Biology subjects

Singh, R.

Publications and source records attributed to Singh, R..

At least 37 records · Page 2Linked to original sources

bioRxiv2026

SLD5/GINS4 controls dynein-dependent centrosome maturation and exposes a candidate mitotic vulnerability in cancer.

Faithful proliferation requires coordinated DNA replication with centrosome maturation and spindle-pole integrity. SLD5, encoded by GINS4, is a core component of the GINS replication complex and is frequently elevated in tumors, but whether it links replication-associated cancer states to centrosome control has remained unclear. Here, we show that GINS4/SLD5 is recurrently upregulated across human cancers at transcript and protein levels and marks tumor programs enriched for DNA replication, chromosome segregation, and mitotic control. In cancer cells, Sld5 depletion dispersed PCM1, AZI1, and CEP290-positive centriolar satellites without eliminating these satellite proteins, reduced dynein heavy chain expression, and destabilized dynein-dynactin localization at spindle poles. Direct depletion of dynein heavy chain, co-depletion analyses, and pharmacological inhibition of dynein motor activity with ciliobrevin D phenocopied Sld5 loss, causing satellite dispersion, defective recruitment of PLK1, Aurora A, CEP192, and CEP215 to centrosomes, and multipolar spindle formation. These defects occurred without detectable DNA damage or checkpoint activation, indicating a non-canonical Sld5 function beyond its role in the replisome. Cancer dependency and kinase network analyses further nominate SLD5-associated mitotic and checkpoint pathways as therapeutic targets. Our findings identify SLD5/GINS4 as a regulator of dynein-dependent centrosome maturation and a candidate vulnerability in replication-driven cancers, with potential value for biomarker-guided therapeutic stratification. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=136 SRC="FIGDIR/small/723511v1_ufig1.gif" ALT="Figure 1"> View larger version (53K): org.highwire.dtl.DTLVardef@525d9org.highwire.dtl.DTLVardef@1f7d7eeorg.highwire.dtl.DTLVardef@815586org.highwire.dtl.DTLVardef@115f286_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2026

Coordinated cell and chloroplast growth and its perturbation by chloroplast DNA replication inhibition in green algae

Coordination among cell growth, chloroplast expansion, and organelle genome dynamics is fundamental to algal physiology, yet its regulation remains unclear. We used time-resolved single-cell analyses to examine scaling relationships among cell size, chloroplast volume, nuclear dynamics, and nucleoid organization in Desmodesmus communis and Chlamydomonas reinhardtii under normal conditions and after inhibition of chloroplast DNA replication with nalidixic acid (NAL). Under control conditions, both species showed coordinated scaling among cell, chloroplast, and nuclear size, while nucleoid dynamics were driven mainly by changes in number. NAL disrupted these relationships in a species- and time-dependent manner. In C. reinhardtii, prolonged treatment uncoupled chloroplast and nuclear growth from cell expansion and led to fewer, enlarged nucleoids, consistent with impaired replication. In contrast, D. communis largely maintained coordinated scaling, with effects mainly limited to reduced nucleoid proliferation and delayed division. Temporal analyses indicated that NAL primarily affected nucleoid replication and segregation, with secondary consequences for chloroplast growth and cell-cycle progression. These findings identify chloroplast genome dynamics as a regulatory link between organelle growth and cell division.

cell biology↗

bioRxiv2026

IL1β/IL1R1/IRAK4 Drives Inflammatory Ovarian Cancer Seeding at the inflamed sites and Is Reversed by an IRAK4 inhibitor UR241-2

Inflammation-driven tumor implantation, such as port-site metastasis (PSM) following laparoscopic gynecologic surgery and peritoneal seeding during post-surgical recurrence, represents an aggressive clinical problem that remains poorly understood and lacks targeted therapies. To address this, we developed a non-surgical Mesothelium-Inflammation/Injury-Metastasis (MIM) model and investigated the role of the IL-1{beta}/IL1R1/MYD88/IRAK1/4 axis and NLRP3 in epithelial ovarian cancer (EOC) seeding at inflamed or injured sites. This model created by a needle injury recapitulates inflammation-driven peritoneal seeding and mimics PSM and inflammation associated dissemination in peritoneum during recurrence. Seeding was dependent on Il1r1 but not Nlrp3, despite its role in regulating IL-1{beta} production, as Il1ra-/- and Nlrp3-/- mice phenocopied wild-type C57BL/6 mice. Given the limited antitumor efficacy of IL-1{beta}-targeting agents such as Anakinra and Canakinumab, we focused on IRAK4 as a therapeutic target. IRAK4 knockdown significantly prolonged survival, reduced tumor cell adhesion, downregulated E-cadherin and Wnt4, and induced S-phase/mitotic arrest. This led to the development of UR241-2, a small-molecule IRAK4 inhibitor, which was validated through molecular simulations, hotspot analysis, nanoBRET, global kinome profiling, and NF-{kappa}{beta} reporter assays. UR241-2 inhibited NF-{kappa}{beta} nuclear translocation and blocked IL-1{beta}-induced IRAK4 phosphorylation. UR241-2 exhibited favorable drug-like properties, including absence of CYP or hERG inhibition, and acceptable CaCo-2 permeability, plasma protein binding, microsomal stability, and pharmacokinetics. In vivo, UR241-2 reduced SKOV3 xenograft growth, suppressed mesothelial seeding, and increased MHC-II macrophages and activated neutrophils in syngeneic high-grade epithelial ovarian HGS3 tumors. RNA-seq revealed enrichment of neutrophil activation signatures and suppression of extracellular matrix (ECM) gene programs. Together, these findings establish a role for the IL-1{beta}/IL1R1/IRAK4 axis in inflammation-driven PSM and peritoneal seeding and ECM regulation in EOC, and demonstrate that IRAK4 inhibition activates antitumor immune responses, providing a therapeutic strategy to block metastatic seeding and improve tumor control.

cancer biology↗

bioRxiv2026

Whole-Genome sequencing of Indigenous Withania somnifera accession and comparative cytochrome P450 phylogenomics

Cytochrome P450 monooxygenases (CYP450s) are key oxidative enzymes that diversify plant specialized metabolites and play a central role in the biosynthesis of bioactive withanolides in Withania somnifera (L.) Dunal. Despite their importance, genome-wide information on CYP450s in W. somnifera has remained elusive. Herein, the first high-quality genome assembly (2.2 Gb, scaffold N50: 47.4 kb) of an Indian W. somnifera cultivar was generated using a hybrid Oxford Nanopore-Illumina sequencing strategy. Comparative analysis with the NCBI reference genome revealed moderate SNP and indel variations, reflecting intraspecific genetic diversity. A comprehensive CYP450 catalog was established and analyzed phylogenomically across nine plant genomes, encompassing both withanolide-producing and non-producing Solanaceae and non-Solanaceae species. Unique CYP families (CYP450A, CYP1194, and CYP705A) were detected exclusively in W. somnifera, suggesting lineage-specific metabolic innovations, while Solanaceae-restricted (CYP82E/M) and absent (CYP81B, CYP6) lineages highlight taxonomic divergence. Across all analyzed genomes, 36 conserved CYP450 subfamilies, including triterpenoid-associated members, were identified, suggesting a shared oxidative framework adaptable to specialized metabolism. Moreover, potential candidate genes in the triterpenoid pathway, including CYP72A692_1, CYP72A560_4, CYP716A48, CYP724B2, and CYP51G1, were identified through phylogenetic integration with functionally validated triterpenoid-modifying enzymes from other plant species. Gene family evolution analysis further revealed contraction of monoterpenoid-related subfamilies (CYP76A), implying a metabolic shift toward triterpenoid specialization. The comprehensive genome assembly and CYPome of W. somnifera offer a valuable resource for functional characterization, evolutionary analysis, and the identification of genes underlying its specialized metabolism. Furthermore, the study advances our understanding of CYP450 diversity and evolution, revealing lineage-specific innovations, conserved subfamilies, and key candidate genes involved in triterpenoid biosynthesis. Together, these findings lay a foundation for future functional studies and pathway engineering aimed at optimizing the metabolic potential of this important medicinal plant.

genomics↗

bioRxiv2026

XRRA1 acts as a molecular brake on radiation-induced DNA damage signaling and immunogenic cell death in tumor cells.

Radiotherapy kills cancer cells by inducing DNA damage, but adaptive responses that buffer injury and limit immunogenic signaling remain incompletely understood. Although ionizing radiation can activate cytosolic DNA sensing and immunogenic cell death, the tumor-intrinsic regulators linking these processes to radioresistance are not well defined. Here, we show that XRRA1 is a stress-adaptive determinant of radioresponse identified by integrating discovery proteomics into chronic myeloid leukemia with clinical tissue validation and functional studies across multiple tumor models. In peripheral-blood proteomes, XRRA1 was quantitatively reduced in chronic myeloid leukemia yet associated with a favorable prognosis and with networks enriched for RNA regulation, apoptosis, and DNA-repair biology. In human biopsy specimens, XRRA1 protein abundance was increased in radiation-exposed tissues, and in cultured cells, ionizing radiation induced XRRA1 more strongly and persistently in normal cells than in cancer cells. Silencing XRRA1 had little effect on basal growth but markedly enhanced radiation-induced loss of viability, apoptosis, spheroid disruption, and clonogenic failure, while increasing {gamma}H2AX- and DNA-PK-associated damage signaling and linking XRRA1 to non-homologous end joining factors. XRRA1 depletion also amplified cGAS-STING-TBK1-IRF3 activation, interferon-stimulated gene expression, extracellular ATP release, and cell-surface calreticulin exposure. These findings identify XRRA1 as a molecular brake that limits the conversion of radiation-induced DNA damage into immunogenic stress responses. XRRA1, therefore, represents a candidate biomarker of radioadaptive stress and a potential target for radiosensitization and radiotherapy-immunotherapy combinations.

cancer biology↗

bioRxiv2026

Saliva Metabolomics Reveals Distinct Metabolic Signatures in Patients with Chronic Obstructive Pulmonary Disease: A GC-MS-based approach.

Chronic obstructive pulmonary disease (COPD), a chronic lung disease, involves complex metabolic disturbances that remain poorly characterized using non-invasive matrices. The metabolic alterations associated with cigarette smoke (CS), one of the major drivers of disease progression in COPD patients, have not been explored in detail. This study primarily aimed to investigate the metabolic signatures in COPD patients categorized into smoker (n=15), ex-smoker (n=11), and non-smoker (n=3) subgroups. Utilizing saliva as a noninvasive sample, we identified 26 metabolites with differential expression in smokers and 31 in ex-smokers. However, no such significant alteration was observed in the non-smokers subgroup. The multivariate analysis distinctly separated the COPD subgroups from healthy controls. Additionally, pathway enrichment analysis revealed perturbations in key metabolic pathways, including unsaturated fatty acid biosynthesis, arginine biosynthesis, the tricarboxylic acid (TCA) cycle, and pyruvate metabolism. Moreover, univariate Random forest analysis identified four metabolites (cyclopentanone, tetradecane 4-methyl, acetophenone, and scyllo-inositol) as potential biomarkers distinguishing COPD subgroups from healthy controls. This study offers novel molecular insights into the association of smoking with disease progression and provides a mechanistic understanding of COPD in different subgroups for better management of the disease. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=163 SRC="FIGDIR/small/717654v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@1c3be84org.highwire.dtl.DTLVardef@10ce0aorg.highwire.dtl.DTLVardef@1470712org.highwire.dtl.DTLVardef@2163b6_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2026

From Light to Acetate: How Trophic Conditions Shape Growth and Cell Cycle Progression in Chlamydomonas reinhardtii

The unicellular green alga Chlamydomonas reinhardtii provides a tractable model for investigating how carbon availability influences metabolic organization and cell-cycle control in photosynthetic eukaryotes. Its capacity for autotrophic (light, CO), mixotrophic (light, CO, acetate), and heterotrophic (acetate, dark) growth enables systematic analysis of trophic-state-dependent regulation. We performed comparative transcriptomic analyses of strain 21gr grown under these three regimes at 30 {degrees}C. Mixotrophy resulted in the highest biomass accumulation and was associated with earlier cell-cycle commitment compared with autotrophy, whereas heterotrophy displayed delayed commitment and reduced growth. Transcriptomic profiling revealed coordinated upregulation of central carbon metabolic pathways under mixotrophy, including photorespiration, glycolysis, the oxidative pentose phosphate pathway, and tricarboxylic acid cycle functions, consistent with enhanced carbon flux and biosynthetic capacity. In contrast, heterotrophy preferentially induced acetate assimilation and glyoxylate cycle genes and was accompanied by elevated expression of cell-cycle regulators, including the CDK-inhibitory kinase WEE1. Together, these findings indicate that trophic mode modulates the coupling between carbon metabolism and cell-cycle progression, with mixotrophy supporting integrated metabolic and proliferative activity, whereas heterotrophy is associated with delayed cell-cycle timing and transcriptional signatures of metabolic adjustment.

plant biology↗

bioRxiv2026

WINDEX: A hierarchical integration of site- and window-based statistics for characterizing the footprint of positive selection in genome-wide population genetic data

Adaptive mutations, or mutations that confer a fitness benefit, can leave behind distinct signals in genetic data. Computational methods have improved the localization of adaptive mutations in genetic samples using a range of statistical and machine learning classification techniques. However, these methods miss the opportunity to jointly integrate statistics at both the site and window-based level, thus failing to harness all available statistical evidence to detect selection. Our method, WINDEX, combines these different resolutions of statistics to improve the detection of adaptive mutations among hitchhiking signals. Our model simultaneously integrates emissions at different resolutions by defining site-based and window-based latent states corresponding to neutral, linked, and sweep regions, with the site-based states and transition models nested within the window-based states. Using evolutionary simulations with varying selection parameters, we validate the ability of WINDEX to classify positive selective sweeps. Using data from the 1000 Genomes Project, we show that WINDEX is able to identify regions harboring signals of selective sweeps, and provides improved localization within those regions over existing methods. In addition, using WINDEX genome-wide allows for estimation of the proportion of whole genomes that are under positive selective pressures; our estimates of between 9.7-10.5% across different populations provide support for other preliminary estimates of these quantities. Author summaryPopulation geneticists often seek evidence for positive selective sweeps, or an evolutionary event in which a beneficial allele increases in frequency over time in a population, resulting in increased fitness of the individuals that have said allele. Positive selective sweeps, however, are difficult to detect due to varying patterns of linkage disequilibrium (LD), or the nonrandom association of alleles, and detecting these signals reliably among differing LD structures remains a challenge in the field. In this work, we present WINDEX, a probabilistic framework designed to leverage signals of positive selective sweeps at both the site- and window-levels in the form of a hierarchical hidden Markov model (HHMM), to localize regions of positive selective sweeps in aligned haplotype data. We validate WINDEX in evolutionary simulations over varying positive selective sweep scenarios, showcasing the improved resolution that the HHMM structure provides. We apply WINDEX in comparative genomic scans of canonical sites of positive selection as well as whole-genome scans to demonstrate the tools power in localizing functionally-validated signals of selection and to offer insights into the proportion of the human genome currently under positive selective pressures. WINDEX is publicly available and easy to apply to many cases of human genetic data.

evolutionary biology↗

bioRxiv2026

Cell type-specific gene regulatory network inference from single cell transcriptomics with ctOTVelo

Inferring gene regulatory networks (GRNs) from gene expression is a crucial task for understanding functional relationships. Gene expression data (transcriptomics) provide a snapshot of gene activity, encoding information about gene regulatory relationships. However, gene regulation is a dynamic process, modulating across time and with different cell types. Temporal GRN inference methods aim to capture these dynamics by utilizing time-stamped transcriptomics, gene expression data of similar samples captured across discrete timepoints, or pseudotime transcriptomics, computationally ordering cells based on an inferred trajectory. These methods can estimate constant or temporal gene regulatory relationships, but may not capture finer, cell type specific relationships. We propose ctOTVelo, an extension to our previous work to account for cell type specificity during GRN inference. ctOTVelo incorporates cell type labels or proportions when inferring the GRN from single cell transcriptomics data. Our methods achieve state-of-the-art performance in GRN prediction in time-stamped and pseudotime-stamped transcriptomics. Furthermore, ctOTVelo is able to generate cell type specific GRNs, allowing cell type resolution analysis of gene regulatory relationships.

genomics↗

bioRxiv2026

Systematic clustering alignment and feature characterization for single-cell omics using ACE-OF-Clust

Clustering is widely used to identify cell types in cellular-resolution transcriptomic data, including single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics (ST). Mixed-membership clustering assigns fractional memberships across clusters and captures continuous variation beyond hard clustering, but integrating and interpreting results from either approach is complicated by the "clustering alignment problem," which arises from label switching, multi-modality, and differences in model settings (including differing numbers of clusters). We introduce ACE-OF-Clust, enabling a four-step workflow for single-cell clustering: multiple clustering, clustering alignment, model comparison, and identification of informative features. ACE-OF-Clust introduces direct comparison of clustering solutions, assesses consistency against annotations, and leverages feature-level clustering profiles to prioritize genes discriminating among cell types, as well as to evaluate how gene sets contribute to clustering patterns. We demonstrate its utility on PBMC scRNA-seq and breast cancer ST data, and on multi-omic single-cell data. ACE-OF-Clust quantifies cross-omic clustering variability and suggests putative cross-omic regulatory links. Overall, ACE-OF-Clust increases the interpretability, flexibility, and robustness of single-cell clustering, providing a scalable tool for studying cellular heterogeneity and gene expression dynamics.

genomics↗

bioRxiv2026

A novel lipid-triggered allosteric site modulates LC3-LIR receptor binding activity

Membrane recruitment is a fundamental regulator of protein function. However, the allosteric mechanisms by which lipid binding controls protein activity remain poorly understood. In autophagy, the ubiquitin-like protein LC3 is lipid-anchored to autophagosomes, where it is essential for receptor recruitment and vesicle formation. While LC3-receptor interactions are structurally well defined, how membrane engagement governs LC3 functional dynamics has remained enigmatic. Here, we uncover that membrane binding triggers a major conformational transition in LC3, exposing functional pockets that are occluded in its cytosolic form. We demonstrate that this shift is mediated by dynamic coupling between the allosteric site (3- loop5-{beta}3-loop6) and the functional binding pockets. To conclusively test this mechanism, we utilised an ensemble-based protein design strategy guided by molecular dynamics to engineer the allosteric site. From a series of mutants, two variants emerged that stabilized LC3 conformation in either active or inactive state on the membrane. X-ray crystal structures of mutant LC3, biophysical assays, super-resolution microscopy, and TEM confirmed that the activated allosteric site mutant facilitates receptor binding and cargo capture. In contrast, the inactive variant is functionally inert on the membrane. Our work identifies a fundamental lipid-triggered allosteric site in LC3 that is critical for autophagy regulation and broader implication of membrane-dependent reprogrammable protein activities.

biophysics↗

bioRxiv2026

Mapping and Genetic Dissection of a Novel Tar Spot Resistance QTL on Maize Chromosome 1

Tar spot, caused by the obligately biotrophic fungus Phyllachora maydis, significantly threatens maize (Zea mays L.) production across the Americas. Host genetic resistance offers the most viable long-term management strategy. Building on observed differential tar spot susceptibility, we utilized 92 recombinant-inbred lines (RILs) from the Intermated B73 x Mo17 (IBM-94) population to characterize the genetic architecture of resistance. Phenotypic analysis of 92 RILs plus the highly susceptible parent Mo17 and the moderately resistant parent B73 confirmed stable differences in susceptibility, with B73 consistently demonstrating moderate resistance compared to Mo17. Analysis of variance revealed highly significant genetic variation within the population (F = 12.96; p < 0.001). A high Pearson correlation (r = 0.8706, p < 0.0001) and coefficient of determination (R2 = 0.7579) across environments indicated that 76% of the phenotypic variance is attributable to genetic factors. Linkage mapping identified a novel, consistent major QTL cluster on chromosome 1. This cluster comprises five regions (qTAR_1.1 through qTAR_1.5) exceeding the significance threshold (LOD 3.8) in both years. We identified 74 candidate genes including bZIP and RING/U-box proteins at significant SNP peaks. Additionally, gene annotations revealed a high concentration of wall-associated kinases and S-locus lectin protein kinases within the qTAR_1.4 and qTAR_1.5 regions, alongside potential defense-related transcription factors (MYB, bZIP, and C2H2 zinc fingers). These results provide a framework for high-resolution mapping and functional validation to accelerate the development of tar spot-resistant maize cultivars.

plant biology↗

bioRxiv2026

Single-Nucleus Transcriptomics Reveals Cell Type-Specific Remodeling and Epilepsy-Associated Microglia

Mesial temporal lobe epilepsy (TLE) is the most common form of acquired epilepsy involving the hippocampus and is a frequent sequelae of head trauma. TLE is associated with refractory seizures and significant cognitive deficits. Yet, the gene expression patterns and cell types driving epileptogenesis and the associated cognitive deficits are poorly understood. To address this, we performed single nucleus RNA sequencing on hippocampal tissue from mice at 3 and 6 weeks following pilocarpine-induced status epilepticus, a robust model of TLE. At these early timepoints, epilepsy samples showed reductions in specific Cck and Lamp5-Lhx6 interneuron subclusters, alongside increases in Cajal-Retzius cells, dentate granule (DG) cell precursors, and a mature DG cell subcluster. Among glia, an astrocyte subcluster and a markedly expanded microglia sublcuster were increased. We term this microglia population epilepsy-associated microglia (EAM). The transcriptomic profile of EAM partially overlaps with microglia described in models of Alzheimers disease and traumatic brain injury, with enrichment of genes including Myo1e and Igf1. EAM display amoeboid morphology, can be found in dense clumps around pyramidal and granule cell body layers, and exhibit enlarged vesicles and mitochondria on electron microscopy. Cell-cell interaction analysis predict that DG cells are the main interaction partners of EAM. This dataset recapitulates known cellular alterations in TLE while defining their underlying transcriptomic programs, enabling mechanistic dissection of the key processes driving epileptogenesis.

neuroscience↗

bioRxiv2026

Diet-Microbiome Analysis in a Healthy Cohort Reveals Potential Role of Intestinal Microbiota in Metabolism

Both preclinical and clinical studies have revealed the indisputable importance of intestinal bacterial community composition in pathogenesis of various disease states, from obesity to neurodegeneration. Diet remains one of the most important factors shaping human intestinal microbiota composition. In this study, we investigated diet-microbiome interactions in a healthy cohort of 88 participants from Atlanta and Calgary. We examine microbial composition (16S rRNA sequencing) with dietary records using Spearman Correlation tests with Benjamini-Hochberg multiple hypothesis correction to make community-level comparisons between dietary scores and microbial diversity index scores. Predictive models were used for molecular-level comparisons between microbial gene pathways and molecules. Among generalized dietary and microbial indices, we identified a negative association between dietary whole grain consumption and a microbial dysbiosis score. Comparisons between dietary food groups and bacterial family abundance reveal significant associations between dairy consumption and Lactobacillaceae abundance, dietary unsaturated to saturated fatty acid ratio and Clostridia Cluster Family XIII, salt intake and Lachnospiraceae, and consumption of greens and beans and Veillonellaceae. Predictive models of microbial gene pathways and molecules reveal significant positive associations between several dietary fatty acids and microbial short-chain fatty acid fermentation pathways, and between dietary lignans and archaeal methanogenesis pathways. Overall, these associations may inform future explorations on specific dietary interventions to impact the gut microbiome. IMPORTANCEIn this study, we compare dietary records and composition of intestinal microbes in a cohort of 88 participants. We identified associations between dietary consumption of dairy and the presence of dairy-consuming bacteria called Lactobacteriaceae and between consumption of dietary fats and the presence of fat-consuming bacteria called Clostridia. Using predictive analysis, we identify specific fatty acids associated with specific biochemical pathways found in Clostridia that might underlie these associations, in addition to an association between archaeal microbes and dietary consumption of estrogen-binding molecules called lignans, which are commonly found in whole grains and vegetables. Overall, our study generates useful associations between diet and intestinal microbes that can be tested in experiments that may help scientists use diet to control intestinal microbes in order to improve human health.

microbiology↗

bioRxiv2026

Non-canonical function of the splicing activator U2AF2 in promoting intron retention in the lncRNAs PURPL and MALAT1

Intron retention (IR) is increasingly recognized as a feature of long noncoding RNAs (lncRNAs), yet the mechanisms that shape IR in lncRNAs and the functional consequences of this process remain largely unexplored. To investigate how IR contributes to lncRNA regulation, we performed a genome-wide screen to identify factors controlling IR in the lncRNA PURPL. This approach uncovered a prominent role for U2AF2, which promotes retention of a specific intron in PURPL through a weak polypyrimidine tract. IR of this intron drives nuclear enrichment of PURPL and enhances cell proliferation, revealing biological relevance. Transcriptome-wide analyses showed that although U2AF2 broadly supports canonical splicing consistent with its well-established function in promoting splicing, it also facilitates IR within a distinct subset of RNAs, including the nuclear speckle-associated lncRNA MALAT1. Loss of U2AF2 disrupts MALAT1 speckle localization and using MALAT1 knockout cells reconstituted with wild-type or intron deleted variants, we identified a single intron critical for MALAT1s speckle localization. Deletion of this intron from endogenous MALAT1 impaired speckle localization and reduced cell migration, phenocopying the loss of MALAT1. Together, these findings reveal IR as a key regulatory mechanism governing lncRNA localization and function and uncover an unexpected role for U2AF2 in promoting IR within specific lncRNA contexts.

cell biology↗

bioRxiv2026

Functional Equivalence of Heat-Inactivated (HI) and Live Probiotic RSB11 in Suppressing Inflammation: Expanding Formulation and Application Potential

The clinical potential of probiotics has been widely recognized, but their translation into reliable therapeutic products has been hindered by major limitations such as undesirable immunogenic responses, the need to maintain viability, instability during storage and transport, and concerns regarding safety in vulnerable populations. Postbiotics, defined as inanimate microbial cells or their components with pro-health activities, overcome many of these limitations by offering enhanced stability, reproducibility, and safety. However, it is very vital to understand if the heat inactivation (conversion of a probiotic to its postbiotic inert form) compromise its functional efficacy. Here, we systematically compared a novel probiotic-derived candidate, Lactiplantibacillus plantarum RSB11 strain, in its live (RSB11 Life, probiotic) and heat-inactivated (RSB11-HI, postbiotic) forms across multiple human epithelial and non-epithelial models relevant to inflammation driven pathologies. To investigate the gut-tissue(s)-axis concept we used gut (Caco-2), lung (HBE), ovary (BG1), bone (osteoblasts, MG-63), kidney (A-498) and liver (HepG2) cells exposed to E-coli or lipopolysaccharide (LPS), and quantified matrix metalloproteinase-9 (MMP-9), an inflammatory mediator, by qPCR and pro-inflammatory cytokines such as tumor necrosis factor- (TNF-), IL-6, and IL-1{beta} by ELISA. In addition, we assessed {beta}-glucuronidase activity and estrogen modulation to explore gut-ovarian axis signaling. Across all models, both RSB11 Life and RSB11-HI robustly suppressed MMP-9, TNF-, IL-6 and IL-1{beta} induction, with equivalent magnitude of effect. The inactivated form retained full cytokine-suppressive capacity and, notably, enhanced {beta}-glucuronidase activity, suggesting additional benefits in microbiome hormone cross-talk. Our findings demonstrate that heat inactivation does not compromise, and may even expand, the functional range of RSB11. By maintaining bioactivity while eliminating the drawbacks of live biotics, heat inactivated RSB11 emerges as a robust, scalable, and versatile postbiotic with potential applications in systemic inflammatory disorders. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=106 SRC="FIGDIR/small/705228v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@7bb1e6org.highwire.dtl.DTLVardef@dc86c7org.highwire.dtl.DTLVardef@147448org.highwire.dtl.DTLVardef@de5232_HPS_FORMAT_FIGEXP M_FIG C_FIG Graphical abstract of RSB11-HI activityHeat-inactivated postbiotic RSB11-HI retains the anti-inflammatory efficacy of its live counterpart (RSB11Life) across diverse organ-relevant cell models. Upon LPS or E-coli stimulation, epithelial, immune, and tissue-specific cells (gut, lung, ovary, bone, kidney, liver) upregulate pro-inflammatory mediators including MMP-9, TNF-, IL-6, and IL-1{beta}. Both RSB11Life and RSB11-HI effectively suppress these inflammatory responses, with RSB11-HI exhibiting more consistent and robust reductions of inflammatory markers across models. Additionally, RSB11-HI uniquely enhances {beta}-glucuronidase activity, facilitating estrogen metabolism and signaling through the gut-ovary axis. Together, these findings highlight RSB11-HI as a stable, safe, and multifunctional postbiotic candidate suitable for therapeutic formulation. Image was designed using ChatGPT.

immunology↗

bioRxiv2026

Confirmation and Transcriptomic Characterization of Glufosinate-ammonium Resistance in Waterhemp (Amaranthus tuberculatus) Populations from Illinois

Glufosinate-ammonium (GA) has been widely used in Midwestern fields, and in recent years a growing number of failures to control waterhemp [Amaranthus tuberculatus (Moq.) Sauer] have raised concerns about the potential evolution of resistance. The goal of this study was to investigate four independent cases of suspected resistance to GA in A. tuberculatus from Illinois using greenhouse, field, and transcriptomics studies. Greenhouse dose-response experiments revealed resistance ratios ranging from 2.2- to 3.4-fold based on survival and from 1.3- to 2.8-fold based on dry biomass relative to a susceptible population. A subsequent field study where one of the populations originated confirmed that twenty percent of treated plants survived the labeled GA field-recommended rate. Screening for other herbicide sites of action revealed that most populations showed reduced sensitivity to atrazine, glyphosate, and imazethapyr, surviving up to three times the field-recommended rates, and to a lesser extent, lactofen and fomesafen. Transcriptomic analysis of plants surviving GA revealed no resistance-associated mutations or differential transcript abundance in the plastidic and cytosolic isoforms of glutamine synthetase. Among the four suspected resistant populations, there were 182 genes differentially expressed relative to two susceptible populations. Different sets of genes were differentially expressed among the populations studied, with only one gene (upregulated relative to two susceptible populations) shared among all four. Many of the differentially expressed genes, including cytochrome P450s, glutathione S-transferases, glycosyltransferases, transporters, and transcriptional regulators, are commonly associated with metabolic resistance. Gene ontology enrichment analyses indicated significant overrepresentation of stress response, defense regulation, and secondary metabolism categories across the populations. Together, these findings provide evidence for the evolution of GA resistance in populations of A. tuberculatus in Illinois. While more in-depth studies are needed to fully characterize the underlying mechanisms, the consistent differential expression of metabolism-related genes and no indication of target-site mechanisms points to a potential metabolic basis for resistance.

plant biology↗

bioRxiv2026

Bioengineered recombinant kisspeptins with extended half-life exhibit novel peripheral function in a large-animal model

Kisspeptins are the small peptide products encoded by the KISS1 gene and physiologically exist in various isoforms of variable length. They are the central regulators of reproduction, being a prominent driver of GnRH hormone secretion. Additionally, they have emerged as an important peripheral therapeutic target for many metabolic diseases like diabetes, obesity, and polycystic ovary syndrome (PCOS). Despite their therapeutic potential, their utility is severely limited by their short half-life. We have rationally bioengineered two versions of native kisspeptins, which we named HSK-1 and HSK-2. HSK-1 (8kDa) and HSK-2 (13kDa) are derived from the fusion of the albumin-binding ZAG domain from Streptococcus zooepidemicus with KP-10 and KP-52 versions of kisspeptins (KPs), respectively. In vitro assays confirmed that the proteins were functionally active and triggered downstream signalling. Molecular dynamics simulations of the proteins revealed their structural features relative to the native kisspeptin isoforms. Both molecules demonstrated stable receptor engagement, and ligand-induced conformational changes were observed, suggesting receptor activation. HSK proteins demonstrated an extended half-life and mostly acted peripherally in young animals. They reduced peripheral luteinizing hormone levels in young animals, likely representing a previously unrecognized mode of peripheral kisspeptin action. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=77 SRC="FIGDIR/small/703727v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@7b02fdorg.highwire.dtl.DTLVardef@13da0org.highwire.dtl.DTLVardef@174d467org.highwire.dtl.DTLVardef@124c653_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗