Biology subjects

Sharma, V.

Publications and source records attributed to Sharma, V..

123 records · Page 7Linked to original sources

bioRxiv2019

Convergent neural representations of acute nociceptive pain in healthy volunteers: A large-scale fMRI meta-analysis

Characterizing a reliable, pain-related neural signature is critical for translational applications. Many prior fMRI studies have examined acute pain-related brain activation in healthy participants. However, synthesizing these data to identify convergent patterns of activation can be challenging due to the heterogeneity of experimental designs and samples. To address this challenge, we conducted a comprehensive meta-analysis of fMRI studies of stimulus-induced pain in healthy participants. Following pre-registration, two independent reviewers evaluated 4,927 abstracts returned from a search of 8 databases, with 222 fMRI experiments meeting inclusion criteria. We analyzed these experiments using Activation Likelihood Estimation with rigorous type I error control (voxel height p < 0.001, cluster p < 0.05 FWE-corrected) and found a convergent, largely bilateral pattern of pain-related activation in the secondary somatosensory cortex, insula, midcingulate cortex, and thalamus. Notably, these regions were consistently recruited regardless of stimulation technique, location of induction, and participant sex. These findings suggest a highly-conserved core set of pain-related brain areas, encouraging applications as a biomarker for novel therapeutics targeting acute pain.\n\nHIGHLIGHTSO_LIPain stimulation recruits a core set of pain-related brain regions.\nC_LIO_LIThis core set includes thalamus, SII, insula and mid-cingulate cortex.\nC_LIO_LIThese regions were recruited regardless of stimulus modality and stimulus location.\nC_LI

neuroscience↗

bioRxiv2019

Protein ubiquitylation is essential for the schizont to merozoite transition in Plasmodium falciparum blood-stage development

Ubiquitylation is a common post translational modification of eukaryotic proteins and in the human malaria parasite, Plasmodium falciparum (Pf) overall ubiquitylation increases in the transition from intracellular schizont to extracellular merozoite stages in the asexual blood stage cycle. Here, we identify specific ubiquitylation sites of protein substrates in three intracellular parasite stages and extracellular merozoites; a total of 1464 sites in 546 proteins were identified (data available via ProteomeXchange with identifier PXD014998). 469 ubiquitylated proteins were identified in merozoites compared with only 160 in the preceding intracellular schizont stage, indicating a large increase in protein ubiquitylation associated with merozoite maturation. Following merozoite invasion of erythrocytes, few ubiquitylated proteins were detected in the first intracellular ring stage but as parasites matured through trophozoite to schizont stages the extent of ubiquitylation increased. We identified commonly used ubiquitylation motifs and groups of ubiquitylated proteins in specific areas of cellular function, for example merozoite pellicle proteins involved in erythrocyte invasion, exported proteins, and histones. To investigate the importance of ubiquitylation we screened ubiquitin pathway inhibitors in a parasite growth assay and identified the ubiquitin activating enzyme (UBA1 or E1) inhibitor MLN7243 (TAK-243) to be particularly effective. This small molecule was shown to be a potent inhibitor of recombinant PfUBA1, and a structural homology model of MLN7243 bound to the parasite enzyme highlights avenues for the development of P. falciparum specific inhibitors. We created a genetically modified parasite with a rapamycin-inducible functional deletion of uba1; addition of either MLN7243 or rapamycin to the recombinant parasite line resulted in the same phenotype, with parasite development blocked at the late schizont stage. These results indicate that the intracellular target of MLN7243 is UBA1, and this activity is essential for the final differentiation of schizonts to merozoites. The ubiquitylation of many merozoite proteins and their disappearance in ring stages are consistent with the idea that ubiquitylation leads to their destruction via the proteasome once their function is complete following invasion, which would allow amino acid recycling in the period prior to the parasites elaboration of a new food vacuole.

microbiology↗

bioRxiv2019

Interspecies transcriptome analyses identify genes that control the development and evolution of limb skeletal proportion

Despite the great diversity of vertebrate limb proportion and our deep understanding of the genetic mechanisms that drive skeletal elongation, little is known about how individual bones reach different lengths in any species. Here, we directly compare the transcriptomes of homologous growth cartilages of the mouse (Mus musculus) and bipedal jerboa (Jaculus jaculus), which has extremely long metatarsals of the feet and mouse-like arms. When we intersected gene expression differences in metatarsals of the two species with expression differences in forearms, we found that about 10% of all orthologous genes are associated with disproportionate elongation of jerboa feet. Among these, Shox2, has gained expression in jerboa metatarsals where it is not expressed in other vertebrates that have been assessed. This transcription factor is necessary for proximal limb elongation, and we show that it is sufficient to increase mouse distal limb length. Unexpectedly, we also found evidence that jerboa foot elongation occurs in part by releasing latent growth potential that is repressed in mouse feet. In jerboa metatarsals, we observed higher expression of Crabp1, an antagonist of growth inhibitory retinoic acid, lower expression of Gdf10, an inhibitory TGF{beta} ligand, and lower expression of Mab21L2, a BMP signaling inhibitor that we show is sufficient to reduce limb bone elongation. By intersecting our data with prior expression analyses in other systems, we identify mechanisms that may both establish limb proportion during development and diversify proportion during evolution. The genes we identified here therefore provide a framework to understand the modular genetic control of skeletal growth and the remarkable malleability of vertebrate limb proportion.

developmental biology↗

bioRxiv2019

Rhes, a Striatal-Enriched Protein, Promotes Mitophagy Via Nix.

Elimination of dysfunctional mitochondria via mitophagy is essential for cell survival and neuronal functions. But, how impaired mitophagy participates in tissue-specific vulnerability in the brain remains unclear. Here we discovered that Rhes, a striatal-enriched protein, is a major regulator of mitophagy in the striatum. Rhes predominantly interact with dysfunctional mitochondria and degrades them via mitophagy, and this function is exacerbated by the striatal toxin, 3-nitropropionic acid (3-NP). 3-NP induces mitochondrial swelling, loss of cristae and neuronal cell death only in WT but not Rhes KO striatum. Mechanistically, Rhes disrupts the mitochondrial membrane potential ({Delta}{Psi}m) and interacts with mitophagy receptor, Nix. In Nix KO cells, Rhes fails to disrupt {Delta}{Psi}m or eliminate dysfunctional mitochondria. Moreover, Rhes travels to the neighboring cell and associates with dysfunctional mitochondria via Nix. Collectively, Rhes is a major regulator of mitophagy via Nix which may determine striatal vulnerability in the brain.

neuroscience↗

bioRxiv2019

Comparative analysis of ChIP-exo peak-callers: impact of data quality, read duplication and binding subtypes

BackgroundChIP (Chromatin immunoprecipitation)-exo has emerged as an important and versatile improvement over conventional ChIP-seq as it reduces the level of noise, maps the transcription factor (TF) binding location in a very precise manner, upto single base-pair resolution, and enables binding mode prediction. Availability of numerous peak-callers for analyzing ChIP-exo reads has motivated the need to assess their performance and report which tool executes reasonably well for the task.\n\nResultsThis study has focussed on comparing peak-callers that report direct binding events with those that report indirect binding events. The effect of strandedness of reads and duplication of data on the performance of peak-callers has been investigated. The number of peaks reported by each peak-caller is compared followed by a comparison of the annotated motifs present in the reported peaks. The significance of peaks is assessed based on the presence of a motif in top peaks. Indirect binding tools have been compared on the basis of their ability to identify annotated motifs and predict mode of protein-DNA interaction.\n\nConclusionBy studying the output of the peak-callers investigated in this study, it is concluded that the tools that use self-learning algorithms, i.e. the tools that estimate all the essential parameters from the aligned reads, perform better than the algorithms which require formation of peak-pairs. The latest tools that account for indirect binding of TFs appear to be an upgrade over the available tools, as they are able to reveal valuable information about the mode of binding in addition to direct binding. Furthermore, the quality of ChIP-exo reads have important consequences on the output of data analysis.

bioinformatics↗

bioRxiv2019

Gene expression-based identification of antigen-responsive CD8+ T cells on a single-cell level

CD8+ T cells are important effectors of adaptive immunity against pathogens, tumors and self antigens. Here, we asked how human cognate antigen-responsive CD8+ T cells and their receptors could be identified in unselected single-cell gene expression data. Single-cell RNA sequencing and qPCR of dye-labelled antigen-specific cells identified large gene sets that were congruently up- or downregulated in virus-responsive CD8+ T cells under different antigen presentation conditions. Combined expression of TNFRSF9, XCL1, XCL2, and CRTAM was the most distinct marker of virus-responsive cells on a single-cell level. Using transcriptomic data, we developed a machine learning-based classifier that provides sensitive and specific detection of virus-responsive CD8+ T cells from unselected populations. Gene response profiles of CD8+ T cells specific for the autoantigen islet-specific glucose-6-phosphatase catalytic subunit-related protein differed markedly from virus-specific cells. These findings provide single-cell gene expression parameters for comprehensive identification of rare antigen-responsive cells and T cell receptors.\n\nOne-sentence summaryIdentification of genes, gene sets, and development of a machine learning-based classifier that distinguishes antigen-responsive CD8+ T cells on a single-cell level.

immunology↗

bioRxiv2019

An unusual amino acid substitution within the hummingbird cytochrome c oxidase alters a key proton-conducting channel

Hummingbirds in flight exhibit the highest metabolic rate of all vertebrates. The bioenergetic requirements associated with sustained hovering flight raise the possibility of unique amino acid substitutions that would enhance aerobic metabolism. Here, we have identified a non-conservative substitution within the mitochondria-encoded cytochrome c oxidase subunit I (COI) that is fixed within hummingbirds, but not among other vertebrates. This unusual change is also rare among metazoans, but can be identified in several clades with diverse life histories. We performed atomistic molecular dynamics simulations using bovine and hummingbird COI models, thereby bypassing experimental limitations imposed by the inability to modify mtDNA in a site-specific manner. Intriguingly, our findings suggest that COI amino acid position 153 (bovine numbering system) provides control over the hydration and activity of a key proton channel in COX. We discuss potential phenotypic outcomes linked to this alteration encoded by the hummingbird mitochondrial genome.

evolutionary biology↗

bioRxiv2019

piNET: a versatile web platform for downstream analysis and visualization of proteomics data

Large proteomics data, including those generated by mass spectrometry, are being generated to characterize biological systems at the protein level. Computational methods and tools to identify and quantify peptides, proteins and post-translational modifications (PTMs) that are captured in modern mass spectrometers have matured over the years. On the other hand, tools for downstream analysis, interpretation and visualization of proteomics data sets, in particular those involving PTMs, require further improvement and integration to accelerate scientific discovery and maximize the impact of proteomics studies by connecting them better with biological knowledge across not only proteomics, but also other Omics domains. With the goal of addressing these challenges, the piNET server has been developed as a versatile web platform to facilitate mapping, annotation, analysis and visualization of peptide, PTM, and protein level quantitative data generated by either targeted, shotgun or other proteomics approaches. Building on our experience with large scale analysis of gene and protein expression profiles as part of the Library of Integrated Network Cellular Signatures (LINCS) project, piNET has been designed as a fast, versatile and easy to use web-based tool with three modules that provide mapping from peptides (with PTMs) to proteins, from PTM sites to modifying enzymes that target those sites, and finally from proteins (with PTMs) to pathways, and for further mechanistic insights to LINCS signatures of chemical and genetic perturbations. piNET is freely available at http://www.pinet-server.org.

bioinformatics↗

bioRxiv2019

Retapamulin-assisted ribosome profiling reveals the alternative bacterial proteome

The use of alternative translation initiation sites enables production of more than one protein from a single gene, thereby expanding cellular proteome. Although several such examples have been serendipitously found in bacteria, genome-wide mapping of alternative translation start sites has been unattainable. We found that the antibiotic retapamulin specifically arrests initiating ribosomes at start codons of the genes. Retapamulin-enhanced Ribo-seq analysis (Ribo-RET) not only allowed mapping of conventional initiation sites at the beginning of the genes but, strikingly, it also revealed putative internal start sites in a number of Escherichia coli genes. Experiments demonstrated that the internal start codons can be recognized by the ribosomes and direct translation initiation in vitro and in vivo. Proteins, whose synthesis is initiated at an internal in-frame and out-of-frame start sites, can be functionally important and contribute to the alternative bacterial proteome. The internal start sites my also play regulatory roles in gene expression.

molecular biology↗

bioRxiv2019

Mesenchymal stem cells (MSCs) offer a drug tolerant and immune-privileged niche to Mycobacterium tuberculosis

Anti-tuberculosis (TB) drugs while being highly potent in vitro require prolonged treatment to control Mycobacterium tuberculosis (Mtb) infections in vivo. We report here, mesenchymal stem cells (MSCs) shelter Mtb to help tolerate anti-TB drugs. MSCs uptake Mtb readily and allow them grow unabated despite having functional innate pathway of phagosome maturation. Unlike macrophage-resident ones, MSC-resident Mtb tolerates anti-TB drugs remarkably well, a phenomenon requiring proteins ABCC1, ABCG2 and vacuolar-type H+ATPases. Additionally, contrary to what is classically known, IFN{gamma} and TNF aid mycobacterial growth within MSCs. Mechanistically, evading drugs and inflammatory cytokines by MSC-resident Mtb is dependent on elevated PGE2 signaling, which we subsequently verified in vivo analyzing sorted CD45-CD73+SCA1+-MSCs from the lungs of infected mice. Moreover granulomas from human pulmonary and extra-pulmonary TB show presence of MSCs co-inhabiting with Mtb. Together the results show targeting the immune-privileged niche, provided by MSCs to Mtb, can revolutionize tuberculosis prevention and cure.

immunology↗

bioRxiv2019

Genes lost during the transition from land to water in cetaceans highlight genomic changes involved in aquatic adaptations

The transition from land to water in whales and dolphins (cetaceans) was accompanied by remarkable anatomical, physiological and behavioral adaptations. To better understand the genomic changes that occurred during this transition, we systematically screened for protein-coding genes that were inactivated in the ancestral cetacean lineage. We discovered genes whose loss is likely beneficial for cetaceans by reducing the risk of thrombus formation during diving (F12, KLKB1), improving the fidelity of oxidative DNA damage repair (POLM), and protecting from oxidative stress-induced lung inflammation (MAP3K19). Additional gene losses may reflect other diving-related adaptations, such as enhanced vasoconstriction during the diving response (mediated by SLC6A18) and altered pulmonary surfactant composition (SEC14L3), while loss of SLC4A9 relates to a reduced need for saliva in aquatic environments. Finally, the complete loss of melatonin synthesis and receptor genes (AANAT, ASMT, MTNR1A/B) may have been a precondition for the evolution of unihemispheric sleep. Our findings suggest that some genes lost in the ancestral cetacean lineage may have been involved in adapting to a fully-aquatic lifestyle.

evolutionary biology↗

bioRxiv2018

Genome-scale metabolic reconstruction and metabolic versatility of an obligate methanotroph Methylococcus capsulatus str. Bath

The increase in greenhouse gases with high global warming potential such as methane is a matter of concern and requires multifaceted efforts to reduce its emission and increase its mitigation from the environment. Microbes such as methanotrophs can assist in methane mitigation. To understand the metabolic capabilities of methanotrophs, a complete genome-scale metabolic model of an obligate methanotroph, Methylococcus capsulatus str. Bath was reconstructed. The model contains 535 genes, 898 reactions and 784 unique metabolites and is named iMC535. The predictive potential of the model was validated using previously-reported experimental data. The model predicted the Entner-Duodoroff (ED) pathway to be essential for the growth of this bacterium, whereas the Embden-Meyerhof-Parnas (EMP) pathway was found non-essential. The performance of the model was simulated on various carbon and nitrogen sources and found that M. capsulatus can grow on amino acids. The analysis of network topology of the model identified that six amino acids were in the top-ranked metabolic hubs. Using flux balance analysis (FBA), 29% of the metabolic genes were predicted to be essential, and 76 double knockout combinations involving 92 unique genes were predicted to be lethal. In conclusion, we have reconstructed a genome-scale metabolic model of a unique methanotroph Methylococcus capsulatus str. Bath. The model will serve as a knowledge-base for deeper understanding, as a platform for exploring the metabolic potential, and as a tool to engineer this bacterium for methane mitigation and industrial applications.

bioengineering↗

bioRxiv2018

A study of phenotypic plasticity of Saccharomyces cerevisiae in natural grape juices shed light on allelic variation under balanced selection.

The ability of a genotype to produce different phenotypes according to its surrounding environment is known as phenotypic plasticity. Within different individuals of the same species, phenotypic plasticity can vary greatly. This contrasted response is due to allelic variations and is caused by gene-by-environment interactions (GxE). Finding the genes and the cellular functions that interact with the environment is a current challenge for better understanding the genetic bases of phenotypic plasticity. In order to study the impact of natural allelic variations having a contrasted but relevant effect in a changing environment, we investigated the phenotypic response of the wine yeast Saccharomyces cerevisiae fermented in various grape juices. In this study we implemented a QTL mapping program using two independent offspring (~100 progeny) in order to investigate the molecular basis of yeast phenotypic response in a wine fermentation context. Thanks to high throughput sequencing approaches, both populations were genotyped, providing saturated genetic maps of thousands of markers. Linkage analyses allowed the detection of 78 QTLs including 21 with significant interaction with the nature of the fermented juice or fermentation conditions. Molecular dissection of a major QTL showed that the sulfite pump Ssu1p has a pleiotropic effect and impacts the phenotypic plasticity of several traits. Both alleles have positive effect according to external condition in phenotypes related to yeast fitness suggesting an example of balanced selection. All together these results pave the way for exploiting and deciphering the genetic determinism of phenotypic plasticity.

genetics↗

bioRxiv2017

Induction of autophagy by trehalose limits opportunistic mycobacterial infections in HIV-infected macrophages

Opportunistic bacterial infections amongst HIV-infected individuals pose serious health challenge. While immediate control of bacterial pathogens is typically attributed to innate defense mechanisms, whether HIV-mediated modulation of innate mechanisms like autophagy promote opportunistic infections, remains obscure. Using U1.1 and U937 macrophages, we show, HIV activation or infection inhibits autophagy and helps survival of pathogenic Mycobacterium tuberculosis and non-pathogenic non-tuberculous mycobacterial strains (NTMs) like Mycobacterium avium complex and Mycobacterium fortuitum. HIV achieves this by blocking xenophagy flux, which could be reversed by the autophagy inducer trehalose that kills intracellular Mtb and NTMs. We found trehalose acts as a PI (3,5) P2 agonist and activates TRPML1 to induce autophagy. Remarkably, trehalose treatment significantly reduced p24 levels in PBMCs infected with clinical HIV strains and in PBMCs derived from treatment-naive HIV patients. Taken together, our study highlights the immense potential of autophagy modulators in the therapeutic intervention of HIV and associated opportunistic infection.

immunology↗

bioRxiv2017

A Library of Phosphoproteomic and Chromatin Signatures for Characterizing Cellular Responses to Drug Perturbations

Though the added value of proteomic measurements to gene expression profiling has been demonstrated, profiling of gene expression on its own remains the dominant means of understanding cellular responses to perturbation. Direct protein measurements are typically limited due to issues of cost and scale; however, the recent development of high-throughput, targeted sentinel mass spectrometry assays provides an opportunity for proteomics to contribute at a meaningful scale in high-value areas for drug development. To demonstrate the feasibility of a systematic and comprehensive library of perturbational proteomic signatures, we profiled 90 drugs (in triplicate) in six cell lines using two different proteomic assays -- one measuring global changes of epigenetic marks on histone proteins and another measuring a set of peptides reporting on the phosphoproteome -- for a total of more than 3,400 samples. This effort represents a first-of-its-kind resource for proteomics. The majority of tested drugs generated reproducible responses in both phosphosignaling and chromatin states, but we observed differences in the responses that were cell line-and assay-specific. We formalized the process of comparing response signatures within the data using a concept called connectivity, which enabled us to integrate data across cell types and assays. Furthermore, it facilitated incorporation of transcriptional signatures. Consistent connectivity among cell types revealed cellular responses that transcended cell-specific effects, while consistent connectivity among assays revealed unexpected associations between drugs that were confirmed by experimental follow-up. We further demonstrated how the resource could be leveraged against public domain external datasets to recognize therapeutic hypotheses that are consistent with ongoing clinical trials for the treatment of multiple myeloma and acute lymphocytic leukemia (ALL). These data are available for download via the Gene Expression Omnibus (accession GSE101406), and web apps for interacting with this resource are available at https://clue.io/proteomics.\n\nHighlightsO_LIFirst-of-its-kind public resource of proteomic responses to systematically administered perturbagens\nC_LIO_LIDirect proteomic profiling of phosphosignaling and chromatin states in cells for 90 drugs in six different cell lines\nC_LIO_LIExtends Connectivity Map concept to proteomic data for integration with transcriptional data\nC_LIO_LIEnables recognition of unexpected, cell type-specific activities and potential translational therapeutic opportunities\nC_LI

systems biology↗