Biology subjects

Sharma, S.

Publications and source records attributed to Sharma, S..

At least 55 records · Page 3Linked to original sources

bioRxiv2026

Early Tracheal and Salivary miRNAs in Extremely Preterm Infants Predict BPD-related Pulmonary Hypertension

Pulmonary hypertension (BPD-PH) associated with bronchopulmonary dysplasia (BPD) in preterm infants associates with high morbidity and mortality within the first two years of life. In a previous unbiased study, we identified a panel miRNAs in tracheal aspirates (TA) that were differentially expressed in extremely low gestational age newborns (ELGANs) with BPD-PH compared to those with BPD but no PH. To explore the predictive potential of these miRNAs, we studied TA exosomes from 7 days old ELGANs and analysed a curated panel of 16 miRNAs through logistic regression and calculated the predictive AUROC to diagnose BPD-PH at 36 weeks PMA. AUROC of TA miRNAs was 0.76 with sensitivity and specificity of 53% and 93%, respectively. Adding sex and gestational age to the variables improved the AUROC to 0.78 with sensitivity and specificity of 61 and 87% respectively. Due to challenges of obtaining TA in non-invasively ventilated infants, we collected saliva samples from ELGANs at 7 days of age and compared the log expression of these 16 miRNAs in both biofluids and found significant correlation in their expression (pearson r=0.92, p<0.001). We calculated the predictive AUROC of the same miRNAs to diagnose BPD-PH at 36 weeks PMA. AUROC of these miRNAs in saliva was = 0.85 with sensitivity and specificity of 82% and 72%, respectively; addition of biological sex and gestational age improved AUROC to 0.86 with sensitivity and specificity of 79% and 76% respectively. Leave-one-sample-out sensitivity analysis demonstrated stable training performance with reduced performance in testing samples, supporting the need for validation in larger independent cohorts. In conclusion, early salivary miRNAs have great potential for risk stratification of ELGANs to develop BPD-PH, while also providing the opportunity to identify target molecules and mechanisms that modulate molecular function.

bioinformatics↗

bioRxiv2026

Tiny Subsamples and Upsampling Tame Big Data Evolutionary Analysis in Phylogenomics

Long runtimes, high memory demands, and reliance on high-performance computing impede phylogenomic analyses. We review a scalable phylogenomic subsampling with upsampling (PSU) framework, in which small subsamples of sites from a concatenated alignment are expanded by upsampling before inference, and the resulting analyses are then aggregated to obtain evolutionary estimates. PSU harnesses the fact that the computational cost of maximum likelihood analysis is strongly influenced by the number of distinct site patterns in the concatenated alignment, whereas statistical power depends primarily on the amount of evolutionary information represented by the total number of sites and substitutions. By reducing the former while restoring the latter through upsampling, PSU can approximate many full-data analyses at substantially lower computational cost. Analysis of simulated and empirical datasets shows that PSU can accurately estimate bootstrap support values, select the optimal substitution model, test evolutionary hypotheses, and infer branch lengths, divergence times, and associated uncertainty measures, while reducing runtime and memory requirements by orders of magnitude. PSU also provides distributions of inferred clade support across independent subsamples, enabling detection of conflicting phylogenetic signals that may remain hidden in conventional bootstrap analysis. Automated tuning of subsample size, the number of subsamples, and the number of upsampling replicates make PSU practical across diverse datasets. We suggest that PSU is a general strategy for scalable phylogenomic inference using a broad range of statistical methods. By enabling analyses of genome-scale alignments on commodity hardware, PSU broadens research access and reduces environmental and infrastructural costs of big-data phylogenomics.

evolutionary biology↗

bioRxiv2026

Cortisol Drives Pregnancy-Associated Induction of Hepatic OAT2, NTCP, and OCT1 in HepaRG cells Through GR-, HNF1α-, and HNF4α-Dependent Signaling

Pregnancy induces or represses hepatic drug metabolism. Whether pregnancy affects hepatic drug transport is unexplored. We previously showed that a cocktail of pregnancy-related hormones (PRHC) induces mRNA expression and activity of sodium/taurocholate cotransporting polypeptide (NTCP), organic anion transporter 2 (OAT2), and organic cation transporter 1 (OCT1, mRNA only) in differentiated HepaRG cells. Here, using HepaRG cells, we identified cortisol as the hormone primarily responsible for this induction and explored the underlying mechanisms. Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9-mediated knockdown studies in HepaRG cells showed that the glucocorticoid receptor (GR) is the primary mediator of this response. GR knockdown markedly attenuated cortisol-induced NTCP, OAT2, and OCT1 mRNA expression and activity. Cortisol also induced the mRNA expression of regulatory factors, including pregnane X receptor (PXR), constitutive androstane receptor (CAR), and hepatocyte nuclear factor (HNF) 4 alpha (HNF4). HNF4 knockdown selectively attenuated OAT2 and OCT1 induction, whereas HNF1 knockdown enhanced NTCP induction, attenuated OCT1 induction, and reduced basal organic anion transporting polypeptide 1B1 (OATP1B1) expression. In contrast, knockdown of CAR or PXR did not significantly alter cortisol-mediated transporter regulation. These data identify cortisol as the principal PRH driving regulation of the hepatic OAT2, NTCP, and OCT1 in HepaRG cells and indicate that this response is mediated primarily by GR, with selective downstream contributions from HNF4 and HNF1. These findings provide mechanistic insights into pregnancy-associated changes in hepatic transporter-mediated drug disposition, including when antenatal corticosteroids are administered to pregnant women to prevent respiratory distress syndrome in their prematurely born infants. Significance StatementThe extent and mechanisms by which pregnancy-related hormones regulate hepatic uptake transporters remain poorly defined. This study identifies cortisol as the principal pregnancy-related hormone driving NTCP, OAT2, and OCT1 induction in HepaRG cells and shows that this response is mediated primarily through GR, with transporter-specific contributions from HNF4 and HNF1.

pharmacology and toxicology↗

bioRxiv2026

Target DNA-Mediated Plasmonic Coupling and Assembly Kinetics of Gold Nanorods for Label-Free Nucleic Acid Detection

DNA-programmed assembly of plasmonic nanostructures provides a powerful route to couple molecular recognition with optical signal generation. Here, we report the sequence-specific assembly of DNA-functionalized gold nanorods using a sesame allergen-derived DNA biomarker as a molecular bridge. Target-induced assembly produces concentration-dependent assembly growth, plasmon coupling, and distinct assembly kinetics that are readily monitored by absorption spectroscopy, enabling label-free detection of the target DNA in the nanomolar concentration range. The assembled nanorods further produce strong surface-enhanced Raman scattering (SERS) signals arising from plasmonic coupling within the assemblies, extending detection to the picomolar regime without the use of Raman reporters. Quantitative analysis reveals that both the extent and rate of assembly formation are governed by target DNA concentration. These results establish a direct relationship between molecular recognition, assembly growth, plasmonic coupling, and spectroscopic response, highlighting DNA-programmed gold nanorod assembly as a versatile platform for investigating hybridization-driven plasmonic self-assembly and nucleic acid detection. Table of Content O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=112 SRC="FIGDIR/small/732610v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@e4fcb9org.highwire.dtl.DTLVardef@18c0d89org.highwire.dtl.DTLVardef@1ccb4bborg.highwire.dtl.DTLVardef@6d84ca_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2026

Novel bile salt analogs reduce lipid accumulation in liver cells with potential to treat both metabolic dysfunction-associated steatotic liver disease and Clostridioides difficile infection

Metabolic dysfunction-associated steatotic liver disease (MASLD) and Clostridioides difficile (C. difficile) infection (CDI) are clinically associated, yet there is limited effective treatment for both diseases. Bile salt analogs (BSAs) have demonstrated potential in treating either MASLD or CDI. We screened a library of BSAs (n=112) previously synthesized as potential inhibitors of C. difficile spore germination, for their therapeutic potential in reducing intracellular accumulation of fatty acids in HepG2 cells as candidates for prevention and treatment of both MASLD and CDI. The screening was based on an in vitro model established by incubating HepG2 cells with free fatty acids, with obeticholic acid (OCA), a known BSA with anti-MASLD activity as a control. Gene and protein expressions were quantified to validate the treatment effect. We found that compounds C13, C24, C25, C74, C98, and C101 demonstrated significant effectiveness in both preventing the intracellular accumulation of lipids and removing pre-loaded cellular lipids. Gene expression analysis showed that C24, C25, and C74 produced a similar pattern characterized by a robust induction of FGF21 expression, while C13, C98, and C101 produced a transcription pattern that mirrors the effect of OCA. Structurally, while C13, C24, and C25 do not display drug-like properties, C74, C98, and C101 are drug-like and share a similar structure. Interestingly, C101 is a potent inhibitor of C. difficile spore germination. OCA shows a weak anti-gemination effect. Our study identified lead compound candidates for the development of novel therapeutics capable of treating both MASLD and CDI. Significance statementThe clinical association between MASLD and CDI remains an unmet need for dual acting therapeutic strategies. Given the reported potential of BSA, we screened 112 previously synthesized as potential inhibitors of C. difficile spore germination, for their therapeutic potential in reducing intracellular accumulation of fatty acids in HepG2 cells. Our study identified compounds that effectively reduce intracellular lipid accumulation and inhibit C. difficile spore germination. These results nominate lead candidates for developing dual-acting therapeutics targeting both MASLD and CDI.

pharmacology and toxicology↗

bioRxiv2026

Scalable generation of pure CD103⁺ cDC1 from iDC1 cultures

Conventional type 1 dendritic cells (cDC1) specialize in cross-presentation and interleukin-12 production and are critical for immunity against intracellular pathogens and tumors, but remain rare in vivo, limiting mechanistic and translational studies. Existing bone marrow-derived dendritic cell (BMDC) methods do not achieve highly selective enrichment of cDC1 or scalable production at high purity. Here, we established a novel in vitro culture system for selective generation of CD103+ cDC1 from mouse bone marrow using defined media conditions together with recombinant FLT3L, GM-CSF, and Kit ligand (KitL), termed iDC1. iDC1 cultures enabled scalable generation of an estimated 1.5 x 109 CD103+ cDC1 at greater than 95% purity from a single mouse, representing at least a 75-fold increase relative to previous recombinant cytokine-based methods. Phenotypic and transcriptional analyses demonstrated that iDC1 closely align with the CD103+ cDC1 lineage while remaining clearly distinct from macrophage populations. Functionally, iDC1 responded robustly to innate stimulation, produced interleukin-12 and inflammatory chemokines, and efficiently cross-presented cell-associated antigen to CD8+ T cells. Mechanistically, KitL and GM-CSF regulated distinct stages of cDC1 generation, whereas proteomic, phospho-proteomic, and functional analyses demonstrated that GM-CSF suppresses apoptosis and oxidative stress while promoting cDC1 proliferation. iDC1 generation was dependent on the +32 kb Irf8 enhancer required for bona fide cDC1 development, and STAT5-and BRD4-associated regulatory programs were identified as important regulators of efficient iDC1 generation. Together, these findings establish iDC1 cultures as a scalable platform for studying cDC1 biology and developing cDC1-based immunotherapeutic strategies.

immunology↗

bioRxiv2026

Vitamin D3 Deficiency Exacerbates Abdominal Aortic Aneurysm Progression Via Complement C3a Activation

Abdominal aortic aneurysm (AAA) is a chronic inflammatory vascular disease characterized by progressive extracellular matrix degradation, vascular smooth muscle cell (VSMC) loss, and immune cell infiltration, ultimately leading to aortic dilation and rupture. Although vitamin 25(OH)D3 deficiency has been associated with cardiovascular inflammation, its mechanistic role in AAA pathogenesis remains poorly defined. Here, we investigated the role of vitamin D{square} mediated signaling to regulate complement pathway activation, particularly the C3a axis, to modulate aneurysm development. Single cell-RNA sequencing analysis of human tissue demonstrated significant differences in Vitamin D and complement pathway-related genes in VSMCs in AAAs compared to control aortic tissue. Using a murine elastase-induced AAA model, we observed that vitamin D3-deficient diet significantly enhances aortic dilation, leukocyte infiltration, proinflammatory cytokine expression and elastin fragmentation, as well as decreases SMC -actin expression compared with vitamin D3-sufficient conditions. Furthermore, vitamin D3 deficiency was accompanied by increased aortic expression of complement component C3a that correlated with vascular inflammation and remodeling during AAA progression. Pharmacological blockade with a C3a receptor antagonist (C3aRA) markedly attenuated AAA formation in two established murine AAA models with concomitant reductions in proinflammatory cytokines and preservation of aortic wall structure. In vitro studies demonstrated that stimulation of VSMCs significantly increased C3a production, which was suppressed by calcitriol (active form of Vitamin D) treatment. These studies suggest that the vitamin D-C3a axis is a critical regulator of vascular inflammation and AAA progression, and postulate that restoring vitamin D{square} sufficiency or targeting C3a signaling may represent a novel therapeutic strategy to limit AAA growth and rupture. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=162 SRC="FIGDIR/small/730431v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@1e6a56dorg.highwire.dtl.DTLVardef@176b1b9org.highwire.dtl.DTLVardef@18e1fcdorg.highwire.dtl.DTLVardef@1c9eb4d_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2026

The beta cell glucocorticoid receptor protects against hyperglycaemia by modulating insulin secretion during glucocorticoid rhythm disruption in mice

Aims/hypothesis: Hyperinsulinaemia is typically viewed as a secondary, compensatory response to insulin resistance or elevated glycaemia. However, we previously found that disrupting the daily glucocorticoid rhythm in mice rapidly increases circulating insulin several-fold while fasting glucose remains normal. This raised the question of what generates and sustains the hyperinsulinaemia. Because glucocorticoids act directly on beta cells through the glucocorticoid receptor (GR), we tested whether beta cell GR is required for this rise in insulin and whether elevated insulin is necessary to maintain glucose homeostasis. Methods: Glucocorticoid rhythms were disrupted in male C57BL/6J mice by subcutaneously implanting corticosterone pellets that raise the trough and lower the peak while maintaining near-physiological mean glucocorticoid exposure, a manipulation we refer to as GC-flattening. Placebo-treated mice served as controls, and high-fat-diet-fed mice provided a metabolic comparison. Beta cell function was assessed by dynamic glucose stimulated insulin secretion and beta cell specific Ca2+ imaging. The requirement for beta cell GR was tested using adult-inducible beta cell specific GR knockout mice, thereby limiting developmental effects of constitutive GR deletion. Combined beta cell and hepatocyte GR knockout mice were used to test the consequences of further reducing systemic insulin availability. Insulin sensitivity and glucose tolerance were assessed in vivo. Insulin clearance was assessed from plasma C-peptide:insulin ratios, direct measurement of the disappearance of intravenously administered human insulin, and hepatic insulin-degrading enzyme abundance and activity. Results: GC-flattening produced rapid, sustained hyperinsulinaemia while blood glucose remained normal, distinct from the more gradual hyperinsulinaemia and hyperglycaemia observed in high-fat-diet-fed mice. Islets from GC-flattened mice retained enhanced insulin secretion and Ca2+ responses to glucose after isolation, indicating a persistent increase in beta cell glucose responsiveness. During GC-flattening, beta cell GR deletion reduced cumulative circulating insulin exposure by approximately 40% (p < 0.001) and worsened glycaemic control despite similar or greater insulin sensitivity, demonstrating that the GR-dependent rise in insulin helps maintain glucose homeostasis. Direct measurement on Day 3 confirmed reduced insulin clearance in GC-flattened mice. This was accompanied by a reduced plasma C-peptide-to-insulin ratio and decreased hepatic insulin-degrading enzyme abundance and activity. Further lowering circulating insulin by combined beta cell and hepatocyte GR deletion worsened glycaemic control further. Conclusions/interpretation: Disruption of glucocorticoid rhythmicity directly initiates hyperinsulinaemia by enhancing glucose-stimulated insulin secretion through beta cell GR signalling and by reducing insulin clearance. The resulting increase in insulin is required to maintain glucose homeostasis, demonstrating that hyperinsulinaemia can be an early adaptive response to altered endocrine timing rather than simply a consequence of insulin resistance or hyperglycaemia.

physiology↗

bioRxiv2026

G quadruplex DNA facilitates a pervasive path to homologous recombination

Homologous recombination (HR) requires efficient homology search and strand invasion, yet how homologous templates are identified within nuclear space remains unclear. Here, we identify G-quadruplex (G4) DNA structures as pervasive effectors of template strand invasion and uncover the G4 helicase DHX36 as a potent suppressor of this process. DHX36 loss stabilizes G4s, enhances HR, and accelerates repair of replication-associated DNA breaks. G4-mediated HR depends on the non-canonical strand invasion factors RAD51AP1, WDR48, and USP1, and requires G4 motifs within the homologous repair template. DHX36 loss partially restores HR and PARP inhibitor resistance in BRCA1-deficient cells, while promoting aberrant recombination and Alternative Lengthening of Telomeres (ALT). Together, our findings establish dynamic G4 regulation as a key determinant of homology search, genome maintenance, and recombination fidelity.

molecular biology↗

bioRxiv2026

Phase composition-specific behaviour of functional RNAs in liquid-liquid phase-separated microenvironment

The internal dynamics of liquid-liquid phase-separated systems are governed primarily by polymer packing, excluded-volume effect, and interactions between polymers and encapsulated macro-molecules. Although one immediate effect of such a constrained microenvironment is diffusion limitation, it remains unclear whether encapsulated macromolecules can also exhibit phase composition-specific functional behaviour that is not observable in a well-mixed aqueous environment. In this regard, different phases in a phase-separated environment can be accessed via a phase diagram that demarcates the region between two-phase (droplets) and one-phase (polymer-rich, no droplets) regimes. While the two-phase region is heterogeneous, most previous work on encapsulating functional macromolecules in phase-separated droplets uses a single point from the phase diagram. This leaves a clear gap in understanding on how the function scales across this landscape of droplets and identifying regions advantageous for the encapsulated macromolecule and its function. Here, using the Spinach light-up RNA aptamer, we show that RNA function does not scale uniformly across the phase diagram. We show that RNA can exhibit phase composition-specific functional behaviour due to constraints imposed by the internal microenvironment of phase-separated droplets. Furthermore, using variants of the Spinach aptamer, we show that fluorescence activity differences among the variants vary differently with phase-separation regimes across the phase map, suggesting that some regions of the phase diagram can confer a selective advantage. Our results highlight the potential of liquid-liquid phase-separated internal microenvironments in guiding the differentiation of functional RNA variants, which could serve as a physical selection pressure in pre-cellular evolution.

evolutionary biology↗

bioRxiv2026

Directional Gene-Level Concordance and Methodological Constraints in Blood Transcriptomic and DNA Methylation Studies of Parkinson's Disease

Assessing reproducibility across different molecular profiling studies is a persistent methodological challenge (Zhang et al., 2009; Sweeney et al., 2017; Ioannidis, 2005). Differences in platform technology, cohort composition, analytical pipelines, and feature definitions often make it difficult to interpret cross-study comparisons based solely on gene-identity overlap. In this study, we conducted a retrospective computational analysis of seven publicly available analytical datasets (including alternative analytical pipelines applied to the same cohort) derived from five biologically independent peripheral blood transcriptomic and DNA methylation cohorts, comprising 3,487 samples (1,824 Parkinsons disease cases and 1,663 controls). Reproducibility was evaluated using gene-identity overlap, enrichment-based comparisons, and a permutation-based framework to assess directional consistency of effect estimates across datasets. We also tested the robustness of results by varying false discovery rate thresholds and applying alternative probe-to-gene collapsing strategies. All analyses were performed using reproducible workflows implemented in R and Python with fixed random seeds. Across independent cohorts, gene-identity overlap was generally limited, with enrichment ratios close to one, especially when datasets were generated using different platforms. In several datasets, limited numbers of statistically significant features further constrained overlap-based comparisons. In contrast, directional consistency showed greater stability. High levels of directional consistency were observed across independent cohort comparisons when restricted to overlapping statistically significant features and remained stable across statistical thresholds (90.0% at FDR < 0.05 and 82.8% at FDR < 0.10). When evaluated across the full shared gene universe without conditioning on statistical significance, directional consistency was substantially lower ([~]30 to 32%) but remained significantly above permutation-based null expectations. Permutation testing confirmed that the observed directional consistency exceeded what would be expected by chance. A combined analysis including methodological replicates (n [≥] 3 datasets) showed 98.3% directional consistency; however, this estimate includes non-independent analytical pipelines applied to the same cohort and reflects analytical stability rather than independent biological replication. Rather than introducing a new statistical method, this study examines how commonly used reproducibility metrics behave under crossstudy heterogeneity and identifies their practical limitations and appropriate use boundaries.

neuroscience↗

bioRxiv2026

Homozygous c.820G>A variant in MGME1 contributes to multi-systemic mitochondrial dysfunction in an Indian patient cohort

Mitochondrial DNA (mtDNA) maintenance disorders arise from defects in mtDNA replication or repair, frequently resulting in extensive deletions or depletion of mtDNA. Mitochondrial genome maintenance exonuclease 1 (MGME1) is a nuclear-encoded nuclease essential for mtDNA replication and genome stability, and biallelic pathogenic variants in MGME1 cause mitochondrial DNA depletion syndrome 11. Here, we report a novel homozygous MGME1 missense variant c.820G>A (p. Ala274Thr) in five affected individuals from unrelated South Indian families presenting with proximal myopathy, chronic progressive external ophthalmoplegia, and cardiac and renal involvement. Patient-derived cells exhibited a significant reduction in mtDNA copy number, consistent with impaired mtDNA maintenance. Mechanistic analyses combining imaging-based and biochemical approaches demonstrated that the MGME1 variant disrupts both mtDNA replication and repair. Functional characterization further revealed defective oxidative phosphorylation and reduced mitochondrial membrane potential, confirming mitochondrial dysfunction. Collectively, our findings establish the pathogenicity of this novel MGME1 variant and expand the clinical and molecular spectrum of MGME1-associated mitochondrial disease, linking impaired mtDNA replication to multisystemic mitochondrial dysfunction. Graphical abstract summarySchematic overview of mitochondrial DNA (mtDNA) replication in wild-type and MGME1 mutant conditions. In wild-type cells, the coordinated activity of the mitochondrial replication machinery (TWNK, POLG, LIG3, TFAM, and MGME1) maintains mtDNA integrity and supports normal oxidative phosphorylation and ATP production. In contrast, the MGME1 mutation disrupts mtDNA replication and processing, leading to replication defects, mtDNA depletion, and mitochondrial dysfunction. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=116 SRC="FIGDIR/small/725852v1_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@406ce7org.highwire.dtl.DTLVardef@1adc863org.highwire.dtl.DTLVardef@3ef92aorg.highwire.dtl.DTLVardef@585559_HPS_FORMAT_FIGEXP M_FIG C_FIG

genetics↗

bioRxiv2026

Mechanistic Insights into TMPyP4 Recognition of the HIV-1 LTR-III G-Quadruplex in Dilute and Protein Condensate Environments Reveal Hidden Dual Binding Modes

G-quadruplex (GQ) structures within the HIV-1 long terminal repeat (LTR) regulate viral transcription and represent promising antiviral targets; however, detailed mechanistic understanding of their ligand recognition at the molecular level remains limited and has largely been investigated under dilute conditions despite the crowded and compartmentalized nature of intracellular environment. Here, we investigate the interaction of the cationic porphyrin TMPyP4 with the HIV-1 LTR-III GQ under dilute conditions and inside protein-rich phase-separated condensates that mimic intracellular biocondensates. Steady-state and time-resolved fluorescence measurements reveal a dual binding behavior that is not discernible from absorption spectroscopy. A high-affinity guanine-rich binding mode leads to efficient fluorescence quenching through electron transfer from ground-state guanine to excited TMPyP4, whereas a weaker non-guanine binding mode gives rise to enhanced and long-lived emission. Nucleotide-specific control experiments validate the origin of these distinct binding environments. Molecular docking and molecular dynamics simulations further support preferential binding of TMPyP4 at the terminal G-quartet together with a secondary binding mode near the quadruplex-duplex junction. Importantly, both TMPyP4 and LTR-III GQ preferentially partition into the condensates, where the hybrid GQ structure, dual binding behavior, and associated excited-state signatures remain preserved despite the crowded and viscous environment. Although a slight reduction in binding affinity is observed inside the condensates, the overall binding mechanism remains largely preserved due to compensatory effects arising from the condensate microenvironment. Overall, this work demonstrates that ligand recognition of viral GQ remains preserved within protein condensates and establishes fluorescence spectroscopy as a sensitive approach for resolving hidden binding heterogeneity in GQ-ligand interactions.

biophysics↗

bioRxiv2026

Distributed Clonal Deletion Prevents Autoimmune Disease Progression

Self-reactive B cells are generated during normal development and can acquire increased pathogenicity through activation-induced cytidine deaminase (AID)-mediated diversification following activation. Clonal deletion is thought to eliminate these cells, yet how deletion is distributed across developmental and activation stages to prevent autoimmune disease remains unclear. Here, we show that clonal deletion is enforced through temporally distinct mitochondrial apoptosis (MOMP) checkpoints that differentially regulate autoreactive B cell fate and disease progression. Using conditional Bcl-2 expression to inhibit MOMP either before or after B cell activation, we find that early inhibition permits the survival and maturation of autoreactive B cells after peripheral egress, expanding the pool of cells available for activation. These cells subsequently undergo AID-dependent diversification, producing class-switched IgG autoantibodies with expanded antigen breadth that target a wider range of self-antigens and drive lethal, female-biased autoimmune disease characterized by complement activation and kidney pathology. In contrast, inhibition of MOMP only after activation allows the accumulation of germinal center, switched memory, and plasma cells and promotes autoantibody production, but results in more restricted IgG autoreactivity, limited complement activation and limited tissue damage, and normal survival. Notably, early MOMP inhibition does not expand immature bone marrow B cells, indicating that a major clonal deletion checkpoint operates in the periphery rather than during initial B cell generation. Together, these findings support a Distributed Clonal Deletion Model in which early checkpoints restrict the entry of autoreactive B cells into diversification pathways, while later checkpoints limit the persistence of diversified autoreactive clones, thereby constraining autoimmune disease progression. One Sentence SummaryDistributed clonal deletion prevents autoimmune disease progression by restricting the breadth of autoreactive clones entering immune responses, with early MOMP checkpoints limiting diversification and later checkpoints constraining persistence.

immunology↗

bioRxiv2026

Antibody Blockade of Ly49/MHC-I interactions enhances Innate and Adaptive Immunity Against Cancer Metastasis

BackgroundAntibody-mediated blockade of innate receptor-MHC-I interactions represents a promising strategy to enhance anti-tumor immunity, particularly against metastatic cancers resistant to conventional checkpoint inhibitors. In this study, we investigated the effects of the pan anti-MHC-I monoclonal antibody M1/42, which targets MHC-I interactions with Ly49, selectively expressed on murine NK cell subsets. MethodsWe administered M1/42 to mice and assayed the proliferation and activation immune cells. Anti-tumor activity of growth and metastasis of checkpoint inhibitor-resistant pancreatic ductal adenocarcoma (PDAC) and B16F10 melanoma were assessed, complemented by extensive cellular phenotypic and RNA expression analysis. Binding and cryo-electron microscopic (cryo-EM) and X-ray crystallographic structural studies of M1/42 complexed with the mouse MHC-I molecule, H2-Dd, examined the Ab interaction site in comparison with those of Ly49 inhibitory receptors. ResultsM1/42 administration in mice robustly unleashed the proliferation and activation of natural killer (NK) cells, memory CD4+ and CD8+ T cells, dendritic cells, and macrophages in both lymphoid and non-lymphoid tissues, independent of Fc{gamma} receptors. M1/42 significantly restricted the growth and metastasis of checkpoint inhibitor-resistant pancreatic ductal adenocarcinoma (PDAC) and B16F10 melanoma in the liver and lungs, accompanied by increased tumor infiltration of effector CD8+ T cells, reduction of T regulatory cells, and a pro-inflammatory cytokine milieu. The anti-tumor effects of M1/42 depend on NK cells and are associated with upregulation of genes involved in antigen processing, interferon gamma responsiveness, and Th1 cytokine production, while downregulating inhibitory PD1/11 signaling. Structural analysis indicated that the effect of M1/42 on Ly49/MHC-I interactions was not due to direct steric competition. ConclusionsCollectively, these findings demonstrate that M1/42 unleashes coordinated innate and adaptive immune responses, overcoming tumor-induced immunosuppression and resistance to checkpoint blockade. This approach represents a paradigm shift in cancer immunotherapy, offering potential for more effective treatment of metastatic cancers that evade immune surveillance through MHC-I modulation. KEY MESSAGESO_ST_ABSWhat is already known on this topicC_ST_ABSA pan anti-mouse MHC-I mAb (M1/42) blocks interaction with several NK inhibitory receptors (Ly49A or Ly49C) resulting in NK cell activation and anti-viral and anti-tumor responses in vitro and in vivo. Other pan anti-human MHC-I mAbs (DX17 and W6/32) function similarly, blocking LILRB inhibitory receptor interaction of myeloid cells and NK cells. These stimulate human immune cells in humanized mouse models. What this study addsThis study analyzes the effects of the pan anti-mouse MHC-I mAb on NK and myeloid cell activation in detail, in the absence of T or B cells, and independent of FcR interaction. Additionally we analyze several mouse models of metastatic tumor progression, indicative of the progressive activation not only of the innate immune response, but also adaptive responses. The molecular mechanism of the mAb blocking of inhibitory receptors is revealed by cryo-EM and X-ray structures of M1/42 Fab/MHC-I (H2-Dd) complexes. How this study might affect research, practice, or policyElucidation of the details of the inhibitory effects of the mouse pan anti-mouse MHC-I mAb provides not only a more advanced understanding of the murine model system, but suggests additional functional avenues to be explored using the parallel an anti-human MHC-I mAbs.

cancer biology↗

bioRxiv2026

An autologous cell-based therapeutic vaccine expressing IL6/1 fusokine drives robust anti-tumor response against ovarian cancer.

BackgroundCytokines are immunomodulatory proteins that play central roles in regulating immune responses and represent attractive targets for cancer therapy. However, as single agents, cytokines have shown limited clinical benefit due to systemic toxicities and a short in vivo half-life. Our group has focused on engineering fusion cytokines (fusokines) that couple two cytokines into a single biologic to reprogram immune cell responses by enforcing non-canonical receptor engagement and signaling. A chimeric IL-6/IL-1{beta} fusokine was engineered to test the hypothesis that enforced co-engagement of IL-6 and IL-1{beta} signaling pathways would confer a gain-of-function phenotype in T cells and promote robust anti-tumor immunity. Here, we describe the immunomodulatory properties of IL6/1 fusokine and a method to deliver this fusokine to produce inhibition of ovarian tumor growth in a pre-clinical mouse model. MethodsLentiviral vectors encoding murine or human IL6/1 were designed using Vector Builder and expressed in either HEK293, CHO or ID8-F3 (p53-/-) cells depending on the downstream experiment to be conducted. IL6/1 expression was validated by ELISA and flow cytometry. Effects of human IL6/1 (hIL6/1) on T cell function (proliferation, memory phenotype, activation induced apoptosis) were monitored by flow cytometry. For in vivo studies, ID8-F3 murine ovarian cancer cells expressing mouse IL6/1 (mIL6/1) were administered intraperitoneally (I.P.) as a cell-based therapy to C57BL/6 female mice bearing established ID8-F3 luciferase tumors. Tumor progression was monitored by bioluminescence (BLI) imaging, and overall survival was evaluated. ResultshIL6/1 significantly enhanced T cell survival and selectively promoted activation and expansion of CD45RO memory T cells. mIL6/1 expressing ID8-F3 cells (ID8IL6/1) demonstrated stable transduction and sustained cytokine secretion. In vivo, ID8IL6/1 cell therapy significantly reduced tumor growth and improved overall survival compared to control groups, with 2 of 8 mice achieving complete tumor clearance. ConclusionThese findings indicate that IL6/1 fusokine enhances T cell survival and proliferation while promoting memory responses. Engineered cancer cells (ID8-F3) expressing mIL6/1 fusokine induced a strong anti-tumor response when delivered as a therapeutic vaccine in ovarian cancer mouse model. What is already known on this topicO_LIFusokines are a class of bifunctional proteins designed to achieve synergistic immune modulation. Previous studies in our lab have shown fusokine exhibit gain-of-function immunomodulating activity. Individually, IL-6 and IL-1{beta} are recognized for their roles in promoting T-cell proliferation and effector function. However, the potential for a fused IL-6/1 fusokine to reprogram the immune system and elicit a superior anti-tumor response in vivo in ovarian cancer model is not yet studied. C_LI What this study addsO_LIThis study develops a novel fusion cytokine (fusokine), combining IL-6 and IL-1{beta}, and demonstrate robust activation of T cells. In a preclinical ovarian cancer model, engineered cancer cells expressing IL6/1 used as a therapeutic vaccine showed significant tumor reduction and improved overall survival. C_LI How this study might affect research, practice or policyO_LIThis study demonstrates that in comparison to individual cytokines, fusokines have greater potential to activate T cell function and when delivered as a cell therapy, achieve clear therapeutic efficacy in an ovarian cancer model. Further translational and clinical studies may enable the development of novel and more effective fusokine cell therapy approaches for patients with ovarian cancer. C_LI

immunology↗

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

Metabiosis underlies a microbiota permissive to Pseudomonadota and increases the risk of gut-borne bloodstream infection

The gut microbiota contains trillions of bacteria essential to health, but also harbors potential pathogens. The phylum Pseudomonadota, which includes Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa, typically composes <1% of the microbiota but causes disproportionate numbers of gut-borne bloodstream infections. Identifying the ecological dependencies that enable Pseudomonadota to cause gut-borne disease is important for human health. Here, we studied microbiota dynamics in patients undergoing allogeneic hematopoietic cell transplantation (allo-HCT) to find that microbiota compositions permissive to Pseudomonadota had, following antibiotic prophylaxis, high levels of Bacteroides--a major reservoir of polysaccharide utilization loci (PULs). We tested the causality of this clinical association in a mouse co-colonization model and discovered that Bacteroides fragilis promotes Pseudomonas gut colonization and survival to ciprofloxacin, a drug commonly used as prophylactic in allo-HCT. In vitro experiments revealed a general mechanism by which diverse Pseudomonadota species depend on Bacteroides polysaccharide breakdown to grow better, form more biofilm, and survive ciprofloxacin treatment under anaerobic conditions, a type of ecological dependency termed metabiosis. Guided by this insight, we used metagenomics to identify the PUL-encoded functions underlying the metabiotic potential of a patients microbiota and establish a link to gut-derived Gram-negative bacteremia in allo-HCT. Together, our findings translate mechanistically based microbiome ecology into a clinically actionable framework for early risk stratification and intervention.

microbiology↗