Biology subjects

Singh, H.

Publications and source records attributed to Singh, H..

101 records · Page 6Linked to original sources

bioRxiv2019

Organizational properties of a functional mammalian cis-regulome

Mammalian genomic states are distinguished by their chromatin and transcription profiles. Most genomic analyses rely on chromatin profiling to infer cis-regulomes controlling distinctive cellular states. By coupling FAIRE-seq with STARR-seq and integrating Hi-C we assemble a functional cis-regulome for activated murine B-cells. Within 55,130 accessible chromatin regions we delineate 9,989 active enhancers communicating with 7,530 promoters. The cis-regulome is dominated by long range enhancer-promoter interactions (>100kb) and complex combinatorics, implying rapid evolvability. Genes with multiple enhancers display higher rates of transcription and multi-genic enhancers manifest graded levels of H3K4me1 and H3K27ac in poised and activated states, respectively. Motif analysis of pathway-specific enhancers reveals diverse transcription factor (TF) codes controlling discrete processes. The cis-regulome strikingly enriches for combinatorial DNA binding regions of lineage determining TFs. Their genomic binding patterns reveal that onset of chromatin accessibility is associated with binding of simpler combinations whereas enhancer function requires greater complexity.

systems biology↗

bioRxiv2019

Genomic programming of antigen cross-presentation in IRF4-expressing human Langerhans cells

Langerhans cells (LCs) in the epidermis present MHC I and MHC II-restricted antigens thereby priming either CD8 or CD4 T cell immune responses. The genomic programs and transcription factors regulating antigen presentation in LCs remain to be elucidated. We show human LCs are highly efficient in MHC I-antigen cross-presentation but lack the transcription factor IRF8 that is critical in dendritic cells. LC migration from the epidermis enhances their ability to cross-present antigens and is accompanied by the induction of the transcription factor IRF4, whose expression is correlated by scRNA-seq with genes involved in ubiquitin-dependent protein degradation. Chromatin profiling reveals enrichment of EICE and AICE composite DNA binding motifs in regulatory regions of antigen-presentation genes, which can be recognized by IRF4 in conjunction with PU.1 or BATF3 expressed in LCs. Thus, the genomic programming of human LCs including inducible expression of IRF4 with enhanced cross-presentation distinguishes them from conventional dendritic cells.

immunology↗

bioRxiv2019

HtrA1 alters endothelial tube formation characteristics in an in vitro model

High temperature requirement factor A1 (HtrA1) is a serine protease of the mammalian HtrA family. It is ubiquitously expressed with high levels in the placenta. Dysregulation of HtrA1 has been linked to a number of diseases, in particular age-related macular degeneration (AMD) and preeclampsia (PE) in which HtrA1 is significantly increased. AMD is the leading cause of irreversible visual impairment in older people, affecting millions across the globe. PE is a life-threatening pregnancy complication, affecting 2-7% of pregnant women worldwide. Although AMD and PE are very different diseases, both are associated with endothelial dysfunction and dysregulation of angiogenesis. Given HtrA1 is up-regulated in both AMD and PE, in this study we examined the impact of excessive HtrA1 on capillary tube formation of HUVECs as an in vitro angiogenesis model. HtrA1 at high concentrations significantly increased the total number of tube branch points and inter-tubular loops, but considerably decreased the mean tube length, resulting in more but much smaller tubes. However, these smaller tubes were incomplete/broken. These data demonstrated that high concentrations of HtrA1 altered endothelial tube formation characteristics of HUVEVs. Our results suggest that HtrA1 over-expression in AMD and PE may directly contribute to the endothelial dysfunction in these diseases.

cell biology↗

bioRxiv2018

Biased visibility in HiC datasets marks dynamically regulated condensed and decondensed chromatin states genome-wide.

Proximity ligation based techniques, like HiC, involve restriction digestion followed by ligation of formaldehyde cross-linked chromatin. We showed that the HiC datasets have significant bias due to differential accessibility of chromatin domains to restriction endonucleases. Through analysis of lamina-associated domains (LADs), inactive X-chromosome in mammals and polytene bands in fly, we established that the DNA in condensed chromatin had lesser accessibility to nucleases used in HiC as compared to that in decondensed chromatin. The observed bias was independent of known systematic biases attributed to length and GC content of the restriction fragments and was not appropriately corrected by computational methods. We identified novel condensed domains outside LADs, which were bordered by insulators and were dynamically associated with the developmentally regulated epigenetic and transcriptional states. Our observations suggested that the corrected read counts of existing HiC datasets can be reliably repurposed to study the dynamics of chromatin condensation and decondensation and that the existing HiC datasets should be interpreted with cautions.

genomics↗

bioRxiv2018

Exponential trajectories, cell size fluctuations and the adder property in bacteria follow from simple chemical dynamics and division control

Experiments on steady state bacterial cultures have uncovered several quantitative regularities at the system level. These include, first, the exponential growth of cell size with time and the balanced growth of intracellular chemicals between cell birth and division, which are puzzling given the nonlinear and decentralized chemical dynamics in the cell. We model a cell as a set of chemical populations undergoing nonlinear mass action kinetics in a container whose volume is a linear function of the chemical populations. This turns out to be a special class of dynamical system that generically has attractors in which all populations grow exponentially with time at the same rate. This explains exponential balanced growth of bacterial cells without invoking any regulatory mechanisms and suggests that this could be a robust property of protocells as well. Second, we consider the hypothesis that cells commit themselves to division when a certain internal chemical population reaches a threshold of N molecules. We show that this hypothesis leads to a simple explanation of some of the variability observed across cells in a bacterial culture. In particular it reproduces the adder property of cell size fluctuations observed recently in E. coli, the observed correlations between interdivision time, birth volume and added volume in a generation, and the observed scale of the fluctuations (CV ~ 10-30%) when N lies between 10 and 100. Third, upon including a suitable regulatory mechanism that optimizes the growth rate of the cell, the model reproduces the observed bacterial growth laws including the dependence of the growth rate and ribosomal protein fraction on the medium. Thus, the models provide a framework for unifying diverse aspects of bacterial growth physiology under one roof. They also suggest new questions for experimental and theoretical enquiry.

systems biology↗

bioRxiv2018

Longitudinal Surveys Show that Urethral Catheters Harbor Recurrent Polymicrobial Biofilms with Cooperative and Competitive Behaviors Among Cohabitating Pathogens

The analysis of catheter biofilms (CBs) from patients with neurogenic bladder disorders revealed persistent colonization by polymicrobial communities. The recurrence of bacterial species in the CBs of sequentially replaced catheters suggests urothelial reservoirs responsible for recolonization of the catheter surface. Proteomic data for CB samples were indicative of chronic inflammation in the patients urinary tracts via neutrophil and eosinophil infiltration and epithelial cell exfoliation. These host defense pathways, effective in killing pathogens during uncomplicated urinary tract infection, failed to eliminate CBs. Intermittent antibiotic drug treatment had different outcomes: either replacement of drug-susceptible by drug-tolerant bacteria or transient microbial biomass reduction followed by resurgence of the previously colonizing bacteria. Proteins that sequester iron and zinc such as lactotransferrin, lipocalin-2 and calprotectin were abundant in the patients CBs and urine sediments. Indicative of a host-pathogen battle for bivalent metal ions, acquisition and transport systems for such ions were highly expressed by many organisms residing in CBs. Species part of the Enterococcaceae and Aerococcaceae families, generally not well-characterized in their ability to synthesize siderophores, frequently cohabitated biofilms dominated by siderophore-producing Enterobacteriaceae. In support of metal acquisition cooperativity, we noticed positive abundance correlations for a Proteus mirabilis yersiniabactin-type siderophore system and two Enterococcus faecalis ABC transporters. Distinct bacterial strains highly expressed known or putative cytotoxins that appeared to compromise the survival of co-resident bacteria, e.g. a P. mirabilis hemolysin and Pseudomonas aeruginosa type 6 secretion and pyoverdin biosynthesis systems. In conclusion, there is support for cooperative and competitive behaviors among bacteria cohabitating CBs.

microbiology↗

bioRxiv2018

Modern botanical analogue of endangered Yak (Bos mutus) dung from India: Plausible linkage with living and extinct megaherbivores

The study present to document the micro and macrobotanical remain on wild Yak dung to understand the diet, habitat, and ecology in relation to determining possible ecological relationships with extant and extinct megaherbivores. Grasses are the primary diet of the yak as indicated by the abundance of grass pollen and phytoliths, though it is obvious. The other associates non-arboreal and arboreal taxa namely, Cyperacaeae, Rosaceae, Chenopodiaceae, Artemisia, Prunus, and Rhododendron are also important dietary plants for their survival. The observation of plant macrobotanical remains especially the vegetative part and seed of the grasses and Cyperaceae also indicates good agreement with the palynodata. The documented micro and macrobotanical data is indicative of both Alpine meadow and steppe vegetation under cold and dry climate which exactly reflected the current vegetation composition and climate in the region. The recovery of Botryococcus, Arcella, and diatom was marked though in trace values and suggestive of the perennial water system in the region which incorporated through the ingestion of water. Energy dispersive spectroscopy analysis marked that the element contained in dung samples has variation in relation to the summer and winter which might be the availability of the food plants and vegetation. This generated multiproxy data serves as a strong supplementary data for modern pollen and vegetation relationship based on surface soil samples in the region. The recorded multiproxy data could be useful to interpret the coprolites of herbivorous fauna in relation to the palaeodietary and paleoecology in the region and to correlate with other mega herbivores in a global context.

paleontology↗

bioRxiv2018

DoubletDecon: Cell-State Aware Removal of Single-Cell RNA-Seq Doublets

Methods for single-cell RNA sequencing (scRNA-Seq) have greatly advanced in recent years. While droplet- and well-based methods have increased the capture frequency of cells for scRNA-Seq, these technologies readily produce technical artifacts, such as doublet-cell and multiplet-cell captures. Doublets occurring between distinct cell-types can appear as hybrid scRNA-Seq profiles, but do not have distinct transcriptomes from individual cell states. We introduce DoubletDecon, an approach that detects doublets with a combination of deconvolution analyses and the identification of unique cell-state gene expression. We demonstrate the ability of DoubletDecon to identify synthetic and cell-hashing cell singlets and doublets from scRNA-Seq datasets of varying cellular complexity. DoubletDecon is able to account for cell-cycle effects and is compatible with diverse species and unsupervised population detection algorithms (e.g., ICGS, Seurat). We believe this approach has the potential to become a standard quality control step for the accurate delineation of cell states.

bioinformatics↗

bioRxiv2017

Evaluation of protein-ligand docking methods on peptide-ligand complexes for docking small ligands to peptides

In the past, many benchmarking studies have been performed on protein-protein and protein-ligand docking however there is no study on peptide-ligand docking. In this study, we evaluated the performance of seven widely used docking methods (AutoDock, AutoDock Vina, DOCK 6, PLANTS, rDock, GEMDOCK and GOLD) on a dataset of 57 peptide-ligand complexes. Though these methods have been developed for docking ligands to proteins but we evaluate their ability to dock ligands to peptides. First, we compared TOP docking pose of these methods with original complex and achieved average RMSD from 4.74[A] for AutoDock to 12.63[A] for GEMDOCK. Next we evaluated BEST docking pose of these methods and achieved average RMSD from 3.82[A] for AutoDock to 10.83[A] for rDock. It has been observed that ranking of docking poses by these methods is not suitable for peptide-ligand docking as performance of their TOP pose is much inferior to their BEST pose. AutoDock clearly shows better performance compared to the other six docking methods based on their TOP docking poses. On the other hand, difference in performance of different docking methods (AutoDock, AutoDock Vina, PLANTS and DOCK 6) was marginal when evaluation was based on their BEST docking pose. Similar trend has been observed when performance is measured in terms of success rate at different cut-off values. In order to facilitate scientific community a web server PLDbench has been developed (http://webs.iiitd.edu.in/raghava/pldbench/).

bioinformatics↗

bioRxiv2017

Microbial Metagenome Of Urinary Tract Infection

Urine culture and microscopy techniques are used to profile the bacterial species present in urinary tract infections. To gain insight into the urinary flora in infection and health, we analyzed clinical laboratory features and the microbial metagenome of 121 clean-catch urine samples. 16S rDNA gene signatures were successfully obtained for 116 participants, while whole genome shotgun sequencing data was successfully generated for samples from 49 participants. Analysis of these datasets supports the definition of the patterns of infection and colonization/contamination. Although 16S rDNA sequencing was more sensitive, whole genome shotgun sequencing allowed for a more comprehensive and unbiased representation of the microbial flora, including eukarya and viral pathogens, and of bacterial virulence factors. Urine samples positive by whole genome shotgun sequencing contained a plethora of bacterial (median 41 genera/sample), eukarya (median 2 species/sample) and viral sequences (median 3 viruses/sample). Genomic analyses revealed cases of infection with potential pathogens (e.g., Alloscardovia sp, Actinotignum sp, Ureaplasma sp) that are often missed during routine urine culture due to species specific growth requirements. We also observed gender differences in the microbial metagenome. While conventional microbiological methods are inadequate to identify a large diversity of microbial species that are present in urine, genomic approaches appear to comprehensively and quantitatively describe the urinary microbiome.

microbiology↗

bioRxiv2016

Glyceraldehyde-3-Phosphate Dehydrogenase and Seven in Absentia Homolog -1 Interactions in Mammalian Cells Visualized Using Proximity Ligation Assay

Proteins seldom function in isolation and thus protein-protein interactions are critical in understanding the molecular basis of diseases and health (1, 2). There are several well established techniques that are used to investigate protein-protein interactions(3). Most of the methods require some form of genetic modification of the target protein and thus always adds extra steps. However, Proximity Ligation Assay(4-6) (PLA) aka Duolink(R) is one such method that requires no genetic modification of the target protein and probes protein-protein interactions in fixed live cells and tissues. Briefly, PLA requires the use of primary antibodies specific to the proteins of interest. Once the sample (fixed cells or tissues) is incubated with species specific primary antibodies, secondary antibodies that are conjugated with oligonucleotides (also known as PLUS and MINUS probes respectively) and connecter oligonucleotides are added. This complex is ligated if the two PLUS and MINUS probes are within 40nm of each other. The resulting nucleic acid is amplified using rolling circle amplification and then probed with appropriate fluorescent probes. If the two proteins are interacting, one could visualize the interaction as a single red foci (for example Far Red Detection) using fluorescent microscopy. Here, we used PLA to probe protein-protein interactions between Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and seven in absentia homolog -1 (Siah-1) - an E3 ubiquitin ligase. We first use PLA to show that GAPDH and Siah-1 proteins exist endogenously in the cytosol of multiple mammalian cell lines. Our data suggest the use of DU145 and T98G cell lines to show translocation of the GAPDH-Siah- 1 complex. Next, we used common nitrosylation agents(7, 8) (S-nitrosoglutathione-GSNO and S-Nitroso-N-acetyl-DL-pencillamine-SNAP) in different concentrations and observed that GAPDH and Siah-1 interact presumably due to the nitrosylation of the former, which is consistent with previous studies(9, 10). Interestingly, no interactions were observed between the two proteins in the absence of GSNO or SNAP indicating that nitrosylation might be critical for GAPDH-Siah1 interactions. Our results suggest that GAPDH-Siah-1 interactions originate in the cytosol and migrate to the nucleus under the conditions tested. We quantify the PLA signal using Duolink(R) Image Tool and observe a clear enhancement of GAPDH-Siah-1 PLA signal upon treating the cells with GSNO or SNAP. Next, we used R-(-)-Deprenyl (deprenyl), a known inhibitor of GAPDH4, and show that it abrogates GAPDH-Siah-1 PLA complex under the conditions tested. Finally, our data suggest that PLA can detect and quantify the GAPDH-Siah1 complex; a well-known protein-protein interaction implicated in neurodegeneration(9-11) and thus could be a method of choice for similar applications.

cell biology↗