Biology subjects

Pal, S.

Publications and source records attributed to Pal, S..

At least 19 recordsLinked to original sources

bioRxiv2026

Exploring recurrent urinary tract infection: paired microbiota and culture-based profiling reveals stable dysbiosis and multidrug-resistant, virulence-enriched uropathogens

**Objectives:** Recurrent urinary tract infection (rUTI) is increasingly complicated by antimicrobial resistance (AMR), yet urinary microbiome dysbiosis and isolate-level resistance-virulence are rarely examined together across different treatment states. In this study, we characterized both within the same sample in rUTI and in healthy controls. **Methods:** In this cross-sectional cohort, mid-stream urine was collected from women with untreated, treated and prophylaxis rUTI and age-matched healthy controls at four centres in South India. Of 80 samples collected, 75 were analysed (26 controls, 20 untreated, 20 treated, 9 prophylaxis) by 16S rRNA V3-V4 sequencing. In addition, 105 culturable isolates from the same samples were identified and tested for antimicrobial susceptibility (VITEK 2) and phenotypic virulence. **Results:** rUTI groups showed approximately half the alpha diversity of controls (median Shannon 1.07-1.18 vs 2.17; Kruskal-Wallis p = 0.041). Community structure differed from controls in every rUTI subgroup (PERMANOVA R-squared = 0.091, p = 0.004; pairwise FDR = 0.008) but not among subgroups. Mean Lactobacillus relative abundance fell from 29.2% in controls to <=13.2% in rUTI groups, while Escherichia-Shigella rose from 7.2% to 25.0-43.0%. Control-referenced dysbiosis z-scores were elevated in all rUTI groups (community distance: FDR <= 0.003; uropathogen: Lactobacillus index: FDR < 0.001 for treated and prophylaxis groups). Among isolates, Escherichia coli predominated (35/105; 16/35, 45.7% multidrug-resistant), followed by Klebsiella spp. (32% multidrug-resistant); carbapenem susceptibility was largely retained, and multidrug-resistant E. coli co-carried more virulence traits. **Conclusions:** rUTI is associated with a stable dysbiotic state that treatment does not restore, combining Lactobacillus loss and uropathogen dominance with a multidrug-resistant culturable reservoir that co-accumulates virulence traits. Reading the community and its isolates together may support microbiome-sparing, resistance-aware management of rUTI.

microbiology↗

bioRxiv2026

CAR T cell targeting of inflammatory myeloid progenitors in the bone marrow remodels border-associated macrophages and reverses cognitive aging

Aging is associated with progressive neuroinflammation and cognitive decline, yet the cellular sources that sustain this process and whether they can be targeted peripherally remain unclear. Here, we identify inflammatory border-associated macrophages (BAMs) as key drivers of neuroinflammation in aging and show that their therapeutic and prophylactic elimination through intrathecal CAR T cell therapy restores cognitive performance in mouse models of aging and Alzheimer's disease. Furthermore, targeting of aged inflammatory bone marrow myeloid progenitors through either intravenous CAR T cells, which do not infiltrate the brain, or through transplantation of CAR T-treated progenitors is sufficient to reduce neuroinflammation and cognitive impairment. These findings reveal that bone marrow myeloid progenitors harbor a heritable inflammatory transcriptional state that is transmitted to their BAM progeny, driving neuroinflammation and cognitive deterioration, and conserved in human aging. Critically, this proinflammatory state is marked by the upregulation of surface proteins, enabling precise peripheral CAR T cell targeting of these progenitors for long-lasting therapeutic effects in cognitive aging.

cell biology↗

bioRxiv2026

Hyaluronan Surface Architecture Dictates Colorectal Cancer Progression and Extracellular Vesicle Communication

Hyaluronan (HA) is a principal component of the tumor glycocalyx in colorectal cancer (CRC). However, how the disease progression is linked to HA abundance and its nanoscale organization remains unclear. Single-molecule measurements of surface glycans on cell membranes and extracellular vesicles (EVs) have not yet been correlated. In this work, using single-molecule force spectroscopy, we mapped HA density and chain length on CRC cells and their EVs across Dukes' stages. HA density increased with stage in both compartments, but their organization diverged. Cell-surface HA became progressively fragmented, whereas EVs remained enriched in short HA chains at every stage. EVs, therefore, appear to select HA during formation rather than inherit it from the parent cell. This divergence had mechanical consequences. Both cells and EVs softened with stage, and removing HA reversed this softening. In addition, coarse-grained membrane simulations revealed that both HA chain length and surface density regulate membrane wrapping, with chain length primarily influencing wrapping kinetics and surface density affecting the final wrapping extent. These findings provide a physical basis for the differences we observed in EV uptake. Reprogramming stage D cells with exogenous high-molecular-weight HA reversed this signature, lowering EV surface HA density, stiffening the vesicles, slowing migration, and suppressing EV uptake by recipient cells. These findings establish HA surface architecture as a stage-encoded and experimentally reversible determinant of CRC progression.

biophysics↗

bioRxiv2026

Phenotypic screens identify biologic regulators of nanoparticle uptake in diffuse midline glioma

Nanoparticle drug delivery systems hold considerable promise for locoregional administration to central nervous system tumors, yet the biological determinants of nanoparticle-cancer cell interactions remain poorly understood. Using patient-derived histone-mutant diffuse midline glioma (DMG) models, we performed a pooled CRISPR-Cas9 perturbation screen to systematically identify regulators of liposomal nanoparticle delivery. The screen identified candidate genes spanning endocytosis, vesicle transport, and metabolic signaling, revealing that nanoparticle delivery is governed by a broader landscape than previously appreciated. Among these, CTNNB1, or {beta}-catenin, emerged as a common negative regulator across two independent DMG models and two distinct nanoparticle surface chemistries. Transcriptomic profiling of CTNNB1-depleted DMG cells revealed upregulation of membrane remodeling and extracellular matrix gene programs, accompanied by reduced cell stiffness measured by a microfluidic acoustic scattering assay. This resulted in a shift in endocytic activity characterized by decreased bulk-phase macropinocytosis and increased receptor-mediated endocytosis. We further identified MAPK and mTOR pathway members as nanoparticle trafficking modulators, and demonstrated concordance between genetic and pharmacologic perturbations in modulating the liposomal nanoparticle interactions in pediatric DMG cells. These findings establish a biology-first screening approach for identifying previously unappreciated regulators with potential relevance to nanoparticle-based therapeutic strategies in pediatric brain tumors.

bioengineering↗

bioRxiv2026

A Covalent Organic Framework-Inspired β-Ketoenamine Crosslinking Strategy for Robust, Injectable Bovine Serum Albumin Hydrogels with pH-Triggered Drug Release

Globular proteins are difficult to convert into robust hydrogels, as their compact, folded structures bury reactive residues, forcing conventional strategies to rely on denaturation or synthetic-polymer reinforcement that compromise the native protein. Inspired by the beta-ketoenamine bond-forming chemistry of covalent organic frameworks (COFs), we report the crosslinking of native bovine serum albumin (BSA) with 1,3,5-triformylphloroglucinol (TFP), a C3-symmetric trialdehyde, into a chemically defined hydrogel. TFP reacts with surface-exposed lysine residues through an irreversible enol-to-keto tautomerization, confirmed by FTIR and NMR spectroscopy, generating stable {beta}-ketoenamine crosslinks under mild aqueous conditions without denaturing the protein, as verified by intrinsic tryptophan fluorescence. The resulting hydrogels are mechanically robust compared to a reversible-imine control, injectable and self-recovering, exhibit reversible shape memory and substantial load-bearing capacity, and remain stable across a broad pH range over extended periods. The network shows consistent swelling behavior at physiological and mildly acidic pH, with modest compaction under strongly basic conditions; scanning electron microscopy reveals a dense, nodular network for the TFP hydrogel versus an open, sheet-like lamellar morphology for the reversible-imine control. The hydrogel efficiently encapsulates doxorubicin and displays pH-triggered, acid-selective release, which comparative kinetic analysis attributes principally to pH-dependent weakening of DOX-BSA binding affinity (linked to the N-to-F conformational transition of BSA near its isoelectric point) rather than to bulk network swelling or degradation. Doxorubicin-loaded hydrogels show enhanced killing of MCF-7 breast cancer cells relative to the free drug while remaining cytocompatible toward normal mammalian cells, and a ciprofloxacin-loaded variant exhibits potent antibacterial activity against both Gram-positive (M. luteus) and Gram-negative (E. coli) bacteria. This work translates reticular {beta}ketoenamine chemistry into a general platform for robust, stimuli-responsive protein biomaterials.

bioengineering↗

bioRxiv2026

Genetic mapping of Citrullus amarus for resistance to gummy stem blight in watermelon

Gummy stem blight affects several cucurbit crops and has emerged as a serious production constraint of watermelon cultivation globally. However, no commercial varieties are available possessing resistance against this disease. Identification of genetic determinants and underlying QTLs are prerequisites for resistant variety development. In this direction, an attempt was made to understand genetics and identify responsible loci using two resistant accessions viz., PI 482276 and IIHR 82. Inheritance analysis indicated that resistance to GSB is governed by polygenes in both accessions. Four QTLs were identified for the population derived from PI 482276 x Arka Manik, while six were observed in the cross IIHR 82 x Arka Manik employing QTL-seq approach. The QTL regions contained important genes putatively involved in resistance reaction such as NAC domain-containing protein 17-like, chitinase 2- like, Pectinesterase, Coiled-coil domain-containing protein 18, Kinase family protein expansin-like B1 protein etc. Our results offer further insight into the disease resistance genetics and its utilization in resistance breeding for gummy stem blight in watermelon.

genomics↗

bioRxiv2026

Evolutionary trade-offs between antimicrobial resistance and virulence in Pseudomonas aeruginosa

Antibiotics impose strong selection on bacteria, resulting in the emergence and spread of antimicrobial resistance. To date, the consequences of resistance evolution for other traits, especially virulence as a key life-history characteristic of high medical relevance remain poorly understood. A central limitation of existing work is reliance on clinical isolates with complex evolutionary histories, hindering causal inferences on resistance-virulence relationships. Such causal information is critical for our understanding of the distribution of resistance-associated evolutionary trade-offs, which may additionally guide optimization of treatment designs. The objectives of our study are to address these knowledge gaps using an experimental evolution framework with the human pathogen Pseudomonas aeruginosa. We quantified changes in resistance, life-history characteristics, in-vivo virulence with a Caenorhabditis elegans infection model, and whole genome sequences for bacterial clones, which had been independently evolved under three antibiotics with distinct cellular targets. We found that the resistance-virulence trade-off depended on the used antibiotic and was additionally driven by the underlying evolutionary path to resistance. Evolved resistance to the fluoroquinolone ciprofloxacin correlated positively with virulence, dependent on genetic changes in either the antibiotic target or efflux regulation. Conversely, evolved resistance to piperacillin/tazobactam, a {beta}-lactam/{beta}-lactamase inhibitor combination, was negatively correlated with virulence, contingent on the coincidental spread of resistance mutations and a large genomic deletion, containing numerous virulence genes. Lastly, evolved resistance to the aminoglycoside streptomycin led to only minor virulence changes. Overall, these antibiotic-specific evolutionary trajectories challenge the assumption of a universal resistance-virulence trade-off and demonstrate that antibiotic choice itself shapes pathogen virulence potential. Significance StatementAntimicrobial resistance and bacterial virulence are typically studied in isolation, yet they are shaped by the same evolutionary pressures and may directly compete for cellular resources. We addressed a fundamental but unresolved question: does evolution of resistance to an antibiotic make a pathogen more or less dangerous to a host? By characterizing experimentally evolved Pseudomonas aeruginosa, a leading cause of drug-resistant infections, we show that the answer depends on which antibiotic drove resistance. A positive resistance-virulence relationship occurred upon adaptation to a fluoroquinolone antibiotic, whereas it was negative upon {beta}-lactam resistance, and unchanged upon aminoglycoside resistance evolution. These antibiotic-specific evolutionary trajectories challenge the assumption of a universal resistance-virulence trade-off and suggest that antibiotic choice shapes pathogen danger beyond drug susceptibility.

evolutionary biology↗

bioRxiv2026

The impact of non-cardiomyocyte MYBPC3 expression on the development of hypertrophic cardiomyopathy

IntroductionHypertrophic Cardiomyopathy (HCM) is a disease defined by the development of left ventricle hypertrophy. One of the most commonly mutated genes in HCM is cardiac myosin binding protein C (MYBPC3). MYBPC3 protein localizes to the cardiomyocyte sarcomere, but studies have reported detection of both MYBPC3 RNA and protein in non-cardiomyocyte cell populations. Therefore, it was unclear if MYBPC3 expression in non-cardiomyocyte cell populations altered the development of cardiomyopathy caused by MYBPC3 protein deficiency. MethodsWe utilized genetically modified murine models with germline deletion of Mybpc3 exons 3 to 5 (Mybpc3-/-) or cardiomyocyte specific deletion of Mybpc3 exons 3 to 5 (Mybpc3fl/fl; Myh6-Cre). Gene expression was assessed using quantitative RT-PCR. Whole tissue protein levels were assessed using immunoblots. Immunohistochemistry and proximity ligation assays were performed to evaluate in situ protein expression. Echocardiography was utilized to measure left ventricular structure and function. ResultsMybpc3 mRNA was detected in multiple organs including the heart, lung and blood from both humans and mice. Utilizing transgenic murine models with germline or cardiomyocyte specific deletion of Mybpc3 exons 3-5, we discovered that the Mybpc3 mRNA detected in extracardiac locations originated primarily from cardiomyocytes. Likewise, MYBPC3 protein was identified in myocardial tissue but not in other organs and cardiomyocytes were the only cell population in myocardial tissue that had detectable MYBPC3 protein. Importantly, cardiomyocyte deletion of Mybpc3 caused similar pathological myocardial remodeling and alterations in left ventricular function compared to germline deletion of Mybpc3 in all cell populations. ConclusionsOur results show that cardiomyocytes are the primary cell source of Mybpc3 mRNA detected in extracardiac organs and they are the principal cell type responsible for the cardiomyopathy caused by MYBPC3 protein deficiency. These results suggest that selective targeting of cardiomyocytes should be the most efficient approach to treat cardiomyopathies associated with MYBPC3 deficiency.

genetics↗

bioRxiv2026

A shape-constrained regression and wild bootstrap framework for reproducible drug synergy testing

High-throughput drug combination screens require methods to identify synergistic pairs, yet widely used synergy scores lack statistical inference and can fail when parametric dose-response fits do not converge. We present SIR (Synergy via Isotonic Regression), a nonparametric framework that defines interaction as deviation from a monotone-additive null, fit by 2D isotonic regression. A degrees-of-freedom-corrected wild bootstrap yields calibrated p-values for each dose-response matrix. On DrugCombDB, SIR interaction surfaces achieve higher replicate concordance (median correlation 0.91 across 1,839 replicate pairs) than all baselines (0.53-0.74), while avoiding Loewes 20.9% and ZIPs 3.6% failure rates. The fitted surface also predicts missing wells (median holdout RMSE 0.040). By replacing heuristic scores with calibrated effect sizes and p-values, SIR enables principled hit calling and error-rate control in large screens.

bioinformatics↗

bioRxiv2026

Widespread data leakage inflates performance estimates in cancer drug response prediction

Drug response prediction models guide biomarker discovery, yet their reported accuracy depends on rigorous cross-validation (CV). We show that supervised feature screening applied to all samples before CV, a widespread practice, introduces data leakage that systematically underestimates prediction error. Across 265 drugs and 1,462 cancer cell lines, leakage-free CV raises mean squared error by 16.6% on average, with near-zero feature overlap between leaked and corrected pipelines (mean Jaccard 0.18; 36.2% of drugs share no features). Despite selecting five times more features, the leaked pipeline recovers known drug targets at nearly the same rate as the corrected pipeline, indicating that the inflated feature sets capture statistical artifacts rather than biological signal. A code-level audit of 32 published methods (2017-2024) confirms leakage in 23 (72%), spanning five distinct modes and cited over 3,000 times. The magnitude of inflation from this single leakage mode is comparable to improvement margins typically claimed over elastic net baselines, raising the possibility that some reported advances reflect evaluation artifacts. We provide a leakage taxonomy, audit guide and reference implementation for leakage-free evaluation.

bioinformatics↗

bioRxiv2025

Random Mutagenesis for the Generation of Repertoire of Aureochrome-based Optogenetic Scaffolds

Engineering of the natural light-regulated transcription factors for the purposeful generation of synthetic photo-switches is a widely adopted strategy in synthetic biology and optogenetics. Aureochrome is one such potential, naturally occurring photoreceptor-cum-transcription factor that can be used for the development of synthetic optogenetic tools, especially meant for light-regulated gene expression. It is however well-known that different biological events demand different duration of signaling state lifetime and diverse affinity towards different DNA substrates. Therefore, in this study, we produce multiple variants of aureochromes via random mutagenesis - mimicking single generation directed evolution. After screening the single generation variants for in-frame transcripts, we sort them on the basis of altered photocycle kinetics as well as DNA-binding affinity. The variants that exhibited reversible photochemistry and light-regulated DNA-binding ability similar/comparable to that of the wild-type despite incurring mutations, are characterized and discussed in this manuscript. These can later be subjected to successive rounds of random mutations to get an eventual superior variety with enhanced functionality. Even the apparently unsuccessful ones, which depicted drastic alteration of photochemical/DNA-binding properties, helped us to identify amino acid residues - lesser known to be indispensable for a certain biological activity. The process of diversification and selection via random mutagenesis not only explains the functional significance of the different amino acids in aureochromes but also generates a repertoire of appropriate aureochrome variants that may be used in optogenetics.

biochemistry↗

bioRxiv2025

Design of Allosteric Inhibitors for Mutant EGFR by Combined use of Machine Learning and Molecular Dynamics Simulations

The non-small cell lung cancer (NSCLC)-associated Epidermal Growth Factor Receptor (EGFR) mutant L858R/T790M confers resistance to first- and second-generation tyrosine kinase inhibitors (TKIs). Allosteric inhibitors, binding outside the ATP-binding site, have emerged as alternative therapeutic agents. Unlike orthosteric inhibitors, they preferentially stabilize EGFR in an inactive conformation. Hence, understanding the mechanistic basis of this inhibition is essential for designing potent allosteric inhibitors. In this study, we performed microsecond-scale molecular dynamics (MD) simulations on the inactive conformations of apo-EGFR and apo-EGFRL858R/T790M to explore how cancer-associated mutations induce a conformational switch toward the active kinase state. Simulations of allosteric inhibitor (EAI001) bound EGFRL858R/T790M revealed that inhibitor binding enhances the inactive state population by disrupting the K745-E762 salt bridge and modulating key structural elements. These findings revealed the structural basis of allosteric inhibition in EGFRL858R/T790M,. It also emphasized the importance of MD simulations in allosteric drug design for assessing the ability of the inhibitor to enhance the population of the inactive state of the mutant EGFR. We have also standardized a virtual screening protocol involving screening of an allosteric TKI library by docking, re-ranking them with a machine learning-based scoring function (SG-ML-PLAP), and evaluating top-scoring molecules by MD and MM/GBSA to identify molecules stabilizing the inactive state of the EGFRL858R/T790M. This approach identified 10 novel allosteric kinase inhibitors predicted to be more potent than EAI001. Overall, our results not only elucidate the mechanism of allosteric inhibition in EGFRL858R/T790M but also offer promising leads for the development of next-generation therapies to overcome TKI resistance.

biophysics↗

bioRxiv2025

DNA methylation reprogramming in marsupial embryos is restricted to the extraembryonic lineage

DNA methylation (5mC) is an epigenetic mark that plays a critical role in defining cell fate. Following fertilisation, DNA methylation inherited from gametes must be reprogrammed to establish totipotency and enable the parental-to-zygotic transition. To accomplish this, non-mammalian vertebrates such as zebrafish and medaka subtly reprogram maternal 5mC profiles while maintaining high methylation levels throughout embryogenesis. In contrast, eutherian mammals such as mouse and human undergo global 5mC erasure in both embryonic and extraembryonic lineages. However, while embryonic 5mC is rapidly re-established to high levels upon implantation, the trophectoderm, which gives rise to the placenta, displays sustained and conserved DNA hypomethylation, suggesting that this drastic 5mC erasure may be functionally linked to complex placentation in mammals. To clarify whether extensive post-fertilisation 5mC erasure co-evolved with placentation, we explored embryonic methylation dynamics in marsupials, a lineage of therian mammals with a short-lived placenta. We produced a near complete telomere-to-telomere (T2T) genome and generated detailed epigenome maps of embryonic development for an Australian marsupial, the fat-tailed dunnart (Sminthopsis crassicaudata). We found the dunnart embryo exhibits genome wide DNA demethylation at the blastocyst stage, but these changes occur in the trophectoderm only, suggesting that 5mC erasure in the placenta is an ancestral state in therian mammals. Furthermore, the T2T-level dunnart genome assembly enabled identification of sex chromosomes, uncovering extensive hypomethylation of the paternally-inherited inactive X chromosome in females and revealing the previously unannotated master regulator of X chromosome inactivation, lncRNA Rsx. Our data indicate that while the use of genome-wide 5mC erasure differs between eutherian and marsupial lineages, 5mC erasure in extraembryonic tissue is ancestral to therian mammals and may be necessary to support placental development. HIGHLIGHTSO_LIFirst embryonic DNA methylation maps in an Australian marsupial C_LIO_LIExtensive global erasure of DNA methylation in the trophectoderm C_LIO_LIMaintenance of high DNA methylation in the embryonic lineage C_LIO_LIHypomethylated paternal X chromosome with methylated escapee genes C_LI

developmental biology↗

bioRxiv2025

Inflammatory Agonists Modulate the Host Response to Type 2 ECM scaffold Immune Environment and Long-Term Remodeling After Severe Traumatic Injury

The immune system is a vital regulator of tissue repair after trauma and the response to implantable scaffolds for regenerative medicine. Decellularized extracellular matrix (ECM) scaffolds promote tissue integration and remodeling following traumatic injury in part due initiating a pro-reparative Type-2 immune response. However, exogenous soluble inflammatory immune signals can be introduced during scaffold implantation, including microbial products in contaminated surgical fields or during immunotherapy to treat autoimmunity and cancer. It remains largely unknown how such immune mediators modulate the ECM scaffold immune environment and subsequent scaffold remodeling. In the present study, we co-delivered 3 distinct inflammatory immune adjuvants (cyclic di-AMP [CDA], monophosphoryl lipid A [MPLA], and granulocyte colony stimulating factor [GM-CSF]) with small intestinal submucosa (SIS) ECM in a murine volumetric muscle loss injury model, evaluating acute (1 week) and long-term (8-week) immune environments and scaffold remodeling. High parameter spectral cytometry, histologic analysis, and PCR revealed differential potentiation of the ECM scaffold microenvironment. Type 2 immune programs including IL-4, eosinophils, CD4 T cells, and CD206/CD86 macrophage ratios were induced in all ECM groups but attenuated by varying amounts with the CDA and MPLA co-delivery. By 8-weeks, inflammation had largely subsided, and histologically ECM with GM-CSF or MPLA showed the greatest degradation and remodeling into adipose tissue. These findings suggest that early pro-inflammatory compound delivery does not abrogate the ECM immune environment but does attenuate some programs while inducing others. These have long-term effects on scaffold remodeling and should be a consideration for surgical reconstruction in patients receiving immune stimulatory therapies.

immunology↗

bioRxiv2025

Roles of G-protein coupled receptors and mechanosensitive ion channels in pressure-induced chronotropy of lymphatic vessels

Active lymph pumping relies on the spontaneous contractions of collecting lymphatic vessels, whose contraction frequencies are exquisitely sensitive to changes in intraluminal pressure. This homeostatic and mechanosensitive mechanism, termed pressure-induced lymphatic chronotropy, enables lymph transport to be matched to the filling state of the lymphatic capillary network. The mechanistic basis of pressure-induced chronotropy was investigated using ex vivo contraction assays of mouse popliteal collecting vessels, in which contraction frequency increases >10-fold with pressure changed from 0.5 to 5 cmH2O. The contractile, electrophysiological and transcriptional similarities between lymphatic muscle cells (LMCs) and arterial smooth muscle led us to hypothesize that pressure-dependent chronotropy shares a parallel signaling process with pressure-induced arterial depolarization/constriction. Thus, we probed two major mechanisms: 1) pressure-induced activation of mechanosensitive cation channels, including TRPC6, TRPM4, PKD1/2, TRPV2 and ENaC, and 2) mechano-activation of GNAQ/GNA11-coupled G-protein receptors (GPCRs) that would generate second messengers to activate those channels. Contraction assays were combined with scRNAseq analysis of the respective targets, with maximum use made of transgenic mice to avoid non-specific effects of pharmacological inhibitors, particularly those used to block TRP channels. Our findings rule out significant roles for the above TRP channels and other putative mechanosensitive channels implicated in arterial myogenic constriction, as well as channels implicated in ionic pacemaking of other tissues. In contrast, smooth-muscle specific knock out or inhibition of ANO1 or IP3R1 significantly blunted the effect of pressure on frequency. Pressure-induced chronotropy was suppressed by [~]70-90% at all pressures in GNAQ/GNA11 double knockout vessels, but with responsiveness partially maintained at pressures above 5 cmH2O. Pressure-induced chronotropy was also suppressed after acute Gq/11 inhibition with YM254890, but was normal in vessels from GNA12/GNA13 double knock out mice. These results support a scheme whereby mechano-activation of one or more GNAQ/GNA11-coupled GPCRs generates IP3, which induces SR Ca2+ release through IP3R1 and drives depolarization through the activation of ANO1 Cl- channels. The major GPCRs expressed in LMCs were subsequently identified and ranked by scRNAseq analysis but knock out or pharmacological inhibition of each of the top 7 candidates failed to significantly affect pressure-induced chronotropy. Our results strongly implicate one or more GNAQ/GNA11-coupled GPCRs in mediating this homeostatic process; however, the specific mechanosensitive GPCRs remain to be identified.

physiology↗

bioRxiv2025

TRIM62 promotes lipid raft-mediated entry of influenza A virus by regulating WASH localization

Viruses employ diverse strategies to gain entry into cells to reach their replication sites. Influenza A virus (IAV), a respiratory pathogen, takes advantage of clathrin-dependent endocytosis or macropinocytosis for its internalization. Here, we report that TRIM62, a member of the tripartite motif (TRIM) protein family, is required for IAV entry, independent of its E3 ubiquitin ligase activity. We find that TRIM62 specifically functions in a clathrin-independent, lipid raft-mediated pathway, which IAV exploits to enter the endocytic network. Additionally, we reveal the involvement of the WASH complex and the retromer component VPS35 in this pathway beyond their canonical functions in endosomal sorting. We observe that a pool of WASH and VPS35 localize to the plasma membrane and associate with lipid rafts, in addition to their typical endosomal presence. While WASH subunits except FAM21 play a proviral role in IAV endocytosis and intracellular trafficking, VPS35 acts antagonistically. We show that TRIM62 counteracts the antiviral function of VPS35 by limiting FAM21-VPS35 interaction. By directly binding WASH in the cytosol, TRIM62 restricts its VPS35-mediated endosomal recruitment and thereby enhances its surface availability to facilitate IAV entry. Together, this study uncovers previously unrecognized roles of TRIM62 and endosomal sorting machinery in IAV entry, offering new antiviral targets.

cell biology↗

bioRxiv2025

Robust antibiotic sensitization of pathogenic Pseudomonas aeruginosa via negative hysteresis in the cell envelope

Antibiotic combination in time and space is a key strategy to combat antimicrobial resistance. The success of such treatment designs requires their robust efficacy across treatment conditions and a pathogens genomic diversity. This study found that an initial treatment with a {beta}-lactam antibiotic causes robust cellular sensitization towards an aminoglycoside antibiotic across the high-risk human pathogen Pseudomonas aeruginosa, including resistant strains. This phenomenon of cellular sensitization, termed negative hysteresis, is modulated by the Cpx envelope stress response system and linked to membrane stress during growth. The increase in efficacy is achieved through a {beta}-lactam induced elevated cellular uptake of the subsequently administered aminoglycoside. Negative hysteresis and the Cpx system are linked in several cases to the expression of synergistic drug interactions, thus enhancing efficacy of antibiotic combinations. Overall, our study identifies the phenomenon of negative hysteresis as a robustly inducible phenotype and thus a unique focus for optimizing antimicrobial therapy.

evolutionary biology↗

bioRxiv2025

Cell type specific allometry controls sex-differences in Drosophila body size

Species and sex-specific differences in organ size are fundamental features of animal biology, yet the mechanisms that drive these differences remain debated. Adult female Drosophila are larger than males. While most organs are present across both sexes, the underlying mechanisms driving sex-specific organ and body size scaling of Drosophila remain unclear. Using single-nucleus transcriptomes from the Fly Cell Atlas, combined with experimental validation, we show that different Drosophila organs scale through distinct strategies, including cell size, cell number, or a combination of both, in an allometric rather than uniform manner. Larger female flight muscles develop from more myoblasts than in males, while cardiomyocyte numbers are the same despite forming a larger heart in females. Female fat body cells are larger and express more ribosomal protein-coding mRNAs, supporting increased cell size. In contrast, males have a greater number of fat body cells. Together, this sex-specific allometry in cell size and number define the cellular basis for differences in body and organ size between sexes in Drosophila. By uncovering how a conserved developmental system produces sex-specific proportions through distinct cellular strategies, our work offers a framework for dissecting sex differences in other species and systems.

cell biology↗