Biology subjects

Acharya, M.

Publications and source records attributed to Acharya, M..

12 recordsLinked to original sources

bioRxiv2026

Human alpha-defensin 5 stabilizes the enterovirus A71 capsid and blocks infection

Human alpha-defensins are antimicrobial peptides abundantly expressed in neutrophils and the small intestine. They block infection of several families of non-enveloped DNA viruses by binding to and stabilizing the viral capsid during entry, thereby preventing the genome from reaching the nucleus to initiate replication. It is unclear if a similar mechanism also applies to RNA viruses. To study this further, we investigated the interaction of human alpha-defensin 5 (HD5) with enterovirus A71 (EV-A71). We found that HD5 disrupts EV-A71 infection in cell culture and blocks viral entry. HD5 binds directly to the EV-A71 capsid and disrupts key conformational changes essential to the initiation of in vitro uncoating as well as downstream viral genome release. Using a suite of HD5 point mutants, we found that these two uncoating blocks are separable, and HD5 must achieve both to fully neutralize EV-A71 infection. This work advances our understanding of alpha-defensin antiviral action and demonstrates several conserved features of HD5 inhibition that expand to a clinically important RNA virus. Author SummaryIn this study, we demonstrated that human defensin 5 (HD5) inhibits infection of pathogenic enterovirus A71 (EV-A71). HD5 binds to EV-A71 and disrupts in vitro viral uncoating by stabilizing the viral capsid and blocking genome release. Using a library of HD5 mutants, we identified a continuous surface within HD5 important for binding to EV-A71 and disrupting infection. This surface includes amino acid residues previously shown to be important for inhibition of human adenovirus 5 and human papillomavirus 16 and represents a conserved interface involved in broad-spectrum HD5 antiviral activity. We determined that capsid stabilization and blocked genome release are independent modes of HD5 action and identified individual HD5 mutants with separable functions. These mutants provide valuable tools to further study the biology of EV-A71 uncoating. Overall, this work demonstrates the first example of an enterovirus inhibited by an -defensin and advances our mechanistic understanding of HD5 antiviral action to a new pathogenic virus.

microbiology↗

bioRxiv2026

Spatial Transcriptomics reveals a T cell-mediated microglial activation axis of neurodegeneration following immune checkpoint inhibition

Immune checkpoint inhibitor (ICI) combinations that block cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death protein 1 (PD-1) signaling have revolutionized cancer care but also exert a range of immune-related adverse events (irAE) in various tissues, including the brain. Our understanding of the mechanisms of irAE in the brain is still evolving, and we recently demonstrated that ICI (blockade of CTLA-4 and PD-1) perturbs hippocampal-dependent memory function by derailing neuro-immune homeostasis and compromising synaptic integrity. However, the spatial patterns and the cell-type-specific molecular mechanisms underlying ICI-related brain dysfunction remain not well-defined. To address this gap, we performed spatial transcriptomic profiling of the hippocampal region using multiplexed error-robust fluorescence in situ hybridization (MERFISH) to map gene expression at single-cell resolution. By integrating spatial single-cell data with bulk RNA-seq, we define the distribution of microglia, astrocytes, synaptic, and neuroinflammatory markers, and determine how ICI reshapes hippocampal cellular composition in a syngeneic murine melanoma model. MERFISH revealed upregulation of microglial, astrocytic, oligodendrocytic, and T cell markers post-ICI treatment, revealing unique pathways driving neuroinflammation, synaptic function, and cellular signaling. Furthermore, immunofluorescence analysis of postmortem brains from patients treated with ICI corroborates our findings of ICI-related immune activation of microglia. Finally, using a conditional deletion model, we show that T cells are indispensable for ICI-driven microglial activation. Altogether, our study provides a high-resolution spatial framework for understanding irAEs in brain function and a T cell-microglia crosstalk axis as a driving mechanism of dysregulated neuro-immune homeostasis during ICI.

immunology↗

bioRxiv2026

Outbred Drosophila populations reveal diet-dependent genetic effects on development time

Metabolic effects of genetic variation often depend on diet, yet the loci underlying diet-dependent developmental responses remain incompletely defined. Here we combine multi-trait phenotyping of Drosophila Genetic Reference Panel lines with genome-wide mapping in newly developed Drosophila Recombinant Populations. High sugar causes genotype- and life-stage-dependent changes in metabolic and life-history traits, with development time emerging as a highly heritable, sugar-sensitive phenotype. Mapping in 16 outbred advanced intercross populations reveals distinct association landscapes under low- and high-sugar diets, with a concentrated low-sugar signal, a more distributed high-sugar pattern, and identified genotype-by-diet loci including tap, Eip75B and Cerk. Functional perturbation supports diet-dependent effects for several prioritized candidates. Allele-frequency analyses identify operationally defined thrifty-like variants associated with delayed development under high sugar and relatively earlier development under low sugar; these variants are enriched for cell-adhesion, neurodevelopmental, and morphogenetic processes. Together, these results establish an outbred Drosophila framework for dissecting how dietary sugar remodels the genetic architecture of development time.

genetics↗

bioRxiv2026

Lineage-specific chromatin poising enforced by ETS-IRF composite elements determines the divergent interferon responses of plasmacytoid dendritic cells and epithelial cells

Plasmacytoid dendritic cells (pDCs) produce robust type I interferons (IFN-I) within hours of viral sensing, while epithelial cells at mucosal surfaces mount a delayed response dominated by type III interferons (IFN-III). Both cell types express pattern recognition receptors that activate similar downstream transcription factors, yet they produce distinct subsets of IFNs. The mechanisms underlying these differences have remained unclear. Here, using Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) in primary human pDCs and intestinal epithelial cells, we show that IFN-I and IFN-III gene loci carry opposing, constitutively established chromatin accessibility landscapes that determine cell-type-specific interferon induction. The IFN-I locus is broadly open in pDCs and constitutively closed in epithelial cells, while the IFN-III locus displays the reciprocal pattern. Motif enrichment analysis of accessible regions at the IFN-I locus in pDCs revealed unexpected and significant enrichment of ETS family binding motifs alongside IRF motifs, which determines the cell-type-specific locus accessibility. The ETS factor PU.1 and IRF8 bind IFN-I promoters, at composite ETS-IRF elements positioned at the Positive Regulatory Domain IV (PRDIV) site of IFNB1 promoter and adjacent to the TATA-proximal IRF motif of IFNA promoters. IFNL gene promoters lack ETS recognition sequences. PU.1-IRF8 composite factor binding extends across intergenic regions of the IFN-I locus, where candidate enhancer elements marked by H3K4me1, H3K27ac, and RNA Pol II occupancy were identified. We propose that this network of ETS-IRF composite-element-anchored enhancers maintains the IFN-I locus in a constitutively poised state in pDCs, licensing the rapid and robust IFN-I response that defines pDCs. In epithelial cells, the absence of PU.1 and IRF8 renders the IFN-I locus epigenetically silent, while the IFN-III locus is constitutively open. Despite this, the delayed IFN-III gene expression in epithelial cells is due to intrinsically weaker promoter activity relative to IFN-I. These findings reveal that the divergent IFN induction of pDCs and epithelial cells is determined by the chromatin architecture prior to infection. Overall, these observations show that lineage-specific ETS-IRF regulatory factors and promoter strength determine the cell-type-specific IFN activation.

immunology↗

bioRxiv2026

KCa3.1 Drives Pro-Fibrotic Activation and Represents a Novel Therapeutic Target in Aortic Stenosis

IntroductionAortic stenosis (AS) is characterised by progressive aortic valve (AV) leaflet fibrosis and calcification, yet no medical therapies exist to slow disease progression. AV interstitial cells (VICs) that differentiate into myofibroblasts are central drivers of fibrosis. The Ca2+-activated K+ channel KCa3.1 promotes pro-fibrotic signalling in several fibrotic diseases, however its role in AS remains unknown. MethodsKCa3.1 protein expression was examined in paraffin embedded tissue by Immunohistochemistry from control and AS valve tissue. VICs were isolated, cultured and phenotypically characterised as myofibroblasts from AV tissue obtained from patients with severe tricuspid AS undergoing surgical AV replacement (n=19). KCa3.1 mRNA and protein expression were assessed by qRT-PCR and immunohistochemistry, and functional channel activity confirmed using patch-clamp electrophysiology. The effects of transforming growth factor-{beta}1 (TGF{beta}1) stimulation and pharmacological inhibition with the selective KCa3.1 blocker senicapoc were examined. ResultsImmunoreactive KCa3.1 channels and smooth muscle actin were detected in both control and AS aortic valve tissue, localised to elongated, nucleated interstitial cells, with significantly higher expression observed in AS tissue compared to control. Isolated VICs exhibited an activated myofibroblast phenotype, expressing THY-1, vimentin, collagen and -smooth muscle actin (SMA) (n=9). Myofibroblasts expressed KCa3.1 mRNA and protein and demonstrated functional plasma membrane channels. TGF{beta}1 stimulation increased KCa3.1, SMA and collagen type I mRNA expression, while KCa3.1 blockade with senicapoc (100 nM) significantly attenuated TGF{beta}1-induced SMA expression, stress fibre formation and collagen gel contraction. Senicapoc had no effect on myofibroblast proliferation or migration. ConclusionsWe show for the first time that functional KCa3.1 channels are expressed in human AS tissue and AV myofibroblasts, where they regulate myofibroblast contraction, -SMA expression, and differentiation, promoting pro-fibrotic activity. These responses are attenuated by the selective KCa3.1 inhibitor senicapoc. Given its established safety in phase 3 clinical trials, KCa3.1 inhibition represents a promising and readily translatable anti-fibrotic therapeutic strategy for AS.

cell biology↗

bioRxiv2025

Novel truncating Desmin mutation Arg150Stop disrupts structural integrity and cellular homeostasis by formation of persistent aggregate-like structure

Desminopathies are a heterogeneous group of myofibrillar myopathies defined by the presence of desmin-positive aggregates that compromise cytoskeletal integrity in skeletal and cardiac muscle. Although desmin knockout models and several truncating mutations typically result in a functional null phenotype without inclusion body formation, the molecular consequences of specific stop-gain variants remain poorly understood. In this study, we investigated the pathogenic mechanism of a novel DES nonsense mutation, NM_001927.4:c.448C>T; p.(Arg150Stop), previously identified in an Indian patient with congenital myopathy. This premature stop codon lies within the Linker 1A domain and is predicted to generate a truncated protein lacking the C-terminal tail. To delineate its functional consequences, we used two complementary experimental approaches: transient overexpression of the R150X mutant in skeletal and cardiac myocytes, and a CRISPR/Cas9-engineered homozygous R150X cardiomyocyte line (Des-R150X-CRISPR). Both models consistently revealed the formation of persistent aggregate-like structures, in striking contrast to desmin knockout systems that do not generate inclusions. These aggregate-like structures disrupted actin filament organization, impaired filament bundling, and induced organelle mislocalization. Biochemical analysis indicated that the aggregates were resistant to proteasomal degradation, yet they were partially cleared by autophagy, underscoring a role for protein quality control pathways in modulating disease severity. Importantly, the Des-R150X-CRISPR line demonstrated aggregate-driven pathology at endogenous levels, confirming that this mutation acts through a toxic gain-of-function mechanism rather than simple loss of desmin function. Our findings establish the Arg150Stop variant as a mechanistically distinct truncating mutation that generates aggregation-prone protein rather than a null state. By reproducing hallmark features of desminopathy in a physiologically relevant human cell models, this work not only broadens the known pathogenic spectrum of DES variants but also highlights aggregate formation as a central driver of cellular dysfunction and a promising therapeutic target in desminopathies.

cell biology↗

bioRxiv2024

TGFb-ANGPT2-Tie2 axis in cancer-associated fibroblasts reprograms oral cancer cells to embryonic-like cell state with predictive significance of poor prognosis

Myofibroblastic cancer-associated fibroblasts (CAFs) in tumor stroma is identified as poor-prognostic indicator in oral cancer; however, biological mechanisms are largely unexplored. Here, we discovered the role of autocrine or exogenous transforming growth factor beta (TGF{beta}) in inducing Tunica Interna Endothelial cell kinase 2 (Tie2) -signaling through histone deacetylase-mediated downregulation of Tie2-antagonist, Angiopoietin-2 in CAFs, responsible for induction and maintenance of myofibroblastic differentiation. To understand the influence of CAF-specific Tie2-signaling on cancer cell properties, we performed CAF-Cancer cell co-culture and its single-cell RNA sequencing (scRNA-Seq). Distinct clustering of CAFs suggested their transcriptional heterogeneity, driven by TGF{beta}-Tie2 activation. Interestingly, CAF-specific Tie2-signaling was responsible to reprogram cancer cells, producing embryonic-like cell state with increased stemness and EMT signatures. Importantly, both the Tie2-specific gene expression signature as well as reprogrammed cancer cell specific gene expression modules were validated respectively in fibroblasts clusters and malignant cell clusters in two independent earlier reported scRNAseq studies of HNSCC tumors. Highlighting the translatability of our study, the gene expression signature derived from reprogrammed cancer cells showed significant association with poor prognosis in HNSCC patient of TCGA cohort. Pharmacological inhibition of Tie2-signaling in CAFs, significantly abrogated the tumor initiating ability of co-cultured oral cancer cell lines. Overall, combining our molecular and computational analysis, we may propose Tie2 as a novel factor responsible for CAF mediated cancer cell plasticity, associated with aggressive nature of oral cancer. TeaserTie2-signaling is activated in myofibroblasts which impacts the behaviour of malignant cells by inducing cancer cell plasticity to acquire stemness.

cancer biology↗

bioRxiv2024

B cell αv integrin regulates germinal center derived lung-resident IgA B cell responses following influenza virus infection

Lung-resident B cells are increasingly recognized as key contributors to protective immunity against respiratory viruses, yet the mechanisms that govern their generation and specialization remain poorly understood. Here, we identify B cell-intrinsic v integrin as a critical negative regulator of germinal center (GC) dynamics and memory B cell formation in the lung following influenza A virus (IAV) infection. Using B cell-specific v knockout mice, we show that loss of B cell v integrin leads to persistent GC activity within the inducible bronchus-associated lymphoid tissue and expansion of lung-resident memory B cells, including IgA+ and cross-reactive B cells capable of recognizing heterologous influenza variants. Single-cell transcriptomic and B cell receptor (BCR) sequence analyses reveal that v restricts clonal expansion and antigenic diversification of GC and memory B cells in the lung, but not in draining lymph nodes, indicating a spatially restricted mechanism of mucosal B cell regulation. These findings position v integrin as a key checkpoint that constrains local mucosal B cell evolution and suggest new strategies to improve mucosal vaccine efficacy by enhancing GC activity directly in the lung.

immunology↗

bioRxiv2024

Post-GWAS functional analyses of CNTNAP5 suggests its role in glaucomatous neurodegeneration

Primary angle closure glaucoma (PACG) affects more than 20 million people worldwide, with an increased prevalence in south-east Asia. In a prior haplotype-based GWAS, we identified a novel CNTNAP5 genic region, significantly associated with PACG. In the current study, we have extended our perception of CNTNAP5 involvement in glaucomatous neurodegeneration in a zebrafish model, through investigating phenotypic consequences pertinent to retinal degeneration upon knockdown of cntnap5 by translation-blocking morpholinos. While cntnap5 knockdown was successfully validated using an antibody, immunofluorescence followed by western blot analyses in cntnap5-morphant (MO) zebrafish revealed increased expression of acetylated tubulin indicative of perturbed cytoarchitecture of retinal layers. Moreover, significant loss of Nissl substance is observed in the neuro-retinal layers of cntnap5-MO zebrafish eye, indicating neurodegeneration. Additionally, in spontaneous movement behavioural analysis, cntnap5-MO zebrafish have a significantly lower average distance traversed in light phase compared to mismatch-controls, whereas no significant difference was observed in the dark phase, corroborating with vision loss in the cntnap5-MO zebrafish. This study provides the first direct functional evidence of a putative role of CNTNAP5 in visual neurodegeneration.

genetics↗

bioRxiv2023

Antigen receptor signaling and cell death resistance controls intestinal humoral response zonation.

Immunoglobulin A (IgA), the main antibody isotype found in the intestine, has evolved to maintain the stability of commensal communities, and prevent dysbiosis. In stark contrast to systemic antibody response against pathogens, the generation of IgA against intestinal resident microbes assures the simultaneous binding to multiple and diverse commensal-derived antigens. However, the exact mechanisms by which B cells mount such broadly reactive IgA response to the gut microbiome at the mucosal barrier remain elusive. Here we show surface IgA B cell receptor (BCR) is required to confer enhanced B cell fitness during the germinal center reaction in Peyers patches and to mediate selection of gut-homing plasma cells with higher efficiency. We demonstrate that, upon antigen stimulation, IgA+ BCR drives greater intracellular signaling in mouse and human B cells and as consequence, IgA+ B cells received higher positive selection cues in the germinal center. Mechanistically, in vivo IgA BCR signaling offsets Fas-mediated cell death to rescue low affinity B cell clones and redirects the humoral response to an increased variety of commensal strains at the intestinal interface. Our findings revealed a new mechanism linking tissue-specific antigen receptor signaling with B cell fate and localization of antibody production; and have implications for understanding how intestinal antigen recognition shapes humoral immunity in health and disease.

immunology↗

bioRxiv2021

A screening pipeline identifies a broad-spectrum inhibitor of bacterial AB toxins with cross protection against influenza A virus H1N1 and SARS-CoV-2

A challenge for the development of host-targeted anti-infectives against a large spectrum of AB-like toxin-producing bacteria encompasses the identification of chemical compounds corrupting toxin transport through both endolysosomal and retrograde pathways. Here, we performed a high-throughput screening of small chemical compounds blocking active Rac1 proteasomal degradation triggered by the Cytotoxic Necrotizing Factor-1 (CNF1) toxin, followed by orthogonal screens against two AB toxins hijacking defined endolysosomal (Diphtheria toxin) or retrograde (Shiga-like toxin 1) pathways to intoxicate cells. This led to the identification of the molecule N-(3,3-diphenylpropyl)-1-propyl-4-piperidinamine, referred to as C910. This compound induces the swelling of EEA1-positive early endosomes, in absence of PIKfyve kinase inhibition, and disturbs the trafficking of CNF1 and the B-subunit of Shiga toxin along the endolysosomal or retrograde pathways, respectively. Together, we show that C910 protects cells against 8 bacterial AB toxins including large clostridial glucosylating toxins from Clostridium difficile. Of interest, C910 also reduced viral infection in vitro including influenza A virus subtype H1N1 and SARS-CoV-2. Moreover, parenteral administration of C910 to the mice resulted in its accumulation in lung tissues and reduced lethal influenza infection.

microbiology↗

bioRxiv2021

Transcriptomic analysis of pathways associated with alpha(v) integrin-related non-canonical autophagy in human B cells

Autophagy proteins have been linked with development of immune-mediated diseases including lupus, but the mechanisms for this are unclear. We have previously shown that non-canonical autophagy induced by v-integrins regulates B cell activation by viral and self-antigens in mice. Here we investigated the involvement of this pathway in B cells from human tissue. Our data revealed that autophagy is specifically induced in germinal-center and memory B cell sub-populations from human tonsil and spleen. Transcriptomic analysis showed that induction of autophagy is related to unique aspects of activated B cells such as mitochondrial metabolism. To understand the function of non-canonical autophagy in B cells, we used CRISPR-mediated knockdown of autophagy genes. Integrating data from primary B cells and knockout cells we found that v-integrin-related non-canonical autophagy limits activation of specific pathways while promoting others. These data provide new mechanistic links for autophagy and immune dysregulation in diseases such as lupus.

immunology↗