Biology subjects

Sharma, S.

Publications and source records attributed to Sharma, S..

At least 73 records · Page 4Linked to original sources

bioRxiv2026

Novel but stable endosymbionts have contrasting effects on aphid dispersal and plant feeding damage in the cereal pest Diuraphis noxia

Endosymbiotic bacteria can affect many ecological attributes of their insect hosts, including (in herbivorous insects) how insects interact with plants where they feed. This raises the issue of whether deliberate endosymbiont introductions could be used to decrease crop damage caused by insect pests. Here we investigate how transinfecting Rickettsiella viridis and Regiella insecticola endosymbionts into a novel pest aphid host, the Russian wheat aphid (Diuraphis noxia), influences population growth, alate production, dispersal ability and crop damage. Both the Rickettsiella (originating from pea aphids) and Regiella (from green peach aphids) were stably maintained in their new host where they had contrasting effects. Rickettsiella increased the severity of aphid damage on wheat and barley, resulting in greater leaf loss, chlorotic streaking, and higher aphid populations, whereas Regiella reduced aphid population growth and the severity of feeding damage by aphids. Their effects on dispersal morphology also differed: Regiella had no detectable impact on alate incidence, while Rickettsiella consistently suppressed wing formation in small cages, and in larger mesocosms with multiple wheat plants this endosymbiont suppressed dispersal. Endosymbiont-mediated changes in feeding damage did not involve the main plant immune response pathways: transinfected and wild type aphids induced similar levels of jasmonic acid, jasmonic acid-isoleucine, and salicylic acid in plant tissues, even though these plant defenses were strongly activated during aphid feeding. Novel endosymbionts can therefore modulate the severity of plant feeding damage by aphids as well as influencing aphid dispersal. Potential applications in controlling pest D. noxia populations are discussed. Significance statementEndosymbiotic bacteria that live within insect cells can have wide-ranging effects on the reproduction and fitness of their insect hosts in different environments. In herbivorous insects this includes effects on host plant use. Here we test if novel endosymbionts in a pest aphid, the Russian wheat aphid, might be used to decrease crop damage and dispersal. We show that the damage caused to wheat and barley plants from aphid feeding is modulated by novel but stably transmitted introduced endosymbionts. One endosymbiont (Rickettsiella) increased the severity of damage but decreased aphid dispersal, while another (Regiella) decreased damage severity without impacting dispersal. These contrasting effects may be associated with changes in aphid population growth and wing formation but were not linked to key plant immune response pathways. We discuss implications of these findings for using endosymbionts in agricultural pest management. Classification: Applied Biological Sciences, microbiology

microbiology↗

bioRxiv2026

NetTracer3D Enables User-Friendly Analysis of Diverse Microscopic and Medical 3D Datasets

Recent advances in high-resolution imaging and spatial transcriptomics have enabled reconstruction of complex 3D tissue maps, providing unprecedented insights into cellular connectivity, organization, and tissue architecture. However, standardization challenges hinder integration, sharing, and analysis of these datasets across research communities. We developed NetTracer3D to simplify three-dimensional image analysis across diverse datasets. NetTracer3D is an integrated tool for defining, processing, and sharing 3D tissue maps with standardized data formats and interactive exploration capabilities. It provides three broadly applicable network analysis modalities: Connectivity networks for analyzing functional tissue units or cells connected via secondary structures such as nerves or vasculature; Branch Adjacency and Branchpoint networks for converting branched anatomical structures into analyzable representations; and Proximity networks for grouping structures by spatial relationships to identify cellular organization patterns. We demonstrate several use cases applying NetTracer3D to analyze multidimensional data from CODEX and label free Raman spectroscopy, multiscalar data encompassing subcellular and anatomical scales and a range of modalities. NetTracer3D was able to characterize neural relationships between functional tissue units in human and mouse kidneys and mouse bronchi. Branchpoint networks were used to identify vascular defects in human brain angiogram and define the innervation structure of a lymph node. Finally, we demonstrate how proximity networks characterize the tumor microenvironment in 3D light sheet cancer images and auto-detect cellular neighborhoods in multiplexed 2D CODEX datasets. Beyond network creation, NetTracer3D enables analysis, spatial statistics, and visual analytics tailored for volumetric tissue data. By establishing interoperable formats and analysis workflows, this work provides accessible and reproducible analytical tools for 3D spatial biology, enabling new discoveries of relationships between structure and physiology.

genomics↗

bioRxiv2026

Diet-conditioned microbiota enhances fecal microbiota transplantation efficacy in alcoholic liver disease through caproic acid-PPARα signaling

Background and AimFecal microbiota transplantation (FMT) in Alcohol-related liver disease (ALD) has shown therapeutic potential, with variable efficacy and unclear mechanism. Because dietary protein influences gut microbiota composition, we hypothesized that donor dietary preconditioning could enhance FMT efficacy. We therefore examined in a murine ALD model if high-protein donor diet improves FMT outcome. MethodsALD was induced in C57BL/6N mice using a Lieber-DeCarli ethanol diet combined with thioacetamide administration for 12 weeks. FMT was performed using stool from diet-modulated donors, and recovery was assessed on day7 post-FMT. Multi-omics analysis using 16s rRNA and mass spectroscopy was performed for Gut microbiota composition, plasma- and stool-metabolome, and hepatic proteomes. Multi-omics outcomes were validated in ALD animal and Huh7 hepatocytes. ResultsBoth protein-based FMTs improved ALD recovery; Veg-FMT demonstrated superior efficacy, significantly reducing hepatic injury (AST 1.2-fold, p=0.002; bilirubin 1.2-fold, p=0.03; steatosis 1.7-fold,p=0.01) and restoring gut barrier integrity (occludin 1.5-fold,p=0.04; mucin 2 2.2-fold, p=002; and plasma endotoxin 1.7-fold, p=0.02). A significant 2-fold increase was observed in Lachnospiraceae NK4A136, Coriobacteriaceae UCG-002, and short-chain fatty acids, particularly caproic acid. Functional validation confirmed that caproic acid promoted hepatic fatty acid {beta}-oxidation through PPAR-dependent mechanisms, reducing triglyceride accumulation and lipogenesis in both cellular and animal models. ConclusionDonor preconditioning with a plant-protein enriched diet enhances FMT efficacy in ALD by gut microbiota modulation with increased metabolites like caproic acid. These findings highlight a microbiota-metabolite-host axis through which diet-modulated FMT improves hepatic lipid metabolism and injury, and identifies a pathway via which FMT imparts its effect. SignificanceThis study identifies a mechanistic basis for improving fecal microbiota transplantation (FMT) efficacy in alcohol-related liver disease (ALD) by demonstrating that dietary preconditioning of donor microbiota improves therapeutic outcomes. We show that plant protein-modulated donor microbiota supplements abstinence-associated recovery through increased production of the microbial metabolite caproic acid, which promotes hepatic fatty acid {beta}-oxidation via PPAR signaling. These findings highlight donor dietary conditioning and microbiota-derived metabolites, rather than microbial composition alone, as important determinants of FMT efficacy. The results suggest that microbial metabolites such as caproic acid may represent potential therapeutic targets or biomarkers to enhance and standardize microbiota-based interventions in ALD. Although the current work is based on a murine model, the identified microbiota-metabolite-host metabolic axis provides a framework for future translational studies aimed at optimizing FMT strategies in liver disease.

microbiology↗

bioRxiv2026

Rpl12 paralog dependent TOR-signaling controls the expression of ribosome preservation factor Stm1

Ribosomes are increasingly recognized as heterogeneous regulators of gene expression, yet how ribosomal protein paralogs interface with nutrient signaling remains poorly understood. In Saccharomyces cerevisiae, ribosomal protein gene expression is governed by duplicated gene pairs, many of which exhibit functional divergence despite high sequence identity. A central regulator of ribosome biogenesis and translational control is the Target of Rapamycin (TOR) pathway, which integrates nutrient signals to modulate growth, stress adaptation, and lifespan. Target of Rapamycin Complex 1 (TORC1) influences ribosome activity by phosphorylating ribosomal protein S6 (Rps6), a modification that links nutrient availability to translational output. Here, we investigated the functional divergence of the Rpl12 ribosomal stalk protein paralogs RPL12a and RPL12b and found that rpl12b{Delta} produces phenotypes consistent with reduced TOR activity, including decreased Rps6 phosphorylation, G2/M cell-cycle accumulation, and significant extension of chronological lifespan as compared to rpl12a{Delta} and wildtype strain. Multi-omics analyses further indicate translational and metabolic reprogramming consistent with activation of a stress-adaptive program associated with Gcn4. Importantly, loss of RPL12b also reduces levels of the ribosome preservation factor Stm1, a TORC1-regulated protein required for stabilization of 80S ribosomes under stress. This finding links ribosomal stalk composition to ribosome stability and nutrient-responsive signaling. Together, our results demonstrate that ribosomal paralog specialization provides an additional regulatory layer connecting translation, TOR signaling, and cellular longevity.

cell biology↗

bioRxiv2026

Molecular genetic characterization of bacterial KH-domain proteins

Post-transcriptional gene regulation is a key mechanism for bacterial stress responses and virulence, and RNA-mediated regulation frequently relies on global RNA-binding proteins (RBPs). A pair of interacting KH-domain proteins (KhpA and KhpB) have recently been identified as global RBPs in several bacterial species. To better understand their molecular functions, we employed bacterial two- and three-hybrid (B2H/B3H) assays in an E. coli reporter system to analyze protein-protein and protein-RNA interactions of KhpA/B orthologs from three human pathogens: Campylobacter jejuni, Helicobacter pylori, and Clostridioides difficile. Protein-protein interactions were conserved across all species, with KhpA-KhpB heterodimers forming more robustly than either homodimer and KhpA homodimerizing more readily than KhpB. On the other hand, protein-RNA interactions were more varied across species: C. jejuni and C. difficile KhpA bound both species-specific and non-specific RNAs, but H. pylori KhpA--and KhpB orthologs from all species--showed no RNA interaction in B3H assays. Site-directed mutagenesis experiments demonstrated that residues in the GXXG motif of KhpA are critical for RNA interaction and differences in these residues account for the distinct RNA-binding behaviors of KhpA orthologs. Collectively, these findings provide a cross-species, molecular view of how KhpA and KhpB recognize one another and RNA ligands to regulate gene expression.

molecular biology↗

bioRxiv2026

In silico analysis reveals the structural basis of TomEP specificity, a tomato extensin peroxidase

BackgroundExtensin peroxidases (EPs) are class III plant peroxidases and are responsible for intermolecular covalent crosslinking of extensin (EXT) monomers to create scaffolds within plant cell walls. The formation of these scaffolds impacts plant development, mechanical wounding, and response to pathogen attacks. Therefore, elucidating the molecular mechanism controlling covalent crosslinking of EXT monomers is crucial for understanding cell wall deposition and potentially improving plant growth and adaptation. The focus of this work is to use in silico analysis to determine the structural characteristics of an EP from tomato (TomEP) to elucidate its specificity for crosslinking of EXT monomers. ResultsIn this study the two-dimensional (2D) and three-dimensional (3D) structures of TomEP were determined using several advanced bioinformatics tools and compared to two other peroxidases: GvEP1 (a known EP) and HRP-C (having a low affinity for EXT substrates). The results revealed that TomEP is a stable and hydrophilic protein with high thermal stability. The heme binding pockets of TomEP and GvEP1 have more hydrophobic residues and larger volume and pocket area compared to HRP-C. Molecular docking at the active site, which includes a heme heteroatom, showed that the ligands consisting of the hydrophobic Tyrosine-X-Tyrosine [-Y-X-Y-] motifs (i.e., [-Y-K-Y-], [-Y-V-Y-], and [-Y-Y-Y-] found in EXTs, and their derivatives, Isodityrosine (IDT), Pulcherosine (Pul), Di-Isodytirosine (diIDT), bind perfectly to the active site of TomEP with dominant interactions of Val54, Ser94, Ala96 and Phe196 residues. Pulcherosine had the highest binding affinity, while [-Y-K-Y-] showed the lowest binding affinity. Molecular dynamics simulations showed that [-Y-X-Y-] motifs (and the derivative substrate ligands) remain bound to the active site of TomEP throughout the 100 ns long simulation. Furthermore, the binding of these substrates stabilized the protein structure. ConclusionThese results may explain why TomEP is particularly well-suited for EXT crosslinking and will have significant implications on biochemistry, biotechnology, and the potential use of these EPs in crops improvement.

plant biology↗

bioRxiv2026

FDA-approved drug library screen identifies antidepressants, antimicrobials, anti-COPD, and anti-CVD agents as blockers of NLRP3 inflammasome and sepsis in a sex-dependent manner.

The NLRP3 inflammasome pathway is central to host defense, but dysregulated activation of inflammasomes promotes diseases associated with metabolic syndrome (diabetes, obesity, CVD, MASLD), neurodegenerative diseases (Alzheimers, Parkinsons), autoinflammatory conditions (CAPS, gout), and respiratory illnesses (asthma/COPD, COVID-19). Therapeutic modulation of NLRP3 is challenging as it requires selective blockade of detrimental inflammasome activation without broadly suppressing innate immunity. Here, we used a phenotypic screen in THP1-ASC-GFP monocytes to identify FDA-approved drugs that can block LPS-induced priming of the NLRP3 inflammasome or inhibit NLRP3 assembly without disrupting upstream priming. Various classes of drugs, such as antidepressants (Fluoxetine, Duloxetine), antihypertensives (Irbesartan, Amlodipine, Nebivolol), antidiabetics (Rosiglitazone), {beta}-adrenergic agonists (Salmeterol), antimalarials (Mefloquine), antifungals (Azoles, Ciclopirox), and antivirals (Saquinavir, Remdesivir), were identified as potent blockers of either priming or assembly of the NLRP3 inflammasome. Secondary validation demonstrated that several compounds suppressed NF-{kappa}B activation, reduced LPS binding to immune cells, decreased pro-inflammatory cytokine production, enhanced efferocytic capacity of macrophages, and enhanced autophagy in vitro and in vivo. Mechanistic analyses further revealed drug-specific effects on lysosomal biogenesis, mitochondrial morphology, and mitochondrial reactive oxygen species. In murine models of acute inflammation and endotoxemia, selected compounds attenuated NLRP3 inflammasome activation, reduced systemic and local inflammatory cytokines, limited tissue injury, and improved survival following LPS-induced sepsis, with efficacy varying by sex. Further studies in primary human cells and in vivo disease models are needed to assess the repurposing and therapeutic relevance of identified drugs.

cell biology↗

bioRxiv2026

Erythropoietin Mediates Glycerophospholipid Remodeling During Human Early Erythropoiesis

The unique biconcave morphology and deformability of mature red blood cells (RBCs) require a specific membrane lipid composition. Disruption of this special lipid profile is seen in multiple types of anemia and bone marrow (BM) failure diseases, yet molecular mechanisms regulating lipid metabolism during normal erythroid differentiation remain poorly defined. Here, we identify a previously undescribed role for erythropoietin (Epo) in contributing to the appropriate lipid composition of differentiating human erythroid cells. Using single-cell transcriptomic profiling of ex vivo cultures of human BM-derived hematopoietic stem and progenitor cells cultured with or without Epo, we delineated transcriptional dynamics across differentiation stages, identifying the Epo-dependent transition of burst-forming unit erythroid (BFU-E) to colony-forming unit erythroid (CFU-E) progenitors. Comparative analysis revealed the activation of canonical erythroid programs involving heme biosynthesis, globin expression, and iron regulation, and additionally, uncovered a transient upregulation of lipid metabolic pathways during the BFU-E to CFU-E transition. Complementary untargeted lipidomics demonstrated Epo-dependent alterations in specific glycerophospholipid (GPL) species, consistent with differential expression of GPL biosynthesis genes in the single cell dataset. Intracellular flow cytometry further confirmed the requirement of Epo for maintaining enzymes critical for phosphatidylcholine and phosphatidylethanolamine synthesis in erythroid cells. Together, these multiomic findings reveal a new role for Epo in modulating lipid metabolism during early erythropoiesis and provide mechanistic insight into how membrane lipid composition is dynamically regulated to support normal red cell development.

cell biology↗

bioRxiv2026

Signalome-wide mapping of the NFκB pathway in T-cells reveals novel targets for immunotherapy

Cell signalling networks govern fundamental cellular processes yet remain incompletely defined. Moreover, what is known is biased toward a limited subset of well-characterised components. Phosphoprotein-based interrogation methods, including mass spectrometry and targeted phosphosite panels, have limited utility in physiological settings dependent on cell-cell interactions because the signalling fluxes can be difficult to detect despite producing robust functional responses. Here we developed a perturbation-based experimental framework that infers signalling pathway architecture using quantitative functional outputs rather than direct measurements of effector state, e.g., phosphorylation levels. Using antigen-specific, NF-{kappa}B-GFP reporter-expressing transformed T-cells co-cultured with cellular targets, we performed an arrayed CRISPR-Cas9 screen targeting a curated signalome of kinases, phosphatases, adaptor and scaffolding proteins, totalling 706 genes. Quantitative effect-size profiling recovered canonical T-cell receptor regulators and revealed unequal, family-specific patterns of control over NF-{kappa}B activation. Comparing T-cell stimulation with low- and high-affinity antigen uncovered signal-strength-dependent buffering of proximal signalling nodes, exemplified by reduced sensitivity to perturbation of LCK under high-intensity stimulation. Targeted perturbation in primary human CD8 T-cells validated our findings and identified TRRAP and CTDSPL2 as negative regulators of T-cell effector output, whose disruption enhanced cytotoxicity, degranulation, and cytokine production in both polyclonal and TCR-engineered T cells. Together, these results establish a scalable strategy for mapping signalling pathway architecture in the setting of physiological T-cell activation.

immunology↗

bioRxiv2026

DNA binding drives phase separation of the Gcn4 bZIP domain and reveals its conformational ensemble in the diluted and condensate phases

Liquid-liquid phase separation is widely invoked in transcriptional regulation, yet prevailing models attribute condensate formation primarily to intrinsically disordered activation domains rather than structured DNA-binding motifs. Here, we overturn this view by demonstrating that the isolated basic leucine zipper (bZIP) domain of the yeast transcription factor Gcn4 undergoes robust DNA-induced phase separation in the complete absence of its activation domain. Using small-angle X-ray scattering in combination with all-atom molecular dynamics simulations and ensemble optimization, we directly resolve the conformational landscape of the Gcn4 bZIP-DNA complex across coexisting dilute and condensed phases. Beyond the canonical uninterrupted helical conformation captured in crystal structures, we identify a previously unrecognized minor population featuring a pronounced helical kink at the basic region-leucine zipper junction. These findings establish DNA binding as a sufficient physical driver of bZIP phase separation and demonstrate that small-angle scattering can quantitatively interrogate protein conformational ensembles within biomolecular condensates, opening new avenues for the structural chemistry of phase-separated systems.

biophysics↗

bioRxiv2026

Ancient intraflagellar transport machinery controls unique spatial distribution of phototropin in an evolutionary important non-flagellated vegetative stage of terrestrial alga

Intraflagellar transport (IFT) is a conserved trafficking system in eukaryotes that moves proteins along microtubules. It is best known for its essential role in building and maintaining cilia and flagella. Intriguingly, several IFT components are still found in organisms that no longer possess flagella, raising important questions about their original functions and how they may have been repurposed during evolution. The filamentous alga Klebsormidium nitens, positioned at the base of the streptophyte lineage, offers a valuable model for exploring this transition. Here, we investigate the IFT machinery in K. nitens and its relationship with the blue-light photoreceptor phototropin. Comparative genomic analyses show that key IFT-A and IFT-B components are retained, despite the complete loss of flagella in vegetative state. Cellular detection and immunofluorescence studies revealed the presence and localisation of IFT components, interestingly, their co-localization with phototropin. Notably, IFT-139 and IFT-20 strongly co-localize with phototropin at plasma membrane-associated regions. Phototropin overlapping localization (plasma membrane associated) with conserved phospho-adaptor protein 14-3-3, pointing to a phosphorylation-dependent signaling network. Unlike in Chlamydomonas reinhardtii, where these proteins localize to flagella, their interaction in K. nitens occurs independently of cilia presence. Together, these results evidenced that IFT components were retained and repurposed early in streptophyte evolution and might support phototropin localization and signalling, revealing an ancestral, non-ciliary role for the IFT system.

cell biology↗

bioRxiv2026

Toxin-triggered activation of regulated exocytosis enhances bacterial egress from the intestinal layer

Bacterial exit from host cells is essential for dissemination yet remains poorly understood. Here, we define a non-lytic egress pathway exploited by the enteric pathogen Yersinia pseudotuberculosis that co-opts the host exocytosis machinery in intestinal epithelial cells. The bacteria secrete a CNF-family toxin that activates the Cdc42-PLC {gamma}1-IP3-IP3R signaling cascade, triggering SNARE-dependent fusion of the Yersinia-containing vacuoles with the cell membrane via VAMP7, Stx4, and SNAP23. This controlled exocytotic release preserves epithelial barrier integrity and occurs infrequently, representing a key rate-limiting step in systemic spread. These findings establish the host exocytotic machinery as an active determinant of bacterial egress, uncover a conserved vesicle trafficking pathway hijacked by intracellular pathogens for dissemination, and assign a new role for bacterial toxins in regulating host cell exit. Authors SummaryMany bacterial pathogens penetrate and cross protective host cell barriers to spread within the body. While the mechanisms by which enteric bacteria enter host cells are well characterized, far less is known about how they exit host cells after invasion. In this study, we investigated how the enteric pathogen Yersinia pseudotuberculosis exits from human gut epithelial cells after crossing them. We found that they egress at the basolateral side of the intestinal cells through a controlled, non-destructive process that preserves the integrity of the epithelial barrier. To achieve this, the bacteria co-opt host signaling pathways involved in host cell vesicle release (exocytosis) to promote controlled egress. These exit events are rare, suggesting that bacterial escape from cells is a major bottleneck during infection. Importantly, we show that this process is enhanced by a secreted toxin of the Cytotoxic Necrotizing Factor (CNF) family (CNFY), which promotes the fusion of the bacteria-containing vacuole with the basolateral cell membrane. This finding uncovers a previously unrecognized role of bacterial toxins in facilitating bacterial cell egress.

microbiology↗

bioRxiv2026

Early face deprivation leads to long-lasting deficits in cortical face processing

Experience-dependent plasticity in early sensory cortex is restricted to critical periods that occur early in development. In contrast, when during development higher-order cortical areas show experience-dependent plasticity remains unclear. In the primate ventral visual stream, face-selective cortex emerges with experience during development, providing a powerful system for testing how the timing of experience shapes higher-order representations. Here, we leveraged a unique macaque model of selective face deprivation followed by years of normal face exposure to causally test late plasticity in higher-order visual cortex. We found that face-deprived monkeys acquire face-directed viewing behavior and neural face selectivity after post-deprivation exposure. However, their neural representations retained lasting signatures of early experience, including mixed face and hand selectivity and impaired tuning for face identity, expression, and viewpoint. These findings demonstrate that higher-order visual cortex remains plastic beyond early development, but that the timing of experience fundamentally constrains the refinement of later-acquired neural representations.

neuroscience↗

bioRxiv2026

Epigenetic Gatekeeping of Intestinal Stem Cell Transformation

Widespread cell plasticity recognized in fetal intestinal epithelium is preserved in limited fashion in Wnt-responsive adult stem cells and contributes to tumour initiation, progression, and relapse. It is unclear which epigenetic features maintain stem-cell properties, restrict adult expression of fetal genes, and are attenuated in tumours, allowing non-stem cells to replenish targeted tumour stem cells. Here we show that reversible stemness in normal adult intestinal crypt cells hinges on a dynamic balance between activating H3K27ac and repressive H3K27me3 marks. Cells that leave the Wnt-rich stem-cell niche normally acquire H3K27me3 at thousands of stemness-associated enhancers. Constitutive tumourigenic Wnt activity transforms Apc-/- intestinal stem cells by gradual erosion of H3K27me3 at select enhancers and extends stem-like properties beyond usual anatomic confines; continued depletion of H3K27me3 reactivates enhancers that control growth and expression of a wider swath of fetal genes than appreciated previously. Subsequent focal DNA demethylation at expanded superenhancer domains is associated with tumour growth. Human colorectal cancers also carry evidence of this epigenetic rewiring. Accelerated H3K27me3 loss in mice hastens, and its preservation delays, activation of stemness-related enhancers, superenhancers, and tumour progression. During transformation, H3K27me3 loss at enhancers erases a crucial distinction between stem and non-stem populations, endowing the latter with stemness and providing an explanation for tumour resistance to cancer stem cell targeting. Thus, H3K27me3 at Wnt-responsive enhancers is an intrinsic barrier to intestinal tumourigenesis and aberrant reactivation of hundreds of fetal genes.

cancer biology↗

bioRxiv2026

Machine Learning Identifies Distinct Treg-Mediated Remodeling in HFpEF Hearts Treated with Neonatal Mesenchymal Stem Cells and Their Secretome

BackgroundHeart failure with preserved ejection fraction (HFpEF) remains a major therapeutic challenge due to its complex pathophysiology and pronounced heterogeneity. Regenerative approaches using neonatal mesenchymal stromal cells (nMSCs) and their secretome (SEC) have shown promise in other heart failure contexts. ObjectivesHowever, the effect of these therapies in HFpEF, and the underlying molecular mechanisms and causal pathways remain poorly understood. MethodsHFpEF was established in two distinct murine models, followed by treatment with either nMSCs or SEC. Functional and histological endpoints were assessed. We developed a novel machine learning framework, VIPcell, which integrates data augmentation, Partial Least Squares (PLS) regression, and causal structure inference to identify genes causally linked to cardiac function using single-nucleus RNA sequencing (snRNA-seq) data. VIPcell was applied to heart tissues from treated HFpEF animals to uncover key regulators of cardiac remodeling. ResultsBoth nMSC and SEC therapies significantly improved diastolic function in two independent rodent HFpEF models. These improvements were associated with reduced inflammation, attenuated myocardial fibrosis, and improved exercise capacity. Intercellular communication analysis revealed widespread, system-level signaling in nMSC-treated hearts, compared to more localized endothelial-cardiomyocyte crosstalk in SEC-treated hearts. Causal inference via VIPcell suggested overlapping upstream regulators in both treatment groups, particularly genes involved in regulatory T cell (Treg) biology and immunomodulatory signaling pathways, including FOXO signaling, NLRP3 inflammasome inhibition, and Tie2 activation. In vivo validation confirmed selective expansion of Tregs following nMSC and SEC therapy. In vitro, nMSCs induced significantly greater Treg expansion compared to multiple adult stem cell types. Critically, chemical depletion of Tregs abrogated the therapeutic effects of both treatments, establishing Tregs as central mediators of diastolic function recovery in the HFpEF preclinical model. ConclusionsnMSC and SEC therapies improve diastolic function in HFpEF through distinct remodeling mechanisms converging on Treg-mediated immune modulation. VIPcell supported identification of causal regulators, highlighting Treg-related signaling as a key driver of myocardial recovery in HFpEF. These findings offer mechanistic insight into cellular therapies for HFpEF and support the development of targeted, Treg-focused interventions.

physiology↗

bioRxiv2026

Drosophila TRAPPC8-Rab1 module regulates retrograde trafficking of Wingless and Evi/Wntless

Lipid-modified Wnt proteins are secreted from polarized epithelial cells through a multi-step pathway involving membrane delivery, internalization and resecretion. Internalization enables dissociation of Wnt from its cargo receptor Evi/Wntless in endosomes and promotes retromer-mediated recycling of Evi to the Golgi, however, the molecular basis of this retrograde Wnt trafficking remains poorly understood. Here, we identify an essential function of TRAPPC8, the TRAPPIII-specific subunit, in the trafficking of Drosophila Wingless (Wg), the Wnt1 homolog. Loss of TRAPPC8 impaired retrograde trafficking of both Wg and Evi, resulting in their intracellular accumulation in Wg-producing cells. Consistent with the role of TRAPPIII as a Rab1-specific GEF, inhibition of Rab1 phenocopied the trafficking defects caused by TRAPPC8 loss, whereas constitutively active Rab1 rescued these defects. Furthermore depletion of either TRAPPC8 or Rab1 increased the levels of Wg-unbound Evi, indicating that they act downstream of Evi-Wg dissociation. Together, these findings identify the TRAPPIII complex and its effector Rab1 as key regulators of retrograde Wg trafficking required for its efficient secretion.

cell biology↗

bioRxiv2026

Reprogramming BCMA-Targeted CAR-T Cells through γ-Secretase Modulation Blocks Antigen Shedding and Extends CAR-T Longevity

B-cell maturation antigen (BCMA) shedding by {gamma}-secretase generates soluble BCMA (sBCMA), which diminishes membrane antigen density, and limits the durability of BCMA-directed immunotherapies in multiple myeloma (MM). Here, using AI-driven diffusion modeling, we identify peptide inhibitors that selectively block {gamma}-secretase-mediated BCMA cleavage, stabilizing membrane-bound BCMA (mBCMA) without compromising cellular viability. The lead peptide, P5, suppresses sBCMA and restores mBCMA in vitro and in xenograft models. To create sustained, cell-intrinsic inhibition, we engineered Shedding Deterrent Locked-in (SHEDLOCK) CAR-T cells that locally secrete {gamma}-secretase-modulatory peptides. SHEDLOCK-1 CAR-T cells secreting P5 exhibits enhanced cytotoxicity and persistence relative to conventional CAR-T cells. Building on this, SHEDLOCK-2 CAR-T cells were generated, which secrete a modified, naturally derived peptide (nxP) that directly engages the {gamma}-secretase catalytic site, concurrently preventing BCMA shedding, and surprisingly enhancing CAR-T cell longevity by preserving telomere integrity and metabolic fitness. In MM xenograft and patient-derived models, SHEDLOCK-2 CAR-T cells demonstrate durable antitumor activity with a favorable safety profile. Together, these findings establish SHEDLOCK as a next-generation CAR-T platform and provide a strong preclinical foundation for Phase I clinical evaluation. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=128 SRC="FIGDIR/small/700575v1_ufig1.gif" ALT="Figure 1"> View larger version (69K): org.highwire.dtl.DTLVardef@3cd38aorg.highwire.dtl.DTLVardef@164167borg.highwire.dtl.DTLVardef@1ffed21org.highwire.dtl.DTLVardef@1294d06_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2026

Small molecule inhibition of the mitochondrial lipid transfer protein STARD7 attenuates influenza viral replication

The increasing appearance of drug-resistant and zoonotic influenza strains highlights an urgent need for host-directed antivirals that offer broad-spectrum activity and a higher barrier to resistance. Here, we describe the characterization of M4, a small-molecule identified from a high-thoughput screen that potently inhibits influenza A and B viruses. Mechanistic studies reveal that M4 suppresses replication by causing nuclear retention of the viral ribonucleoprotein (vRNP) complex. Chemoproteomic profiling identified the lipid transfer protein STARD7 as the primary cellular target, and genetic depletion of STARD7 phenocopies the antiviral effects of M4. Additional studies localizes the M4 binding site to cysteine 302 within the lipid-binding domain of STARD7, supporting a model in which STARD7-dependent lipid transfer activity promotes efficient vRNP trafficking and nuclear export. Combining M4 with baloxavir enhances antiviral efficacy in a murine infection model, providing in vivo support for a host-directed strategy. Together, these results identify STARD7 as a metabolic checkpoint licensing vRNP export and establish a proof of concept for therapeutic intervention with small molecule inhibitors. Author SummaryInfluenza viruses continue to cause widespread illness and pose an ongoing pandemic threat, in part because existing antiviral drugs can lose effectiveness as the virus evolves resistance. To address this challenge, we focused on identifying therapies that target host cell processes required for viral replication, rather than viral proteins themselves. In this study, we describe a small molecule, M4, that inhibits replication of both influenza A and B viruses by blocking a critical step in the viral life cycle. We found that M4 acts on a host protein called STARD7, which helps move certain lipids to the right places inside cells. When STARD7 is inhibited, the influenza viral ribonucleoprotein (vRNP) complex becomes trapped in the cell nucleus and cannot reach the cytoplasm, preventing the virus from completing its replication cycle. Disrupting STARD7 genetically produces the same effect, confirming that this host protein is important for influenza replication. These findings point to a previously unrecognized host gating mechanism that controls nuclear export of the vRNP complex during infection. Although M4 alone showed limited activity in animals, combining it with an existing antiviral drug strongly improved antiviral efficacy. Together, our results reveal a new host pathway that influenza viruses rely on and support host-directed combination approaches to strengthen antiviral treatment and help counter drug resistance.

microbiology↗