Biology subjects

Kumar, M.

Publications and source records attributed to Kumar, M..

161 records · Page 9Linked to original sources

bioRxiv2020

Human Telomerase Expression is under Direct Transcriptional Control of the Telomere-binding-factor TRF2

Tight regulatory mechanisms to maintain repression of human Telomerase (hTERT), the sole protein that synthesizes telomeres, is crucial for normal adult somatic cells. In contrast, enhanced telomerase activity and resulting pathological maintenance of telomeres, is widely understood as causal in >90% of human cancers. These implicate underlying mechanisms connecting hTERT regulation and telomeres, possibly through telomeric proteins, that remain unclear. In light of of recent work by us and others showing non-telomeric function of the telomere-binding protein TRF2, here we examined whether and how TRF2 affected hTERT regulation. Direct binding of TRF2 - spanning [~]450 bp of the hTERT promoter from the Transcriptional Start Site (TSS) - led to TRF2-dependent recruitment of the polycomb repressor complex PRC2 in both normal and cancer cells. This induced repressor histone modifications resulting in TRF2-dependent hTERT repression. Mutations in the hTERT promoter, found frequently in aggressive glioblastoma and reported to destabilize the G-quadruplex structure, resulted in loss of TRF2 binding and consequent hTERT over-expression. Conversely, using G-quadruplex-stabilizing ligands we regained TRF2 binding, hTERT re-suppression, in highly proliferating glioblastoma cells with telomerase hyperactivation due to hTERT promoter mutations. Together, results herein demonstrate direct control of hTERT through TRF2 in a G-quadruplex-dependent manner - implicating mechanisms of how telomerase regulation might be linked to telomeres in normal and cancer cells.

cancer biology↗

bioRxiv2019

Differential propagation of ripples along the proximo-distal and septo-temporalaxes of dorsal CA1 of rats

The functional connectivity of the hippocampus with its primary cortical input, the entorhinal cortex, is organized topographically. In area CA1 of the hippocampus, this leads to different functional gradients along the proximo-distal and septo-temporal axes of spatial/sensory responsivity and spatial resolution respectively. CA1 ripples, a network phenomenon, allows us to test whether the hippocampal neural network shows corresponding gradients in functional connectivity along the two axes. We studied the occurrence and propagation of ripples across the entire proximo-distal axis along with a comparable spatial range of the septo-temporal axis of dorsal CA1. We observed that ripples could occur at any location, but their probability of co-occurrence and amplitude decreased with increasing distance from the reference tetrode. This reduction was greater along the proximo-distal axis than the septo-temporal axis. Furthermore, we found that ripples propagate primarily along the proximo-distal axis. Thus, over a spatial scale of ~1.5 mm, the network is anisotropic along the two axes, complementing the topographically organized cortico-hippocampal connections.

neuroscience↗

bioRxiv2019

Messenger RNAs with large number of upstream ORFs are translated via leaky scanning and reinitiation in the asexual stages of Plasmodium falciparum

The genome of Plasmodium falciparum has one of the most skewed base pair compositions of any eukaryote, with an AT content of 80-90%. As start and stop codons are AT-rich, the probability of finding upstream open reading frames (uORFs) in messenger RNAs (mRNAs) is high and parasite mRNAs have an average of 10 uORFs in their leader sequences. Similar to other eukaryotes, uORFs repress the translation of the downstream gene (dORF) in P. falciparum, yet the parasite translation machinery is able to bypass these uORFs and reach the dORF to initiate translation. This can happen by leaky scanning and/or reinitiation.\n\nIn this report, we assessed leaky scanning and reinitiation by studying the effect of uORFs on the translation of a dORF, in this case the luciferase reporter gene, and showed that both mechanisms are employed in the asexual blood stages of P. falciparum. Furthermore, in addition to codon usage of the uORF, translation of the dORF is governed by the Kozak sequence and length of the uORF, and inter-cistronic distance between the uORF and dORF. Based on these features whole genome data was analyzed to uncover classes of genes that might be regulated by uORFs. This study indicates that leaky scanning and reinitiation appear to be widespread in asexual stages of P. falciparum, which may require modifications of existing factors that are involved in translation initiation in addition to novel, parasite-specific proteins.

molecular biology↗

bioRxiv2019

Proteome-wide characterization and biomarker identification of intracranial aneurysm

The scientific basis of intracranial aneurysm (IA) formation, its rupture and further development of cerebral vasospasm remains incompletely understood. Deciphering the molecular mechanisms underlying these events will lead to identification of early detection biomarkers and in turn, improved treatment outcomes. Aberrant protein expression may drive structural alterations of vasculature found in IA. To unravel these aberrations, we performed untargeted, global, quantitative proteomic analysis of aneurysm tissue and serum from patients with IA. Samples were derived from patients of three clinical sub groups- 1) unruptured aneurysm 2) ruptured aneurysm without vasospasm 3) ruptured aneurysm who developed vasospasm. A total of 937 and 294 proteins were identified in aneurysm tissue and serum samples respectively. Several proteins that are known to maintain the structural integrity of vasculature were found to be dysregulated. ORM1, a glycoprotein, was significantly upregulated in both the aneurysm tissue and serum samples of unruptured IA patients. We employed a larger cohort of patients and validated ORM1 as a potential biomarker for screening of unruptured aneurysm using ELISA. Samples from ruptured aneurysm with vasospasm showed significant upregulation of MMP9 as compared to ruptured aneurysm without vasospasm. Using a cohort of ruptured aneurysm patients with and without vasospasm, we validated MMP9 as a potential biomarker for vasospasm. This study reveals pathophysiology underlying different clinical sub groups of IA and also suggests potential biomarkers.

neuroscience↗

bioRxiv2019

A Pan-Cancer assessment of alterations of ULK1 kinase, an upstream regulator of autophagy

Autophagy is a key clearance process to recycle damaged cellular components. One important upstream regulator of autophagy is the ULK1 kinase. Several structures of the ULK1 kinase domain have been solved, but a comprehensive study, including molecular dynamics, is missing. Also, an exhaustive description of ULK1 alterations found in cancer samples is presently lacking. We here applied a framework which links -omics data to structural protein ensembles to study ULK1 alterations from genomics data available for more that 30 cancer types. Moreover, we predicted the effects of mutations on ULK1 function and structural stability, accounting for protein dynamics and the different layers of changes that a mutation can induce in a protein at the functional and structural level. We find that ULK1 is down-regulated in gynecological tumors. In other cancer types, ULK2 could compensate for ULK1 downregulation and, in the majority of the cases, no marked changes in expression have been found. 36 missense mutations of ULK1 are co-occurring with mutations in a large number of ULK1 interactors, suggesting a pronounced effect of the upstream steps of autophagy in many cancer types. Moreover, our results pinpoint that more than 50% of the ULK1 mutations that we studied are predicted to affect protein stability. Three mutations (S184F, D102N, and A28V) are predicted with only impact on kinase activity, either modifying the functional dynamics or the capability to exert effects from distal site to the functional and catalytic regions. The framework here applied could be extended to other protein targets to aid the classification of missense mutations from cancer genomics studies, as well as to prioritize variants for experimental validation, or to select the appropriate biological readouts for experiments.

bioinformatics↗

bioRxiv2019

The conformational and mutational landscape of the ubiquitin-like marker for the autophagosome formation in cancer

Autophagy is a cellular process to recycle damaged cellular components and its modulation can be exploited for disease treatments. A key autophagy player is a ubiquitin-like protein, LC3B. Compelling evidence attests the role of autophagy and LC3B in different cancer types. Many LC3B structures have been solved, but a comprehensive study, including dynamics, has not been yet undertaken. To address this knowledge gap, we assessed ten physical models for molecular dynamics for their capabilities to describe the structural ensemble of LC3B in solution using different metrics and comparison with NMR data. With the resulting LC3B ensembles, we characterized the impact of 26 missense mutations from Pan-Cancer studies with different approaches. Our findings shed light on driver or neutral mutations in LC3B, providing an atlas of its modifications in cancer. Our framework could be used to assess the pathogenicity of mutations by accounting for the different aspects of protein structure and function altered by mutational events.

cancer biology↗

bioRxiv2019

De novo assembly and annotation of Asiatic lion (Panthera leo persica) genome

We report the first draft of the whole genome assembly of a male Asiatic lion, Atul and whole transcriptomes of five Asiatic lion individuals. Evaluation of genetic diversity placed the Asiatic lion in the lowest bracket of genomic diversity index highlighting the gravity of its conservation status. Comparative analysis with other felids and mammalian genomes unraveled the evolutionary history of Asiatic lion and its position among other felids. The genome is estimated to be 2.3 Gb (Gigabase) long with 62X sequence coverage and is found to have 20,543 protein-coding genes. About 2.66% of the genome is covered by simple sequence repeats (SSRs) and 0.4% is estimated to have segmental duplications. Comparison with seven well annotated genomes indicates the presence of 6,295 single copy orthologs, 4 co-orthologs, 21 paralogs uniquely present in Asiatic lion and 8,024 other orthologs. Assessment of male and female transcriptomes gave a list of genes specifically expressed in the male. Our genomic analyses provide candidates for phenotypes characteristic to felids and lion, inviting further confirmation of their contribution through population genetic studies. An Asiatic lion-specific expansion is detected in the Cysteine Dioxygenase-I (CDO-I) family that is responsible for taurine biosynthesis in cats. Wilms tumor-associated protein (WT1) family, a non-Y chromosome genetic factor underlying male-sex determination and differentiation is found to have undergone expansion, interestingly like that of the human genome. Another protein family, translation machinery-associated protein 7 (TMA7) that has undergone expansion in humans, also expanded in Asiatic lion and can be further investigated as a candidate responsible for mane in lions because of its role in hair follicle morphogenesis.

genomics↗

bioRxiv2019

Comparison of expression profiling through microarray and RNA-seq analysis for Nipah virus

The Nipah virus is responsible various outbreaks among countries of south east Asia, most recent is in Kerala, India. It is considered to be highly contagious and having a range of vectors for transmission. The condition worsens due to the lack of effective inhibitors. This study is first study, which focused to detect the differentially expressed genes among two different NiV studies from 2012 and 2017. The transcriptomic profiling data were retrieved from the sequence archives. The multivariate gene enrichment analyses were performed on the log transformed data from them using pathway, gene ontology, disease, reactome, etc. The comparison study suggests that the down regulated differentially expressed genes are common among them as compared to up regulated ones with statistical significance. However, among the diseased category the upregulated genes are mostly from metabolic pathways and diseased category like metabolic pathways, heart failure, cholesterol metabolism while the downregulated genes linked to various cancers, and viral diseases like hepatitis, dengue, influenza, etc. We found various small molecules mapped in the pathways which are differentially expressed among the studies, which could be targeted so as to control the Nipah infection. In order to design the inhibitors, our study would be useful to extract the effective and broad-spectrum drug targets.

bioinformatics↗

bioRxiv2018

Activation of Multiple Signalling Pathways by P152Lp53 Mutant Reveals New Gain-of-function Implicating Tumorigenesis

TP53 is the most frequently mutated tumor suppressor gene in context of all varieties of cancer, yet biochemical characterization of several reported mutations with probable biological significance have not been accomplished. We have identified a relatively rare proline to leucine mutation (P152L) in p53 in an Indian oral cancer patient sample at the fade end of its DNA binding domain (DBD). Although P152Lp53 DBD potentially binds to DNA, the full length protein is completely devoid of DNA binding ability at its cognate site. Interestingly, P152Lp53 can efficiently tetramerize. Significantly, this mutant when expressed in p53 null cell line, was found to induce cell mobility, proliferation, and invasion as compared to vector control. Also, enhanced tumorigenic potential was observed when cells expressing P152Lp53 were xenografted into nude mice, the mechanistic details of which were also investigated upon where several of the pathways were found to be upregulated such as Cell-Cell/Cell-ECM signalling, EGFR signalling, Rho-GTPase signalling. Taken together, this study establishes P152Lp53 as a new gain of function mutant.

cancer biology↗

bioRxiv2018

Andaman mangrove sediments: source of nutrients and sink of heavy metals

Andaman Islands (AI) of India is a biodiversity hotspot of mangroves but biogeochemical dynamics of AI is less understood. We collected sediment samples of four AI mangrove sites and one site without mangroves for nutrients and trace metal analysis. Samples were collected from each site at the inlet of seawater (Zone A) and the other 500m into the mangrove creek (Zone B). Nutrients (sulphate, ammonium, nitrite and nitrate) levels, organic matter (OM) and carbon content were higher at Zone B of mangrove ecosystem due to the higher OM content from mangrove leaf litter decomposition and microbial degradation. Metal (Pb &Cd) content of zones with and without mangroves were similar and Igeo values indicated moderate contamination of mangrove zones of AI due to lack of anthropogenic pollution. Our results suggest mangrove ecosystems of AI are uncontaminated from heavy metals and are source of nutrients to the oligotrophic coastal ecosystems of Andaman Sea.

ecology↗

bioRxiv2018

Conserved SQ and QS motifs in bacterial effectors suggest pathogen interplay with the ATM kinase family during infection

Understanding how bacteria hijack eukaryotic cells during infection is vital to develop better strategies to counter the pathologies that they cause. ATM kinase family members phosphorylate eukaryotic protein substrates on Ser or Thr residues followed by Gln. The kinases are active under oxidative stress conditions and/or the presence of ds-DNA breaks. While examining the protein sequences of well-known bacterial effector proteins such as CagA and Tir, we noticed that they often show conserved (S/TQ) motifs, even though the evidence for effector phosphorylation by ATM has not been reported. We undertook a bioinformatics analysis to examine effectors for their potential to mimic the eukaryotic substrates of the ATM kinase. The candidates we found could interfere with the hosts intracellular signaling network upon interaction, which might give an advantage to the pathogen inside the host. Further, the putative phosphorylation sites should be accessible, conserved across species and, in the vicinity to the phosphorylation sites, positively charged residues should be depleted. We also noticed that the reverse motif (QT/S) is often also conserved and located close to (S/TQ) sites, indicating its potential biological role in ATM kinase function. Our findings could suggest a mechanism of infection whereby many pathogens inactivate/modulate the host ATM signaling pathway.

bioinformatics↗

bioRxiv2017

BMP signalling is critical for maintaining homeostasis of hair follicles and intestine in adult mice

BMP signalling play critical roles during embryonic development, however, its roles have not been extensively studied in the maintenance of adult tissue homeostasis. In this study we have used temporal knock out of Bmp2 and Bmp4 to uncover the role of BMP signalling in adult mice. We observed rapid changes in the adult hair follicles and the intestine within two days of depleting BMP signalling, which demonstrates the critical and acute requirement of BMP signalling in maintaining homeostasis in these tissues. In addition, our study demonstrates that while BMP signalling is required for maintenance of quiescence in telogen hair follicles, it is needed for survival and proliferation in late anagen hair follicles. Our study also reveals differential requirement of BMP signalling in differentiation of distinct intestinal cell types. In addition, Loss of BMP signalling rapidly promotes early neoplastic transformations in mouse intestine.

developmental biology↗

bioRxiv2017

MS Western, a method of multiplexed absolute protein quantification is a practical alternative to western blotting

Absolute quantification of proteins elucidates the molecular composition, regulation and dynamics of multiprotein assemblies and networks. Here we report on a method termed MS Western that accurately determines the molar abundance of dozens of user-selected proteins at the sub-femtomole level in whole cell or tissue lysates without metabolic or chemical labelling and without using specific antibodies. MS Western relies upon GeLC-MS/MS and quantifies proteins by in-gel co-digestion with an isotopically labelled QconCAT protein chimera composed of concatenated proteotypic peptides. It requires no purification of the chimera and relates the molar abundance of all proteotypic peptides to a single reference protein. In comparative experiments, MS Western outperformed immunofluorescence Western blotting by the protein detection specificity, linear dynamic range and sensitivity of protein quantification. To validate MS Western in an in vivo experiment, we quantified the molar content of zebrafish core histones H2A, H2B, H3 and H4 during ten stages of early embryogenesis. Accurate quantification (CV<10%) corroborated the anticipated histones equimolar stoichiometry and revealed an unexpected trend in their total abundance.

biochemistry↗

bioRxiv2017

A high-throughput assay to identify robust inhibitors of dynamin GTPase activity

Clathrin-mediated endocytosis is the major pathway by which cells internalize materials from the external environment. Dynamin, a large multidomain GTPase, is a key regulator of clathrin-mediated endocytosis. It assembles at the necks of invaginated clathrin-coated pits and, through GTP hydrolysis, catalyzes scission and release of clathrin-coated vesicles from the plasma membrane. Several small molecule inhibitors of dynamins GTPase activity, such as Dynasore and Dyngo-4a, are currently available, although their specificity has been brought into question. Previous screens for these inhibitors measured dynamins stimulated GTPase activity due to lack of sufficient sensitivity, hence the mechanisms by which they inhibit dynamin are uncertain. We report a highly sensitive fluorescence-based assay capable of detecting dynamins basal GTPase activity under conditions compatible with high throughput screening. Utilizing this optimized assay, we conducted a pilot screen of 8000 compounds and identified several \"hits\" that inhibit the basal GTPase activity of dynamin-1. Subsequent dose-response curves were used to validate the activity of these compounds. Interestingly, we found neither Dynasore nor Dyngo-4a inhibited dynamins basal GTPase activity, although both inhibit assembly-stimulated GTPase activity. This assay provides the basis for a more extensive search for robust dynamin inhibitors.

biochemistry↗

bioRxiv2017

Competition between histone and transcription factor binding regulates the onset of transcription in zebrafish embryos

SUMMARYUpon fertilization, the genome of animal embryos remains transcriptionally inactive until the maternal-to-zygotic transition. At this time, the embryo takes control of its development and transcription begins. How the onset of zygotic transcription is regulated remains unclear. Here, we show that a dynamic competition for DNA binding between nucleosome-forming histones and transcription factors regulates zebrafish genome activation. Taking a quantitative approach, we found that the concentration of non-DNA bound core histones sets the time for the onset of transcription. The reduction in nuclear histone concentration that coincides with genome activation does not affect nucleosome density on DNA, but allows transcription factors to compete successfully for DNA binding. In agreement with this, transcription factor binding is sensitive to histone levels and the concentration of transcription factors also affects the time of transcription. Our results demonstrate that the relative levels of histones and transcription factors regulate the onset of transcription in the embryo.

developmental biology↗

bioRxiv2017

Towards the human cellular microRNAome

microRNAs are short RNAs that serve as master regulators of gene expression and are essential components of normal development as well as modulators of disease. MicroRNAs generally act cell autonomously and thus their localization to specific cell types is needed to guide our understanding of microRNA activity. Current tissue-level data has caused considerable confusion and comprehensive cell-level data does not yet exist. Here we establish the landscape of human cell-specific microRNA expression. This project evaluated 8 billion small RNA-seq reads from 46 primary cell types, 42 cancer or immortalized cell lines, and 26 tissues. It identified both specific and ubiquitous patterns of expression that strongly correlate with adjacent super-enhancer activity. Analysis of unaligned RNA reads uncovered 207 unknown minor strand (passenger) microRNAs of known microRNA loci and 2,632 novel putative microRNA loci. Although cancer cell lines generally recapitulated the expression patterns of matched primary cells, their isomiR sequence families exhibited increased disorder suggesting Drosha and Dicer-dependent microRNA processing variability. Cell-specific patterns of microRNA expression were used to deconvolute variable cellular composition of adipose tissue samples highlighting one use of this cell-specific microRNA expression data. Characterization of cellular microRNA expression across a wide variety of cell types provides a new understanding of this critical regulatory RNA species.

genomics↗

bioRxiv2016

High-spatial-resolution transcriptome profiling reveals uncharacterized regulatory complexity underlying cambial growth and wood formation in Populus tremula

Trees represent the largest terrestrial carbon sink and a renewable source of ligno-cellulose. There is significant scope for yield and quality improvement in these largely undomesticated species, and efforts to engineer new, elite varieties will benefit from an improved understanding of the transcriptional network underlying cambial growth and wood formation. We generated high-spatial-resolution RNA Sequencing data spanning the secondary phloem, vascular cambium and wood forming tissues. The transcriptome comprised 28,294 expressed, previously annotated genes, 78 novel protein-coding genes and 567 long intergenic non-coding RNAs. Most paralogs originating from the Salicaceae whole genome duplication were found to have diverged expression, with the notable exception of those with high expression during secondary cell wall deposition. Co-expression network analysis revealed that the regulation of the transcriptome underlying cambial growth and wood formation comprises numerous modules forming a continuum of active processes across the tissues. The high spatial resolution enabled identification of novel roles for characterised genes involved in xylan and cellulose biosynthesis, regulators of xylem vessel and fiber differentiation and lignification. The associated web resource (AspWood, http://aspwood.popgenie.org) integrates the data within a set of interactive tools for exploring the expression profiles and co-expression network.

plant biology↗