Biology subjects

Rawat, S.

Publications and source records attributed to Rawat, S..

13 recordsLinked to original sources

bioRxiv2026

Tiled base editing screens uncover rare second-site mutations that rescue a misfolded disease protein

Therapeutic base editing can directly revert ~30% of disease-causing missense mutations to the wild-type sequence. One strategy to broaden its reach is to install second-site corrector mutations that improve protein stability or function across multiple pathogenic alleles. Here, we use tiled adenine and cytosine base editing screens to identify such corrector mutations in NPC1, a lysosomal cholesterol transporter whose misfolding causes Niemann-Pick type C disease. Tiling mutagenesis of the endogenous NPC1 locus in haploid human cells carrying the prevalent NPC1I1061T disease allele, combined with phenotypic reporters for cholesterol transport and protein abundance, uncovered rare intragenic suppressor mutations distributed across multiple domains. Unexpectedly, we identified a promiscuous second-site variant that can correct many different pathogenic NPC1 mutations. These correctors improve protein stability, promote ER exit and lysosomal trafficking, and restore cholesterol clearance in patient-derived fibroblasts and induced neurons. Our results establish in situ base editing screens for the discovery of second-site corrector mutations that are actionable for therapeutic base editing when the original mutation cannot be corrected.

cell biology↗

bioRxiv2026

Dengue Virus Serotype 2 Infection Transcriptionally Reprograms Host Neutrophils to Establish an Immunosuppressive Microenvironment in Mice

Dengue virus (DV) infection triggers profound alterations in host immune responses, yet the transcriptomic reprogramming of innate immune cells during disease progression remains incompletely understood. Here, we demonstrated that Dengue virus serotype 2 (DV-2) infection transcriptionally reprograms host neutrophils, establishing an immunosuppressive microenvironment that facilitates pathogenesis. Through scRNA transcriptomic profiling of bone marrow and spleen cells from the DV-2-infected AG129 mouse model, we delineated a distinct neutrophil signature that shows dynamic shifts in immature and activated populations over the course of dengue infection. We also characterized a specific Ly6Glow subpopulation, notably Fibrinogen-like protein 2 (FGL2)-expressing subsets, that appeared with disease progression. Serum FGL2 levels were significantly elevated in both DV-2-infected mice and human dengue-positive patients. Furthermore, treatment of splenocytes with FGL2 or FGL2-enriched neutrophil secretomes suppressed T cell functional activity and promoted an immunosuppressive phenotype. Together, these findings reveal how DV-2-driven neutrophil reprogramming shapes an immunosuppressive network, highlighting the importance of studying FGL2-expressing neutrophil subsets to understand dengue immunopathogenesis.

immunology↗

bioRxiv2026

Polypharmacology of an Optimal Kinase Library

Despite decades of research, current understanding of the spectrum of targets bound by kinase inhibitors remains incomplete. This complicates mechanism of action studies, drug repurposing, and development of new therapies. Here, we describe kinome-wide profiling of an optimal kinase library (OKL) comprising 192 small molecules selected based on stage of clinical development, chemical diversity, and target coverage. Our results show that polypharmacology is widespread and independent of regulatory approval. The generally understood ("assigned") targets of approved molecules are not necessarily the most potently inhibited and off-targets include multiple understudied kinases. Moreover, median selectivity has not increased over time We illustrate how an OKL in combination with detailed kinome profiling can be used to identify potential toxicity targets, repurpose anti-inflammatory drugs for neurodegenerative and infectious diseases, and perform chemical genetic studies. Our studies also highlight how much remains to be discovered about the chemistry and biology of one of the largest classes of human therapeutics.

pharmacology and toxicology↗

bioRxiv2025

Time-dependent processing of dengue virus polyprotein yields multiple capsid forms that disrupt cellular homeostasis

Dengue virus infections spread to new geographical regions every year, cause significant morbidity and mortality, yet the progress in drug/vaccine development is limited due to an incomplete understanding of its life cycle. While a few of the proteins coded by this virus exist in multiple forms, the sequential events of polyprotein processing to yield them and the consequential effects on host cells are unclear. In this study, the virus infected cell culture data suggest that the polyprotein undergoes a time-dependent processing to yield multiple capsid forms, ie, capsid, capsid-anchor, capsid-anchor-pr, capsid-anchor-prM. Among them, the c-anc and c-anc-pr were found to localize to the mitochondria upon transfection into the HEK cells. The transmission electron microscopy studies suggest that c-anc induces mitochondrial fragmentation. Further studies showed that c-anc affects the mitochondrial fusion-related proteins and the genes involved in their functions. The pulldown experiments indicate that c-anc also interacts with 1-syntrophin (SNTA1), a cytoplasmic protein that plays a role in redox potential, oxidative stress and mitochondrial biogenesis. Importantly, the mitostress analyses suggest that c-anc triggers impaired mitochondrial dysfunctions like potential, respiration and ATP generation. In this study, we also identify ursonic acid (UNA) as a c-anc binding compound that restores mitochondrial functions and suppresses virus multiplication in vitro, ex vivo and in mice. The revealed disrupted mitochondrial homeostasis appears to be common to DENV, ZIKV, JEV, YFV, HCV, ASFV and SARS-CoV-2; hence, UNA or its related compounds could be considered as inhibitors for the above virus infections.

microbiology↗

bioRxiv2025

Hierarchical Neural Circuit Theory of Normalization and Inter-areal Communication

The primate brain exhibits a hierarchical, modular architecture with conserved microcircuits executing canonical computations across reciprocally connected cortical areas. We present a hierarchical neural circuit theory with feedback connections that dynamically implements divisive normalization across its hierarchy. In a two-stage instantiation (V1 {leftrightarrow} V2), increasing feedback from V2 to V1 amplifies responses in both areas. We analytically derive power spectra (V1) and coherence spectra (V1-V2) as functions of frequency (f), and compare them with experimental observations: peaks in both spectra shift to higher frequencies with increased stimulus contrast, and power decays as 1/f4 at high frequencies. The closed-form spectra are validated against direct stochastic simulation of the full nonlinear circuit across contrasts. The theory further predicts distinctive spectral signatures of feedback and input gain modulation. Crucially, the theory offers a unified view of inter-areal communication, defined as the ability to linearly predict neural activity in one brain area from neural activity in another, with emergent features consistent with empirical observations of both communication subspaces and inter-areal coherence. It admits a low-dimensional communication subspace, where inter-areal communication is lower-dimensional than within-area communication. It further predicts that: i) increasing feedback strength enhances inter-areal communication and diminishes within-area communication, without altering the subspace dimensionality; ii) communication is strongest, and the subspace dimensionality lowest, at the frequencies at which V1-V2 coherence peaks; iii) normalization reduces the subspace dimensionality. Finally, a three-area (V1 {leftrightarrow} V4 and V1 {leftrightarrow} V5) instantiation of the theory demonstrates that differential feedback from higher to lower cortical areas dictates their dynamic functional connectivity. Altogether, our theory provides a robust and analytically tractable framework for generating experimentally-testable predictions about normalization, inter-areal communication, and functional connectivity.

neuroscience↗

bioRxiv2025

Stabilization of recurrent neural networks through divisive normalization

Stability is a fundamental requirement for both biological and engineered neural circuits, yet it is surprisingly difficult to guarantee in the presence of recurrent interactions. Standard linear dynamical models of recurrent networks are sensitive to the precise values of the synaptic weights, since stability requires all eigenvalues of the recurrent matrix to lie within the unit circle. Here we demonstrate, both theoretically and numerically, that an arbitrary recurrent neural network can remain stable even when its spectral radius exceeds 1, provided it incorporates divisive normalization, a dynamical neural operation that suppresses the responses of individual neurons. Sufficiently strong recurrent weights lead to instability, but the approach to the unstable phase is preceded by a regime of critical slowing down, a well-known early warning signal for loss of stability. Remarkably, the onset of critical slowing down coincides with the breakdown of normalization, which we predict analytically as a function of the synaptic strength and the magnitude of the external input. Our findings suggest that the widespread implementation of normalization across neural systems may derive not only from its computational role, but also to enhance dynamical stability. I. SIGNIFICANCE STATEMENTNeural circuits must remain stable to function correctly, yet strong recurrent connections, which are essential for complex computations, often hinder this stability. We demonstrate that divisive normalization, a canonical neural operation found across many sensory systems, can actively stabilize neural networks. By analyzing a biologically plausible model (ORGaNICs), we find that normalization allows circuits to remain stable even when recurrent interactions are strong enough to otherwise cause exploding neural dynamics. Furthermore, we identify a theoretical link between the breakdown of normalization and critical slowing down, a state where the brain recovers slowly from perturbations. This suggests that loss of normalization may serve as an early warning signal for the onset of pathological instabilities, such as seizures.

neuroscience↗

bioRxiv2025

Changes in free-roaming dog population demographics and health associated with a catch-neuter-vaccinate-release program in Jamshedpur, India

Indias large free-roaming dog populations contribute to significant human health, environmental, and social challenges. Population management strategies, such as capture-neuter-vaccinate-return (CNVR), aim to reduce dog numbers, improve their welfare, and reduce human-animal conflict. The Humane Society International, in partnership with the Animal Health Foundation, implemented a CNVR program in Jamshedpur, neutering and vaccinating over 20,000 dogs. This study evaluates the impact of this program on dog health, population structure and size. The study areas encompassed 10 sites within the Jamshedpur Metropolitan Region, including both intervention sites where CNVR was directly applied and sites without direct intervention. Data was collected from May 2014 until December 2018, including bi-annual street surveys, as well as clinical data from the dogs captured and treated. We fit logistic regression, negative binomial, and binomial mixed effects models to assess changes in dog population characteristics, health, and reproductive conditions over time in relation to the CNVR intervention. We found that, over time, the CNVR program significantly reduced the probability of dogs entering the clinic with mange, transmissible venereal tumours, and pregnant. Street surveys showed an increase in sterilised dogs, with higher proportions observed in CNVR-treated sites, although the counts of dogs observed increased overall. The age-structure of free-roaming dogs remained stable over time. In CNVR-treated areas, the probability of observing lactating female dogs decreased, whereas it increased in untreated sites. This work contributes to the growing body of knowledge investigating the impact of dog population management interventions. Continued monitoring and evaluation of CNVR programs are required to identify optimal coverage required to reduce population size effectively.

ecology↗

bioRxiv2024

Autoimmunity and clinical pathology amelioration in SLE by Dexamethasone primed Mesenchymal Stem Cell derived conditioned media

This study proposes a novel approach, utilizing cell-free dexamethasone (Dex) primed Whartons jelly mesenchymal stem cells derived conditioned media (DW), offering improved efficacy, simplicity, and alternative medicine for addressing complications associated with systemic lupus erythematosus (SLE). This study explores the immunomodulatory effects of DW treatment on immune cell populations in SLE patients and a pristane-induced lupus (PIL) mouse model. DW induces significant expansion of Tregs, Bregs, suppressing Th17, double-negative T cells, and inflammatory neutrophils through modulating IL-10 and IL-17A production. Comparisons with the standard drug hydroxychloroquine reveal similar effects, suggesting TGF beta; pathway mediation in DWs actions. Compared with the immunosuppressive drug Dex, DW better attenuated autoantibody production, increased anti-inflammatory cytokines and maintained a balanced Th17/Treg ratio. In the preclinical in vivo studies, DW exhibits therapeutic efficacy, reducing mortality, preventing proteinuria, and reversing limb inflammation, seizure and alopecia. Organ specific evaluations using advanced live imaging or histopathological analysis highlighted DWs protective effects on kidneys, liver, lungs, heart, and spleen. This provided insight into the immunomodulatory benefits of DW at various levels and suggested that it could be a potential therapeutic avenue for managing complications related to SLE.

immunology↗

bioRxiv2023

Limb regeneration is blastema dependent in a ladybird beetle, Cheilomenes Sexmaculata (Fabricius)

Holometabolous insects undergoes metamorphosis which involves an intercalated pupal stage between larva and adult. The body plan of the adult is established during pupal stage and larval systems are de-differentiated and reorganized in insects undergoing complete metamorphosis. In ladybird beetles, limbs amputated in larval stages are regenerated in adults. This occurs during pupation. Given that changes in pupa are akin to embryogenesis, does the lost limbs are redeveloped as a part of metamorphosis or has some pre-patterning initiated prior pupation? To study this we hypothesised that limb regeneration in a holometabolous ladybird beetles, Cheilomenes sexmaculata is the result of the recapitulation of the embryonic gene programs in the pupa. To test this, we exposed third larval stages of C. sexmaculata to amputation and prior to amputation the tissues on the amputated site was scraped off every 24hrs. It was found that limb regeneration does not occur in the treatment where scrapping was done. Assuming that these epidermal cells correspond to blastema, for limb regeneration in ladybird beetles blastema is essential and does not occur in its absence.

developmental biology↗

bioRxiv2023

In-silico Discovery of Genetic Diversity in Cucumis sativus var. hardwickii: A Wild Relative of Cultivated Cucumber

Genome-wide sequencing data play an important role in evaluating the genomic level differences between superior and poor-quality crop plants and improving our understanding of molecular association with desired traits. We analyzed the obtained 92,921,066 raw reads from genome-wide resequencing of Cucumis sativus var. hardwickii through in-silico approaches and mapped to the reference genome of Cucumis sativus to identify the genome-wide single nucleotide polymorphisms (SNPs) and Single nucleotide variations (SNV). Here, we report 19, 74,213 candidate SNPs including 1,33,468 insertions and 1,43,237 deletions and 75 Indels genome-wide. A total of 2228224 identified variants were classified into four classes including 0.01% sequence alteration, 5.94% insertion, 6.37% deletion and 87.66% SNV respectively. These variations can be a major source of phenotypic diversity and sequence variation within the species. Overall, the discovery of SNPs and genomic variants may help predict the plant response to certain environmental factors and can be utilized to improve crop plants economically important traits.

bioinformatics↗

bioRxiv2021

Saturated Proteostasis and Nuclear Injuries Defeat Homeostatic Potentials of α-Synuclein Filaments

Biogenesis of inclusion bodies (IBs) facilitates protein quality control (PQC). Canonical aggresomes execute degradation of misfolded proteins while non-degradable amyloids quarantine into Insoluble Protein Deposits. Lewy Bodies (LBs) are well-known neurodegenerative IBs of -Synuclein associated diseases like Dementia with Lewy bodies (DLB) and Parkinsons disease. Intriguingly, the PQC benefits and drawbacks associated with LBs remain underexplored. Here, we report that the crosstalk between LBs and aggresome-like IBs of -Synuclein (Syn-aggresomes) buffers amyloidogenic -Synuclein load in primary neurons and mitotic cell models. Using cellular biochemistry, genetic knockdown, and microscopy tools we find that LBs possess unorthodox PQC-capacities of self-quarantining the Syn-amyloids, and being degradable upon receding fresh amyloidogenesis. Syn-aggresomes equilibrate biogenesis of LBs by facilitating spontaneous and opportunistic turnover of soluble -Synuclein and Syn-amyloids, respectively. However, LBs overgrow at the perinucleus once amyloidogenesis sets in and are misidentified by cytosolic BICD2 as cargos for motor-protein dynein. Simultaneously, microtubules surrounding the perinuclear LBs are distorted, misbalancing the dynein motor-force on nucleoskeleton leading to lamina injuries. Like typical Laminopathies, nucleocytoplasmic mixing, DNA-damage, and deregulated transcription of stress chaperones defeat the proteostatic purposes of LBs. We confirmed the lamina disintegrities in brain sections of Parkinsons disease patients. Together, our study provides insights into the intricate complexities of proteostatic possibilities associated with -Synuclein inclusions and offers understanding on the proteostasis-sensitivity of LB-containing aging neurons via lamina-injuries. Significance statementAmyloid inclusions of -Synuclein called Lewy Bodies (LBs) are hallmark of multiple neurodegenerative diseases like Dementia with Lewy Bodies and Parkinsons disease. A significant reason for the degeneration of LB-containing aging neurons in these diseases is their sensitivity to proteostasis stresses. We find two distinct inclusions of -Synuclein in the same neurons/cells. First, the Syn-aggresomes titrate the biogenesis of the other i.e., the LBs, by facilitating degradation of soluble -Synuclein. Syn-amyloids deposited in LBs remain degradable but LBs overgrow at the perinucleus once the kinetics of amyloid-biogenesis exceeds aggresome-assisted degradation of -Synuclein. Perinuclear LBs destabilize local tubulin-meshwork and are misidentified as cargo for cytosolic motor dynein. Aberrant cytoskeleton-nucleoskeleton tension disintegrates lamina, deregulates stress-responsive transcription, and triggers proteostasis-sensitivity in LB-containing neurons.

cell biology↗

bioRxiv2020

Improved Supra-Organization Is A Conformational And Functional Adaptation Of Respiratory Complexes

Multiple surveillance mechanisms accelerate proteasome mediated degradation of misfolded proteins to prevent protein aggregation inside and outside mitochondria. But how cells safeguard mitochondrial function despite increased protein aggregation during proteasome inactivation? Here, using two-dimensional complexome profiling, we extensively characterize the dynamic states of respiratory complexes (RCs) in proteasome-inhibited cells. We report that RC-subunits are increasingly integrated into supra-organizations to optimize catalytic activity simultaneous to their aggregation inside mitochondria. Complex-II (CII) and CV are incorporated into oligomers. CI, CIII, and CIV subcomplexes are associated into holocomplexes followed by integration into supercomplexes. Time-course experiments reveal that the core (CI+CIII2) stoichiometry of supercomplex (I+III2+IV) is preserved during early-stress while CIV composition varies. Simultaneously, increased CI-activity suggests conformational optimization of supercomplexes for better function. Re-establishment of steady-state stoichiometry and relative increase in supercomplex-quantity consolidates functional adaptation during prolonged proteasome-inhibition. Together, we name this pre-emptive adaptive mechanism as improved Supra-organization of Respiratory Complexes (iSRC). We find that iSRC is active in multiple protein-unfolding stresses, in multiple cell-types that differ in proteostatic and metabolic demands, and reversible upon stress-withdrawal.

biochemistry↗

bioRxiv2018

Proteostasis perturbation destabilizes respiratory complex assembly-intermediates via aggregation of subunits

Proteostasis is maintained by optimum expression, folding, transport, and clearance of proteins. Deregulation of any of these processes triggers widespread protein aggregation and loss of function. Here, we perturbed proteostasis by blocking proteasome-mediated protein degradation and investigated proteome partitioning from soluble to insoluble fraction. Aggregation of Respiratory Chain Complex (RCC) subunits highlights the early destabilization event as revealed by proteome redistribution. Sequence analyses followed by microscopy suggest that low complexity regions at the N-terminus are capable to facilitate aggregation of RCC subunits. As a result, respiratory complex assembly process is impaired due to destabilization of sub-complexes marking the onset of mitochondrial dysfunction and ROS accumulation. Redistribution of Histone proteins and their modifications indicated reprogramming of transcription as adaptive response. Together, we demonstrate susceptibility of RCC subunits to aggregation under multiple proteotoxic stresses providing an explanation for the simultaneous deregulation of proteostasis and bioenergetics in age-related degenerative conditions.

molecular biology↗