Biology subjects

Sarkar, R.

Publications and source records attributed to Sarkar, R..

At least 19 recordsLinked to original sources

bioRxiv2026

Bio-Templated Zinc Oxide Nanotherapeutics from Flemingia macrophylla kuntze Ex Merr: In Vivo Anti-Diabetic and Anti-Inflammatory Efficacy in Drosophila melanogaster

Nanotechnology has a major impact on the creation of novel and effective therapies for the treatment of several diseases, including diabetes and cancer. The current work is a report on the use of the aqueous extract from the leaves of the Flemingia macrophylla plant as a capping and reducing agent in the green synthesis of zinc oxide nanoparticles (ZnO-NPs).The synthesized ZnO-NPs were subjected to characterization through X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), transmission electron microscopy (TEM) scanning electron microscopy (SEM), and ultraviolet-visible spectroscopy (UV-Vis). Dose-dependent in vivo effects were investigated using Drosophila melanogaster subjected to a high-sugar diet (HSD) to induce a diabetic-like model. In vitro anti-oxidant, anti-diabetic, anti-inflammatory assay, survival, locomotor function, neuroanatomical integrity, ovary and testis morphology, egg-laying capacity, and male fertility were evaluated. The most prominent UV-visible peak was detected at around 370 nm. Structural and morphological analysis via XRD and electron microscopy confirmed the hexagonal phase of the ZnO-NPs, indicating an average calcined particle size of around 47 nm at 500 degree C. The manuscript reports significant anti-diabetic and anti-inflammatory effects in vitro and in the in vivo Drosophila model, together with antioxidant activity. The in vivo results indicate that green-synthesized ZnO-NPs mitigate adverse phenotypes triggered by a high-sugar diet and also potential as an anti-diabetic intervention and warrant further mechanistic and preclinical investigation. Keywords: Flemingia macrophylla; Green ZnO-NPs; Drosophila melanogaster; high-sugar diet; nanobiotechnology

pharmacology and toxicology↗

bioRxiv2026

Systematic Engineering of Loss-of-Function Alleles in the Zebrafish Mitochondrial Proteome

Pathogenic variants in the 13 protein-coding genes of the mitochondrial genome underlie clinically and biochemically heterogeneous disorders. Most mtDNA-encoded genes lack defined loss-of-function (LOF) models in vivo. To address this gap, we have generated Z-Terminator, a systematic in vivo atlas of loss-of-function alleles covering all the mtDNA-encoded OXPHOS subunits in zebrafish (Danio rerio). We used mitochondrial TALE base editors to introduce premature termination codon (PTC) alleles via C-to-T transitions across Complexes I, III, IV, and V. Larvae harboring PTC alleles displayed bioenergetic defects and elevated lactate. While mtDNA mutations are associated with sensorineural hearing loss, the cellular basis has remained unclear. We show that engineered mtDNA LOF alleles directly impair hair cell function in proportion to heteroplasmy in a living vertebrate. We investigated the germline transmission and tissue-specific heteroplasmy of these LOF alleles and observed that a subset of variants was transmitted to the F1 generation and displayed distinct mutant loads across organs. These findings establish Z-Terminator as a vertebrate platform for interrogating the role of mtDNA protein-coding genes in cellular dysfunction and for elucidating the pathophysiology of mitochondrial disorders.

genetics↗

bioRxiv2026

Gut Microbiota-derived Acetate Safeguards the Colonic Epithelial Acetyl-CoA Reserve to Avert Colonic Senescence

Aging is a major driver of tissue senescence, but little is known about the development of age-unrelated tissue senescence. Here we show that nucleocytosolic acetyl-CoA deficiency in colon epithelial cells, caused by Acly ablation and bacterial depletion, triggers age-independent, p53-dependent colonic senescence leading to severe systemic inflammation. Acetate supplementation, acetate-producing bacterial transplantation, targeted depletion of senescent cells or treatment with lysine deacetylase inhibitors blocks colonic senescence and inflammatory injury. Spontaneous colonic senescence develops following simultaneous deletion of epithelial Acly and Acss2, confirming that microbe-derived acetate maintains the epithelial acetyl-CoA pool via ACSS2 to avert colonic senescence. Mechanistically, acetyl-CoA deficiency deprives a cohort of mitochondrial and nuclear proteins of acetylation, leading to increased oxidative stress and DNA repair stress that trigger cellular senescence. Among these proteins, ATP5F1A-K161 acetylation and H4-K5/8 acetylation are required to protect colonic epithelial cells from developing senescence. These observations unveil a previously unknown mechanism that governs colonic senescence.

physiology↗

bioRxiv2025

Mapping Allosteric Rewiring in Viral RNA: Sequence-Encoded Control of Protein Binding Mechanisms

RNA recognition by proteins is governed not only by static structure but also by allostery encoded within non-local dynamic motifs. In this study, we systematically identify allosteric communication hubs in RNA and map multiple residue-connected pathways, revealing how these networks are rewired upon mutation and protein binding. To capture these effects under physiological salt conditions, we performed tens of microseconds of atomistic and steered molecular dynamics simulations and computed binding free energies for Tat-TAR complexes across three immunodeficiency virus variants, BIV, HIV-1, and HIV-2. Allosterically coupled sites were identified using contact-based principal component analysis, and communication pathways were traced through an extended graph-network algorithm--the first such application to RNA systems. Two distant motifs--the bulge and the apical loop--emerge as allosteric switches and information hubs: the bulge engages Tat, while the loop interacts with another protein partner, CycT1, both essential for transcriptional activation and antiviral targeting. We find that HIV-2 TAR, with strong loop-bulge coupling and high self-integrity, favour conformational selection and exhibits lower Tat-binding affinity. In contrast, a single C24 insertion in HIV-1 TAR reconfigures communication pathways, enabling an induced-fit mechanism with enhanced affinity. The study not only elucidates an allosteric rewiring between the loop and bulge but also highlights how this communication is dynamically reconfigured upon protein binding. Tat association at the bulge reorganizes and reorients loop residues, thereby promoting the subsequent recruitment of CycT1. This work overall underscores how sequence (even a single mutation) encoded RNA allostery can modulate not only a proteins binding mechanism and affinity but also influence downstream molecular events within transcriptional signalling cascades. STATEMENT OF SIGNIFICANCEIn vitro selection strategies such as SELEX have long underscored the therapeutic relevance of the TAR RNA-Tat protein interface, yet they remain experimentally demanding and often overlook how subtle sequence variations reshape molecular recognition and RNA-protein binding affinity. By integrating atomistic and free-energy simulations with a graph-network-based framework, the study reveals that even a single-nucleotide change can rewire RNAs allosteric communication pathways, switching between conformational selection and induced-fit mechanisms and alter binding affinity. These findings bridge molecular dynamics with experimental selection, offering a predictive, thermodynamics-based framework to rationalize sequence-structure-function relationships and guide the rational design of high-affinity RNA-protein complexes and next-generation RNA therapeutics.

biophysics↗

bioRxiv2025

Microglial metabolic reprogramming drives cognitive decline in heart failure with preserved ejection fraction

Heart failure with preserved ejection fraction (HFpEF) is a rapidly growing public health concern and an emerging contributor to dementia, yet the mechanisms linking cardiometabolic dysfunction to neurodegeneration remain poorly understood. Here, we demonstrate that HFpEF drives a sustained neuroinflammatory state through microglial metabolic reprogramming. Using a clinically relevant murine model of HFpEF, we identified robust induction of HIF-1 signaling in microglia via integrated transcriptomics and metabolomics, coupled with increased glycolytic metabolism revealed by extracellular flux analysis. Conditional deletion of Hif1a in microglia during HFpEF attenuated neuroinflammation, preserved white matter integrity, and rescued cognitive performance. We further identify Sema4D as a HIF-1-dependent, microglia-derived effector linking metabolic stress to white matter injury. These findings establish a mechanistic bridge between cardiovascular disease and cognitive dysfunction and reveal microglial HIF-1 signaling as a tractable therapeutic strategy for preventing cognitive decline in cardiometabolic disease.

immunology↗

bioRxiv2024

EVOLVE: A Web Platform for Evolutionary Phase Analysis and New Variant Exploration from Multi-Sequence Data

Recurrent updates from the World Health Organization (WHO) on refining definitions for Variants of Concern (VOCs), Variants of Interest (VOIs), and Variants Under Monitoring (VUMs) underscore the need for systematic investigations to develop quantifiable metrics that differentiate critical phases of variant evolution. This study illustrates how protein data can be systematically analyzed by applying phase transition principles from statistical mechanics, where quantifying mutational response function (MRF) helps identify concerning variants along evolutionary paths. To support the exploration of metrics like mutational entropy, MRF, and other relevant indicators for novel or uncharacterized viruses and pathogenic bacteria, we introduce EVOLVE--a web platform that empowers researchers across diverse fields to access sequence-based analyses. EVOLVE streamlines data upload and analysis with a user-friendly interface and comprehensive tutorials. Overcoming the challenges with large sequence-space analysis its AI-driven data-learning module integrates evolutionary dynamics with microscopic mutational information, enabling the prediction of future site-specific mutations in prospective missense variants. Access EVOLVE for free at https://www.evolve-0fm3.onrender.com.

biophysics↗

bioRxiv2024

Gravitational effect on the cell in bio-fluid suspension

The growing interest in space exploration and human spaceflight has highlighted the critical challenges posed by microgravity on human physiology. One such challenge is the behavior of immune and red blood cells, which primarily rely on hematic or lymphatic streams (biofluids) for transport. This study aims to quantitatively address the puzzle of how red blood cells are influenced by gravity when they are suspended in bio-fluid. The Dissipative Particle Dynamics (DPD) approach was used to model blood and the cell by applying gravity as an external force along the vertical axis and varied from 0g to 2g during parameter sweeps. Key metrics, including Elongation and Deformation indices, pitch angle, and normalized center of mass, were utilized to assess cellular behavior. Results revealed that gravity induces shape changes and spatial alignment in red blood cells. The Elongation Index and the normalized center of mass declined linearly with the applied gravity. Correlation analysis showed a strong correlation between applied gravity and the aforementioned variables. Additionally, forces acting on the cell, such as drag, shear stress, and solid forces, diminished as gravitational force increased. Further analysis indicates that increasing gravity affected the cells velocity, resulting in prolonged proximity to vessel walls and intensified viscous interactions with surrounding fluid particles, thereby triggering morphological changes. This study provides crucial insights into the biophysical effects of gravity on the red blood cell and presents a significant step toward understanding cellular dynamics under altered gravitational conditions.

biophysics↗

bioRxiv2024

CELL CYCLE STATE PREDICTION USING GRAPH NEURAL NETWORKS

Mitosis is a crucial process ensuring the faithful transmission of the genetic information stored in the cell nucleus. Aberrations in this intricate process pose a significant threat to an organisms health, leading to conditions like cancer and various diseases. Hence, the study of mitosis holds paramount importance. Recent investigations have involved manual and semiautomated analyses of time-lapse microscopy images to understand mitosis better. This paper introduces an approach for predicting mitosis stages, employing a Convolutional Neural Network (CNN) as the initial feature extractor, followed by a Graph Neural Network (GNN) for predicting cell cycle states. A distinctive timestamp is incorporated into the feature vectors, treating this information as a graph to leverage internal interactions for predicting the subsequent cell state. To assess performance, experiments were conducted on three datasets, demonstrating that our method exhibits comparable efficacy to state-of-the-art techniques.

cell biology↗

bioRxiv2023

Leveraging Graph Machine Learning for Moonlighting Protein Prediction: A Local PPI Network and Physiochemical Feature Approach

Moonlighting proteins are those proteins that perform more than one distinct function in the body. They are pivotal in various metabolic pathways and disease mechanisms. Identifying moonlighting proteins remains a challenge in Computational Biology. In this work, we propose the first graph neural network based models to identify moonlighting proteins. Our models work on large protein-protein interaction (PPI) networks with sparse labels of moonlighting and non-moonlighting proteins. In addition to PPI network, the models make use of features derived from the amino acid sequences of the proteins. We propose two frameworks: one as graph classification based on the local neighborhood of the query protein; and the other node classification based on the entire graph. These GNN-based methods outperform traditional machine learning methods that have previously been used for moonlighting prediction. The global full network-based model, operating on Homo sapiens data achieves accuracy of 88.4% and F1 score of 88.8%. The local neighborhood method is more lightweight and can be applied to larger protein sets with multiple species. CCS CONCEPTS* Applied computing [->] Computational proteomics.

bioinformatics↗

bioRxiv2022

Glucocorticoid signaling mediates CD8+ T cell memory differentiation

We provide evidence on the TRM potentiation by glucocorticoids in stimulated CD8+ T cells. Signaling via glucocorticoid receptor reciprocally regulated short-lived effector cells and memory precursor effector cells. Influenza A virus infected mice treated transiently with dexamethasone preferentially generated multipotent TRM cells that efficiently responded to the subsequent infection. Compromised memory following abrogation of glucocorticoid signaling further confirmed their role in memory potentiation. Transcriptomic and biochemical analysis of dexamethasone treated cells revealed a metabolic switch to oxidative phosphorylation via an engagement of AMPK signaling due to reduced glucose uptake. These cells exhibited an accumulation of phosphorylated STAT3 and STAT5 to drive memory differentiation. Therefore, glucocorticoids mediate tissue homing memory T cell differentiation. One-Sentence SummaryGlucocorticoid signaling in responding CD8+ T cells promotes memory differentiation

immunology↗

bioRxiv2021

Correlation in Domain Fluctuations Navigates Target Search of a Viral Peptide along RNA

Biological macromolecules often exhibit correlation in fluctuations involving distinct domains. This study decodes their functional implications in RNA-protein recognition and target-specific binding. The target search of a peptide along RNA in viral TAR-Tat complex is closely monitored using atomistic simulations, steered molecular dynamics simulations, free energy calculations, and a machine-learning-based clustering technique. An anti-correlated domain fluctuation is identified between the tetraloop and the bulge region in the apo form of TAR RNA that sets a hierarchy in the domain-specific fluctuations at each binding event and that directs succeeding binding footsteps. Thus, at each binding footstep, the dynamic partner selects an RNA location for binding where it senses higher fluctuation, which is conventionally reduced upon binding. This event stimulates an alternate domain-fluctuation which then dictates sequential binding footstep/s and thus, the search progresses. Our cross-correlation maps show that the fluctuations relay from one domain to another specific domain till the anti-correlation between that inter-domain fluctuations sustains. Artificial attenuation of that hierarchical domain fluctuation inhibits specific RNA binding. The binding is completed with the arrival of a few long-lived water molecules that mediate slightly distant RNA-protein sites and finally stabilizes the overall complex. The study underscores the functional importance of naturally designed fluctuating RNA motifs (bulge, tetraloop) and their interplay in dictating the directionality of the search in a highly dynamic environment.

biophysics↗

bioRxiv2021

Neutralization of SARS-CoV-2 lineage B.1.1.7 pseudovirus by BNT162b2 vaccine-elicited human sera

Recently, a new SARS-CoV-2 lineage called B.1.1.7 has emerged in the United Kingdom that was reported to spread more efficiently than other strains. This variant has an unusually large number of mutations with 10 amino acid changes in the spike protein, raising concerns that its recognition by neutralizing antibodies may be affected. Here, we investigated SARS-CoV-2-S pseudoviruses bearing either the Wuhan reference strain or the B.1.1.7 lineage spike protein with sera of 16 participants in a previously reported trial with the mRNA-based COVID-19 vaccine BNT162b2. The immune sera had equivalent neutralizing titers to both variants. These data, together with the combined immunity involving humoral and cellular effectors induced by this vaccine, make it unlikely that the B.1.1.7 lineage will escape BNT162b2-mediated protection.

immunology↗

bioRxiv2021

Targeting conserved viral virulence determinants by single domain antibodies to block SARS-CoV2 infectivity

We selected SARS-CoV2 specific single domain antibodies (sdAbs) from a previously constructed phage display library using synthetic immunogenic peptides of the virus spike (S) protein as bait. The sdAbs targeting the cleavage site (CS) and the receptor binding domain (RBD) in S protein efficiently neutralised the infectivity of a pseudovirus expressing SARS-CoV2 S protein. Anti-CS sdAb blocked the virus infectivity by inhibiting proteolytic processing of SARS-CoV2 S protein. Both the sdAbs retained characteristic structure within the pH range of 2 to 12 and remained stable upto 65{degrees}C. Furthermore, structural disruptions induced by a high temperature in both the sdAbs were largely reversed upon their gradual cooling and the resulting products neutralised the reporter virus. Our results therefore suggest that targeting CS in addition to the RBD of S protein by sdAbs could serve as a viable option to reduce SARS-CoV2 infectivity and that proteolytic processing of the viral S protein is critical for infection.

immunology↗

bioRxiv2020

Modeling PPRV pathogenesis in mice to assess the contribution of innate cells and anti-viral T cells

We demonstrate a rapid induction of type I IFN response in PPRV stimulated cells and the susceptibility of mice, genetically ablated of interferon response, to PPRV infection. Following PPRV infection, IFNR KO mice gradually reduced their body weights and succumbed to the infection within 10 days. While the infecting inoculum size did not alter the outcome of infection, the nature of the induced disease was qualitatively different. Immunopathological lesions were characterized by the expansion and infiltration of innate immune cells distinctly evident at the lower infecting dose of PPRV infection. The replicating virus particles as well as the viral antigens were abundant in most of the critical organs of PPRV infected IFNR KO mice. Neutrophils and macrophages transported the replicating virus to central nervous system and contributed to pathology while the NK cells and T cells were protective against the virus. Using an array of fluorescently labeled H-2Kb tetramers PPRV specific CD8+ T cells responses were identified and measured in the infected as well as the peptide immunized mice. Our study therefore established and employed a laboratory animal model for investigating PPRV pathogenesis and the contribution of virus specific CD8+ T cells during the virus infection to pave the way for elucidating protective or pathological roles of immune cells during PPRV infection. ImportanceWe developed a laboratory animal model for investigating the pathogenesis and immunity induced by PPRV. IFNR KO animals succumbed to the infection irrespective of the dose and the route of infection. Neutrophils and macrophages served as the Trojan horse and helped transport the virus to CNS to cause encephalitis while the NK cells and CD8+ T cells provided the protection against PPRV infection. We additionally identified class I restricted immunogenic epitopes of PPRV in C57BL/6 mice. Our study therefore paves the way for an optimal utilization of this model to unravel PPRV pathogenesis and assessing the host correlates of protection.

immunology↗

bioRxiv2020

Amyloidogenic core of a human {lambda}-III immunoglobulin light chain fibril and their germline variants probed by MAS solid state NMR

Systemic antibody light chains (AL) amyloidosis is characterized by deposition of amyloid fibrils derived from a particular antibody light chain. Cardiac involvement is a major risk factor for mortality. Using MAS solid-state NMR, we study the fibril structure of a recombinant light chain fragment corresponding to the fibril protein from patient FOR005, together with fibrils formed by protein sequence variants that reflect the closest germline (GL) sequence. Both analyzed fibril structures were seeded with ex-vivo amyloid fibrils purified from the explanted heart of this patient. We find that residues 11-42 and 69-102 adopt {beta}-sheet conformation in patient protein fibrils. We identify glycine-49 that is mutated with respect to the germline sequence into arginine-49 as a key residue that forms a salt bridge to aspartate-25 in the patient protein fibril structure. Fibrils from the GL protein and from the patient protein harboring the single point mutation R49G can be both heterologously seeded using patient ex-vivo fibrils. Seeded R49G fibrils show an increased heterogeneity for the C-terminal residues 80-102 which is reflected by the disappearance of all resonances of these residues. By contrast, residues 11-42 and 69-77, which are visible in the MAS solid-state NMR spectra show 13C chemical shifts that are highly similar to patient fibrils. The mutation R49G thus induces a conformational heterogeneity at the C-terminus in the fibril state, while the overall fibril topology is retained.

biophysics↗

bioRxiv2020

Localized Proteotoxic Stress in Mitochondrial Intermembrane Space and Matrix Elicits Sub-compartment Specific Response Pathways Governed by Unique Modulators

The double-membrane-bound architecture of mitochondria, essential for ATP production, sub-divides the organelle into inter-membrane space (IMS) and matrix. IMS and matrix possess contrasting oxido-reductive environments and distinct protein quality control (PQC) machineries resulting different protein folding environments. To understand the nature of stress response elicited by equivalent proteotoxic stress to sub-mitochondrial compartments, we fused well-described bipartite or matrix-targeting signal sequences to misfolding and aggregation-prone stressor proteins to target and impart stress to yeast mitochondrial IMS or matrix. We show, mitochondrial proteotoxicity leads to growth arrest of yeast cells of varying degrees depending on nature of stressor proteins and the intra-mitochondrial location of stress. Next, using transcriptomics and proteomics, we report a comprehensive stress response elicited by two types of targeting signal-fused stressor proteins. Among global responses by mitochondria-targeted stressors by both types of signal sequences, an adaptive response of abrogated mitochondrial respiration and concomitant upregulation of glycolysis is uncovered. Beyond shared stress responses, specific signatures due to stress within mitochondrial sub-compartments are also revealed. We report that bipartite signal sequence-fused stressor proteins eliciting stress to IMS, leads to specific upregulation of IMS-chaperones and TOM complex components. In contrast, matrix-targeted stressors lead to specific upregulation of matrix-chaperones and cytosolic PQC components. Finally, by systematic genetic interaction using deletion strains of differentially upregulated genes, we found prominent modulatory role of TOM complex components during IMS-stress response. In contrast, VMS1 markedly modulates the stress response originated from matrix.

cell biology↗

bioRxiv2020

Japanese encephalitis virus capsid protein interacts with non-lipidated MAP1LC3 on replication membranes and lipid droplets

Studies have shown that Japanese encephalitis virus (JEV), replicates on ER derived membranes that are marked by autophagosome negative non-lipidated MAP1LC3 (LC3-I). Depletion of LC3 exerts a profound inhibition on virus replication and egress. Here, we further characterize the role of LC3 in JEV replication, and through immunofluorescence and immunoprecipitation show that LC3-I interacts with the virus capsid protein in infected cells. This association was observed on capsid localized to both the replication complex and lipid droplets (LDs). JEV infection decreased the number of LDs per cell indicating a link between lipid metabolism and virus replication. This capsid-LC3 interaction was independent of the autophagy adaptor protein p62/SQSTM1. Further, no association of capsid was seen with the GABARAP protein family, suggesting that this interaction was specific for LC3. High resolution protein-protein docking studies identified a putative LC3-interacting region (LIR) in capsid, 56FTAL59, and other key residues that could mediate a direct interaction between the two proteins.

microbiology↗

bioRxiv2020

Comprehensive analysis of genomic diversity of SARS-CoV-2 in different geographic regions of India: An endeavour to classify Indian SARS-CoV-2 strains on the basis of co-existing mutations

Accumulation of mutations within the genome is the primary driving force for viral evolution within an endemic setting. This inherent feature often leads to altered virulence, infectivity and transmissibility as well as antigenic shift to escape host immunity, which might compromise the efficacy of vaccines and antiviral drugs. Therefore, we aimed at genome-wide analyses of circulating SARS-CoV-2 viruses for the emergence of novel co-existing mutations and trace their spatial distribution within India. Comprehensive analysis of whole genome sequences of 441 Indian SARS-CoV-2 strains revealed the occurrence of 33 different mutations, 21 being distinctive to India. Emergence of novel mutations were observed in S glycoprotein (7/33), NSP3 (6/33), RdRp/NSP12 (4/33), NSP2 (2/33) and N (2/33). Non-synonymous mutations were found to be 3.4 times more prevalent than synonymous mutations. We classified the Indian isolates into 22 groups based on the co-existing mutations. Phylogenetic analyses revealed that representative strain of each group divided themselves into various sub-clades within their respective clades, based on the presence of unique co-existing mutations. India was dominated by A2a clade (55.60%) followed by A3 (37.38%) and B (7%), but exhibited heterogeneous distribution among various geographical regions. The A2a clade mostly predominated in East India, Western India and Central India, whereas A3 clade prevailed in South and North India. In conclusion, this study highlights the divergent evolution of SARS-CoV-2 strains and co-circulation of multiple clades in India. Monitoring of the emerging mutations would pave ways for vaccine formulation and designing of antiviral drugs.

microbiology↗