Biology subjects

Chowdhury, S.

Publications and source records attributed to Chowdhury, S..

At least 19 recordsLinked to original sources

bioRxiv2026

Effect of Neck Muscle Strength and Passive Cervical Viscoelasticity on Regional Brain and Axonal Tissue Response

This study evaluated how neck muscle strength and passive cervical viscoelasticity influence global brain motion, intracranial pressure, and regional predicted axonal strain during rapid head loading. An MRI-derived human head-neck finite element model with 5000 diffusion-derived axonal tracts represented by 90,692 embedded beam elements was evaluated using a 4 by 4 factorial design, with four levels each of muscle strength and passive ligament/disc viscoelasticity. The 16 cervical configurations were simulated under a 15 g acceleration-driven inertial load and a 7000 N frontal impact, resulting in 32 simulations. Increasing muscle strength from S25 to S95 at the reference passive-viscoelasticity level reduced mean regional 95th-percentile predicted axonal strain by 22% under inertial loading and 18% under frontal loading, with regional reductions ranging from 10% to 58% and 7% to 31%, respectively. Increasing passive cervical viscoelasticity from D25 to D95 at the reference strength level produced larger reductions in mean regional axonal strain of 35% and 33%, with regional reductions ranging from 14% to 70% and 16% to 56%. Passive viscoelasticity also reduced brain center-of-mass angular acceleration by 16% and 13%, compared with reductions of only 1% and 2% with increased muscle strength. Regional intracranial pressure decreased by 9%~21% under inertial loading and 5%~12% under frontal loading with increased passive viscoelasticity, whereas greater muscle strength reduced axonal strain without a corresponding reduction in pressure. The effects of the two cervical factors also depended on one another: under inertial loading, the mean axonal-strain reduction from increasing strength increased from 21% at D25 to 28% at D95, while the corresponding passive-viscoelasticity effect increased from 32% at S25 to 38% at S95. These findings show that cervical mechanical properties can substantially alter regional brain and axonal response, with passive cervical viscoelasticity producing larger effects than muscle strength across the ranges evaluated.

bioengineering↗

bioRxiv2026

Topological Complementarity Predicts Synergistic Drug Pairs in Branched Metabolic Networks.

Drug discovery typically ranks targets by individual sensitivity. Branched metabolic networks, in contrast, have redundant flux routes that buffer single-enzyme inhibition possibly deviating the best target from the most sensitive node. We developed a topology-driven framework for identifying optimal dual-target pairs and applied it to the E. coli folate pathway. A kinetic model of the folate pathway and its extended one-carbon pool showed that single-node inhibition is compensated by flux redistribution. Pairwise synergy scoring, a metric to measure drug-pair potency as compared to single drug, identified DHPS (folP) and DHFR (folA) as a top-ranked complementary pair. Experimental validation in E. coli MG1655 using sulfamethoxazole and trimethoprim confirmed strong dose-dependent synergy at intermediate inhibition and attenuation at saturation. Our results support topological complementarity as a key predictor of combination efficacy and provide a rationale for drug-pair design in branched biosynthetic pathways.

systems biology↗

bioRxiv2026

Xist-mediated gene silencing promotes T cell-driven autoimmunity

Four out of five individuals affected by autoimmune disease are female. Yet, sex-specific factors that drive autoimmune predisposition in females remain poorly understood. Recent studies have implicated the female-specific long non-coding RNA Xist in autoimmunity through its ability to activate immune responses as a TLR7 ligand and through the immunogenic properties of Xist-associated proteins, stimulating dendritic cells and B cells. Here, we identify a T cell-intrinsic role for Xist in promoting autoimmunity. T cell-specific Xist deletion in mice had minimal effects on T cell populations at rest but impaired T cell fitness and increased the frequency of regulatory T cells (Tregs) in a competitive transfer setting. Xist deficiency derepressed X-linked genes, including Foxp3, the lineage-defining transcription factor for Tregs and Sh2d1a, encoding the SLAM-associated adaptor SAP, which regulates T cell activation and contraction and is linked to monogenic colitis. Finally, Xist deficiency impaired the colitogenic capacity of CD4+ T cells in an adoptive transfer model. Together, these findings reveal a previously unrecognized role for Xist-mediated gene silencing in supporting CD4+ T cell persistence and colitogenic capacity.

immunology↗

bioRxiv2026

Multiplexed hormone immunoassays for women's health on a regenerable silicon photonic chip

Hormone dynamics are central to a multitude of conditions in women's health; however, limitations in the cost, convenience, and multiplexing capacity of current hormone assays preclude routine longitudinal hormone testing. This limitation severely restricts our understanding in this historically neglected field, presenting a need for improved bioanalytical tools to support hormone-informed care. Silicon photonic resonator sensors have excellent multiplexing capacity and scalable fabrication, and offer strong potential to meet this important need. Here we demonstrate, for the first time, multiplexed silicon photonic detection of two urinary hormone biomarkers, follicle-stimulating hormone (FSH) and pregnanediol-3-glucuronide (PdG), using sub-wavelength grating ring resonators and a polydopamine-based, regenerable surface chemistry. We combine a sandwich immunoassay for FSH and an antigen-down competitive immunoassay for PdG on the same chip, both amplified using a commercially available quantum-dot-streptavidin conjugate and with on-chip regeneration over multiple measurement cycles. In artificial urine, this proof-of-principle multiplexed assay achieves limits of detection of 0.594 ng/mL for FSH and 19.1 ng/mL for PdG. This architecture is inherently scalable to tens of hormone targets on a millimetre-scale chip (a >10x multiplexing improvement over typical lateral-flow immunoassays) enabling future low-cost, quantitative at-home hormone panels that could transform research, diagnostics, and treatment in women's health.

bioengineering↗

bioRxiv2026

KDM6A Loss Confers an Invasive Phenotype in Osteosarcoma by Activating Cytoplasmic YAP-Dependent β-catenin Stabilisation

Osteosarcoma (OS) is the most common primary malignant bone tumour and is characterised by aggressive growth, early metastasis, and a very stagnant clinical outcome. Although epigenetic dysregulation has been implicated in OS progression, the mechanisms linking epigenetic alterations to metastatic signalling remain unclear. Here, we identified the lysine-protein demethylase 6A (KDM6A/UTX) as a critical suppressor of OS metastasis and uncovered a novel regulatory axis involving the Hippo/YAP and Wnt/{beta}-catenin signalling. Initial bioinformatics analyses revealed frequent KDM6A alterations and significantly reduced expression in OS patient datasets, which correlated with metastatic disease and poor prognosis. Functional inhibition of KDM6A by pharmacological inhibitors and siRNA induced a hyper-invasive phenotype, marked by elevated mesenchymal markers, enhanced cytoskeletal remodelling, increased transendothelial adhesion and decreased chemotherapeutic drug sensitivity. Importantly, restoration of KDM6A expression effectively counteracted these effects. Mechanistically, KDM6A loss activated Wnt/{beta}-catenin signalling, resulting in nuclear translocation of {beta}-catenin and transcriptional activation of genes associated with stemness and invasion. Therefore, inhibition of {beta}-catenin reversed the invasive phenotype. Further analysis revealed that KDM6A regulated Hippo signalling through epigenetic control of the negative regulator of Yes-Associated Protein (YAP)-LATS1. KDM6A inhibition led to enrichment of H3K27me3, a repressive mark, at the LATS1 promoter. Accumulated YAP was predominantly localised in the cytoplasm, where it interacted with GSK3{beta} and contributed to the stabilisation of {beta}-catenin by preventing its proteasomal degradation. Collectively, our findings identify a novel KDM6A-LATS1-YAP-{beta}-catenin signalling axis that drives metastatic progression in OS.

cancer biology↗

bioRxiv2026

Co-targeting Metabolic Neighbours Constraints Bacterial Adaptive Evolution

Antibiotic resistance often arises through genetic changes that reduce drug susceptibility, with mutation representing one of the primary mechanisms. Here, we report a dual targeting strategy that restricts these escape routes by simultaneously inhibiting two metabolic neighbours in folate biosynthesis pathway. To establish a general framework for limiting adaptive escape in bacteria, we screened the folate pathway to identify enzymes with shared conserved structural features. This analysis identified dihydroneopterin aldolase (folB) and dihydroneopterin triphosphate 2'-epimerase (folX) as optimal dual targets due to their shared pocket architecture and their roles early in the folate pathway. We then screened a large chemical space of FDA-approved small molecules to identify candidates predicted to bind both folB and folX, followed by experimental validation. We identified Arbutin showing the strongest growth inhibition in E. coli and emerging as the potent dual-target inhibitor, validated by metabolic rescue assays that confirmed direct inhibition of both folB and folX. Experimental evolution further revealed that populations exposed to Arbutin exhibit constrained adaptive trajectories and minimal increases in tolerated drug concentration, consistent with the evolutionary bottleneck imposed by dual-enzyme inhibition. Together, these results establish a framework for developing evolution-resistant antibacterials by targeting structurally convergent enzymes within metabolic pathways.

biophysics↗

bioRxiv2026

Endothelial cell RpL17-dependent translational control mediates intima-media thickening in response to disturbed flow

BackgroundCarotid intima-media thickening (IMT) is a major risk factor for cardiovascular disease (CVD). The large ribosomal subunit protein 17 (Rpl17) was recently reported as a CVD-associated gene; however, ribosomal mutations generally are not associated with vascular dysfunction. We have created a novel genetic model of decreased RpL17 in endothelial cells (EC) to determine how changes in endothelial ribosome expression cause IMT. MethodsEC-restricted RpL17 heterozygous mice (Cdh5-Cre; RpL17fl/wt, or Rpl17-Het), were generated and subjected to sham or partial carotid ligation (PCL) surgery of the left artery to induce acute disturbed (d)-flow in vivo. Carotids were harvested on day 14 for quantitative tissue immunostaining. Purified mouse and human EC in vitro were exposed to steady (s)-flow or d-flow using cone viscometry, and collected for flow cytometry, protein expression, electron microscopy, or purification of ribosomes. Human carotid samples from healthy and endarterectomy patients were used for tissue analysis. ResultsCarotids from RpL17-Het mice with PCL-induced d-flow showed increased IMT relative to RpL17-WT controls. In addition, RpL17 protein levels were decreased in regions of d-flow compared to s-flow. Increased levels of ER stress markers were observed by carotid immunostaining, as well as activation of the integrated stress response (ISR) in RpL17-Het EC. Analysis of mRNAs bound to polysomes vs. monosomes in EC-RpL17-Het revealed increased translational efficiency of key regulators of glycolysis, redox, inflammation, matrix, and endothelial-to-mesenchymal transition (EndMT). Metabolic profiling by Seahorse assay showed enhanced anaerobic glycolysis and decreased oxidative respiration in RpL17-Het EC, consistent with the translational efficiency data. Immunostaining of carotids identified upregulated EC inflammation and EndMT. ConclusionsOur data support RpL17 as a key mediator of EC phenotypic modulation that causes IMT in response to d-flow. We show a novel pathway for d-flow-mediated IMT: endoplasmic reticulum stress and activation of the ISR. These changes alter translational efficiency and reprogram EC cell cycle, metabolism, and redox state in the presence of d-flow to cause IMT, a precursor to cardiovascular pathology.

cell biology↗

bioRxiv2026

Infection drives localised and individualised antibody repertoires, whereas immunisation promotes repertoire convergence in pig

Respiratory viruses elicit mucosal and systemic immunity, yet how tissue compartmentalisation and exposure route shape B cell repertoires remain unclear. Using the pig model, we characterised antibody repertoires in bronchoalveolar lavage, tracheobronchial lymph nodes, spleen, and blood following intranasal infection with pandemic influenza virus (pH1N1) or porcine respiratory coronavirus (PRCV), and after intramuscular PRCV immunisation. Infection induced highly compartmentalised responses, with antigen-associated clones enriched in lung, lymph nodes and spleen but with limited presentation in blood. These clones were largely private, indicating individualised responses, and displayed tissue-specific diversity. The similar tissue distributions between pH1N1 and PRCV infections suggest a conserved spatial organisation, although differential lymph node involvement indicates pathogen-specific effects. In contrast, intramuscular immunisation generated more convergent, public repertoires in blood, characterised by reduced clonal size diversity. Monoclonal antibody analysis revealed functional heterogeneity and limited overlap with bulk repertoires. Together, these findings show that despite stable germline usage, functional antibody responses are shaped by the route of antigen exposure, clonal selection, and host specific factors, resulting in distinct repertoire architectures between infection and immunisation. Author summaryRespiratory viruses such as influenza and coronaviruses infect the airways and lungs, where they trigger immune responses that help the body fight infection. Most studies of immunity rely on blood, although many immune cells function in tissues like the lungs. Therefore, we may be missing important parts of the immune response. We used pigs, a large animal model that closely resembles humans in respiratory biology and is naturally infected with influenzas and coronaviruses, to investigate how immune responses are shaped by infection compared with intramuscular vaccination. We examined B cells in lungs, nearby lymph nodes, spleen, and blood after infection with influenza or a porcine coronavirus and compared these with responses after vaccination. We found that infection generates distinct, individual immune responses in the lung and lymphoid organs. Vaccination by injection led to more similar responses across animals, mostly in the blood. This suggests the way the immune system encounters an antigen influences the response generated. We also showed that antibodies with similar binding properties can behave very differently in their ability to neutralise viruses. Our findings show that infection and vaccination shape immunity in fundamentally different ways, with important implications for designing vaccines that better protect against respiratory diseases.

immunology↗

bioRxiv2026

Transient efferocytosis-induced activation of IKKβ reprograms macrophages to promote tissue resolution

The clearance of apoptotic cells by macrophages, termed efferocytosis, reprograms macrophages to a resolution/repair phenotype, and pathologic defects in efferocytosis drive many chronic inflammatory diseases. Previous studies have elucidated numerous downstream pro-resolving pathways activated by efferocytosis, but whether there exists a common upstream trigger of these pathways remains unknown. Here, we report that efferocytosing macrophages surprisingly use a signaling module typically associated with inflammation to carry out this key initiating role in tissue resolution. The binding of apoptotic cells to the MerTK receptor triggers a rapid and transient activation of inhibitor of nuclear factor (NF) kappa-B kinase subunit beta (IKK{beta}), leading to NF{kappa}B and p38-signal transducer and activator of transcription 3 (STAT3) signaling and then activation of several key downstream pro-resolving pathways, including interleukin-10 (IL-10) production, continuing efferocytosis, and regulatory T (Treg) cell expansion. The upstream IKK{beta} pathway and the downstream resolution pathways are linked through several intermediary molecules, including the transcription factor Myc, the epigenetic modifier ten-eleven translocation-2 (TET2), and the immune checkpoint protein programmed cell death ligand 1 (PD-L1). Deletion of macrophage IKK{beta} in vivo blocks the above resolution pathways and compromises tissue repair in two efferocytosis-mediated repair settings: resolution of thymic injury after dexamethasone-induced thymocyte apoptosis; and, most importantly, atherosclerosis regression induced by low-density lipoprotein (LDL)-lowering, which is highly relevant to the prevention of cardiovascular disease in humans. These findings illustrate the existence of a unifying upstream signal for efferocytosis-induced resolution, which could suggest new therapeutic strategies to enhance multiple tissue resolution pathways and to optimize anti-inflammatory therapies by avoiding blocking IKK{beta}-NF{kappa}B/p38-mediated resolution.

immunology↗

bioRxiv2026

Integron-Mediated Convergence of Carbapenemase and Disinfectant Resistance in Acinetobacter spp. from Critical Care Units

Acinetobacter spp. represents critical opportunistic pathogens driving severe bloodstream infections (BSIs) in intensive care unit (ICU) and neonatal intensive care unit (NICU) settings. The convergence of carbapenem resistance and emerging biocide tolerance, often mediated by mobile genetic elements, has intensified concerns regarding co-selection and persistence in clinical environments. A total of 90 molecularly confirmed Acinetobacter isolates (ICU = 44; NICU = 46) from bloodstream infections were analyzed. Antimicrobial susceptibility was determined using the Kirby-Bauer disk diffusion method in accordance with CLSI M100 (2024) guidelines and extended-spectrum {beta}-lactamase production was assessed by combined disc diffusion. Polymerase chain reaction (PCR) was employed to detect carbapenemase genes (blaVIM, blaNDM, blaIMP, blaOXA-23, blaOXA-58), biocide resistance determinants (qacE, qac{Delta}E1), and the class 1 integron-integrase gene (intI1). Multidrug-resistant (MDR) and extensively drug-resistant (XDR) phenotypes were identified in 71.1% (64/90) and 22.2% (20/90) of isolates, respectively. High resistance (>71%) was observed against meropenem and cephalosporins, whereas colistin (58.8%, 53/90) and amikacin (47.8%, 43/90) showed moderate susceptibility. The most prevalent genotypes were qac{Delta}E1 (76.6%, 69/90) and blaVIM (56.6%, 51/90). Statistical and network analyses revealed significant correlations between biocide and carbapenemase genes, identifying IntI1 as a primary driver of co-resistance. The findings indicate a significant co-occurrence of carbapenemase genes, biocide resistance determinants, and class 1 integrons among Acinobacter spp. isolates. These associations suggest that mobile genetic elements may contribute to the dissemination of resistance traits.

microbiology↗

bioRxiv2026

Inverse-scattering of absorptive samples via beam propagation

Inverse-scattering methods enable label-free, quantitative visualization of a samples three-dimensional (3D) refractive index (RI), providing intrinsic and volumetric morphological contrast without exogenous labels. This is achieved by developing computational frameworks that reconstruct the samples 3D RI from a series of scattering measurements acquired under different data-capture conditions. Recent advances have demonstrated successful 3D RI reconstructions in multiple-scattering samples using angle-varying illuminations; however, these studies have primarily focused on non-absorptive samples. Here, we extend the multi-slice beam propagation (MSBP) inverse-scattering framework to reconstruct complex-valued RI, encompassing both the samples conventional RI (real part) and absorptivity (imaginary part). We show that reconstructing complex-valued RI makes the inverse problem ill-posed under angle-varying illumination alone, and that incorporating measurement diversity from both angle-varying illumination and sample defocus is necessary to ensure stable and accurate convergence. Experimental demonstrations were conducted on 1) dyed microsphere samples to characterize accuracy of reconstructed RI and absorptivity; and 2) diverse absorptive scattering samples to demonstrate biological utility. These results represent an important step for label-free volumetric imaging in biological tissue, which typically exhibits both scattering and absorption.

bioengineering↗

bioRxiv2026

KRAS inhibition is an effective therapy for appendiceal adenocarcinoma

BackgroundAppendiceal adenocarcinoma (AA) is a rare cancer with limited treatment options. KRAS is the most commonly mutated gene in AA and a promising therapeutic target, but its preclinical and translational relevance in AA remains unclear. MethodsWe evaluated KRASG12D-specific (MRTX1133) and pan-KRAS inhibitor (RMC-6236) in KRASmut organoid and orthotopic PDX models of AA. Tumor-intrinsic and microenvironmental responses were characterized using multi-omics profiling. Clinical outcomes were also assessed in six heavily pre-treated AA patients treated with KRAS inhibitors. ResultsMRTX1133 was highly effective for KRASG12D organoids (IC50=4.1 nM); both KRASG12D and KRASG12V organoids were sensitive to RMC-6236 (IC50=4.4 nM vs 0.5 nM, respectively). In orthotopic PDX models of peritoneal carcinomatosis from AA, MRTX1133 significantly reduced tumor growth in the KRASG12D model TM00351, and RMC-6236 reduced tumor growth in KRASG12V model AAPDX-16. Pathologic evaluation showed dramatically reduced tumor cellularity, proliferation, and pERK expression as well as induction of apoptosis. Gene Sets Enrichment Analysis (GSEA) revealed significant downregulations of E2F targets (NES=-1.9, p-adj=0.06) and the newly developed RAS/ERK (NES=-2.3, p-adj=0.06) gene set, consistent with the observed decrease in cell proliferation. There was marked upregulation of EMT (NES=2.7, FDR<0.001) and TGF-{beta} signaling (NES=2.3, FDR=0.004) in remaining tumor cells, suggesting these pathways could confer resistance. scRNA-seq analysis of TME showed dramatic shifts in cancer-associated fibroblasts (CAFs), with KRAS inhibition driving a shift from normal fibroblasts to inflammatory CAFs, and upregulation of interferon alpha and gamma pathways, suggesting that KRAS inhibition can activate innate immune response in the setting of peritoneal metastases. In a cohort of 6 heavily pre-treated patients with AA treated with KRAS inhibitors (1 G12D, 3 G12C, 2 pan-KRAS), all had biochemical response based on CEA/Ca19-9 or ctDNA and clinical benefit by RECIST criteria (1 CR, 1 PR, 4 SD). ConclusionsWhile effective suppression of RAS/ERK signaling by KRAS inhibitors reduces tumor growth, adaptive activation of EMT and TGF-{beta} pathways may mediate resistance in KRASmut AA. Additionally, KRAS inhibition remodels TME and may enhance innate immune signaling. These findings support continued clinical development of KRAS inhibitors in AA and provide a rationale for combination strategies targeting resistance pathways and stromal remodeling.

cancer biology↗

bioRxiv2026

Adaptive Landscapes of Plasmodium Falciparum Dihydrofolate Reductase Reveal Pathways to Antifolate Resistance

Antifolate resistance in Plasmodium falciparum dihydrofolate reductase (pfDHFR) remains a major challenge for malaria control. To understand how this enzyme maintains function under antifolate selection, we developed PfPATH, a computational framework that integrates mutational fitness measurements, evolutionary interactions, and structural information to map adaptive trajectories. PfPATH adaptive walks reveal that evolution is constrained to a narrow ridge in sequence space defined by a small subset of residues. Most resistance mutations are individually deleterious and become viable only when supported by stabilizing changes, restricting adaptation to a few high-resistance pathways. Structure network analyses show that these mutations do not disrupt the global enzyme architecture but instead reorganize internal communication to preserve catalytic function. Consistent with this, molecular dynamics and free-energy analyses indicate that resistant variants remain stable while sampling a broader ensemble of low-energy conformational substates. Together, these results reveal a narrow and predictable route to antifolate escape.

biophysics↗

bioRxiv2026

Mettl3-catalyzed m6A methylation determines CD8+ T cell differentiation fate in tumor

The heterogeneity in patient responses to immune checkpoint blockade (ICB) is dictated by the relative abundance of exhausted CD8 T cell (Tex) subsets with distinct therapeutic responsiveness. Progenitor exhausted (pTex) cells remain sensitive to ICB, whereas terminally exhausted (tTex) cells are refractory; however, the molecular cues that bias differentiation toward these divergent fates remain poorly defined. Here, we identify the RNA methyltransferase Mettl3 as a central regulator of Tex fate. Across murine tumor models, human T cells, and adoptive transfer systems, Mettl3 expression is selectively enriched in tTex cells and inversely correlated with TCF1 pTex populations. Mechanistically, Mettl3 drives terminal exhaustion by stabilizing DNMT3B transcripts via mA modification, enforcing CpG methylation and chromatin compaction at memory-associated loci. Inhibition of the Mettl3-Dnmt3b axis reprograms chromatin accessibility toward memory-like states, thereby preserving progenitor potential and effector function. Consequently, T cells lacking Mettl3-Dnmt3b activity persist longer, mount robust recall responses, and achieve superior tumor control with enhanced responsiveness to PD-1 blockade. These findings establish the Mettl3-mA-Dnmt3b axis as a molecular rheostat of CD8 T cell fate, coupling epitranscriptomic regulation to epigenetic remodeling, and reveal a tractable pathway to improve the durability of cancer immunotherapy.

immunology↗

bioRxiv2025

DRP1-mediated mitochondrial dynamics orchestrate EMT in glioblastoma cells

BackgroundEpithelial to mesenchymal transition (EMT), a differentiation process, frequently imparts invasive properties in Glioblastoma Multiforme (GBM), which leads to a poor prognosis. Cells lose apical-basal polarity, cell-cell connections, and/or chemo-resistance during EMT, which can result in the spread of cancer and the acquisition of additional stem cell-like traits. It is unclear how organelle dynamics influence EMT in this respect. The interaction between cytoskeletal and mitochondrial regulators governing GBM cell EMT is explored in this article. Results and DiscussionIn GBM cells, we observed that TGF-{beta}-induced EMT led to a proliferative arrest, which was accompanied by a fragmented mitochondrial morphology, elevated expression of fission markers such as DRP1, MFF, and FIS1, and most importantly, localization of mitochondria near the cell boundaries. An increase in mitochondrial ROS accompanied this, but their functional status was indicated by a higher oxygen consumption rate (OCR). Additionally, cytoskeleton re-distribution and EMT reversal were the outcomes of si-RNA-mediated elimination of the fission-marker DRP-1 or pharmacological inhibition of fission by Mdivi-1. On the other hand, drugs that disrupt the cytoskeleton, shifted the spatial distribution of mitochondria to the perinuclear area, which had an adverse effect on EMT. Notably, it was shown that RhoA, a protein that helps organize the actin cytoskeleton, co-immunoprecipitates with DRP1 and governs both cytoskeletal dynamics and mitochondrial fission in its presence. ConclusionOur research sheds substantial insight on the current interactions between the cytoskeleton and mitochondrial spatial dynamics that control EMT in GBM cells, which may have significant therapeutic implications. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=164 SRC="FIGDIR/small/694080v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@81b796org.highwire.dtl.DTLVardef@3bec9aorg.highwire.dtl.DTLVardef@2a79c3org.highwire.dtl.DTLVardef@9ff14a_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2025

A switch from histone methyltransferase-EZH2 to demethylase KDM6A activity marks reinitiation of proliferation of drug treated colorectal cancer cells

Colorectal cancer (CRC) is one of the deadliest cancers, ranking third in cancer incidence worldwide. These tumor cells often adopt unique strategies under drug stress to attain a reversible drug-tolerant state and evade cell death. However, the molecular adaptations associated with this transitory emergence of the drug-tolerant state remain elusive. Herein, epigenetic alterations often dictate such reversible dynamic changes, and this study aims to characterize the role of specific epigenetic modifiers governing CRC cell survival under drug pressure and their subsequent relapse. We observed that under drug stress there is a drastic increase in the histone-repressive mark-H3K27me3, linked to an enhanced expression of EZH2 driving transcriptional inhibition of cell proliferation-associated genes and a proliferative arrest. Interestingly, drug-induced oxidative stress increased the expression of the P65 protein, which was found to interact and regulate EZH2 expression. Quenching of ROS, drug vacation, or P65 inactivation compromised EZH2 activity concurrent with a re-initiation of cell proliferation. Interestingly, this reversal to proliferative state was associated with an elevated activity of the histone lysine demethylase-KDM6A. The promoter elements of the proliferative genes were now occupied by KDM6A instead of EZH2. Accordingly, a genetic knockdown or pharmacological inhibition of KDM6A in vitro not only resulted in increased cell death but also prevented emergence of the re-proliferative CRC cells. Furthermore, KDM6A inhibition in combination with chemotherapy drug, resulted in an increased tumor regression in vivo. Our study thus highlights the importance of KDM6A as a therapeutic target in preventing CRC growth and relapse which can have future therapeutic implications.

cancer biology↗

bioRxiv2025

Deep conservation of mitochondrial HSP60 structure with lineage-specific and context-dependent regulation reflects thermal resilience in cnidarians

Heat shock proteins (HSPs) are ubiquitous molecular chaperones that safeguard proteostasis under stress. We first investigated the expression dynamics of the mitochondrial chaperonin HSP60 across diverse cnidarians to understand its stress-responsive regulation. Using immunoblotting, we quantified HSP60 expression in Pocillopora acuta (reef-building coral), Exaiptasia diaphana (sea anemone), and Cassiopea xamachana (upside-down jellyfish). In P. acuta, HSP60 was not detected at the fragment scale under either control or heat stress, whereas isolated cells exhibited transient HSP60 expression during exposure to both control and heated temperatures (+5 {degrees}C above optimum), indicating that HSP60 regulation in this coral is strongly context-dependent and potentially suppressed at the tissue level. In contrast, E. diaphana and C. xamachana showed gradual, and temperature-dependent accumulation of HSP60 over 24 h under heated conditions (+5 {degrees}C above its thermal optimum), however C. xamachana also displayed constitutive basal expression under control conditions. These contrasting profiles highlight clear lineage-specific differences in HSP60 regulation among cnidarians. The consistent antibody cross-reactivity observed across all three species then prompted us to explore the evolutionary basis of this conservation. Phylogenetic analyses of HSP60 sequences confirmed that cnidarian proteins are orthologous to the canonical vertebrate HSP60 (human HSPD1), demonstrating deep structural and evolutionary conservation of this chaperonin across Metazoa. Collectively, these findings reveal that while HSP60 is evolutionarily ancient and conserved, its regulation under thermal stress varies across lineages and physiological context, reflecting complex modulation of mitochondrial proteostasis in early-diverging metazoans. This lineage- and context-dependent regulatory framework provides new insight into how chaperone plasticity contributes to cnidarian thermal tolerance and the differential susceptibility of reef taxa to bleaching under ocean warming. Significance StatementHeat shock protein 60 (HSP60) is a highly conserved mitochondrial chaperonin critical for maintaining protein homeostasis, yet its regulatory dynamics across early-diverging animal lineages are poorly understood. By first comparing the expression responses of three phylogenetically and ecologically distinct cnidarians--the coral Pocillopora acuta, the sea anemone Exaiptasia diaphana, and the upside-down jellyfish Cassiopea xamachana--we uncovered clear lineage-specific differences in HSP60 regulation. P. acuta showed no detectable HSP60 induction in intact tissue, underscoring strong context-dependence that may prevent the deployment of this critical molecular defense mechanism, reflecting its high thermal susceptibility. In contrast, E. diaphana and C. xamachana displayed gradual, temperature-dependent accumulation aligning with their thermal flexibility, with C. xamachana also displaying constitutive basal levels under control condition. The consistent antibody cross-reactivity across all three species then led us to investigate evolutionary conservation, revealing that cnidarian HSP60s are orthologous to the canonical HSP60 (human HSPD1). This demonstrates that HSP60 is deeply conserved from cnidarians to mammals, yet its stress-responsive regulation has diversified across lineages and physiological contexts. This lineage- and context-dependent regulatory framework illuminates how fundamental differences in chaperone control shape cnidarian stress physiology, offering new mechanistic insight into the cellular basis of coral bleaching susceptibility under ocean warming.

biochemistry↗

bioRxiv2025

What Could Go Wrong? Promoting Success by Planning for Failure in Label-Free Biosensor Assay Development

Label-free biosensors offer powerful platforms for detecting molecular interactions, but developing robust assays on these systems presents several challenges due to the complexity of the testing systems and intersection of disciplines. In this work, we describe a lightweight, 3-step experimental workflow supporting troubleshooting and root cause analysis that we developed during the design and optimization of silicon photonic biosensor assays in an academic research setting. Because such environments often lack the resources and formal quality-management infrastructure available in industry, our approach emphasizes practicality and ease of adoption. Drawing from our own assay failures and successes, we identify common failure modes, propose structured troubleshooting workflows, and provide case studies illustrating the application of established frameworks, including the Five Whys, the Plan-Do-Check-Act cycle, Open-Narrow-Close and the Ishikawa (fishbone) diagram. By applying this framework to our research, we increased our assay yield by a factor of 2, from ~45% to ~90%. This paper aims to support other teams engaged in label-free assay development by equipping them with practical tools and experimental best practices. HighlightsO_LIWe doubled assay yield from ~45% to ~90% by planning for failure, not just success. C_LIO_LITailored troubleshooting frameworks help pinpoint and solve various assay failures. C_LIO_LIApplying innovative problem-solving to workflows accelerates biosensor development. C_LIO_LISystematic troubleshooting improves assay development across a range of lab settings. C_LIO_LIA 3-step workflow highlighting what could go wrong supports continuous improvement. C_LI

bioengineering↗