Biology subjects

Sahu, A.

Publications and source records attributed to Sahu, A..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

Retention of ES cell-derived 129S genome drives NLRP1 hypersensitivity and transcriptional deregulation in Nlrp3-/- mice

Immune response genes are highly polymorphic in humans and mice, with heterogeneity amongst loci driving strain-specific host defense responses. The inadvertent retention of polymorphic loci can introduce confounding phenotypes, leading to erroneous conclusions, and impeding scientific advancement. In this study, we employ a combination of RNAseq and variant calling analyses and identify a substantial region of 129S genome, including the highly polymorphic Nlrp1 locus proximal to Nlrp3, in one of the most commonly used mouse models of NLRP3 deficiency. We show that increased expression of 129S NLRP1b sensitizes Nlrp3-/- macrophages to NLRP1 inflammasome activation. Furthermore, the presence of 129S genome leads to altered gene and protein regulation across multiple cell-types, including of the key tissue-resident macrophage marker, TIM4. To address the challenge of resolving NLRP3-dependent phenotypes, we introduce and validate a conditional Nlrp3 allele, enabling precise temporal and cell-type-specific control over Nlrp3 deletion. Our study establishes a generic framework to identify functionally relevant SNPs and assess genomic contamination in transgenic mice. This allows for unambiguous attribution of phenotypes to the target gene and advances the precision and reliability of research in the field of host defense responses.

immunology↗

bioRxiv2023

Ecdysone Orchestrates Notch and Broad Symphony to Craft Epithelial Cell Shape Change

Morphogenesis in the metazoans relies on cell shape transformations that forms an integral component of organ development, form generation and maintenance of tissue homeostasis. Nevertheless, a comprehensive grasp of how the epithelial morphogenesis is modulated in the metazoans remain still elusive. The Steroid hormones play a pivotal role in morphogenesis spanning several organs including the gonads, urogenital tracts, and mammary glands. Employing the Drosophila oogenesis model, we investigated the the role of steroid hormone receptor, Ecdysone receptor (EcR) involvement in transforming anterior epithelial follicle cells (AFCs) from cuboidal to squamous shape. Consistent with the fact that the activity of EcR in the AFCs coincides with the timing of cuboidal-to-squamous shape transition, we found that depletion of EcR function impedes the shape transformation of AFCs. We report that EcR doesnt impair the follicle cell fate, but impedes the morphological change by restricting the remodelling of lateral and adherens junctions. Employing the classical genetic tools and immnohistochemistry, we show that EcR limits the Notch-Broad axis to facilitate alteration of the shape of AFCs. Our study suggests a mechanistic model where Ecdysone signalling, via the Notch pathway, finetunes the activity of non-muscle myosin heavy chain zipper, prompting AFC shape transition. In sum, our work illuminates how Ecdysone signalling orchestrates epithelial follicle cell morphogenesis during metazoan development.

developmental biology↗

bioRxiv2023

Drug repurposing approach for potential Pfmrk inhibitors as antimalarial agents: an In-silico analysis

Malaria is a major global health issue due to the emergence of resistance to most of the available antimalarial drugs. There is an urgent need to discover new antimalarials to tackle the resistance issue. A CDK-like protein, Pfmrk from Plasmodium falciparum, plays a crucial role in regulating cell proliferation and shares 36.28% homology with humans CDK (hCDK7). Pfmrk complex with Pfcyc-1 and stimulates kinase activity. Also, Pfcyc-1 from P. falciparum, which has the highest sequence homology with human cyclin (Cyclin H), binds to and activates Pfmrk in a cyclin-dependent way. This is the first indication that cyclin subunits regulate human and plasmodial CDKs in a similar manner. In this study, molecular docking analysis of Pfmrk against the selected FDA-approved drugs acquired from the ZINC15 database. The top five drugs, Lurasidone, Vorapaxar, Donovex, Alvesco, and Orap, were screened based on binding energies of best-docked scores ranging between -8 kcal/mol and -12 kcal/mol. Based on Molecular dynamics simulations for 100ns, Lurasidone showed the highest binding affinity (-105.90 {+/-} 57.72 kJ/mol), followed by Donovex (-92.877 {+/-} 17.872 kJ/mol) and exhibited stable interactions with the amino acid residues present in the active site of Pfmrk. The outcomes of in silico investigation putatively suggested that Lurasidone and Donovex exhibit antimalarial potency and could be translated as potential Pfmrk inhibitors and developing new drugs based on further in-vitro studies. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=130 SRC="FIGDIR/small/547694v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@1e14204org.highwire.dtl.DTLVardef@1b522e8org.highwire.dtl.DTLVardef@106df75org.highwire.dtl.DTLVardef@389899_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsWe investigated the potential FDA-approved drugs for repurposing against the modelled protein Pfmrk. Alvesco, Donovex, Lurasidone, Orap, and Vorapaxar are potential FDA-approved drugs firmly binding with Pfmrk. Molecular docking and simulation studies show that Donovex and Lurasidone are potential inhibitors of the modelled Pfmrk protein. Donovex and Lurasidone are potential drugs that act as kinase inhibitors by binding to the ATP -binding site of the enzyme Pfmrk.

biochemistry↗

bioRxiv2023

Functional Relations of CSLD2, CSLD3, and CSLD5 Proteins during Cell Wall Synthesis in Arabidopsis

Plant cell expansion is a dynamic process that is physically constrained by the deposition of new cell wall components. During tip growth, new cell wall materials are delivered at a restricted plasma membrane domain which results in a highly polarized expansion within that specified domain. Previous studies demonstrated that this process requires the activities of members of the Cellulose Synthase-Like D (CSLD) subfamily of cell wall synthases. CSLD3 displays {beta}-1,4 glucan synthase activity, but whether other members of CSLD subfamily share this conserved biochemical activity, and whether CSLD proteins form higher-order complexes to perform {beta}-1,4 glucan synthase activities have not been determined. Here, we use genetic methods to demonstrate that CSLD2 and CSLD3 functions are interchangeable during root hair elongation and cell plate formation, while CSLD5 provides a unique and irreplaceable function in the formation of cell plates. Importantly, genetic analysis with inactivated versions of CSLD3 show that, unlike CESA proteins, CSLDs do not require the simultaneous presence of different isoforms to perform catalytic cell wall synthase activities. In vitro biochemical activity experiments confirmed that CSLD2, CSLD3, and CSLD5 proteins displayed {beta}-1,4 glucan synthases activities. Taken together, these results indicated that while all three vegetatively expressed CSLD proteins possess conserved {beta}-1,4 glucan synthase activities, that CSLD5 has a more complicated and specialized role during cell plate formation.

cell biology↗

bioRxiv2023

Dynamical modeling reveals RNA decay mediates the effect of matrix stiffness on aged muscle stem cell fate

Loss of muscle stem cell (MuSC) self-renewal with aging reflects a combination of influences from the intracellular (e.g., post-transcriptional modifications) and extracellular (e.g., matrix stiffness) environment. Whereas conventional single cell analyses have revealed valuable insights into factors contributing to impaired self-renewal with age, most are limited by static measurements that fail to capture nonlinear dynamics. Using bioengineered matrices mimicking the stiffness of young and old muscle, we showed that while young MuSCs were unaffected by aged matrices, old MuSCs were phenotypically rejuvenated by young matrices. Dynamical modeling of RNA velocity vector fields in silico revealed that soft matrices promoted a self-renewing state in old MuSCs by attenuating RNA decay. Vector field perturbations demonstrated that the effects of matrix stiffness on MuSC self-renewal could be circumvented by fine-tuning the expression of the RNA decay machinery. These results demonstrate that post-transcriptional dynamics dictate the negative effect of aged matrices on MuSC self-renewal. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=125 SRC="FIGDIR/small/529950v2_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@1a840corg.highwire.dtl.DTLVardef@d354cforg.highwire.dtl.DTLVardef@650556org.highwire.dtl.DTLVardef@41587c_HPS_FORMAT_FIGEXP M_FIG Graphical abstract description: The balance of self-renewal and differentiation in young muscle stem cells (MuSCs) is robust to perturbations of the biophysical microenvironment. In contrast, aged MuSCs are highly sensitive to extrinsic perturbations, and exposure to a youthful microenvironment rejuvenates the self-renewing potential of aged MuSCs by modulating post-transcriptional RNA dynamics. C_FIG

cell biology↗

bioRxiv2021

Data-driven discovery of targets for bipotent anticancer drugs identifies Estrogen Related Receptor Alpha

Drugs that kill tumors through multiple mechanisms have potential for broad clinical benefits, with a reduced propensity to resistance. We developed BipotentR, a computational approach to find cancer-cell-specific regulators that simultaneously modulate tumor immunity and another oncogenic pathway. Using tumor metabolism as proof-of-principle, BipotentR identified 38 candidate immune-metabolic regulators by combining epigenomes with bulk and single-cell tumor transcriptomes from patients. Inhibition of top candidate ESRRA (Estrogen Related Receptor Alpha) killed tumors by direct effects on energy metabolism and two immune mechanisms: (i) cytokine induction, causing proinflammatory macrophage polarization (ii) antigen-presentation stimulation, recruiting CD8+T cells into tumors. ESRRA is activated in immune-suppressive and immunotherapy-resistant tumors of many types, suggesting broad clinical relevance. We also applied BipotentR to angiogenesis and growth-suppressor pathways, demonstrating a widely applicable approach to identify drug targets that act simultaneously through multiple mechanisms. BipotentR is publicly available at http://bipotentr.dfci.harvard.edu/. One-Sentence SummaryBipotentR identifies targets for bipotent anticancer drugs, as shown by the energy and immune effects of ESRRA inhibition.

cancer biology↗

bioRxiv2021

Phenotyping the tumor-immune microenvironment (TiME) in vivo by cellular-level optical imaging reveals unique combination phenotypes with variable inflammation and endothelial anergy

Immunotherapies have shown unprecedented clinical benefits in several malignancies1-3. However, clinical responses remain variable and unpredictable, indicating the need to develop predictive platforms that can improve patient stratification4. Phenotyping of tumors into hot, altered, or cold5 based on assessment of only T-cell infiltration in static tumor biopsies provides suboptimal prediction of immunotherapy response6,7. In vivo dynamic mechanisms within the tumor microenvironment such as tumor angiogenesis and leukocyte trafficking5,8,9 also play a central role in modulating anti-tumor immunity and therefore immunotherapy response. Here, we report novel tumor immune microenvironment (TiME) phenotyping in vivo in patients with non-invasive spatially-resolved cellular-level imaging based on endogenous contrast. Investigating skin cancers as a model, with reflectance confocal microscopy (RCM) imaging10, we determined four major phenotypes with variable prevalence of vasculature (Vasc) and inflammation (Inf) features: VaschiInfhi, VaschiInflo, VascloInfhi and Vascmed/hiInflo. The VaschiInfhi phenotype correlates with high immune activation, exhaustion, and vascular signatures while VaschiInflo with endothelial anergy and immune exclusion. Automated quantification of TiME features demonstrates moderate-high accuracy and correlation with corresponding gene expression. Prospectively analyzed response to topical immunotherapy show highest response in VascloInfhi, and reveals the added value of vascular features in predicting treatment response. Our novel in vivo cellular-level imaging and phenotyping approach can potentially advance our fundamental understanding of TiME, develop robust predictors for immunotherapy outcomes and identify novel targetable pathways in future.

cancer biology↗

bioRxiv2021

Differential Regulation of Rice Transcriptome to Rhizoctonia solani infection

Sheath Blight (SB) disease in rice crop caused by the infection of the fungal pathogen Rhizoctonia solani (R. solani) is one of the severe rice diseases that can cause up to 50% yield losses. Naturally occurring rice varieties resistant to SB have not been reported yet. We have performed a Time-Series RNA-Seq analysis on a widely cultivated rice variety BPT-5204 for identifying its transcriptomic response signatures to R. solani infection at 1st, 2nd and 5th day post inoculation (dpi). In total, 428, 3225 and 1225 genes were differentially expressed in the treated rice plants post 1, 2 and 5 dpi, respectively. GO and KEGG enrichment analysis identified significant processes and pathways differentially altered in the rice plant after the fungal infection. Machine learning and network based integrative approach was used to construct Transcriptional Regulatory Networks (TRNs) of the rice plant at the three Time Points. Regulatory network analysis identified SUB1B, MYB30 and CCA1 as important regulatory hub Transcription Factors in rice during R. solani infection. Jasmonic acid signaling pathway was activated and in contrast, photosynthesis and carbon fixation processes were significantly compromised. Involvement of MAPK, CYPs, Peroxidases and PAL genes was observed in response to the fungal infection. Circadian clock was also strongly influenced by R. solani infection. Our integrative analysis identified 7 putative SB resistant genes altered in rice after R. solani infection and provided a better understanding of rice plant response to R. solani infection. One sentence summaryTime series expression analysis of rice variety BPT-5204 identifies key molecular signatures involved in rice plant response to R. solani infection.

systems biology↗

bioRxiv2021

Notch signaling via Hey1 and Id2b regulates Müller glia's regenerative response to retinal injury

Zebrafish Muller glia (MG) respond to retinal injury by suppressing Notch signaling and producing progenitors for retinal repair. A certain threshold of injury-derived signal must be exceeded in order to engage MG in a regenerative response (MGs injury-response threshold). Pan-retinal Notch inhibition expands the zone of injury-responsive MG at the site of focal injury, suggesting that Notch signaling regulates MGs injury-response threshold. We found that Notch signaling enhanced chromatin accessibility and gene expression at a subset of regeneration-associated genes in the uninjured retina. Two Notch effector genes, hey1 and id2b, were identified that reflect bifurcation of the Notch signaling pathway, and differentially regulate MGs injury-response threshold and proliferation of MG-derived progenitors. Furthermore, Notch signaling component gene repression in the injured retina suggests a role for Dll4, Dlb, and Notch3 in regulating Notch signaling in MG and epistasis experiments confirm that the Dll4/Dlb-Notch3-Hey1/Id2b signaling pathway regulates MGs injury-response threshold and proliferation.

developmental biology↗

bioRxiv2020

Cardiac overexpression of microRNA-7 is associated with adverse cardiac remodeling

Role of microRNA-7 (miRNA-7) in targeting Epidermal growth factor receptor (EGFR/ERBB) family is known in dividing cancer cells while less is known about its role in terminally differentiated cardiac cells. We generated transgenic (Tg) mice with cardiomyocyte-specific overexpression of miRNA-7 to determine its role in regulating cardiac function. Despite similar survival, expression of miRNA-7 results in cardiac dilation as measured by echocardiography, instead of age-based cardiac hypertrophy observed in littermate controls. In contrast to the classical adaptive hypertrophy in response to TAC, miRNA-7 Tg mice directly undergo cardiac dilation post-TAC that is associated with increased fibrosis. Interestingly, significant loss in ERBB2 expression was observed in cardiomyocytes with no changes in ERBB1 (EGFR). Gene ontology and cellular component analysis using the cardiac proteomics data showed significant reduction in mitochondrial membrane integrity reflecting the differential enrichment/loss of proteins in miRNA-7 Tg mice compared to littermate controls. Consistently, electron microscopy showed that miRNA-7 Tg hearts had disorganized and rounded mitochondrial morphology indicating mitochondrial dysfunction. These findings show that expression of miRNA-7 uniquely results in cardiac dilation instead of adaptive hypertrophic response to cardiac stress providing insights on adverse remodeling in physiology and pathology.

physiology↗

bioRxiv2020

Insulin inhibits protein phosphatase 2A to impair β-adrenergic receptor function

Insulin impairs {beta}2-adrenergic receptor ({beta}2AR) function through G protein-coupled receptor kinase 2 (GRK2) by phosphorylation but less is known about dephosphorylation mechanisms mediated by protein phosphatase 2A (PP2A). Pharmacologic or genetic inhibition of phosphoinositide 3-kinase {gamma} (PI3K{gamma}) unexpectedly resulted in significant reduction of insulin-mediated {beta}2AR phosphorylation. Interestingly, {beta}2AR-associated phosphatase activity was inhibited by insulin but was reversed by knock-down of PI3K{gamma} showing negative regulation of PP2A by PI3K{gamma}. Co-immunoprecipitation and surface plasmon resonance studies using purified proteins showed that GRK2 and PI3K{gamma} form a complex and could be recruited to {beta}2ARs as GRK2 interacts with insulin receptor substrate following insulin treatment. Consistently, {beta}-blocker pretreatment did not reduce insulin-mediated {beta}2AR phosphorylation indicating agonist- and G{beta}{gamma}-independent non-canonical regulation of receptor function. Mechanistically, PI3K{gamma} inhibits PP2A activity at the {beta}AR complex by phosphorylating an intracellular inhibitor of PP2A (I2PP2A). Knock-down or CRISPR ablation of endogenous I2PP2A unlocked PP2A inhibition mediating {beta}2AR dephosphorylation showing an unappreciated acute regulation of PP2A in mediating insulin-{beta}2AR cross-talk. SummaryInsulin impairs {beta}2-adrenergic receptor ({beta}2AR) function through G protein-coupled receptor kinase 2 (GRK2). We show that insulin simultaneously inhibits protein phosphatase 2A (PP2A) sustaining {beta}2AR functional impairment. Unexpectedly, releasing PP2A inhibition by PI3K{gamma} preserves {beta}2AR function despite intact insulin-driven GRK2-mechanisms.

biochemistry↗

bioRxiv2020

PAN-INDIA 1000 SARS-CoV-2 RNA Genome Sequencing Reveals Important Insights into the Outbreak

The PAN-INDIA 1000 SARS-CoV-2 RNA Genome Sequencing Consortium has achieved its initial goal of completing the sequencing of 1000 SARS-CoV-2 genomes from nasopharyngeal and oropharyngeal swabs collected from individuals testing positive for COVID-19 by Real Time PCR. The samples were collected across 10 states covering different zones within India. Given the importance of this information for public health response initiatives investigating transmission of COVID-19, the sequence data is being released in GISAID database. This information will improve our understanding on how the virus is spreading, ultimately helping to interrupt the transmission chains, prevent new cases of infection, and provide impetus to research on intervention measures. This will also provide us with information on evolution of the virus, genetic predisposition (if any) and adaptation to human hosts. One thousand and fifty two sequences were used for phylodynamic, temporal and geographic mutation patterns and haplotype network analyses. Initial results indicate that multiple lineages of SARS-CoV-2 are circulating in India, probably introduced by travel from Europe, USA and East Asia. A2a (20A/B/C) was found to be predominant, along with few parental haplotypes 19A/B. In particular, there is a predominance of the D614G mutation, which is found to be emerging in almost all regions of the country. Additionally, mutations in important regions of the viral genome with significant geographical clustering have also been observed. The temporal haplotype diversities landscape in each region appears to be similar pan India, with haplotype diversities peaking between March-May, while by June A2a (20A/B/C) emerged as the predominant one. Within haplotypes, different states appear to have different proportions. Temporal and geographic patterns in the sequences obtained reveal interesting clustering of mutations. Some mutations are present at particularly high frequencies in one state as compared to others. The negative estimate Tajimas D (D = -2.26817) is consistent with the rapid expansion of SARS-CoV-2 population in India. Detailed mutational analysis across India to understand the gradual emergence of mutants at different regions of the country and its possible implication will help in better disease management.

genomics↗

bioRxiv2020

Phylogenomic analysis of SARS-CoV-2 genomes from western India reveals unique linked mutations

India has become the third worst-hit nation by the COVID-19 pandemic caused by the SARS-CoV-2 virus. Here, we investigated the molecular, phylogenomic, and evolutionary dynamics of SARS-CoV-2 in western India, the most affected region of the country. A total of 90 genomes were sequenced. Four nucleotide variants, namely C241T, C3037T, C14408T (Pro4715Leu), and A23403G (Asp614Gly), located at 5UTR, Orf1a, Orf1b, and Spike protein regions of the genome, respectively, were predominant and ubiquitous (90%). Phylogenetic analysis of the genomes revealed four distinct clusters, formed owing to different variants. The major cluster (cluster 4) is distinguished by mutations C313T, C5700A, G28881A are unique patterns and observed in 45% of samples. We thus report a newly emerging pattern of linked mutations. The predominance of these linked mutations suggests that they are likely a part of the viral fitness landscape. A novel and distinct pattern of mutations in the viral strains of each of the districts was observed. The Satara district viral strains showed mutations primarily at the 3' end of the genome, while Nashik district viral strains displayed mutations at the 5' end of the genome. Characterization of Pune strains showed that a novel variant has overtaken the other strains. Examination of the frequency of three mutations i.e., C313T, C5700A, G28881A in symptomatic versus asymptomatic patients indicated an increased occurrence in symptomatic cases, which is more prominent in females. The age-wise specific pattern of mutation is observed. Mutations C18877T, G20326A, G24794T, G25563T, G26152T, and C26735T are found in more than 30% study samples in the age group of 10-25. Intriguingly, these mutations are not detected in the higher age range 61-80. These findings portray the prevalence of unique linked mutations in SARS-CoV-2 in western India and their prevalence in symptomatic patients. ImportanceElucidation of the SARS-CoV-2 mutational landscape within a specific geographical location, and its relationship with age and symptoms, is essential to understand its local transmission dynamics and control. Here we present the first comprehensive study on genome and mutation pattern analysis of SARS-CoV-2 from the western part of India, the worst affected region by the pandemic. Our analysis revealed three unique linked mutations, which are prevalent in most of the sequences studied. These may serve as a molecular marker to track the spread of this viral variant to different places.

genomics↗

bioRxiv2020

The biphasic and age-dependent impact of Klotho on hallmarks of aging and skeletal muscle function

Aging is accompanied by a disrupted information flow, which results from accumulation of molecular mistakes. These mistakes ultimately give rise to debilitating disorders such as skeletal muscle wasting, or sarcopenia. To estimate the growing "disorderliness" of the aging muscle system, we employed a statistical physics approach to estimate the state parameter, entropy, as a function of genes associated with hallmarks of aging. Although the most prominent structural and functional alterations were observed in the oldest old mice (27-29 months), we found that the escalating network entropy reached an inflection point at old age (22-24 months). To probe the potential for restoration of molecular "order" and reversal of the sarcopenic phenotype, we overexpressed the longevity protein, -Klotho. Klotho overexpression modulated genes representing all hallmarks of aging in both old and oldest-old mice. However, whereas Klotho improved strength in old mice, intervention failed to induce a benefit beyond the entropic tipping point.

physiology↗

bioRxiv2019

Genome-wide CRISPR screens identify essential cell growth mediators in BRAF-inhibitor resistant melanoma

BRAF is a serine-threonine kinase that harbors activating mutations in [~]7% of human malignancies and [~]60% of melanomas. Despite initial clinical responses to BRAF inhibitors (BRAFi), patients frequently develop drug resistance. To identify candidate therapeutic targets for BRAFi-resistant melanoma, we conducted CRISPR screens in melanoma cells harboring an activating BRAF mutation that had also acquired resistance to BRAFi. The screens identified pathways and genes critical for BRAFi resistance in melanoma cells. To investigate the mechanisms and pathways enabling resistance to BRAFi in melanomas, we integrated expression data, ATAC-seq, and CRISPR screen results. We identified the JUN family of transcription factors and the ETS family transcription factor ETV5 as key regulators of CDK6 that enabled resistance to BRAFi in melanoma cells. Our findings reveal genes whose loss of function conferred resistance to a selective BRAF inhibitor, providing new insight into signaling pathways that contribute to acquired resistance in melanoma.

cancer biology↗

bioRxiv2019

EPIGENE: genome wide transcription unit annotation using a multivariate probabilistic model of histone modifications

BackgroundUnderstanding transcriptome is critical for explaining functional as well as regulatory roles of genomic regions. Current methods for the identification of transcription unit (TU) uses RNA-seq which, however, requires large quantities of mRNA limiting the identification of inherently unstable TUs e.g. for miRNA precursors. This problem can be resolved by chromatin based approaches due to a correlation between histone modifications and transcription. ResultsHere we introduce EPIGENE, a novel chromatin segmentation method for the identification of active TUs using transcription associated histone modifications. Unlike existing chromatin segmentation approaches, EPIGENE uses a constrained, semi-supervised multivariate hidden markov model (HMM) that models the observed combination of histone modifications using a product of independent Bernoulli random variables, to identify active TUs. Our results show that EPIGENE can identify genome-wide TUs unbiasedly. EPIGENE predicted TUs showed an enrichment of RNA Polymerase II in transcription start site and gene body indicating that they have been transcribed. Comprehensive validation with existing annotations revealed that 93% of EPIGENE TUs can be explained by existing gene annotations and 5% of EPIGENE TUs in HepG2 can be explained by microRNA annotations. EPIGENE outperforms existing RNA-Seq based approaches in TU prediction precision across human cell lines. Finally, we identify 381 novel TUs in K562 and 43 novel cell-specific TUs all of which are supported by RNA Polymerase II data. ConclusionsWe demonstrate the applicability of HMM to identify genome-wide active TUs and provides valuable information about unannotated TUs. EPIGENE is an open-source method and is freely available at: https://github.com/imbeLab/EPIGENE.

bioinformatics↗

bioRxiv2019

Non-axisymmetric shapes of biological membranes from locally induced curvature

In various biological processes such as endocytosis and caveolae formation, the cell membrane is locally deformed into curved configurations. Previous theoretical and computational studies to understand membrane morphologies resulting from locally induced curvature are often limited to axisymmetric shapes, which severely restricts the physically admissible morphologies. Under the restriction of axisymmetry, past efforts predict that the cell membrane buds at low resting tensions and stalls at a flat pit at high resting tensions. In this work, we lift the restriction of axisymmetry by employing recent theoretical and numerical advances to understand arbitrarily curved and deforming lipid bilayers. Our non-axisymmetric morphologies reveal membrane morphologies which agree well with axisymmetric studies--however only if the resting tension of the membrane is low. When the resting tension is moderate to high, we show that (i) axisymmetric invaginations are unstable; and (ii) non-axisymmetric ridge-shaped structures are energetically favorable. We further study the dynamical effects resulting from the interplay between intramembrane viscous flow and induced curvature, and find the rate at which the locally induced curvature increases is a key determinant in the formation of ridges. In particular, we show that axisymmetric buds are favored when the induced curvature is rapidly increased, while non-axisymmetric ridges are favored when the curvature is slowly increased: The rate of change of induced curvature affects the intramembrane viscous flow of lipids, which can impede the membranes ability to transition into ridges. We conclude that the appearance of non-axisymmetric ridges indicates that axisymmetry cannot be generally assumed when understanding processes involving locally induced curvature. Our results hold potentially relevant implications for biological processes such as endocytosis, and physical phenomena like phase separation in lipid bilayers.

biophysics↗

bioRxiv2018

Structural basis of hypoxic gene regulation by the Rv0081 transcription factor of Mycobacterium tuberculosis

The transcription factor Rv0081 of M. tuberculosis controls the hypoxic gene expression and acts as a regulatory hub in the latent phase of tuberculosis infection. We report here the crystal structure of Rv0081 at 3.3 [A] resolution revealing that it belongs to the well-known ArsR/SmtB family proteins. ArsR/SmtB family transcriptional repressors exert gene regulation by reversible metal binding. Hypoxia in general is sensed by bacterial transcriptional regulators via metals or Cys-mediated thiol switches. Oxygen sensing typically leads to transcriptional repressor changing its conformational state with altered DNA-binding property under different oxygen levels. Surprisingly Rv0081 neither has a metal binding domain nor does it possess Cys residues suggesting an alternate mechanism of gene regulation. Our structural analysis identified Ser 48, Ser 49, Ser 52 and Gln 53 as potential residues of Rv0081 involved in DNA binding. We probed DNA-binding of Rv0081 with electrophoretic mobility shift assay (EMSA) as well as surface plasmon resonance (SPR), where the Alanine mutants of these residues showed diminished DNA binding. Similarly, Aspartate mutants of these Ser residues was shown to fail to bind to DNA. Since, phosphorylation of various regulatory proteins is one of the important controlling mechanisms, we expected the role of Ser-phosphorylation of Rv0081 in hypoxic condition. Probing Rv0081 with anti-phosphoserine antibodies in M. tuberculosis cell lysate showed marked enhancement in the phosphorylation of Rv0081 protein under hypoxia. Overall, our structural and biochemical analysis provides the molecular basis for the regulation of Rv0081 in the latent phase of tuberculosis infection.\n\nIMPORTANCETuberculosis is one of the deadliest infectious diseases caused by the bacterium Mycobacterium tuberculosis. In about 90% of the infected people, M. tuberculosis exists in a dormant or a latent stage which can be reactivated in favorable conditions. Hypoxia (low oxygen pressure) is one of causes of dormancy. Understanding hypoxic gene regulation in M. tuberculosis is therefore an important step to understand latency. Rv0081 is a transcriptional regulator of genes expressed during hypoxia. In order to understand the mechanism by which Rv00081 regulates gene expression during hypoxia, we have solved the crystal structure of Rv0081 and identified amino acid residues which are critical in its transcriptional regulator activity. The crystal structure is suggestive of mechanism of gene regulation under hypoxia.

biophysics↗