Biology subjects

Gupta, A.

Publications and source records attributed to Gupta, A..

At least 55 records · Page 3Linked to original sources

bioRxiv2026

NaP-TRAP: A versatile and accessible workflow to dissect principles of translational regulation and mRNA stability

The translation of mRNA into protein is tightly regulated by both cellular trans-factors and cis-regulatory elements encoded within transcripts. Although transcript fate can be measured by transcript abundance or translation efficiency, separating the contribution of each individual cis-element within a single transcript is an ongoing challenge. Current massively parallel reporter assay (MPRAs) approaches enable systematic interrogation of cis-regulatory elements that control transcript stability, but translation-focused MPRAs remain technically limited and often inaccessible. Here we present Nascent Peptide Translating Ribosome Affinity Purification (NaP-TRAP), a reporter-based approach that simultaneously measures translation and mRNA abundance. Unlike previous methods, NaP-TRAP captures translation directly through the immunoprecipitation of epitope-tagged nascent peptide chains, providing instantaneous, frame-specific readouts without specialized instrumentation. The method is highly scalable from single reporters to complex libraries, and adaptable across in vivo and in vitro systems. NaP-TRAP is versatile, allowing assessment of cis-regulatory impact of elements distributed throughout the mRNA, from cap-to-tail. This protocol covers experimental design, reporter construction, sample processing, and computational analysis for both low- and high-throughput applications. Bench work can be completed in 4- 5 days, with qPCR-based readouts requiring only basic Excel skills for data processing. Sequencing-based readouts require skills in command-line tools and Python scripting and add an additional 2-3 days. NaP-TRAP thus offers an accessible, robust, and quantitative platform to decode the regulatory logic of mRNA translation and stability in diverse biological contexts. Basic Protocol 1Design, assembly, and synthesis of NaP-TRAP reporter libraries. Support Protocol 1Design, assembly, and synthesis of NaP-TRAP individual reporters and spike-ins. Basic Protocol 2NaP-TRAP delivery by micro-injection in zebrafish embryos. Alternate Protocol 1NaP-TRAP delivery by transfection in cultured mammalian cells. Basic Protocol 3NaP-TRAP pulldown and RNA extraction. Basic Protocol 4Preparation of NaP-TRAP cDNA Sequencing Libraries. Alternate Protocol 2NaP-TRAP-qPCR module for low-cost validation. Basic Protocol 5Computational analysis of NaP-TRAP MPRA data.

molecular biology↗

bioRxiv2026

Decoupled calcium homeostasis and signaling associated with cytoskeletal instability in YWHAG R132C induced pluripotent stem cell-derived cortical neurons

YWHAG Syndrome (Developmental and Epileptic Encephalopathy 56, DEE56) is an ultra- rare childhood epilepsy associated with neurodevelopmental delays, with no therapeutic intervention available. Multiple de novo mutations in the YWHAG gene, encoding for the 14-3-3{gamma} protein, have been identified as causative for YWHAG Syndrome. 14-3-3{gamma} interacts with various targets, including major neurodevelopmental signaling proteins such as components of the ROCK pathway. Despite substantial evidence of the essential role of 14-3-3{gamma} in neurite outgrowth, cytoskeletal rearrangements, and neuronal migration during cortical development, little is known regarding the molecular consequences of YWHAG mutations and their effect on neuronal function and survival. Here, we characterized an isogenic, pluripotent stem cell (iPSC) model of YWHAGR132C/+ cortical neurons. The YWHAGR132C/+iPSC-derived neurons exhibited early cytoskeletal phenotypes, coupled with an elevated calcium baseline, lower frequency of calcium spikes, and reduced network activity. The widespread alterations in the transcriptome of mutant neurons revealed a biphasic dysregulation in the core genes and modulators associated with the ROCK pathway that resulted in maturation-dependent changes to cytoskeletal protein stability and calcium phenotypes. Direct inhibition of ROCK with Y27632 further increased the calcium baseline compared to the isogenic control. Exposure of YWHAGR132C/+ neurons to Trypsin-EDTA revealed underlying cytoskeletal instability, which was partially reversed by lovastatin treatment. Further, lovastatin partially rescued the elevated calcium baseline, but not the frequency or amplitude of calcium spikes. Together, these results suggest decoupling of calcium homeostasis and calcium signaling associated with cytoskeletal instability in YWHAGR132C/+ neurons. These findings lay the groundwork for future mechanistic studies of YWHAG function and molecular therapeutic targets for YWHAG Syndrome and YWHAG-associated conditions.

neuroscience↗

bioRxiv2026

Secretome-Mediated Antimicrobial and Immunomodulatory Activity of Lactobacillus johnsonii Against Multidrug-Resistant Enteroaggregative Escherichia coli

Enteroaggregative Escherichia coli (EAEC) is a leading cause of persistent diarrhea in children in low- and middle-income countries, and the emergence of multidrug-resistant (MDR) strains necessitates non-antibiotic therapeutic strategies. This study evaluates Lactobacillus johnsonii, previously characterized by our group, as a probiotic candidate against a clinically confirmed MDR EAEC isolate resistant to ampicillin, ciprofloxacin, azithromycin, amoxicillin, and gentamicin. L. johnsonii demonstrated robust gastrointestinal resilience, high cell surface hydrophobicity, phenol tolerance, and rapid autoaggregation reaching 80.4 {+/-} 2.3% by 4 hours, collectively supporting mucosal colonization potential. In antimicrobial assays, L. johnsonii produced zones of inhibition against MDR EAEC substantially exceeding those of gentamicin, reduced viable biofilm-associated EAEC by over 80%, and displaced pre-adhered EAEC from HCT-116 intestinal epithelial cells in a time-dependent manner. L. johnsonii also attenuated MDR EAEC-induced gas production and reduced nitric oxide levels by 67.7% in infected RAW 264.7 macrophages, suggesting immunomodulatory activity. Nutrient competition did not appear to contribute to EAEC suppression under tested conditions, indicating inhibition is predominantly secretome-dependent. Fractionation of the L. johnsonii cell-free supernatant by fast protein liquid chromatography yielded five fractions below 75 kDa; fractions S5 and S6 exhibited sustained bactericidal activity at 6 hours. Gram staining confirmed that both fractions reduced viable EAEC cell numbers, with S6 producing a greater reduction than S5, indicating quantitatively distinct bactericidal potencies. These in vitro findings support the potential of L. johnsonii as a biotherapeutic candidate for antibiotic-resistant enteric infections. In vivo validation and chemical characterization of active fractions remain important next steps.

microbiology↗

bioRxiv2026

A nicotine biosensor derived from microbial screening

Physiologically relevant biosensors are in increasingly high demand, yet existing ones are severely limited in the number and type of biomarkers that are detected. The lack of biorecognition elements for most medically relevant biomarkers restricts the development of next generation single and continuous use monitors. Over billions of years, microbes have evolved a vast array of proteins to sense and metabolize small molecules, including those pertinent to human health. Of particular interest to us is the identification and subsequent integration of new microbial redox enzymes into electronic biosensors building off the established electrochemical technology of the continuous glucose monitor. Here we deploy genomic screening to identify analyte specific redox enzymes for biosensor development. As a proof of concept, we report the first electrochemical enzyme-based nicotine biosensor from a novel microbial enzyme, and use a variant with improved catalytic performance to enhance sensor performance. The biosensor detects nicotine over 0.4-100 M, a range relevant to nicotine concentrations present in active smoker sweat, saliva, gastric juice, and urine. This microbial mining approach for discovering redox enzymes expands the sensing parts toolbox available over conventional antibodies and aptamers.

bioengineering↗

bioRxiv2026

Spatial transcriptomics for gene discovery identifies Slc13a5 as a modulator of bone mechanoadaptation

Bone is a dynamic tissue that continuously adapts its structure in response to mechanical loading, an essential process for maintaining skeletal health. However, this adaptive capacity declines with aging, contributing to increased fragility and fracture risk. Developing therapeutic strategies that preserve or restore bone mechanoadaptation in patients with increased bone fragility requires identifying key molecular regulators of this process. We applied spatial transcriptomics (GeoMx, NanoString) to characterize gene expression changes induced by mechanical loading in the murine tibia, focusing on periosteal and bone compartments in regions under tension and compression. Spatial data were validated and cross-compared with previously published bulk RNA-seq and laser-capture microdissection datasets, identifying a set of 12 genes consistently regulated by loading across independent platforms and laboratories. As part of a functional analysis, we selected Slc13a5, a citrate transporter implicated in bone mineralization and metabolism. Conditional deletion of Slc13a5 in osteolineage cells using Osteocalcin-Cre significantly increased the loading-induced mineralizing surface in tensile regions compared with Cre- Slc13a5fl/fl littermates. In addition, Slc13a5 cKO mice exhibited lower resorption around the neutral axis after loading compared to controls. Together, these findings identify Slc13a5 as a regulator of bone adaptation in regions experiencing low mechanical stimulation and suggest it as a potential therapeutic target for conditions characterized by impaired mechanoadaptive responses. This study highlights spatial transcriptomics as a powerful gene discovery framework for bone, enabling identification of novel targets to understand mechanisms and develop therapies. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=181 SRC="FIGDIR/small/711126v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@14fc0e9org.highwire.dtl.DTLVardef@1023a16org.highwire.dtl.DTLVardef@133aa0eorg.highwire.dtl.DTLVardef@e19c86_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2026

ACTIVATED CASO₄-INDUCED VACUOLATION AS A QUANTITATIVE PLATFORM FOR PHAGOCYTOSIS-DRIVEN DRUG SCREENING

Cytoplasmic vacuolization is a fundamental process associated with phagocytosis, lysosomal acidification, and autophagy, yet robust in-vitro models for its quantification and pharmacological screening remainlimitedor insufficiently established. In this study, we demonstrate that thermally activated calcium sulfate (ACS) induces extensive vacuolation across mammalian cell lines including HeLa, RAW 264.7, 3T3-L1, and SH-SY5Y, thereby establishing a versatile platform to study vacuole biogenesis. To ensure reproducibility, particle heterogeneity was addressed using sedimentation-based fractionation, with homogeneous suspensions obtained at the 5th minute producing stable and consistent vacuole formation. Vacuolation was subsequently quantified by Neutral Red (NR) uptake, dose and time dependent response analyses confirmed direct correlation between ACS concentration and vacuole induction. The assay was validated with bafilomycin A1 (BFA1), a selective V-ATPase inhibitor, which served as a positive control and demonstrated concentration and time dependent inhibition of vacuole formation and acidification. Building on this framework, ten commercially available drugs were screened, revealing distinct profiles ranging from early cytotoxicity, strong vacuole inhibition to partial suppression or negligible effects. This dual capacity to discriminate between vacuole inhibition and cytotoxic responses highlights the utility of ACS-induced vacuolization as a sensitive and scalable in vitro platform. Collectively, our findings position this system as a tractable assay for mechanistic studies of vacuole biology and a functional screening tool for identifying modulators of lysosomal and phagocytic pathways relevant to infection, Lysosomal Disorders, and Phagocytotic dysfunction disorders. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/711143v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@e5b920org.highwire.dtl.DTLVardef@1dd1072org.highwire.dtl.DTLVardef@62ae59org.highwire.dtl.DTLVardef@a468bf_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2026

A Multispecies, Modality-Agnostic Scalable In Vivo Mosaic Screening Platform for Therapeutic Target Discovery

Validating therapeutic targets for complex diseases requires investigating gene functions within native tissue architectures rather than reductionist in vitro models. Here we present a modality-agnostic AAV-based in vivo high-throughput screening platform capable of delivering knockouts, gain-of-function, and synthetic miRNA knockdowns directly to cells within the diseased environment. This system scales to hundreds of perturbations and is adaptable to diverse species and organ systems. To translate high-dimensional screen data into therapeutic assessment, we established a curated analysis framework that scores single-cell transcriptomes against human disease-specific molecular signatures. This method enables quantitative ranking of targets across distinct biological domains ranging from structural fibrosis to inflammatory signaling, to narrow in on the therapeutic potential of each intervention. We applied this strategy to screen loss- and gain-of-function libraries in a murine pulmonary fibrosis model and within the spontaneously osteoarthritic joints of aged horses, identifying metabolic, antifibrotic, and immunomodulatory targets. Importantly, our analysis framework successfully predicted functional outcomes in orthogonal human ex vivo tissue models, including soluble collagen reduction in lung slices and glycosaminoglycan restoration in cartilage, thus establishing a powerful paradigm for prioritizing therapeutic targets by uniting human disease signatures with highly multiplexed in vivo functional genomics.

systems biology↗

bioRxiv2026

Oncogenic chromodomain mutations allosterically impair TIP60 acetyltransfearse function preventing activation of DNA repair genes under genotoxic stress

TIP60 is a tumor suppressor with histone acetyltransferase activity that regulates chromatin accessibility in diverse processes including DNA repair, apoptosis, mitosis, transcription, and autophagy. Structurally TIP60 contains a C-terminal MYST domain that mediates its HAT activity, while the N-terminal chromodomain are conserved modules that facilitate its association with chromatin by recognizing histone modifications. Mutations within the chromodomain have been implicated in various cancers, yet their functional consequences remain poorly understood, particularly with respect to TIP60s role in maintaining genomic integrity. Here, we uncover a novel allosteric mechanism whereby cancer-associated chromodomain mutations impair TIP60s catalytic activity without disrupting its chromatin binding, underscoring critical interdomain communication between the chromodomain and the MYST domain. Through structural modelling and molecular dynamics simulations, we identified two missense mutations (R53H and R62W) in TIP60s chromodomain that not only altered TIP60s conformation but also destabilized its trimeric assembly, thereby impairing acetyl-CoA docking. Importantly, we found that TIP60 engages acetyl-CoA exclusively in its trimeric state, and the R62W mutation perturbs the trimeric interface, thereby altering the docking sites for acetyl-CoA. Consistent with these structural changes, biochemical assays revealed that chromodomain mutant TIP60 variants, while retaining chromatin loading, exhibited markedly reduced autoacetylation and histone acetyltransferase activity. Moreover, these mutants failed to activate the p21 gene in response to DNA damage, thereby predisposing the genome to the accumulation of mutations and leaving cells unable to arrest the cell cycle for repair of genomic lesions. Together, our findings establish that distal chromodomain mutations allosterically destabilize TIP60 oligomerization, impair acetyl-CoA utilization, and compromise DNA damage responses. The mechanism establishes a link between chromodomain mutations and genomic instability, shedding light on how reader domain alterations may underlie cancer progression.

molecular biology↗

bioRxiv2026

Predicting Post-Stroke Aphasia Speech Performance from Multimodal Data with Explainable Machine Learning

Aphasia, an acquired language deficit, is the most common post-stroke focal cognitive impairment, and roughly 60% cases become chronic (duration >6 months). Aphasia therapies could be optimized if clinicians could make personalized predictions of how individual persons with aphasia (PWA) would be likely to perform on particular language tasks. However, current approaches relying on imaging, lesion volume, patient demographics, and clinical scores achieve less than 50% accuracy in predicting performance in PWA. Research algorithms using complex imaging and fMRI can make binary predictions about the presence or absence of aphasia but do not give more clinically relevant information. We aim to predict word-by-word speech accuracy in PWA to better enable personalized speech therapies. To be clinically informative, machine learning models developed for this purpose should use clinically available inputs, explain key features behind a prediction, and generalize to new PWA and previously unseen words. This study combines multimodal input features from clinical testing scores and structural MRI neuroimaging with a novel data source: word-by-word linguistic difficulty. We computed metrics of cognitive burden, such as semantic selection and recall demands, and articulatory burden, such as word length in phonemes and syllables, using naturalistic corpora containing over a billion words of English text. Retrospective training, ten-fold cross validation and 500-run bootstrapping of different machine learning models with various combinations of input features was conducted using 4620 trials. A simplified version of the best model using widely available inputs was deployed clinically through a web app, and prospective generalization was tested on 570 trials with unseen words and different naming tasks in new PWA. We found the best performances with random forest classifiers using linguistic difficulty combined with either clinical information (AUROC {+/-} SEM = 0.87 {+/-} 0.07), or all together with structural imaging connectivity (0.90 {+/-} 0.04). Classifiers using multimodal inputs significantly outperformed others employing single inputs (range 0.66-0.85, p<0.05). Extracting feature importances from the best model showed that Western Aphasia Battery scores, semantic demands, number of phonemes, and syllables were predictive of PWA speech accuracy. Structural integrity in peri-lesional brain regions predicted better language performance whereas higher connectivity of select contralateral homotopes contributed to prediction of worse speech. Without the inclusion of MRI data, lesion volume was a key predictor of PWA speech as well. A simplified, clinically ready, explainable model (publicly available as AphasiaLENS web application) predicted PWA accuracy for any user-entered word, not restricted to a standardized battery. Its prospective generalization performance was not significantly different from the best model using full inputs (AUROC ranges 0.81-0.89, p>0.05). Thus, our research can help inform individualized treatment planning for PWA, while also suggesting research targets through better understanding of brain-behavior relationships.

bioengineering↗

bioRxiv2026

Phage-Mediated Iron Acquisition by Pseudomonas aeruginosa

Iron acquisition is critical to bacterial growth and pathogenesis. Here, we describe a previously unrecognized mechanism by which the major human pathogen Pseudomonas aeruginosa acquires iron through a unique partnership with bacteriophages (phages). Pf is a filamentous phage that infects P. aeruginosa and is associated with chronic infections. We reveal that Pf contributes to P. aeruginosa pathogenesis by promoting iron uptake. We demonstrate that Pf phage is highly induced under iron-deplete growth conditions and that once induced, Pf virions directly bind and locally concentrate free iron. Pf-bound iron is more efficiently utilized by P. aeruginosa than unbound iron, enhancing bacterial growth. We further demonstrate that Pf-mediated iron acquisition depends on type IV pili, which facilitate Pf attachment and confers strain selective uptake of phage-bound iron, providing a competitive fitness advantage in polymicrobial settings. Together, these findings identify an unrecognized role for filamentous phages in mediating iron acquisition and reveal a novel phage-bacterium partnership that operates through selective kin cooperation.

microbiology↗

bioRxiv2026

Platelet-derived CXCL7 induces neutrophil extracellular traps via CXCL7/CXCR2 axis, exacerbating the pathogenesis of diabetic retinopathy

BackgroundProlonged hyperglycemia in diabetes activates platelets and immune cells, forming platelet-immune complexes that damage blood vessels in the retina. However, the role of platelet-neutrophil interactions and neutrophil extracellular traps(NETs) in the development of diabetic retinopathy (DR) was not well studied. In this study, we investigated the mechanisms underlying platelet-mediated NET formation in DR. MethodologyPlatelet activation markers, platelet-neutrophil aggregates (PNA), and NETs markers were assessed by flowcytometry, and the circulatory level of inflammatory markers was measured by Luminex assays in healthy control(HC), type 2 diabetes mellitus(T2DM), non-proliferative DR(NPDR) and proliferative DR(PDR) subjects. In vitro studies investigated platelet-neutrophil interaction in NETs formation using an immunofluorescence assay. Proteomics analysis identified the mechanistic regulators of platelet-induced NETs in DR. Platelet pellet and plasma CXCL7 were quantified using western blot and ELISA, respectively. The role of the CXCL7/CXCR2 axis in inducing NETs formation was examined using CXCL7 recombinant protein, anti-CXCL7 antibody and CXCR2 antagonist (SB225002). ResultsPlatelet activation markers (p-selectin & PF4), PNA, and NETs markers (%NETs, proteinase-3 (PR3), neutrophil elastase (NE)) were significantly increased in the DR group. In vitro studies confirmed that DR-platelets aggregate with healthy neutrophils and form NETs compared to T2DM and HC-platelets. Furthermore, platelet activation and NETs markers were positively correlated with pro-angiogenic (ANGPT2, VEGFA) and inflammatory markers (IL18, ICAM1). In vitro studies reveal that NETs induce inflammation, endothelial dysfunction, disrupt the endothelial monolayer and exacerbate angiogenesis in RF/6A endothelial cell spheroids. Proteomics analysis of platelet-induced NETs in DR revealed dysregulation of proteins involved in platelet activation and NET formation, including CXCL7. Furthermore, increased CXCL-7 levels were observed in platelet pellet and plasma samples from the DR group. Additionally, CXCL7-treated neutrophils formed NETs via the CXCR2 receptor, and inhibition of NETosis was observed in neutrophils exposed to an anti-CXCL-7 antibody and a CXCR2 antagonist. ConclusionOur findings revealed that platelets released CXCL-7 induce NETs formation via the CXCL7/CXCR2 axis and blockade of CXCL7/CXCR2 axis inhibits the NETosis in DR, thereby inhibiting the pathogenesis of DR. Circulating CXCL7 serves as a potential prognostic marker, and the CXCL7/CXCR2 axis may be a therapeutic target for the treatment of DR. What Are the Clinical Implications?Platelets have emerged as immune cells, and platelet-neutrophil interactions are reported to play a significant role in the pathogenesis of various metabolic diseases. The role of platelet-neutrophil interactions and neutrophil extracellular traps (NETs) in the development of diabetic retinopathy (DR) remains poorly understood. Investigating the mechanistic regulators of platelet-mediated NETs in DR is crucial for identifying new therapeutic approaches. Our study observed increased platelet activation, platelet-neutrophil aggregates and NETs among DR subjects. Further, DR-platelet induces NET in healthy neutrophils, and NETs induce inflammation, angiogenesis, and disrupt endothelial barrier function in RF/6A cells in vitro. These findings strengthen the evidence that platelet-neutrophil interactions play a major role in DR pathogenesis. Proteomic analysis identified CXCL7 as a mechanistic regulator of platelet-induced NETs formation in DR. Inhibitors that target the platelet-derived CXCL7/CXCR2 axis for NETosis can be used for the prevention of retinal injury in DR. Overall, our work emphasises the mechanistic understanding of platelet-neutrophil interactions and CXCL7/CXCR2 axis as a therapeutic target for inhibiting the pathogenesis of DR. Graphical abstractScheme for the platelet-derived CXCL7 regulation of NETs in DR. Activated platelets release CXCL7 and platelet aggregates with neutrophils to form NETs via the CXCL7:CXCR2 axis. Blockade of the CXCL7:CXCR2 axis by anti-CXCL7 antibody and CXCR2 inhibitor prevents NETs formation in DR.

immunology↗

bioRxiv2026

Actin-dependent mechanotransduction controls nucleocytoplasmic partitioning of DNMT3a through ERK1/2 signaling during cutaneous wound healing

Cutaneous wound healing is a multifaceted physiological process that requires a cell state transition from homeostasis to tissue repair. An important contributor to this reprogramming is the wound-induced mechanical cues that are perceived by epidermal keratinocytes. Previously we identified that the nuclear translocation of the de novo DNA methyltransferase 3A (DNMT3a) upon wounding as an important regulator of this cell-state transition. However, the molecular mechanisms linking mechanotransduction to epigenetic regulation remain incompletely understood. Here we show that under homeostasis, active ERK1/2 phosphorylates DNMT3a, intramolecularly masking its nuclear localization signal (NLS), resulting in its cytoplasmic sequestration. Upon wounding actin cytoskeleton remodeling leads to the downregulation of the Extracellular Signal-Related Kinase 1/2 (ERK 1/2) pathway. Wound-induced ERK1/2 inactivation unmasks the NLS, enabling DNMT3a nuclear translocation. Collectively, these findings define a mechanotransduction-driven signaling axis linking cytoskeletal dynamics to epigenetic regulation and confirms an active role for differentiated keratinocytes in initiating early wound repair programs.

cell biology↗

bioRxiv2026

Outlier Detection in Single-Cell Transcriptomics Reveals Disease-Enriched Cytotoxic Immune Populations

Single-cell RNA sequencing (scRNA-seq) enables detailed analysis of cellular heterogeneity, but standard clustering approaches can miss rare cell populations with important disease relevance. We present an outlier-aware framework that complements clustering by identifying statistically unusual cells. Applied to COVID-19 bronchoalveolar lavage fluid (BALF) samples, this approach revealed strong disease-associated enrichment of outliers: 9.62% in moderate disease and 6.16% in severe disease, compared with 1.14% in healthy controls. These cells exhibited elevated cytotoxic marker expression (NKG7, GZMB, PRF1, GNLY; 2.0-2.9x enrichment) consistent with activated effector immune states, with replication across independent datasets. To verify that out-liers were not low-quality cells, we confirmed superior QC metrics: outliers showed 68% more genes and 78% higher UMI counts than normal cells. Application to rheumatoid arthritis PBMCs showed modest enrichment (5.40% in RA vs. 4.66% in healthy), consistent with chronic inflammation. Comparison of four outlier detection algorithms identified Isolation Forest as the best-performing method. Subclustering revealed diverse functional states within outliers, including NK cytotoxic cells, activated T cells, and proliferating cells. Overall, outlier detection reveals rare, functionally distinct immune populations overlooked by conventional clustering.

bioinformatics↗

bioRxiv2026

Genome wide analysis of YBX1-mediated redistribution of JMJD6 in ER+ breast cancer cells

The molecular mechanisms by which cancer cells proliferate under diminished ER expression leading to intrinsic and acquired resistance to endocrine therapy remain incompletely understood. Here, we identify a previously unrecognized transcriptional axis centred on JMJD6 and YBX1 that cooperatively reprograms chromatin, redistributes enhancer engagement and sustains tumor proliferation when JMJD6 levels are elevated (JOE cells). Through proximity ligation, ChIP-reChIP assays, KO-based ablation experiments, and genome-wide profiling, we demonstrate that YBX1 is required for JMJD6 enrichment at regulatory sites. [~]20% of JMJD6 and YBX1 peaks physically superimpose, and remaining peaks are contained within a neighbourhood of {+/-}50kb. A shared extended binding site (EBS) containing (i) flanking poly-A/poly-T tracts; (ii) a canonical YBX1 recognition sequence; and (iii) a second complementary motif enriched for zinc-finger proteins defines JMJD6-YBX1 regulated enhancers. Histone modification landscapes in JMJD6 overexpressing cells supported the presence of a transcription permissive chromatin. Further, JMJD6/YBX1 siRNA and overexpression analysis in MCF7 cells identified estrogen and interferon gamma signalling as prominent pathways. The most compelling observation was that ER loss was transcriptional in JOE cells and was accompanied by the emergence of JMJD6-YBX1 enhancer sites at alternate genomic positions relative to canonical ER-JMJD6 sites described in estrogen-stimulated cells. These findings offer a mechanistic insight into earlier reports from our group showing that high JMJD6 with YBX1 suppresses ER expression and this could promote endocrine resistance.

cancer biology↗

bioRxiv2026

Bayesian Inference of Gene Regulatory Networks at Stochastic Steady State

Gene Regulatory Networks (GRNs) form the regulatory back-bone that coordinates gene expression. The architecture of GRNs shapes their function and constraints the biochemical pathways through which information flows. Inferring the structure of regulatory interactions is thus essential for understanding biological systems, and designing targeted therapies. Despite substantial progress in GRN inference, most approaches - from statistical methods to deep learning - do not take into account fundamental biochemical processes that drive regulatory dynamics. To address this shortcoming, here we present a novel Bayesian inference approach based on using the Chemical Langevin Equation (CLE) as a model of gene expression dynamics at stochastic equilibrium. Inter-actions in GRNs are sparse, and we thus use a regularized horseshoe prior enabling selective shrinkage of unsupported interactions while identifying strong regulatory edges. We evaluate our method using synthetic gene expression data, allowing for benchmarking against a known ground truth. Our approach allows us to infer kinetic parameters, identify network structure, and infer regulatory cycles without the need to observe transient dynamics. This Bayesian alternative to current methods thus provides both biological interpretability and structural identifiability in GRN inference.

bioinformatics↗

bioRxiv2025

Altered aperiodic EEG spectral power during speech perception task is associated with verbal communication in youths with Autism Spectrum Disorder

Most children with Autism Spectrum Disorder (ASD) have co-occurring language impairment, but its neural mechanisms are not well known. Excitation (E) / inhibition (I) imbalance is considered as a key neurobiological mechanism of ASD, and several electroencephalography (EEG)-based E/I balance metrics have been proposed in the previous studies. The goal of the present research was to focus on these metrics abstracted from the speech perception task to investigate their relation to language/communication in autistic youths. We used a high-density 128-channel EEG to register neural responses during speech perception task in the sex- and age-matched groups of youths with ASD (N = 162) and typically developing (TD) controls (N = 144), aged 7-18 years old. The results revealed alterations in the E/I measures in the ASD group, pointing to a higher level of excitation or neural noise in the cortex as well as broadband reduction of spectral power during speech perception. A greater neural noise reflected in the reduction of aperiodic exponent and offset was associated with lower verbal communication skills in youths with ASD. The findings suggested that the higher noisiness in the cortical systems may be a relevant marker to monitor in relation to language/communication in ASD.

neuroscience↗

bioRxiv2025

A transition zone enriched WIF1⁺ basal cell subtype is associated with benign prostatic hyperplasia

ABSTRACT/SUMMARYThe cellular composition and disease susceptibilities of the distinct zones of the human prostate remain incompletely understood. Through extensive single-cell RNA sequencing (scRNA-seq) of benign regions from prostatectomy specimens, we identified a basal cell population expressing WIF1, VCAN, and NRG1, among other genes, that was significantly enriched in the transition zone (TZ). Benign prostatic hyperplasia (BPH) is a common condition that causes widespread morbidity and is nearly exclusively localized to the TZ. Analysis of previously published scRNA-seq datasets further confirmed that WIF1+ basal cells were significantly enriched in BPH compared to normal prostate. Pathway and cell-cell communication analyses revealed that this basal subtype is associated with programs related to cell proliferation, epithelial-mesenchymal transition (EMT), angiogenesis, and hormone response. Together, the molecular signature, zonal distribution, and pathway enrichment suggest that TZ-enriched WIF1+ basal cells may contribute to BPH pathogenesis by promoting epithelial and stromal remodeling.

cell biology↗

bioRxiv2025

Gut microbial interaction networks control autoimmunity to neuroretina

The gut microbiome influences the development of immune-mediated inflammatory diseases, including autoimmune uveitis, a sight-threatening ocular inflammation driven by retina-specific T cells1. Using a model of spontaneous autoimmune uveitis (sEAU) we showed that gut commensals provide immune stimuli that trigger disease2. Here we report that uveitis-promoting microbes are present in human gut flora and that colonization of germ-free (GF) mice with commensals from healthy human donors was sufficient to provoke disease. Severity of sEAU correlated with expansion of Akkermansia and contraction of short-chain fatty acid (SCFA)-producing Firmicutes, followed by decreased SCFA levels and a dominant gut Th1 effector response. Mechanistic gain-of-function experiments, enriching GF sEAU mice with Akkermansia, reproduced these microbiome, metabolite and immune phenotype shifts, and exacerbated disease, suggesting that Akkermansia promotes autoimmunity by outcompeting SCFA-producers and enhancing Th1-type responses. An inverse correlation between Akkermansia (Verrucomicrobia) and Firmicutes was also present in patients with uveitis, multiple sclerosis and Crohns disease. These findings reveal a stereotypic gut microbial interaction network that regulates systemic immune balance, and may represent an ecologically conserved mechanism through which the gut microbiome modulates autoimmune and inflammatory diseases.

immunology↗