Biology subjects

Chakraborty, S.

Publications and source records attributed to Chakraborty, S..

At least 37 records · Page 2Linked to original sources

bioRxiv2026

Reduced tubulin polyglutamylation perturbs neuronal function and causes late-onset neurodegeneration

Microtubules are essential for neuronal functions and are involved in many neurodegenerative disorders. A posttranslational modification (PTM) of neuronal microtubules, polyglutamylation, was shown to control cargo transport, Tau protein binding to microtubules and to induce early-onset neurodegeneration when exceeding physiological levels. To determine the physiological importance of this PTM, we characterised mouse models with reduced polyglutamylation. We demonstrate that these mice develop late-onset atrophy of the olfactory bulb and frontal cortex, progressively reduced dendritic spine density, and deficits in olfaction, memory, and cognitive control. This indicates an important role of polyglutamylation in neuronal function. To test whether the observed defects are linked to neurodegeneration, we combined mice with reduced polyglutamylation with a transgenic model for tauopathy. Strikingly, reduced polyglutamylation accelerated the premature demise of mice transgenic for human Tau carrying the P301S mutation. Our work thus reveals key roles of microtubule polyglutamylation in neuronal function with implications for late-onset neurodegeneration linked to Tau pathology.

neuroscience↗

bioRxiv2026

Assessing the physiological S nitrosoproteome reveals nitric oxide mediated regulatory networks in rice

Nitric oxide metabolism-based protein post-translational modifications, such as reversible S- nitrosylation, have been at the pinnacle of plant redox research owing to their significant correlation with seed dormancy, interaction with other signaling molecules, plant development and metabolism, biotic and abiotic plant stress responses, immune defense responses against plant pathogens, and senescence. The rapid interconversion of reactive nitrogen species, the abrogation of nitric oxide homeostasis by exogenous supplementation of NO donors and scavengers, the lack of spatio-temporal specificity of NO signaling, and the limited bioavailability or assay sensitivity for detection often limit the effectiveness of identifying and characterizing S-nitrosothiols in plants. Hitherto unknown, we report the first experimental evidence of the total in vivo S-nitrosoproteome in Oryza sativa L. subsp. indica, comprising 134 PSNOs, enriched with 169 putative sites susceptible to S-nitrosylation, without any exogenous supplementation of NO donors. In the present study, mercuric salt- driven facile decomposition of S-nitrosoproteins in the presence of nitrone spin trap 5,5- dimethyl-1-pyrroline N-oxide, resulting in the synthesis of DMPO-nitrone adducts with PSNO-derived protein thiyl radicals in O. sativa, has been demonstrated as an efficient and novel strategy for characterizing the PSNOs using mass spectrometry analysis. The evidence of physiological levels of PSNOs was further re-examined in a bi-directional qualitative and quantitative approach involving the 2,3-diaminonaphthalene assay in tandem with fluorescence-based visualization and fluorometric quantification. In silico analyses, involving both functional enrichment and pathway prediction analyses, have furthermore revealed unique protein-protein interaction networks and signaling pathways among the S- nitrosoproteome candidates and their predictable physiological roles in O. sativa indica, awaiting further in vitro validation for their functional correlation in response to S- nitrosylation. In conclusion, the present study provides novel evidence of nitric oxide signaling in rice cultivars under physiological conditions, bringing new insights into the potential in vivo transnitrosylation of regulatory or active-site cysteine thiols.

biochemistry↗

bioRxiv2026

Rabies virus antagonizes interferon signaling by targeting phosphorylated STAT1 tetramers

AO_SCPLOWBSTRACTC_SCPLOWRabies virus phosphoprotein (P protein) antagonizes interferon signalling by selectively targeting activated STAT1 protein, but the structural basis for the selective recognition of activated STAT1 has remained unclear. Here we report cryo-EM structures of tyrosine-phosphorylated STAT1 (pY-STAT1) in complex with the C-terminal domain of P protein (P-CTD). P-CTD engages the pY-STAT1 tetramer through three discrete interfaces, contacting two DNA-binding domains within a pY-mediated STAT1 dimer and the N-terminal domain of a third STAT1 protomer. This binding mode explains how P-CTD selectively recognizes activated STAT1, occludes surfaces required for importin 5 and DNA binding, and stabilizes the compact tetrameric state, thereby restricting the conformational transition required for cooperative DNA binding. Cell-based assays further indicate that distinct P-CTD surfaces differentially contribute to antagonism of type I and type II interferon signalling. Further, by acting as an anchoring molecule, P-CTD also enabled structural elucidation of the complete STAT1 structures in the tetramer. Together, these data reveal critical mechanisms in viral immune evasion and pathogenesis, and fundamental innate immune signaling.

molecular biology↗

bioRxiv2026

Progranulin haploinsufficiency remodels the cerebral microvasculature and neurovascular unit

Progranulin (PGRN) deficiency is a major genetic cause of frontotemporal dementia (FTD), yet its impact on cerebrovascular function remains understudied. Here, we show that PGRN deficiency contributes to cerebral microvascular perfusion and induces alterations within the neurovascular unit. In vivo two-photon imaging revealed increased capillary stalling and in cerebral blood flow (CBF), driven in part by increased leucocyte-capillary interactions and elevated endothelial ICAM-1 expression. Transcriptomic profiling of isolated cerebral microvessels demonstrated coordinated upregulation of immune and extracellular matrix pathways alongside suppression of angiogenic and stress-response programs, indicative of endothelial activation. Cross-species analyses identified partial conservation of these vascular signatures in endothelial cells from human FTD-GRN patients, associated with dysregulated angiogenic and inflammatory signaling. Despite altered tight junction organization and reduced solute carrier transporter expression, blood-brain barrier (BBB) permeability remained largely intact, suggesting functional rather than structural BBB disruption, as well as. These vascular changes were accompanied by broad alterations in the morphology of astrocytes, pericytes, and microglial cells. Here we determined a novel role for progranulin in cerebrovascular homeostasis and established microvascular dysfunction as a key driver of FTD-GRN pathophysiology.

neuroscience↗

bioRxiv2026

Loss of Tubulin Tyrosination in Purkinje Neurons Does Not Cause Their Degeneration

Posttranslational modifications (PTMs) of tubulin have been suggested to form a tubulin code to coordinate microtubule functions. While at the molecular level PTMs were demonstrated to act in a target-specific manner, an outstanding question is the selectivity with which they control cellular and physiological functions. For instance, several tubulin PTMs have been linked to neurodegeneration, suggesting that whichever PTM is dysregulated could be sufficient to induce microtubule perturbation and cause neuronal death. Here we test whether perturbation of tyrosination can cause degeneration of Purkinje neurons similar to what happens when the PTM polyglutamylation is abnormally accumulated. Strikingly, this is not the case: deletion of tubulin tyrosine ligase in Purkinje cells, while resulting in loss of tyrosinated tubulin in these neurons, does not affect their survival nor function for more than one year. Our results thus reveal a highly specific role of polyglutamylation in neuronal homeostasis that is not replicated by other tubulin PTMs, suggesting stringent functional specialisation of those PTMs at the physiological level.

neuroscience↗

bioRxiv2026

Hybrid breakdown is temporary and not expressed in a novel environment during 50 generations of experimental evolution in Tetranychus urticae hybrids

Hybridization, the interbreeding between species or genetically distinct populations, can lead to deleterious fitness consequences, but simultaneously it can boost adaptive potential by increasing genetic variation, especially in novel environments. However, how incompatibilities and beneficial genetic combinations interplay across generations remains poorly understood. Here, we tracked how two fitness proxies, the absolute number of adult offspring and the proportion of eggs that reached adulthood, evolve in parental and hybrid populations at three different time points over 50 generations, in both novel and ancestral environments. To test this, we used two geographically distinct populations with low divergence (Dxy=0.002) of the two-spotted spider mite (Tetranychus urticae). Across the first three generations, hybrids showed significantly lower fitness than parental populations in the ancestral environment, indicating incompatibilities between the parental genomes. In contrast, hybrid and parental fitnesses were similar in the novel environment, indicating that the impact of incompatibilities was minor compared to the selection imposed by the novel environment. However, after 50 generations, hybrids displayed similar fitness relative to parental populations in all environments, suggesting resolution of the incompatibilities. Furthermore, around generation 45, hybrids temporarily outperformed parental populations in a novel environment, suggesting a transient window of higher adaptive potential, before fitness stabilized again by generation 50. In conclusion, we show that hybrid populations of T. urticae can swiftly purge incompatibilities when genetic divergence is low. These findings suggest that the dynamics of incompatibility resolution and adaptive potential of novel haplotypes play out over a long time frame, highlighting the importance of tracking hybrid fitness past the first few generations.

evolutionary biology↗

bioRxiv2026

Optimized Urine Metagenomic Methods Reveal Longitudinal Microbial Community Dynamics and Predictors of Transition from Asymptomatic Colonization to CAUTI

BackgroundUrinary tract infections (UTIs) rank among the most common infections globally, with many linked to indwelling urinary catheters. Our prior culture-based longitudinal evaluation of long-term catheterized nursing home residents revealed persistent asymptomatic colonization by pathogens and demonstrated that CAUTI onset was not necessarily due to new pathogen acquisition. In this study, we optimized metagenomics methods to examine the ecological structure underlying persistent colonization and the transition to infection. ResultsWe present a comprehensive longitudinal metagenomic analysis of catheterized urine specimens, revealing colonization dynamics of 69 microbial species across 198 samples from 9 individuals. Descriptive ecological metrics were combined with Bayesian mixed-effects models that accounted for repeated within-participant sampling to identify clusters of co-occurring species, determine the impact of perturbations such as antibiotic exposure and catheter changes on community structure, and identify taxa predictive of infection sign and symptom onset. Longitudinal specimens clustered into three main ecological phenotypes: 1) moderate diversity, unstable communities (3 participants); 2) high diversity, stable communities that resisted disruption even after multiple catheter changes (3 participants); and 3) low diversity, pathogen-dominated communities (3 participants). Catheter changes alone did not significantly disrupt community composition, while antibiotic exposures induced major shifts often followed by re-colonization with the same genera within subsequent weeks. Six clusters of species were identified for which relative abundances correlated across perturbations to the microbial community, including a mutually exclusive Enterobacterales cluster and fastidious-anaerobe group cluster. 24 species were found to correlate with onset of signs and symptoms of infection, 11 of which were missed by standard urine culture. ConclusionsThe catheterized urinary tract represents a novel ecosystem that is resilient to disruption by catheter changes but susceptible to antibiotic perturbation. Antibiotic exposure did deplete all species associated with signs and symptoms but also depleted potentially benign microbes. Our findings have direct implications for catheter management protocols and antibiotic stewardship in long-term catheterized patients. Prospective evaluation using this framework in a larger cohort can help translate these ecological insights into clinical decision-making tools.

microbiology↗

bioRxiv2026

Molecular Basis of Core Fucosylation-Dependent Modulation of IgG1-FcCD16a Binding

Core fucosylation of the IgG1 Fc N297 glycan is known to reduce binding affinity to the Fc{gamma}RIIIa (CD16a) receptor and attenuate antibody-dependent cellular cytotoxicity (ADCC), yet the structural mechanisms underlying this effect remain incompletely understood. Here, we use extensive all-atom molecular dynamics simulations to systematically investigate how Fc glycosylation modulates the structural, energetic, and dynamical landscape of the IgG1 Fc-CD16a complex across multiple systems with fucosylation and galactosylation. Relative binding free energy calculations reproduce experimentally established trends, showing that afucosylation consistently strengthens Fc-CD16a interactions. Mechanistically, dual fucosylation (on both Fc arms) increases inter-glycan packing between the Fc N297 glycans, restricts Fc glycan conformational sampling, and destabilizes the conformational organization of the CD16a N162 glycan. These glycan-mediated perturbations propagate to the protein interface. The result is reduced Fc-CD16a contact persistence, redistribution of energetically important residues away from the canonical binding interface, and broader, less stable receptor-bound conformational states. Dynamic cross-correlation analysis further reveals that afucosylated systems maintain substantially stronger coordinated motions across the Fc-CD16a assembly, whereas fucosylation disrupts long-range dynamic coupling between the receptor and antibody domains. Across these different energetic, structural, conformational, and dynamical readouts, fucosylation systematically shifts the Fc-CD16a assembly from a compact, interface-stabilized binding mode toward a more heterogeneous and weakly coupled receptor-bound ensemble. Together, our findings set forth a mechanistic basis for Fc glycosylation regulating receptor engagement through ensemble-level conformational and dynamical reorganization rather than simple local steric effects. These results provide mechanistic design principles for rational Fc glycoengineering and the development of therapeutic antibodies with enhanced effector functions. More broadly, this work highlights how glycan composition can be leveraged as a tunable molecular design parameter for engineering protein recognition, conformational stability, and immune effector function in therapeutic glycoproteins.

biophysics↗

bioRxiv2026

In search of intralocus sexual conflict in the multivariate and local genetic architecture of metabolic traits in humans

Despite a common genome, males and females are remarkably different across species. Intralocus Sexual Conflict (IASC) arises when selection favours different trait values in the sexes but a shared genetic architecture constrains their evolutionary divergence. IASC is typically studied at the single-trait level, and genome-wide architectures can obscure conflict localized to specific genomic regions. We investigated IASC in the multivariate and local genetic architecture of 17 human metabolic traits and lifetime reproductive success by estimating the sex-stratified additive genetic (co)variance matrix (Gmf) and local cross-sex-cross-trait genetic correlations. Genome-wide, between-sex covariance matrix (B) showed only 1.15% asymmetry, and sexually concordant (SC) genetic variation exceeded sexually antagonistic (SA) variation by 18.5-fold. Indirect evolutionary response to SC selection was 1.6-fold stronger than direct responses to SA selection. In contrast, local analyses revealed a more heterogeneous structure - among the regions with significant local correlations, 12% were consistently SA, showing opposite fitness effects in the sexes, with functional enrichment in WNT signalling, while 20% exhibited both SA and SC effects. Our results demonstrate that in human metabolic traits, SC variation predominates genome-wide, whereas SA variation is concentrated in specific local regions, highlighting the importance of integrating multivariate and local frameworks in understanding conflict.

evolutionary biology↗

bioRxiv2026

Development of GS-441524 Derivatives as Potent SARS-CoV-2 Mac1 Inhibitors via a Direct-to-Biology Approach

Targeting viral macrodomains (Mac) has emerged as a promising strategy for antiviral drug development, especially after the outbreak of COVID-19 that claimed millions of lives worldwide. Several severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Mac1 inhibitors have been reported in the past few years. In the present work, we converted GS-441524 (IC50 of [~]10 M for SARS-CoV-2 Mac1) to KP-S54 (18c), a potent inhibitor of both SARS-CoV-2 Mac1 (IC50: 44 nM) and Middle East respiratory syndrome coronavirus (MERS-CoV) Mac1 (IC50: 91 nM) through an iterative direct-to-biology approach. This approach leverages efficient amide-coupling reaction and the mix-and-read fluorescence polarization (FP) assays where reaction mixtures could be screened directly without purification. Cocrystal structure of a selected derivative (12p) binding to SARS-CoV-2 Mac1 revealed the binding mode, which will guide future drug development against viral macrodomains.

pharmacology and toxicology↗

bioRxiv2026

Urbanization effects are trait-specific and city-dependent across a widespread spider's global range

Urban environments impose strong selective pressures through biotic and abiotic factors, driving changes in behavior, physiology, and morphology. Yet, responses vary across taxa and cities, and it remains unclear which traits respond consistently and what factors moderate this variation. We addressed these questions using the widespread European garden spider (Araneus diadematus) as a model, measuring size, color, and web-building traits along urban-rural transects in 22 cities across its distribution range. Using a meta-analytic framework, we assessed how city-specific characteristics influenced trait variation. Urbanization consistently reduced relative abdomen surface area, a proxy for body condition. Exploratory meta-regressions suggest that web-building response was predicted by temperature: compared to their non-urban surroundings, urban webs are larger in colder regions and smaller in warmer regions. In contrast, body size and abdomen brightness varied among cities without clear environmental predictors. These findings show that urbanization effects are trait- and context-dependent, likely influenced by local factors such as heat island intensity, microclimate, or prey availability. Linking within- and between-city variation will improve understanding of species phenotypic responses to urban environments.

ecology↗

bioRxiv2026

A Facile and Versatile Technique for Creating Antifibrotic Coatings on Biomedical Implants

Foreign body response is a common yet serious challenge for biomedical implants. It can trigger inflammation and eventually lead to the formation of a fibrotic capsule, which compromises device function. Although significant efforts have been made to develop antifibrotic surface coatings for implantable materials, developing broadly applicable solutions remains challenging due to the diversity of materials used in biomedical implants. Here, we propose a simple and versatile strategy to develop antifibrotic coatings for biomedical implants. Photoreactive benzophenone groups are incorporated into designer polymers to enable covalent attachment to various substrates. The effect of benzophenone group density within polymer chains on surface coating efficiency was investigated, and an optimal BP incorporation ratio was identified. Polymers incorporating varying ratios of an anti-fibrotic small molecule and anti-fouling zwitterionic moieties were synthesized and successfully attached to silicone implants. In vivo evaluation of these implants in C57BL/6 mice identified an optimized polymer composition that reduced fibrotic capsule thickness by around 60%. Coating of commercial medical catheters with this optimized polymer reduced collagen deposition by over 3.5-fold following 4 weeks of implantation in the peritoneal space of C57BL/6 mice. Finally, we demonstrated that the optimized polymer coating can be readily applied to a variety of commonly used biomedical materials using this straightforward method, highlighting the versatility of the approach. This work provides a facile and broadly applicable strategy for developing antifibrotic coatings, which has the potential to expand the design of surface modifications aimed at improving the performance of biomedical implants.

bioengineering↗

bioRxiv2026

Tracking multi-site somatic voltage dynamics via high-speed fiber photometry

Investigating neural circuit dynamics across distributed brain regions in awake, behaving animals is crucial for understanding complex behavior. Genetically encoded voltage indicators (GEVIs) offer a powerful approach to tracking transmembrane voltage with high temporal and cellular specificity. However, scaling high-sensitivity GEVI recordings across multiple brain regions and multiple animals simultaneously remains a major technical challenge. Furthermore, it is unclear whether soma-targeted GEVIs - typically used for single-cell resolution imaging - can be effectively adapted for fiber photometry. Here, we show that a sCMOS-based widefield imaging system achieves sensitive dual-color multi-site fiber photometry using soma-targeted GEVI indicators with high temporal resolution. We validated this approach in the mouse hippocampal CA1, capturing theta (3-10Hz) and gamma (30-80Hz) rhythms and theta-gamma cross-frequency coupling. Additionally, we recorded high-frequency neural entrainment (>100 Hz) and somatic depolarization induced by electrical stimulation in CA1. Lastly, we tracked synchronized neural activity between the bilateral CA1s as well as multi-site dual-color imaging across CA1 and cortex simultaneously in three freely running mice. This work provides a scalable, accessible platform for high-speed optical electrophysiology in distributed neural circuits. Key points- Implementation of a sCMOS-based widefield imaging setup for sensitive and scalable fiber photometry and cellular imaging. - Demonstration of population multi-site and inter-animal voltage imaging with soma-targeted genetically encoded voltage indicators. - Tracking of high-frequency gamma and high-gamma (>100Hz) neural entrainment

neuroscience↗

bioRxiv2026

Spatially confined niches support hypoxia-associated transcriptional plasticity contributing to malignant progression in IDH-mutant gliomas

BackgroundWhile hypoxia is a well-established driver of glioblastoma progression, its role in IDH-mutant gliomas, characterized by localized hypoxic microenvironments rather than overt necrosis, remains poorly understood. Here, we investigate how hypoxia and microenvironmental-adaptations shape cellular heterogeneity and transcriptional plasticity in these tumors. MethodsWe integrated bulk, single-cell, and spatial-transcriptomics datasets from IDH-mutant glioma patients (Astrocytomas and Oligodendrogliomas) to characterize cellular-states and map the localization of hypoxic niches. To uncover tumor microenvironment effects, we established co-culture models using primary IDH-mutant glioma cells with human microglia and astrocytes, maintained under hypoxic and normoxic conditions, followed by bulk RNA-sequencing. ResultsWe identified a hypoxia-associated astrocyte-like (AC-like) program that defines a quiescent, non-cycling population with a distinct transcription factor profile indicative of functional plasticity in IDH-mutant gliomas. These cells harbor glioma stem cell (GSC)-like features and are poised for a quiescent-to-activated (Q-to-A) transition that drives tumor progression. Mechanistically, co-culture models reveal that microglia promote this Q-to-A transition by enhancing HBEGF/EGFR paracrine signaling. Spatial transcriptomics uncovers the co-localization of hypoxic niches within quiescent AC-like cells, whereby the activated subpopulation forms discrete niches defined by localized HBEGF/EGFR communication gradients. Notably, tumors exhibiting elevated EGFR-driven activation signatures correlate with higher histological grade and poorer patient survival, implicating the Q-to-A transition as a critical driver of malignant progression. ConclusionQ-to-A transition within the hypoxic niche represents a critical driver of malignant progression in IDH-mutant gliomas, providing a microenvironment-driven mechanism for the transition to higher-grade disease and identifying targetable-vulnerabilities for therapeutic intervention. Key pointsO_LIHypoxic niches spatially confine quiescent, astrocyte-like cellular state characterized by glioma stem cell features in IDH-mutant gliomas. C_LIO_LIMicroglia triggers the quiescent-to-activated (Q-to-A) transition via paracrine EGFR signaling crosstalk. C_LIO_LIEGFR-driven Q-to-A plasticity serves as a microenvironmentally-regulated driver of malignant progression and adverse patient survival. C_LI Importance of the studyWhile high-grade glioblastomas feature well-defined hypoxic and necrotic regions that drive tumor progression and therapy resistance, IDH-mutant gliomas lack these distinct hallmarks, instead exhibiting disorganized hypoxic niches. Consequently, the functional contribution of these niches to tumor progression has remained poorly understood. Although genetic and epigenetic alterations are established drivers of progression in IDH-mutant gliomas, we provide an additional, essential layer of complexity: microenvironment-driven transcriptional plasticity. By uncovering the relationship between tumor hypoxia and heterogeneous glioma cell states, we uncover that these niches are essential for such plasticity. We also show that the tumor microenvironment provides critical molecular cues necessary to initiate a transcriptional shift enabling glioma cells to transition from a quiescent reservoir into an activated state primed for rapid proliferation. By identifying this paracrine mechanism, our work uncovers a targetable vulnerability that dictates how dormant cell reservoirs are mobilized to fuel malignant transformation.

cancer biology↗

bioRxiv2026

De novo steroidogenesis maintains female-specific Th2 identity and constrains effector function

T helper 2 (Th2) lymphocytes drive type-2 immunity and allergic diseases that disproportionately affect females, yet the mechanisms underlying this sexual dimorphism remain poorly understood. Current models attribute sex-biased immune differences to circulating gonadal hormones and to sex chromosome composition, leaving unresolved whether immune cells possess cell-autonomous mechanisms acting independently of both. Here we show that female and male Th2 cells differentiated under identical, hormone-free conditions are transcriptionally distinct but functionally equivalent: female cells carry a heightened inflammatory and interferon-response programme, yet produce IL-13 and IL-4 at rates indistinguishable from male cells, with only a modest baseline difference in proliferative capacity. Using T cell-specific deletion of Cyp11a1 that encodes the first and rate-limiting enzyme of de novo steroidogenesis, we identify the constraint that maintains this equivalence. Cyp11a1 loss acts almost exclusively in females, where it simultaneously dampens the female-specific inflammatory programme and drives proliferation and IL-13 production above the level seen in either sex. Male Th2 cells are essentially unaffected. The effect requires differentiation, being absent in naive precursors, indicating that the steroidogenic machinery becomes functionally consequential only in the mature effector state. Cross-species analysis of two human atopic dermatitis single-cell datasets identifies a shared directional signature of gene expression. Cell-intrinsic steroidogenesis therefore constitutes a third axis of immunological sexual dimorphism, one that operates by uncoupling transcriptional identity from effector output rather than by specifying identity itself.

immunology↗

bioRxiv2026

Cryo-EM structure and biochemical characterization of a BRAF/CRAF heterodimer: Negative charge in the NtA motif is not required for RAF activation

Upon RAS-driven membrane recruitment, RAF kinases ARAF, BRAF and CRAF are activated via formation of homo- or hetero-dimers to initiate signaling through the MAP kinase cascade. Although RAF heterodimers are important for both physiologic and oncogenic signaling, they have been little studied at a structural and biochemical level. Here we report the preparation, biochemical characterization, and the cryo-EM structure of a 14-3-3-bound BRAF/CRAF heterodimer complex. The heterodimer exhibited kinetic parameters and sensitivity to a panel of twelve structurally diverse RAF inhibitors that were closely similar to, or intermediate between, those of BRAF and CRAF homodimers. Cryo-EM structures of the heterodimer with and without MEK1 revealed an overall organization essentially identical to that of RAF homodimers, but with an asymmetric interaction in the MEK1-bound structure in which the BRAF N-terminal acidic (NtA) motif extends across the dimer interface to engage the CRAF RKTR motif. Mutagenesis of this interface unexpectedly revealed that replacing the acidic NtA sequence with a basic RARA sequence yields highly active RAF homodimers and heterodimers, demonstrating that negative charge in the NtA motif is not required for activity. Collectively, our findings suggest that the charge state of the NtA motif influences RAF activity through effects on local backbone dynamics and the stability of the inactive kinase conformation, rather than via stereospecific recognition across the dimer interface.

biophysics↗

bioRxiv2026

PFAS triggers a SpoT-associated metabolic switch that promotes persister-like phenotype in Salmonella Typhi

Per- and polyfluoroalkyl substances (PFAS) are new pollutants in the environment whose effects on bacterias physiology is not well understood. In this study, we show that exposure to PFAS causes membrane depolarization in Salmonella enterica serovar Typhi. This works as a metabolic uncoupler that breaks down proton motive force without immediately killing the cells. This disturbance results in a significant elevation of intracellular NADH and NAD levels while preserving redox equilibrium, signifying an augmented metabolic flux. At the same time, we see that {beta}-oxidation pathways are turned on, which suggests that the cells are shifting toward breaking down fats to make up for the lack of energy. Even though there are more reducing equivalents, ATP levels go down, which is what happens when respiration is uncoupled. This puts the cells in a state of "pseudo-starvation." This metabolic stress triggers the SpoT-dependent stringent response, leading to the accumulation of (p)ppGpp. Genetic analysis employing {Delta}relA and {Delta}relA{Delta}spoT mutants confirm that SpoT is necessary for this adaptive response. Functionally, PFAS-treated populations show an enhanced proportion of persister-like cells, which connects exposure to environmental pollutant in the environment to antibiotic tolerance. Our findings reveal a previously unidentified mechanism by which PFAS alters bacterial metabolism and stress responses, facilitating persistence through membrane depolarization, metabolic reconfiguration, and stringent response activation. This study underscores the potential influence of environmental pollutants on bacterial survival mechanisms and antibiotic resistance.

microbiology↗

bioRxiv2026

Interpretable Antibody-Antigen Structural Interface Prediction via Adaptive Graph Learning and Cyclic Transfer

Experimental structural methods can identify antibody-antigen interfaces with high precision, but they remain time-consuming and resource-intensive, limiting their application across the rapidly expanding space of antibody and antigen sequences. Computational models capable of predicting these interfaces could therefore accelerate antibody discovery and provide insight into the principles governing immune recognition. However, this problem remains challenging due to limited structural datasets, severe class imbalance, and the complex, non-local nature of biomolecular interactions. Here we present VASCIF (Variable-domain Antibody-antigen Structural Complex Interface Finder), a structure-aware framework built on a Masked Graph Attention (MGA) architecture that represents protein complexes as residue graphs and captures long-range structural dependencies through attention-based message passing. The framework is straightforward to implement and enables efficient inference, allowing substantially faster predictions than other existing structure-based approaches. Evaluated on curated structural complexes across multiple benchmark datasets using rigorous cross-validation, VASCIF achieves state-of-the-art performance for residue-level interface prediction. Interpretability analyses reveal that the model recovers biophysically meaningful interaction patterns consistent with known principles of antibody recognition, and redefining interfaces using larger residue distance thresholds ([~]10 [A]) significantly improves predictive performance. Together, VASCIF provides a practical predictive framework and new insights into antibody-antigen molecular recognition.

bioinformatics↗