (S10), harboring half of ribosomal protein genes, to different chromosomal positions in the fast-growing pathogen V. cholerae. We found that growth rate, fitness and infectivity inversely correlated the distance between S10 and oriC. To gain insight into the evolutionary effect of RP genomic position, we evolved strains bearing S10 at its current oriC-proximal location or derivatives harboring the locus far from it (i.e. 1.5Mbp). Populations deep sequencing on average 1 mutation fixed each 100 generations, mainly at genes linked to flagellum regulation, lipopolysaccharide synthesis, chemotaxis, biofilm and quorum sensing. Along the experiment, populations showed an increment in biofilm forming capacity. All populations increased their growth rate. However, growth rate advantage of populations bearing S10 at an oriC-proximal persisted along the experiment over those where the main ribosomal protein gene cluster locates at an oriC-distal position. This indicates that suppressor mutations cannot compensate S10 genomic location. We selected fast-growing clones displaying a [~]10% growth rate increment finding that they harbored inactivating mutations at, among other sites, flagellum master regulators flrAB regardless S10 genomic location. The introduction of these mutations on naive V. cholerae strains resulted in a [~]10% increase in growth rate. Our study therefore demonstrates that the location of ribosomal protein genes conditions the evolutionary trajectory of growth rate in the long term. While genomic content is highly plastic in prokaryotes, gene order is an underestimated factor that conditions cellular physiology and lineage evolution. The lack of suppression enables artificial gene relocation for genetic circuit reprogramming. | microbiology |
Characteristics of intestinal microflora and dysbiosis in relation to the disease duration in patients with Meniere's disease Menieres disease is an intractable disease characterized by paroxysms of intense rotatory dizziness, hearing loss, and other auditory symptoms. It is believed to be caused by endolymphatic edema of the vestibule and cochlea. The mechanism underlying endolymphatic edema is believed to be stress-related. Endolymphatic edema identification using cochlear contrast-enhanced MRI (hybrid of reversed image of positive endolymph signal and native image of positive perilymph signal [HYDROPS]) is an objective test for Menieres disease, enabling reliable diagnosis of this disease. Since dysbiosis of the intestinal microflora has been reported in stress-induced depression and irritable colitis, a similar abnormality may also occur in stress-triggered Menieres disease. Therefore, we investigated the gut microbiota of 10 patients (6 males and 4 females; mean age, 49.6 {+/-} 8.1 years) diagnosed with Menieres disease and marked endolymphatic edema on HYDROPS. These 10 patients underwent intestinal microbiota examination, and the results of the examination were evaluated in relation to the duration of the disease, the results of auscultation of the affected side, and Dizziness Handicap Inventory (DHI) scores. A significant negative correlation was found between the disease duration and Shannon diversity index and Faiths phylogenetic diversity, which indicated dysbiosis of the intestinal microflora. No correlation was found between the indicators of microbial diversity and the results of audiometry or the DHI on the affected side. Dysbiosis of the intestinal microbiota worsened with increasing duration of Menieres disease. Moreover, Akkermansia muciniphila was not detected in any patient with Menieres disease. Thus, despite the small number of cases in this study (n = 10), the findings indicate the possibility of abnormalities of the intestinal microflora in Menieres disease. | microbiology |
Local adaptation of Aedes aegypti mosquitoes to Wolbachia-induced fitness costs Aedes aegypti mosquito eggs can remain quiescent for many months before hatching, allowing populations to persist through unfavorable conditions. Aedes aegypti infected with the Wolbachia strain wMel have been released in tropical and subtropical regions for dengue control. wMel reduces the viability of quiescent eggs, but this physiological cost might be expected to evolve in natural mosquito populations that frequently experience stressful conditions. We therefore compared the costs of wMel infection for quiescent egg viability in field-derived and laboratory populations. Quiescent egg viability was highly variable in wMel-infected populations, with greater costs of wMel in field-derived populations. In contrast, there was little variation between matched field-derived and long-term laboratory populations lacking wMel, suggesting that laboratory adaptation does not influence this trait and that differences are due to wMel infection. Comparisons of populations collected a year apart show a decline in costs under laboratory rearing conditions involving a rapid turnover of mosquito generations; this pattern was consistent across populations despite their origin, suggesting adaptation of mosquitoes to the wMel infection under laboratory conditions. Reciprocal crossing experiments confirm that differences in quiescent egg viability were mainly due to the genetic background and not Wolbachia alone. wMel-infected mosquitoes hatching from long-term quiescent eggs showed partial loss of cytoplasmic incompatibility and female infertility, highlighting additional costs of long-term quiescence. Our study provides the first evidence for a shift in Wolbachia phenotypic effects following deliberate field release and establishment and it highlights interactions between Wolbachia infections and local adaptation. The unexpected changes in fitness costs observed here suggest potential tradeoffs with undescribed fitness benefits of the wMel infection. | evolutionary biology |
Structure of C. elegans TMC-1 complex illuminates auditory mechanosensory transduction The initial step in the sensory transduction pathway underpinning hearing and balance in mammals involves the conversion of force into the gating of a mechanosensory transduction (MT) channel. Despite the profound socioeconomic impacts of hearing disorders and the fundamental biological significance of understanding MT, the composition, structure and mechanism of the MT complex has remained elusive. Here we report the single particle cryo-EM structure of the native MT TMC-1 complex isolated from C. elegans. The 2-fold symmetric complex is composed of 2 copies each of the pore-forming TMC-1 subunit, the calcium-binding protein CALM-1 and the transmembrane inner ear protein TMIE. CALM-1 makes extensive contacts with the cytoplasmic face of the TMC-1 subunits while the single-pass TMIE subunits reside on the periphery of the complex, poised like the handles of an accordion. A subset of particles in addition harbors a single arrestin-like protein, ARRD-6, bound to a CALM-1 domain. Single- particle reconstructions and molecular dynamics simulations show how the MT complex deforms the membrane bilayer and suggest crucial roles for lipid-protein interactions in the mechanism by which mechanical force is transduced to ion channel gating. | biochemistry |
A curated data resource of 214K metagenomes for characterization of the global resistome The growing threat of antimicrobial resistance (AMR) calls for new epidemiological surveillance methods, as well as a deeper understanding of how antimicrobial resistance genes (ARGs) have transmitted around the world. The large pool of sequencing data available in public repositories provides an excellent resource for monitoring the temporal and spatial dissemination of AMR in different ecological settings. However, only a limited number of research groups globally have the computational resources allowing analyses of such data. We retrieved 442 Tbp of sequencing reads from 214,095 metagenomic samples from the European Nucleotide Archive (ENA) and aligned them using a uniform approach against ARGs and 16S/18S rRNA genes. Here, we present the results of this extensive computational analysis and share the counts of reads aligned. Over 6.76 {middle dot} 108 read fragments were assigned to ARGs and 3.21 {middle dot} 109 to rRNA genes, where we observed distinct differences in both the abundance of ARGs and the link between microbiome and resistome compositions across various sampling types. This collection is another step towards establishing a global surveillance of AMR and can serve as a resource for further research into the environmental spread and dynamic changes of ARGs. | bioinformatics |
Effects of anti-fibrotic standard of care drugs on senescent human lung fibroblasts RationaleCellular senescence is crucial in the progression of idiopathic pulmonary fibrosis (IPF), but it is yet unclear whether the standard-of-care (SOC) drugs nintedanib and pirfenidone have senolytic properties.
ObjectivesWe attempted to illuminate the effects of SOC drugs on senescent normal and IPF lung fibroblasts in vitro.
MethodsColorimetric/fluorimetric assays, qRT-PCR, and western blotting were used to evaluate the effect of SOC drugs on senescent normal and IPF lung fibroblasts.
ResultsSOC drugs did not induce apoptosis in the absence of death ligands in either normal or IPF senescent cells. Nintedanib increased caspase-3 activity in the presence of Fas Ligand (FasL) in normal but not in IPF senescent fibroblasts. Conversely, nintedanib enhanced B cell lymphoma (Bcl)-2 expression in senescent IPF lung fibroblasts. Moreover, in senescent IPF cells, pirfenidone alone induced mixed lineage kinase domain-like pseudokinase (MLKL) phosphorylation, provoking necroptosis. However, fragmented gasdermin D, indicating pyroptosis, was not detected under any condition. In addition, SOC drugs increased transcript levels of fibrotic and senescence markers in senescent IPF fibroblasts, whereas D+Q inhibited all these markers. Finally, D+Q enhanced growth differentiation factor 15 (GDF15) transcript and protein levels in both normal and IPF senescent fibroblasts.
ConclusionsIn the presence and absence of the extrinsic pro-apoptotic ligands, SOC drugs failed to trigger apoptosis in senescent fibroblasts, possibly due to enhanced Bcl-2 levels and the activation of the necroptosis pathway. SOC drugs elevated fibrotic and senescence markers in IPF lung fibroblasts. Together, these data demonstrated the inefficacy of SOC in targeting senescent cells. Further investigation is required to fully elucidate the therapeutic implications of SOC drugs on other senescent cell types in IPF. | pharmacology and toxicology |
The peptide GOLVEN10 controls nodule and lateral root organogenesis and positioning along the longitudinal root axis O_LIGLV/RGF peptide encoding genes can be identified in genomes of all plants that can form roots or root-like structures suggesting they were essential for transition of plants to land.
C_LIO_LIIn Medicago truncatula, five of fifteen GOLVEN(GLV)/ROOT MERISTEM GROWTH FACTOR (RGF) peptide coding genes were induced during nodule organogenesis and to a varying extent under nitrogen deficiency and auxin treatment. Expression of MtGLV9 and MtGLV10 at nodule initiation sites was dependent on the transcription factor NODULE INCEPTION.
C_LIO_LIOverexpression of all five nodule-induced GLV genes in M. truncatula hairy roots as well as application of the corresponding synthetic peptides resulted in a 25-50% reduction in nodule number indicating GOLVENs are negative regulators of nodule organogenesis.
C_LIO_LIThe peptide GOLVEN10 shifted the position of the first formed lateral root (rhizotaxis) as well as the first formed nodule along the longitudinal primary root axis, a phenomenon we term nodulotaxis, thereby reducing the absolute length of the zone of lateral organ formation on roots.
C_LIO_LIApplication of synthetic GOLVEN10 peptide caused an increase in cell number but not cell length in each root cortical cell layer causing an increase in root length and a consequent spatiotemporal delay in formation of the first lateral organ.
C_LI
Plain Language SummaryNodule positioning is an understudied trait, yet it determines the length of the root that can support nodule formation and consequently the total number of functional nodules formed. We identify for the first time, genetic factors called GOLVEN peptides that alter nodule and lateral root positioning on the primary root along with several other traits including nodule organ initiation and root architecture. | plant biology |
Insulin docking within the open hemichannel of connexin 43 may reduce risk of amyotrophic lateral sclerosis BackgroundType 2 diabetes (T2D), characterized by hyperinsulinemia, protects motor neurons against amyotrophic lateral sclerosis (ALS). Type 1 diabetes and a total lack of insulin are associated with increased risk of ALS. Connexin 43 (Cx43), an astrocyte protein, operates as an open pore via which toxic substances from the astrocytes reach the motor neurons.
MethodsIn the current study we performed molecular docking of insulin with monomeric Cx31, monomeric Cx43, and hexameric Cx31 to assess whether insulin might affect the pore. Hexameric Cx31 and hexameric Cx43 have hemichannels composed of 6 subunits that work as transmembrane channels, binding together to form gap junction intercellular channels that open and close. We used the program AutoDock Vina Extended for the molecular docking study.
ResultsCx31 shares amino acid and structural similarity to Cx43 and insulin docks to the same position of the N-terminal domain of monomeric Cx31 and monomeric Cx43. Insulin docks within the open hemichannel of hexameric Cx31, potentially blocking it. The block may be responsible for the protective relationship of T2D to ALS.
ConclusionInsulin might be a treatment for ALS, especially since insulin enhances glucose-stimulated insulin secretion in healthy humans. An insulin secretogogue such as oral sulfonylurea or glinide might also be of value. | neuroscience |
Synovial fibroblasts assume distinct functional identities and secrete R-spondin 2 to drive osteoarthritis ObjectivesSynovium is acutely affected following joint trauma and contributes to post-traumatic osteoarthritis (PTOA) progression. Little is known about discrete cell types and molecular mechanisms in PTOA synovium. We aimed to describe synovial cell populations and their dynamics in PTOA, with a focus on fibroblasts. We also sought to define mechanisms of synovial Wnt/{beta}-catenin signaling, given its emerging importance in arthritis.
MethodsWe subjected mice to non-invasive anterior cruciate ligament rupture as a model of human joint injury. We performed single-cell RNA-sequencing to assess synovial cell populations, subjected Wnt-GFP reporter mice to joint injury to study Wnt-active cells, and performed intra-articular injections of the Wnt agonist R-spondin 2 (Rspo2) to assess whether gain-of-function induced pathologies characteristic of PTOA. Lastly, we used cultured fibroblasts, macrophages, and chondrocytes to study how Rspo2 orchestrates crosstalk between joint cell types.
ResultsWe uncovered seven distinct functional subsets of synovial fibroblasts in healthy and injured synovium, and defined their temporal dynamics in early and established PTOA. Wnt/{beta}-catenin signaling was overactive in PTOA synovium, and Rspo2 was strongly induced after injury and secreted exclusively by Prg4hi lining fibroblasts. Trajectory analyses predicted that Prg4hi lining fibroblasts arise from a pool of Dpp4+ mesenchymal progenitors in synovium, with SOX5 identified as a potential regulator of this emergence. We also showed that Rspo2 orchestrated pathological crosstalk between synovial fibroblasts, macrophages, and chondrocytes.
ConclusionsSynovial fibroblasts assume distinct functional identities during PTOA, and Prg4hi lining fibroblasts secrete the Wnt agonist Rspo2 to drive pathological crosstalk in the joint after injury. | cell biology |
Messenger-RNA Modification Standards and Machine Learning Models Facilitate Absolute Site-Specific Pseudouridine Quantification Enzyme-mediated chemical modifications to mRNA are important for fine-tuning gene expression, but they are challenging to quantify due to low copy number and limited tools for accurate detection. Existing studies have typically focused on the identification and impact of adenine modifications on mRNA (m6A and inosine) due to the availability of analytical methods. The pseudouridine ({Psi}) mRNA modification is also highly abundant but difficult to detect and quantify because there is no available antibody, it is mass silent, and maintains canonical basepairing with adenine. Nanopores may be used to directly identify {Psi} sites in RNAs using a systematically miscalled base, however, this approach is not quantitative and highly sequence dependent. In this work, we apply supervised machine learning models that are trained on sequence-specific, synthetic controls to endogenous transcriptome data and achieve the first quantitative {Psi} occupancy measurement in human mRNAs. Our supervised machine learning models reveal that for every site studied, different signal parameters are required to maximize {Psi} classification accuracy. We show that applying our model is critical for quantification, especially in low-abundance mRNAs. Our engine can be used to profile {Psi}-occupancy across cell types and cell states, thus providing critical insights about physiological relevance of {Psi} modification to mRNAs. | genomics |
Atomic protein structure refinement using all-atom graph representations and SE(3)-equivariant graph neural networks Three-dimensional (3D) protein structures reveal the fundamental information about protein function. The state-of-art protein structure prediction methods such as Alphafold are being widely used to predict structures of uncharacterized proteins in biomedical research. There is a significant need to further improve the quality and nativeness of the predicted structures to enhance their usability. Current machine learning methods of refining protein structures focus mostly on improving the backbone quality of predicted structures without effectively leveraging and enhancing the conformation of all atoms including sidechains, while molecular simulation methods are computationally intensive and time-consuming.
SummaryIn this work, we develop ATOMRefine, a deep learning-based, end-to-end, all-atom protein structural model refinement method. It uses a SE(3)-equivariant graph transformer network that is equivariant to the rotation and translation of 3D structures in conjunction with a novel graph representation of all atoms to directly refine protein atomic coordinates of all the atoms in a predicted tertiary structure represented as a molecular graph. The method is first trained and tested on the structural models in AlphafoldDB whose experimental structures are known, and then blindly tested on 69 CASP14 regular targets and 7 CASP14 refinement targets. ATOMRefine improves the quality of both backbone atoms and all-atom conformation of the initial structural models generated by AlphaFold. It also performs better than the state-of-the-art refinement methods in multiple evaluation metrics including an all-atom model quality score - the MolProbity score based on the analysis of all-atom contacts, bond length, atom clashes, torsion angles, and sidechain rotamers. As ATOMRefine can refine a protein structure quickly, it provides a viable, fast solution for improving protein geometry and fixing structural errors of predicted structures through direct coordinate refinement. | bioinformatics |
Metagenomic analysis of coprolites from three Late Pleistocene megaherbivores from the Southwestern United States. 1.BackgroundDetermining the life-history traits of extinct species is often difficult from skeletal remains alone, limiting the accuracy of studies modeling past ecosystems. However, the analysis of the degraded endogenous bacterial DNA present in paleontological fecal matter (coprolites) may enable the characterization of specific traits such as the hosts digestive physiology and diet. An issue when evaluating the microbial composition of coprolites is the degree to which the microbiome is representative of the hosts original gut community versus the changes that occur in the weeks following deposition due to desiccation. Analyses of paleontological microorganisms are also relevant in the light of recent studies linking the Late Pleistocene and Early Holocene extinctions with modern-day zoonotic pathogen outbreaks.
MethodsShotgun sequencing was performed on ancient DNA (aDNA) extracted from coprolites of the Columbian mammoth (Mammuthus Columbi), Shasta ground sloth (Nothrotheriops shastensis) and paleontological bison (Bison sp.) collected from caves on the Colorado Plateau, Southwestern USA. The novel metagenomic classifier MTSv, parameterized for studies of aDNA, was used to assign bacterial taxa to sequencing reads. The resulting bacterial community of coprolites was then compared to those from modern fecal specimens of the African savannah elephant (Loxodonta africana), the brown-throated sloth (Bradypus variegatus) and the modern bison (Bison bison). Both paleontological and modern bison fecal bacterial communities were also compared to those of progressively dried cattle feces to determine whether endogenous DNA from coprolites had a microbiome signal skewed towards aerobic microorganisms typical of desiccated fecal matter.
ResultsThe diversity of phyla identified from coprolites was lower than modern specimens. The relative abundance of Actinobacteria was increased in coprolites compared to modern specimens, with fewer Bacteroidetes and Euryarchaeota. Firmicutes had a reduced relative abundance in the mammoth and bison coprolites, compared to the African savanna elephants and modern bison. There was a significant separation of samples in NMDS plots based on their classification as either paleontological or modern, and to a lesser extent, based on the host species. Increasingly dried cattle feces formed a continuum between the modern and paleontological bison samples.
ConclusionOur results reveal that any coprolite metagenomes should always be compared to desiccated modern fecal samples from closely related hosts fed a comparable diet to determine the degree to which the coprolite metagenome is a result of desiccation versus true dissimilarities between the modern and paleontological hosts. Also, a large-scale desiccation study including a variety of modern species may shed light on life-history traits of extinct species without close extant relatives, by establishing the proximity of coprolite metagenomes with those from dried modern samples. | paleontology |
Highly efficient healing of critical sized articular cartilage defect in situ using a chemically nucleoside-modified mRNA-enhanced cell therapy Critical sized cartilage defects heal poorly and MSC-based therapies holds promise functional cartilage regeneration either used alone or in combination with growth factors. However, Recombinant protein growth factors were proven to have minimal benefits while to have adverse side effects and high cost. Nonviral mRNA delivery provides a promising, alternative approach to delivering therapeutic proteins within defect lesion for an extended period of time. Despite successful therapeutic outcome in bone and other vascularized tissues, the therapeutic application of mRNA in poorly vascularized tissues such as cartilage is still facing many challenges and rarely studied. We report here using chemically modified messenger RNA encoding TGF-{beta}3(TGF-{beta}3 cmRNA) to enhance the therapeutic efficacy of BMSCs to efficient repair of cartilage defect. Local administration of TGF-{beta}3 cmRNA enhanced BMSCs therapy restored critical-sized cartilage defects in situ in a rat model within 6 weeks with structural and molecular markers similar to its nature counterparts. In addition, the development of osteoarthritis caused by cartilage damage was prevented by this mRNA-enhanced BMSCs therapy evidenced by minimal late-stage OA pharmacological presentations. This novel mRNA enhanced-MSC technology extend the development of new therapeutic approaches for treating functional cartilage repair. | synthetic biology |
Developmental Morphogens Direct Human Induced Pluripotent Stem Cells Towards an Annulus Fibrosus-like Cell Phenotype Therapeutic interventions for intervertebral disc herniation remain scarce due to the inability of endogenous annulus fibrosus (AF) cells to respond to injury and drive tissue regeneration. Unlike other orthopaedic tissues, such as cartilage, delivery of exogenous cells to the site of annular injury remains underdeveloped, largely due to a lack of an ideal cell source and the invasive nature of cell isolation. Human induced pluripotent stem cells (iPSCs) can be differentiated to specific cell fates using biochemical factors and are, therefore, an invaluable tool for cell therapy approaches. While differentiation protocols have been developed for cartilage and fibrous connective tissues (e.g., tendon), the signals that regulate the induction and differentiation of human iPSCs towards the annulus fibrosus fate remain unknown. Here, we screened a number of candidate factors (and their combinations) and assessed the transcriptomic signatures of key signaling factors involved in embryonic AF development and differentiated function. The transcriptional signatures of treated cells were compared to those of mature human AF cells, and conditions that promoted expression of annulus fibrosus extracellular matrix genes and key transcription factors involved in embryonic AF development were identified. These findings represent an initial approach to guide human induced pluripotent stem cells towards an annulus fibrosus-like fate for cellular delivery strategies. | cell biology |
Modulation of Tau Tubulin Kinases (TTBK1 and TTBK2) Impacts Ciliogenesis Tau tubulin kinase 1 and 2 (TTBK1 and TTBK2) are highly homologous kinases that are expressed and mediate disease-relevant pathways predominantly in the brain. Distinct roles for TTBK1 and TTBK2 have been delineated. While efforts have been devoted to characterizing the impact to TTBK1 inhibition in diseases like Alzheimers disease and amyotrophic lateral sclerosis, TTBK2 inhibition has been less explored. TTBK2 serves a critical function during cilia assembly. Given the biological importance of these kinases, we designed a targeted library from which we identified several chemical tools that engage TTBK1 and TTBK2 in cells and inhibit their downstream signaling. Indolyl pyrimidinamine 10 significantly reduced the expression of primary cilia on the surface of human induced pluripotent stem cells (iPSCs). Furthermore, analog 10 phenocopies TTBK2 KO in iPSCs, confirming an essential role for TTBK2 in ciliogenesis. | cell biology |
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