Biology subjects

Kumar, A.

Publications and source records attributed to Kumar, A..

At least 109 records · Page 6Linked to original sources

bioRxiv2026

NMDAR-mediated shift of neuronal gain across the cortical hierarchy

Neuronal gain is a critical feature of neuronal information processing, reflecting a cells excitability in response to presynaptic inputs. Evidence from spine counts in non-human primates suggests that the number of presynaptic inputs increases across the cortical hierarchy. Therefore, understanding the regulation of the neuronal gain can yield important insights into different integration strategies across the cortex. Along with the increasing spine counts, there is further evidence for a similar increase in NMDA-receptor-mediated currents. Here, we show, using detailed simulations of a thick-tufted layer 5 pyramidal neuron, that the amount of current mediated through NMDA receptors (NMDARs) strongly affects the neuronal gain function with balanced presynaptic inputs. Without NMDA, postsynaptic activity quickly saturates and declines with increasing inputs. However, with NMDARs, the gain of postsynaptic activity shifts into higher input regimes, enabling integration of dense inputs. We show that, with NMDARs, a significant fraction of postsynaptic activity arises from feedback between the voltage dependence of NMDARs and voltage-gated sodium channels. Lastly, we show, using a network model, that NMDAR knockout in single cells has differential effects on postsynaptic activity depending on network size (a proxy for cortical hierarchy). Thus, we propose that an NMDA gradient across the cortical hierarchy may be necessary to maintain excitability amid increasing presynaptic input.

neuroscience↗

bioRxiv2026

TAK1 regulates skeletal muscle mass, hypertrophic signaling, and metabolic homeostasis in male and female mice

Skeletal muscle is the most abundant tissue in the human body and is essential for locomotion and the regulation of whole-body metabolism. The maintenance of skeletal muscle mass is essential for health, yet the molecular and signaling mechanisms that control skeletal muscle mass remain poorly understood. Transforming growth factor-{beta}-activated kinase 1 (TAK1) is a key signaling protein that regulates multiple intracellular pathways. Recent studies have demonstrated that TAK1 is a critical regulator of skeletal muscle mass. However, the mechanisms by which TAK1 regulates muscle mass and whether its role is sex dependent remain incompletely understood. In this study, we show that targeted inactivation of TAK1 induces muscle atrophy more rapidly in male than in female mice. Loss of TAK1 activity also abolished mechanical overload-induced phosphorylation of p70S6K and rpS6, and the induction of myofiber hypertrophy in both sexes. RNA-Seq analysis further revealed that TAK1 inactivation in skeletal muscle disrupts the gene expression of various molecules involved in catabolic processes, calcium signaling, muscle structure development, and aerobic respiration. Moreover, TAK1 inactivation impairs fatty acid oxidation and promotes lipid accumulation in skeletal muscle of adult mice in a sex-independent manner. Collectively, our findings demonstrate that TAK1 regulates skeletal muscle mass and growth by coordinating distinct intracellular pathways in both male and female mice.

physiology↗

bioRxiv2026

Regions of genome plasticity are systematically organized into recurrent integration spots that shape accessory-genome functional architecture: insights from a complete genome of strain F1C1 and pangenomic analysis of the Ralstonia solanacearum species complex

1The Ralstonia solanacearum species complex (RSSC) is a highly diverse plant pathogen whose evolution is shaped by horizontal gene transfer. We generated a complete, gap-free hybrid genome assembly of F1C1, a South Asian Phylotype I strain classified as R. pseudosolanacearum. The closed assembly resolves a bipartite genome (3.73 Mb chromosome; 2.03 Mb megaplasmid), enabling precise localization of mobile genetic elements. Using F1C1 together with 142 complete RSSC genomes, we implemented a lineage-stratified pangenome framework that reveals a hierarchically structured gene repertoire and shows that accessory gene content can discriminate host-associated lineages beyond core-genome. Pangenome-informed mapping of genome plasticity identified 651 conserved integration spots that concentrate accessory turnover and are enriched for adaptive functions, including type III secretion system effectors and antiviral defense systems (e.g., Wadjet and CRISPR-Cas). Genes within these spots display elevated Ka/Ks relative to housekeeping functions, consistent with conflict-driven diversification and/or relaxed constraint. Together, these results link RSSC genome architecture to adaptive potential and provide a spot-based framework for genomic surveillance and resistance breeding in bacterial wilt pathosystems.

genomics↗

bioRxiv2026

The sugar-beet cyst nematode effector Hs2B11 targets the Arabidopsis serine protease inhibitor AtPR-6 to favor parasitism

Cyst nematodes secrete effector proteins to manipulate host cell biology and suppress immunity, yet the mechanisms underlying these interactions remain largely unexplored. In this study, we characterize the function of Hs2B11, a Heterodera schachtii effector that was previously shown to be expressed in the dorsal gland of sugar-beet cyst nematodes (BCN). Here, we report that in Arabidopsis thaliana Hs2B11 functions as an immune regulator that modulates the production of elicitor-induced oxidative species, likely to favor parasitism. To elucidate the molecular basis of this immune suppression, we performed a yeast-two-hybrid screen and identified the host serine protease inhibitor AtPR-6 as a direct interactor of Hs2B11. We show that AtPR-6 acts as a positive regulator of plant immunity; its expression is induced upon nematode infection and knock-out of AtPR-6 compromises oxidative species production leading to higher susceptibility to H. schachtii infection. Conversely, AtPR-6 overexpression enhances immune responses resulting in increased resistance to BCN infection. Detailed analysis of this interaction demonstrated that Hs2B11 interacts with AtPR-6 using its carboxyl-terminal domain. AlphaFold2 predicts that the C-terminal domain forms a beta-solenoid-like structure with a ladder of serine residues organized across one of its surfaces. We propose that using this interface, Hs2B11 targets AtPR-6 via molecular titration, preventing the inhibitor from regulating host proteases that control immune signaling. These findings highlight a counter-defense strategy where a nematode effector neutralizes a specific host protease inhibitor to subvert plant immunity.

plant biology↗

bioRxiv2026

Loss of the Coronary Artery Disease Risk Gene Leiomodin1 in Vascular Smooth Muscle Cells Triggers Rapid Onset Coronary Atherosclerosis

BackgroundAtherosclerosis is the primary underlying cause of coronary artery disease (CAD). Leiomodin1 is a vascular smooth muscle cell (VSMC)-restricted CAD risk gene whose role in coronary artery pathophysiology is unknown. Global loss of Leiomodin1 causes lethal neonatal visceral myopathy, requiring unique approaches for study in VSMCs. MethodsSeveral distinct Leiomodin1 mutant mouse models were generated by clustered regularly interspaced short palindromic repeats (CRISPR). Control (Lmod1WT) and VSMC-restricted Lmod1 knockout (Lmod1SMKO) mice were subjected to various atherogenic regimens. Atherosclerosis and LMOD1 expression in mouse and human coronary arteries were assessed by histopathology and confocal immunofluorescence microscopy. Coronary arteries from Lmod1WT and Lmod1SMKO mice were analyzed with assorted stains and antibodies, immunogold lineage tracing, spatial metabolomics/transcriptomics, and single-cell RNA sequencing (scRNA-seq). Mouse aortic SMCs from Lmod1WT and Lmod1SMKO mice were subjected to lipid loading with lentiviruses expressing wild-type Lmod1, a nucleation deficient Leiomodin1 (Lmod1ND), or a short hairpin RNA (shRNA) targeting Thrombospondin (Thbs1). ResultsUnder atherogenic conditions, Lmod1SMKO mice displayed unremarkable vessels in several organs but developed diffuse and occlusive coronary atherosclerosis. No such disease was observed in Lmod1WT mice. Time-course studies documented lipid insudation and VSMC foam cell formation in the coronary arteries of Lmod1SMKO mice as early as six days post-regimen. Immunogold lineage tracing demonstrated 46% of coronary plaque cells being of VSMC origin, with most showing evidence of lipid uptake. An intronic deletion of Lmod1, containing a conserved region where the single nucleotide variant associated with CAD exists, showed attenuated LMOD1 expression; heterozygous Lmod1SMKO mice, with a similar reduction in LMOD1, showed no CAD. Spatial metabolomics uncovered multiple lipid species within coronary atheromata of Lmod1SMKO mice, and spatial/scRNA-seq of similar coronary lesions disclosed altered lipid pathways with a consistent elevation in Thbs1. In vitro mechanistic studies revealed lipid accumulation in Lmod1SMKO VSMCs that was rescued by Lmod1WT, Lmod1ND, and Thbs1 shRNA. VSMC-restricted expression of Lmod1ND in mice resulted in negligible coronary atherosclerosis. ConclusionsUnder proatherogenic conditions, Lmod1SMKO mice present with rapidly manifesting coronary atherosclerosis that appears to be independent of the actin nucleation function of LMOD1. Targeting Thbs1 represents a viable strategy to mitigate VSMC foam cell formation. Clinical PerspectiveO_ST_ABSWhat is new?C_ST_ABSO_LIVascular smooth muscle cell (VSMC) loss of Leiomodin1 (Lmod1) causes diffuse and occlusive coronary atherosclerosis in mice, with little or no such disease in other vascular beds. C_LIO_LIA novel immunogold lineage tracing assay shows VSMC migration to the intima as early as six days following an atherogenic regimen, and quantitative studies demonstrate that 46% of coronary plaque cells are of SMC origin. C_LIO_LIThe coronary phenotype appears to be independent of LMOD1s actin nucleation activity, but VSMC lipid uptake is thrombospondin-dependent. C_LI What are the clinical implications?O_LILMOD1 is an annotated smooth muscle cell-restricted risk allele for human coronary artery disease (CAD), offering new insight into the role of smooth muscle cells in atherogenesis. C_LIO_LIThe rapidly manifesting CAD phenotype in Lmod1 knockout mice enables expedited testing of novel therapeutics to mitigate disease progression. C_LIO_LINew insight into LMOD1 pathobiology will help inform further SNV interrogation of the LMOD1 locus for CAD risk in patients. C_LI

pathology↗

bioRxiv2026

Multiscale Analysis of PNPLA2 and PNPLA3 Membrane Targeting

Lipid droplets (LDs) are dynamic organelles that regulate cellular lipid storage and mobilization through the coordinated action of LD-associated proteins. Patatin-like phospholipase domain-containing proteins PNPLA2 (ATGL) and PNPLA3 are central regulators of lipid metabolism, yet the molecular mechanisms underlying their membrane targeting and distinct enzymatic activities remain poorly understood. Here, we combine coarse-grained and all-atom molecular dynamics simulations with enhanced sampling to investigate how PNPLA2 and PNPLA3 associate with endoplasmic reticulum (ER) and LD membranes. Despite sharing a conserved N-terminal patatin domain, the two proteins exhibit distinct membrane-binding modes driven by divergent C-terminal amphipathic helices. In both proteins, membrane association is mediated primarily by deep insertion of C-terminal helices, while the patatin domain provides surface contact. PNPLA2 forms a deeply embedded U-shaped helical bundle on LDs that induce pronounced membrane curvature and promote opening of the catalytic dyad, consistent with its high triglyceride lipase activity. In contrast, PNPLA3 engages membranes through a more flexible helical arrangement that maintains a compact catalytic geometry and limits substrate accessibility. Membrane composition further modulates these interactions and leads to protein-specific lipid redistribution and curvature remodeling. Fluorescence microscopy experiments validate the computational predictions and demonstrate that mutation of a single arginine residue within the C-terminal region is sufficient to reduce LD targeting of both proteins. These results establish a mechanistic connection between membrane binding, conformational plasticity, and catalytic regulation in PNPLA2 and PNPLA3. Our work provides molecular insights into how lipid environments tune the function of LD-associated enzymes. Author SummaryLDs are essential cellular organelles that control how fats are stored and released, a process that relies on the precise recruitment and regulation of lipid-metabolizing enzymes. Our work focuses on two closely related enzymes, PNPLA2 (ATGL) and PNPLA3, which play central but distinct roles in lipid metabolism and metabolic diseases. Using a combination of multiscale modeling simulations and fluorescence microscopy, we examine how these proteins recognize and bind to ER and LD membranes. Although PNPLA2 and PNPLA3 share a conserved catalytic core, we show that they interact with membranes in different ways due to differences in their C-terminal amphipathic helices. We find that PNPLA2 forms a deeply embedded helical arrangement that reshapes the membrane and promotes access to its catalytic site, which explains why it typically shows strong lipase activity. In contrast, PNPLA3 adopts a more compact membrane-bound catalytic geometry that limits substrate access and enzymatic activity. We further applied fluorescence microscopy to experimentally validate the computational predictions. The results show that mutation of a single arginine residue within the membrane-binding helix reduces LD targeting. These findings reveal how membrane association and protein conformational dynamics jointly regulate catalytic accessibility and activity.

biophysics↗

bioRxiv2026

Discovering Novel PETase Enzymes for Enhanced PET Plastic Degradation Using in Silico Approaches

Accounting for 12% of global solid waste, Poly (ethylene terephthalate) (PET) is one of the most abundantly produced synthetic polymers. While PET offers substantial commercial benefits, its widespread use has led to disproportionate environmental hazards due to its resistance to degradation. To address this problem, several solutions have been proposed, including enzymatic degradation via PETase, MHETase, and Cutinase. Among these, PETase exhibited significant PET-degrading activity. However, the application of PETase has been hampered by its lack of robustness to pH, temperature ranges, and slow reaction rates. Hence, it has become novel enzymes that can overcome these limitations and function efficiently. In this study, we utilised an integrated in silico bioinformatics pipeline to identify and characterise novel PETase candidates from the Thermophilic actinobacteria Thermobifida cellulosilytica and Thermobifida halotolerans species. The PlasticDB database contains 228 plastic-degrading enzyme sequences. In which PETase (00188) is significantly homologous with two putative proteins, Hydrolase (ALF00495.1) and hypothetical protein (WOZ56011.1). The discrete optimized protein energy (DOPE) scores, stereochemical assessments, and homology modeling results closely mirrored our findings for both proteins, supporting their structural stability. The molecular dynamics simulations revealed that the putative ALF00495 variant exhibited more extensive and robust hydrogen-bonding networks, enhanced conformational stability, and increased structural compactness compared to the reference enzyme. The present in silico investigation underscores the potential of putative ALF00495 as a highly effective PETase biocatalyst for polyethylene terephthalate (PET) degradation. Collectively, these findings illustrate the utility of computational approaches of novel PET-degrading enzymes, thereby facilitating the development of sustainable biotechnological strategies to mitigate global plastic pollution.

bioinformatics↗

bioRxiv2026

Wax-printing-free fabrication of paper-supported 3D cancer cell culture

Three-dimensional (3D) in vitro tumor models are critical for studying transport-limited drug efficacy in solid tumors; however, many existing platforms are technically complex and remain difficult to access. Stacked paper-based tumor models ("cells-in-gel-in-paper", CiGiP) address this challenge by enabling formation of diffusion-limited microenvironments while allowing direct access to cells from distinct tissue depths. Nevertheless, current CiGiP implementations rely on wax or hydrophobic barrier patterning of paper, which has become increasingly inaccessible. Here, we present a wax-printing-free approach for fabricating stacked paper-supported 3D tumor tissues using a simple 3D-printed press-fit enclosure that holds circular paper layers snugly together, thereby enforcing one-dimensional transport without lateral leakage. Using MDA-MB-231 breast cancer cells embedded in Matrigel, we demonstrate the formation of nutrient-limited microenvironments across the tissue depth, as evidenced by layer-dependent cell viability. The platform enables direct quantification of spatial and temporal drug responses, demonstrated using doxorubicin and paclitaxel, both individually and in combination. Layer-dependent cytotoxicity was measured, and combination treatment analysis revealed antagonistic interactions consistent with prior reports. By eliminating the need for hydrophobic patterning, this approach substantially lowers the technical barriers to constructing stacked paper tumor models and is expected to facilitate broader adoption of paper-supported 3D tissues for drug screening and mechanistic studies. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=77 SRC="FIGDIR/small/705119v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@55f56aorg.highwire.dtl.DTLVardef@1633288org.highwire.dtl.DTLVardef@18aae9eorg.highwire.dtl.DTLVardef@1ce1532_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2026

A Functional Genetic Atlas of Parkin Resolves Variants of Uncertain Significance and Predicts Parkinson's Disease Age at Onset

Autosomal recessive mutations in the Parkin gene (PRKN) cause early-onset Parkinsons disease (PD). Parkin functions as a ubiquitin E3 ligase acting downstream of the PINK1 kinase to promote phosphorylated ubiquitin at sites of mitochondrial damage. Yet the functional effects of most PRKN gene variants remain unknown. Here we use a pooled cellular assay measuring phosphorylated ubiquitin accumulation to quantify the activity of [~]9,200 PRKN missense and nonsense variants. Our screen identifies thousands of loss- and gain-of-function variants, including activating variants mainly residing at autoinhibitory interfaces. Functional scores accurately distinguish known PD pathogenic and benign variants, and enable reclassification of 173/184 variants of uncertain significance (VUS). When combined into biallelic genotype scores, these data predict the age at disease onset in patients, revealing a quantitative link between Parkin activity and clinical manifestation. Our results provide a comprehensive functional genetic map of Parkin and demonstrate the power of multiplexed assays of variant effects (MAVEs) for variant interpretation and precision medicine in PD.

genetics↗

bioRxiv2026

Adenine Base Editing Potently Suppresses Hepatitis B Surface Antigen Expression and Inhibits Hepatitis D Virus Release

Background and AimsNovel antiviral approaches capable of permanently inactivating the intrahepatic HBV DNA reservoir, the covalently closed circular DNA (cccDNA) and HBV DNA integrated into the host genome, are urgently needed. This study evaluated adenine base editing as a strategy to disrupt HBV replication by introducing mutations in the overlapping HBs/polymerase open reading frame (ORF). MethodsAn adenine base editor (ABE) and 3 guide RNAs (gS1-gS3) were designed to introduce missense mutations within the HBs/polymerase ORF. ABE mRNA and individual gRNAs were co-transfected into HBV-infected HepG2-hNTCP cells and primary human hepatocytes. Antiviral efficacy was further assessed in HepG2.2.15 and PLC/PRF/5 cells harboring integrated HBV DNA. In vivo, lipid nanoparticles (LNP)-mediated delivery of ABE mRNA and gRNAs was evaluated in HBVcircle DNA-transduced mice and in HBV-infected human liver-chimeric mice. The impact of HBs editing on hepatitis D virus (HDV) release was assessed using PLC/PRF/5 and Huh7 cell-based HDV replication models. ResultsAdenine base editing efficiently reduced HBsAg production and HBV replication in vitro by targeting both cccDNA and integrated HBV DNA. A single LNP injection of ABE-gS2 resulted in undetectable HBsAg in HBVcircle mice, while two injections achieved a 90% reduction in serum HBsAg in HBV-infected human liver chimeric mice. HBV DNA replication was also inhibited in vivo. Furthermore, HBs ORF base editing markedly suppressed HDV release in vitro. ConclusionsAdenine base editing of the HBs ORF effectively impairs HBV replication and HBsAg production in vitro and in vivo and concomitantly inhibits HDV release, highlighting its therapeutic potential.

molecular biology↗

bioRxiv2026

A Systematic Review of the Ocean Acidification Research in India: Research Trends, Gaps and Recommendations

Ocean acidification, a consequence of climate change, has become a significant threat to marine organisms. Globally, tremendous efforts have been made to understand its impact on different ecological and biological processes. In India, this research area is still not fully explored, but expanding at an exponential rate. Hence, it is essential to consolidate the fragmented knowledge into a systematic review, which will assist future researchers to develop their work. In this study, we utilized the Scopus, Web of Science and Ocean Acidification - International Coordination Centre bibliography to conduct a systematic review of ocean acidification research in India. We used the Biblioshiny package in R to conduct a bibliometric analysis, identify spatial and temporal research trends, and highlight the growth of literature in ocean acidification research, as well as existing knowledge gaps. We used the following keywords: ocean acidification, lowered pH, acidifying ocean, elevated carbon dioxide, elevated CO2, marine carbonate chemistry, shell decalcification and affiliation as India to obtain relevant publications. We selected 353 publications by applying relevance filtering and adherence to PRISMA guidelines. Almost one-third of the publications were non-primary articles. Among research articles, only 71 publications were found to investigate the response of marine organisms to ocean acidification. Majority of them involved single stressors, for a short term on very limited taxa. Lack of molecular-level investigation, multifactorial experimental design, and long-term observations were major gaps. This review aims to support researchers, policymakers, and other stakeholders involved in the planning, monitoring, and developing adaptation strategies. Finally, it provides recommendations for future research and policy development.

ecology↗

bioRxiv2026

Brackish Beginnings: Rethinking the Role of Salinity in Shaping Mosquito Proboscis Morphology and Disease Risk

Climate change driven saltwater intrusion is expanding coastal brackish water habitats, promoting the proliferation of salinity-tolerant mosquitoes such as Culex sitiens, a vector of lymphatic filariasis and Japanese encephalitis. This study investigated whether environmental salinity affects mosquito morphology, specifically proboscis length, a trait of ecological significance related to feeding efficiency and vector competence. Late fourth-instar larvae and pupae of Cx. sitiens were collected from habitats with varying salinity levels in the Muttukadu Backwater, Tamil Nadu, India, and reared under controlled conditions using habitat specific water. Adult female body and proboscis lengths were measured, and water quality parameters were analysed to characterize environmental variation. Statistical analyses (Welchs ANOVA, regression, and ANCOVA) revealed a significant positive relationship between salinity and body length (R2 = 0.94, p=0.0003) as well as with proboscis length (R2 = 0.90, p=0.001). Additionally, ANCOVA indicated that the proboscis elongation remained significant after adjusting for body length (F{square}, {square}{square}{square} = 32.36, p < 0.001, partial 2 = 0.257). This confirmed that the salinity exerts an independent effect on this morphological trait. These findings provide the first field-based evidence that the environmental salinity drives proboscis elongation in Cx. sitiens, indicating an adaptive response under saline stress. This may have implications for disease transmission in climate-affected coastal regions.

zoology↗

bioRxiv2026

Prioritizing DNA methylation biomarkers using graph neural networks and explainable AI

DNA methylation is a significant epigenetic modification involving the addition of a methyl group to the position 5' of the cytosine residues. The modification is responsible for disease progression, immune response, and outcomes in diseases such as breast cancer (BC) and acute myeloid leukemia (AML). Illuminas HumanMethylation450 BeadChip (450K) and EPIC BeadChip (850K) methylation arrays are heavily used for such cancer studies to determine differentially expressed and differentially methylated genomic regions. Many of these are biomarkers used effectively for exploring therapeutic targets. Several studies report a few potential biomarkers, but the enormous numbers of largely unexplored probe-level (CpG sites) methylation signals may contain additional significant biomarkers. To prioritise the under-explored and disease-specific CpG sites from DNA methylation arrays and potentially uncover novel biomarkers, we present the novel approach GraphMeX-plain, a graph neural network (GNN)-based approach with explainable AI module. The underlying graph neural network is a principal neighbourhood aggregation (PNA). The approach uses the biomarkers reported in recent studies to rank biomarkers from the unexplored set. A similarity graph between CpG sites (known and unexplored sets) is constructed using DNA methylation {beta} values from arrays, producing an interaction graph. Biomarkers from recent studies are used as seeds and from the unexplored CpG sites, highly-variable ones (excluding the seeds) are selected that vary significantly between conditions (BC patients and normal controls for breast cancer arrays). Using the combination of seed and highly-variable CpG sites, a positive-unlabeled approach, network-informed adaptive positive-unlabeled learning (NIAPU), is utilized to assign a set of soft labels to unknown CpG sites such as likely positive, weakly negative, likely negative, and reliable negative in the descending order of likelihood of CpG sites being potential biomarkers. The graph neural network, a multi-layer PNA, refines the soft label assignments and achieves a high F1 classification score (weighted) of 0.93 for BC and 0.91 for AML. The most likely set of CpG sites, classified under "likely positive", are further explored using GNNExplainer, an explainable AI approach. Subgraphs for likely positive CpG sites, predicted with high probabilities, are computed and their proximities to the original seed CpG sites are analysed. The CpG sites which are predicted as likely positives have close interactions to the seeds. The top likely positive CpG site for BC is cg13265740 (C6orf115) where gene C6orf115 is strongly associated with BC. For AML, the top likely positive predicted CpG site is cg23281527 (KLHDC7A) where gene KLHDC7A plays a strong role in the mechanism of AML. A high percentage of these likely positive predicted CpG sites for both BC and AML, which remained unseen by the GNN model during training, are highly relevant to them and can serve as potential therapeutic targets and prognostic values.

bioinformatics↗

bioRxiv2026

Molecular Crowding-Driven Nucleosome Interactions Revealed Through Single-Molecule Optical Tweezers

Molecular crowding causes the compaction of chromatin fibers, contributing to the formation of the nuclear architecture. However, the molecular mechanism of compaction under crowded conditions is not yet fully understood. In this study, we employed the single-molecule optical tweezer method to investigate the effect of molecular crowding on chromatin structure. Force-extension experiments on a 12-mer polynucleosome in the presence of different sizes and concentrations of polyethylene glycol (PEG) as a crowding agent showed that at low concentrations of low-molecular-weight (MW) PEG, the compaction of the polynucleosome was not significant. In this respect, nucleosomes predominantly remained separated, while DNA-histone interactions within individual nucleosomes were slightly stabilized. In contrast, high concentrations of high-MW PEG significantly promote internucleosomal interactions, leading to highly compact polynucleosome conformations. Under these conditions, approximately 30 pN of force was required to disrupt the internucleosomal interactions and release DNA; this force was 36% higher than that required for DNA unwrapping in the absence of PEG. These findings suggest that molecular crowding impacts cellular processes by mechanically regulating chromatin accessibility for regulatory proteins and the passage of motor molecules such as RNA polymerase. Significance StatementChromatin condensation is closely related to biological processes such as transcription and replication. Molecular crowding has recently attracted attention as a factor regulating chromatin condensation. In this study, we used the optical tweezer method to analyze the molecular mechanisms underlying chromatin condensation. We found that high-molecular weight and high-concentration crowders (polyethylene glycol) induced significant compaction, which involved internucleosomal interactions that markedly reduced DNA accessibility. Our results suggest that molecular crowding not only alters the condensation state, but also mechanically regulates chromatin accessibility.

biophysics↗

bioRxiv2026

Selective identification of the herb Picrorhiza kurroa and probiotic Lactobacillus fermentum as a synbiotic with fermentation-enhanced physicochemical, biological, and metabolomic properties

PurposeAlthough secondary metabolites of medicinal plants can modulate the gut microbiota, their prebiotic and synbiotic potential is not fully explored. This study aimed to identify novel medicinal plant-probiotic combination(s) that show prebiotic and synbiotic effects through mutual synergism and reciprocal interactions. MethodsTen Himalayan region medicinal plants were screened for their selective prebiotic efficacy by studying in vitro proliferation of three species of lactic acid bacteria (LAB) and concurrent suppression of E. coli. The top-performing plants were evaluated for synergism with LAB through fermentation of pure extracts, followed by physicochemical, biological, and metabolomic profiling. ResultsAmong the ten plant species, Picrorhiza kurroa (PK) and Adhatoda vasica (AV) exhibited promising prebiotic attributes with PAS scores of 0.50 and 0.44, respectively, while Lactobacillus fermentum (LF) emerged as the most compatible probiotic strain. Unlike AV, fermentation of PK with LF demonstrated superior probiotic growth (2.2-fold increase), elevated total flavonoids (21.6% increase), and improved antioxidant capacity (24.02% increase) compared to unfermented PK. Fermented PK significantly enhanced cytoprotective effects and mitigated oxidative (ROS levels/lipid peroxidation) and inflammatory damage (NO/IL-6 levels) in macrophages and muscle cells exposed to separate exogenous stressors (LPS and H2O2). Elemental (ICP-OES) and metabolomic analysis (LC-MS/UHPLC) revealed that LF metabolised major elements of PK (Ca/Fe/K/Na/Cu) and induced biotransformation of large secondary metabolites, including the characteristic iridoid glycosides of PK, into smaller, more abundant metabolites. ConclusionsPK-LF combination is identified as a synbiotic, and its fermented product is a superior bioactive product that can be developed into innovative nutraceuticals or herbal formulations.

biochemistry↗

bioRxiv2026

High-throughput mapping of 6,888 RAD51D variants identifies distinct biochemical functions needed for homologous recombination and olaparib response

The tumor suppressor RAD51D is essential for homologous recombination (HR). Pathogenic variants in RAD51D are associated with breast and ovarian cancers. However, most clinical missense variants are of unknown significance. We performed a multiplex assay of variant effect to test 6,888 RAD51D coding variants for loss-of-function. The resulting variant-to-function map perfectly separates known pathogenic and benign variants and is validated by orthogonal HR and biochemical assays across 70 clinical variants. Our screen shows that variants in the DNA-binding or ATPase core most severely compromise HR, and we identify the RAD51D-RAD51C interface within the BCDX2 complex as essential for regulating its ATPase activity. We hypothesize that, paradoxically, the primary function of RAD51D is to slow the ATPase activity of BCDX2, thereby allowing sufficient time and space for RAD51 filament assembly. Together, we identify hotspots of deleterious RAD51D variants and uncover the mechanisms by which variants compromise its biochemical functions. HighlightsO_LIUsed a multiplexed assay of functional effect (MAVE) to assess the functionality via olaparib sensitivity of 6,888 RAD51D coding variants, which can be used for variant classification C_LIO_LIProvided cellular functional analysis for 70 clinically-identified breast and ovarian cancer RAD51D variants C_LIO_LIIdentified key regions and enzymatic activities of RAD51D critical for its function in the BCDX2 and the X3CDX2 complexes C_LIO_LIDetermined mechanism of RAD51D-mediated regulation of BCDX2 ATPase activity C_LI

cancer biology↗

bioRxiv2026

CELLWHISPER: INFERENCE OF DIRECT CELL-CELL COMMUNICATION FROM SPATIAL TRANSCRIPTOMICS

AbstractDirect, contact-dependent cell-cell communication shapes tissue physiology and disease. Spatial transcriptomics offers a unique opportunity to infer such communication mechanisms, but the few existing tools for such inference are prone to high error rates due to spatial structure in distribution of cell types and gene expression. We present CellWHISPER, a statistical framework for inferring contact-mediated cell-cell communication, including gap junctions and ligand-receptor mechanisms, from single-cell resolution spatial data. CellWHISPER achieves strict error control via a specialized hypothesis-testing procedure that accounts for the confounding effects of cell- type-specific expression and spatial organization. Its use of an exact closed-form significance score obviates expensive permutation testing and enables scalable analysis of large tissues an extensive signaling compendia. To uncover salient communication patterns, CellWHISPER also includes a latent variable model that distills recurrent patterns into maps of mutual preferences among cell types and signaling genes. Applied to mouse brain STEREO-seq and Xenium datasets, CellWHISPER identifies cell type-specific gap-junction coupling and yields a comprehensive connexin-interaction map. We experimentally confirmed Connexin 43-mediated interfaces among astrocytes, microglia and endothelial cells, which were among the highest- ranked predictions; in contrast, existing approaches fail to prioritize these confirmed interactions. Differential analysis of an Alzheimers disease model versus wild-type identifies preserved astroglial coupling but increased microglia-associated gap-junction communication. In summary, CellWHISPER disentangles direct cell-cell communication from structural proximity, enabling statistically robust and computationally scalable inference from spatial transcriptomics, leading to the discovery of tissue- and disease-associated contact-mediated signaling programs.

bioinformatics↗

bioRxiv2026

Bedaquiline inhibits the ATP synthase leak channel and prevents glutamate-induced neuronal death

FOF1-ATP synthase is one of the most abundant proteins of the mitochondrial inner membrane and the primary enzyme responsible for ATP production in eukaryotic cells. Nevertheless, it was recently reported to play a prominent role in cell death by forming a large-conductance leak channel in the mitochondrial permeability transition pore (mPTP), making it a promising therapeutic target. Bedaquiline (BDQ), a member of the diarylquinoline class of drugs, was shown to selectively inhibit the catalytic activity of Mycobacterium tuberculosis ATP synthase with no effect on the mammalian enzyme. Here, we report a new role for BDQ as a potent inhibitor of the ATP synthase c-subunit leak channel in mammals. BDQ inhibited the single-channel activity of porcine heart ATP synthase in planar lipid bilayer recordings and prevented glutamate-induced cell death in primary hippocampal neurons. These findings reveal the potential new application of BDQ for treating mPTP-related diseases by targeting the ATP synthase c-subunit leak channel. Why it mattersBedaquiline (BDQ) is the only FDA-approved drug to treat pulmonary multidrug-resistant tuberculosis (TB), caused by the Mycobacterium tuberculosis. BDQ cures TB by specifically targeting mycobacterial ATP synthase and inhibiting ATP production. Recently, BDQ was also reported to bind to mammalian ATP synthase at the interface between the a and c-subunits and to inhibit its catalytic activity. However, the effect of BDQ on ATP synthase leak channel activity has not been explored. Here, we report that BDQ inhibits the ATP synthase c-subunit leak channel (ACLC) activity with an IC50 of [~]24 nM and prevents glutamate-induced neuronal death, suggesting a new therapeutic repurposing of BDQ for treating ACLC-related diseases.

biophysics↗