Biology subjects

Liao, X.

Publications and source records attributed to Liao, X..

At least 19 recordsLinked to original sources

bioRxiv2026

Impaired glycolysis in bone underlies age-related bone loss in mice

Bone loss and metabolic dysregulation often coexist in older adults, yet the relationship between age-related changes in bone cell metabolism and bone-forming activity remains unclear. By modeling both conditions in aged mice and using in vivo stable-isotope tracing, we demonstrated that age-related decline in bone formation was coupled with an intrinsic impairment of glycolysis within bone tissue. Impaired glycolysis was associated with reduced protein levels of key glycolysis enzymes, and transcriptomics analyses revealed compensatory upregulation of glycolysis genes in aged bone. Metabolic intervention through long-term caloric restriction prevented age-related trabecular bone loss. Furthermore, osteoblast-specific genetic enhancement of glycolysis, via overexpression of HIF1 or the rate-limiting enzyme PFKFB3, significantly improved osteoblast function and increased bone mass in aged mice. Together, the study identifies glycolytic decline as a cell-autonomous driver of osteoblast deficiency and provides proof of principle that reactivating glycolysis can promote bone accrual in aging.

physiology↗

bioRxiv2026

Bio-Babel: autonomous cross-language reconstruction of agent-ready computational biology ecosystems

Scientific software is often locked in its source language, callable from others but not natively extensible or built upon. We present Bio-Babel (biobabel.stanford.edu), an autonomous framework that rebuilds software natively in target ecosystems and makes it agent-callable, shipping each package with an agent-readable contract of its usage. Reconstructing a hierarchical, interdependent R-to-Python single-cell stack, Bio-Babel reproduced the originals faithfully and revealed pancreatic differentiation dynamic defects under graded SWI/SNF loss.

bioinformatics↗

bioRxiv2026

Pressure-induced membrane tension mechanically opens the germinant receptor GerA ion channel to trigger bacterial spore germination

High pressure (HP) can trigger bacterial spore germination, acting either through germinant receptors (GRs) or the SpoVA channel. However, the mechanism by which HP activates these membrane-embedded proteins remains elusive. Here, using Bacillus subtilis, we demonstrate that the GerA germinant receptor (GR) is the primary target of moderate HP (50-300 MPa). Mutagenesis reveals that pore-lining residues within the GerA ion channel are essential for the pressure response, whereas canonical ligand-binding and intramembrane signaling residues are dispensable. We then propose a s tretch- t o- o pen (STO) model, in which HP differentially compresses the more compliant inner membrane (IM) relative to the rigid spore core, generating lateral membrane tension that promotes opening of the GerA channel. In situ membrane tension measurements indicate HP-induced compression of IM phospholipids and elevated membrane tension. This tension-dependent gating is further supported by the pressure-dependent phenotypic rescue of GerA channel mutants. Consistently, HP increases IM permeability to water-soluble and membrane-impermeable agents (propidium iodide and formaldehyde), an effect potentiated by GerA, indicating concomitant opening of GerA by HP. Furthermore, modulating IM fluidity via heat activation or decoating altered membrane physical properties and delayed HP-induced germination, establishing the IM as the critical mechanical transducer. Additionally, computational modeling and calculations support faster compression of the IM than of the core under HP, rationalizing the source of tensile stress. Together, our findings establish a novel mechanism of HP-induced GerA activation via the STO model: HP compresses the IM, generates lateral tension, and promotes opening of the GerA ion channel to trigger bacterial spore germination.

microbiology↗

bioRxiv2026

Functional and evolutionary insights into the emerging tet(X4)-carrying non-O1/O139 Vibrio cholerae from retail pork

The tigecycline resistance gene tet(X4) is prevalent in Enterobacteriaceae, particularly in Escherichia coli. To our knowledge, no study has reported the dissemination dynamics of tet(X4) in Vibrio spp. Herein, we isolated and characterized a first tet(X4)-positive non-O1/O139 Vibrio cholerae isolate from retail pork. Genomic sequencing identified a novel tet(X4) variant in the V. cholerae chromosome, harboring a G568A nucleotide substitution that resulted in an Ala190Thr (A190T) amino acid substitution in Tet(X4). While this Tet(X4)-A190T variant conferred lower phenotypic resistance to tetracyclines (including tigecycline) than the wild-type Tet(X4), its overall catalytic efficiency against these antibiotics was paradoxically enhanced despite a reduced substrate affinity. Genomic comparisons revealed that two copies of ISCR2 flanked the variant gene, and the structure was ISCR2-hp-hp-abh-tet(X4)G568A -ISCR2, which is highly homologous to the reported E. coli plasmids carrying tet(X4). In addition, it confirmed the presence of an ISCR2-mediated circular intermediate, proving this modules capacity for horizontal transfer of the tet(X4)G568A variant. Furthermore, the ISCR2-tet(X4) genetic structure carrying the G568A substitution was integrated within a chimeric SXT/R391-like integrative and conjugative element (ICE), which is also serving as a vehicle for genetic dissemination. As per our knowledge, this is the first report on the emergence of SXT/R391-like ICE carrying tet(X4) in Vibrio strains. Our finding demonstrates that the clinically relevant tigecycline resistance gene tet(X4), previously confined mainly to Enterobacterales from humans and livestock, is now actively spreading into environmental Vibrio populations. This cross-species transfer highlights a previously underappreciated ecological and public health concern in aquatic ecosystems.

microbiology↗

bioRxiv2026

Intrinsic ignition-based propagation networks reveal hierarchical propagation pathways of spontaneous activity in the human brain

Directed communication across brain regions is fundamental to human brain function, yet how local spontaneous activity links to directed whole-brain propagation patterns remains elusive. Prior studies have characterized local propagation events and global wave-like dynamics, but a framework linking local events, interregional propagation pathways, and canonical large-scale propagation patterns remains lacking. Using resting-state functional MRI data from the Human Connectome Project 7T cohort and an intrinsic ignition framework, we quantified cascades of suprathreshold activity events and constructed directed propagation probability networks. We found spatially heterogeneous regional propagation preferences, with high outgoing propagation preferences predominantly localized in somatomotor, visual, and default-mode regions. At the functional system level, propagation modules broadly separated visual, somatomotor, dorsal attention, and ventral attention networks from association systems. Regional stepwise propagation pathways were aligned with the principal functional gradient, and greater hierarchical separation between regions was associated with longer propagation distance. These pathways formed two canonical propagation patterns linking primary and association systems, with somatomotor regions preferentially initiated in bottom-up propagation, default-mode regions in top-down propagation, and attention networks occupying intermediate positions. Notably, the two patterns were not simple mirror images, with top-down propagation showing substantial deviations within somatomotor and visual systems. These findings were replicated in an independent cohort. Moreover, individual propagation architecture predicted cognitive performance and tobacco-use behavior. Collectively, our findings provide cross-scale insights into spontaneous activity propagation in the human brain by bridging local ignition events with hierarchical whole-brain propagation pathways and behaviorally relevant individual differences. SignificanceSpontaneous brain activity is often quantified by the temporal synchronization between regions, but this approach does not show how local activity propagates across the brain. We used an interpretable ignition framework to link local spontaneous events and their cascades to directed whole-brain propagation patterns. This approach revealed outgoing propagation preferences of primary and default-mode regions, a modular architecture separating primary and attentional systems from association systems, and two canonical propagation patterns aligned with the brains functional hierarchy. These two patterns were not simple mirror images, indicating partially distinct bottom-up and top-down routes. Individual propagation architecture also predicted cognitive performance and tobacco-use behavior. These findings provide a path-based view of spontaneous brain dynamics.

neuroscience↗

bioRxiv2026

Differentiation of Bacterial, Fungal, and Algal Communities on Coastal Concrete Versus Drainage Pipes and the Coexistence of Corrosion and Healing Potentials

Coastal concrete structures and drainage pipes are prone to microbially influenced deterioration. However, differences in microbial communities and their corrosion/healing potentials between these habitats remain unclear. Here, we compared bacterial(16S), fungal (ITS) and algal(18S) communities on coastal concrete(C) and drainage pipe(P) surfaces. Fungal and algal -diversity were significantly higher in P than in C, while bacterial diversity did not differ. {beta}-Diversity strongly separated bacterial and algal communities between habitats, but not fungi. A shared core "seed bank" of 575 bacterial, 520 fungal and 40 algal ASVs was identified. Students t-test revealed that P enriched oligotrophic degraders (Sphingomonas) and acid-producing fungi (Arxiella, Bisifusarium), whereas C selected for halotolerant EPS-producing bacteria (Tunicatimonas, Muricauda) and the extremotolerant alga Coelastrella. db-RDA linked these differences to salinity, NH4+-N, NO3--N, and COD. Functional prediction indicated a shift from metabolism pathways in C to signaling in P. Co-occurrence networks revealed cross-kingdom competition and within-kingdom cooperation, especially among algae. Importantly, both habitats harbored microorganisms with documented corrosion and healing potentials, but under natural conditions, net deterioration dominated, microbial healing is hardly to counteract the negative effects. This study provides a functional taxonomic framework for understanding and managing concrete microbiomes in coastal and sewer infrastructure. ImportanceConcrete is the most widely used construction material, and its deterioration in coastal and sewer environments poses significant economic and safety challenges. Microorganisms play a dual role in concrete durability -- they can both corrode and heal concrete, but the net outcome under natural conditions is poorly understood. Most studies have focused on bacteria alone, overlooking the contributions of fungi and algae, and few reports on the of coastal concrete which also exist the microbially influenced concrete corrosion (MICC) similar to sewer. Here, we simultaneously analyzed all three microbial kingdoms on coastal concrete and drainage pipes. We found that while concrete surfaces harbor a diverse "seed bank" of microorganisms potentially involved in both corrosion and healing, under natural conditions, net deterioration dominated, indicating microbial healing is hard to counteract the negative effects from the environment and microbial. Our work provides a functional framework to guide the development of microbiome-based strategies for enhancing concrete durability, such as activating rare healing taxa in drainage pipes or selecting for endogenous healers in marine environments.

microbiology↗

bioRxiv2026

Polyploid cardiomyocytes define disease-specific transcriptional states in the mammalian heart

The adult mammalian heart has a limited regenerative capacity. Following injury, cardiomyocytes undergo a hypertrophic response accompanied by polyploidization, which has been described as a barrier to proliferation and regeneration of the heart1,2. However, the unique molecular programs of polyploidy, or genome multiplied cardiomyocytes, and their influence on the disease-related myocardial remodelling process remains unclear. Here, we integrate single-nuclei and high-resolution spatial multi-omics across human, rat, and mouse hearts to define novel cardiac cell states and their tissue niches in ischemic and non-ischemic heart disease. Computational analysis across scales allowed us to generate detailed networks of the cardiac tissue remodelling process as well as tissue and sub-cellular environments uniquely enriched in polyploid cardiomyocytes or their diploid origins. We identify a conserved, dichotomous transcriptional program distinguishing diploid from polyploid cardiomyocytes. Polyploid cardiomyocytes demonstrated rewired metabolic and chromatin-remodeling transcriptional programs and recapitulate the gene signature of immature human fetal cardiomyocytes. Notably, we observe that polyploid cardiomyocytes--rather than the general myocyte population--are the primary sites of enrichment for major heart-failure drug targets, including the mineralocorticoid, {beta}1-adrenergic, and glucagon-like peptide-1 receptors. Based on our cross-species dataset we further identified TNIK, a Wnt-pathway regulator expressed in polyploid cardiomyocytes across species, as a potential therapeutic target and demonstrate that pharmacological TNIK inhibition improves cardiac function after myocardial infarction in rats. Together, this species-spanning, disease-resolved study redefines cardiomyocyte heterogeneity in heart disease and suggests a therapeutic path to heart failure treatment by targeting polyploid cardiomyocytes.

genomics↗

bioRxiv2026

A proteomic signature of oocyte quality from models of varying oocyte developmental competence

Study questionWhich proteins underpin oocyte developmental competence, as modelled by oocytes of variable competence matured in vivo, or matured in vitro under different conditions (capacitation in vitro maturation (CAPA) or standard in vitro maturation (IVM))? Summary answerSignificant differences in the global proteome were observed in both oocytes and their corresponding cumulus cells depending on the mode of oocyte maturation, with key variations in eukaryotic translation, autophagy and endocytosis pathways within oocytes, and changes in reactive oxygen species detoxification and serine biosynthesis in cumulus cells. What is known alreadyWithin the ovarian follicle, mammalian oocytes must acquire the necessary molecular machinery to support successful fertilisation and embryonic development. Close contact with the surrounding cumulus cells ensures coordinated nuclear and cytoplasmic maturation of the oocyte, along with the accumulation of proteins stored within the oocyte in cytoplasmic lattices and endo-lysosomal vesicular assemblies. Study design, size, durationThis basic science study utilised a mouse model to assess proteomic changes across three oocyte competence models. Key proteins identified in mouse oocytes were also assessed in discarded immature human germinal vesicle (GV) oocytes and MII oocytes following rescue-IVM. Three oocyte maturation methods were tested: i) in vivo maturation, (ii) CAPA and (iii) standard IVM. In vivo maturation served as a positive control group, whereby metaphase II (MII) mature oocytes were collected from mice stimulated with pregnant mare serum gonadotropin (PMSG) and triggered with human chorionic gonadotropin (hCG), simulating full ovarian stimulation. For the in vitro maturation groups, immature cumulus oocyte complexes (COCs) were collected from mildly stimulated (23 hr PMSG) mice. For the standard IVM group, immature COCs were matured in media containing amphiregulin and epiregulin for 18 hours. For the CAPA group, COCs were held for 24 hours in pre-IVM conditions in the presence of c-type natriuretic peptide (CNP), oestradiol, insulin and follicle stimulating hormone (FSH), and then matured via IVM in media containing FSH, amphiregulin and epiregulin. Four biological replicates were performed for mouse proteomics experiments, three biological replicates performed for mouse immunocytochemistry experiments and six replicates were performed for embryology experiments. Participants/materials, settings, methodsFour to six-week-old C57BL/6JAusb mice were used for all mouse experiments. Embryology outcomes were used to confirm the variation in oocyte developmental competence between the three maturation groups. For the in vivo, CAPA and IVM groups, mature MII COCs were collected and separated into oocytes and cumulus cells. Oocytes and cumulus cells were subjected to mass spectrometry and bioinformatic analysis was performed using Proteome Discoverer and Ingenuity Pathway Analysis, with data validated by immunofluorescence. To assess conservation of proteins in human oocytes, 49 oocytes were collected from 36 patients following assisted reproduction technology (ART) cycles and subject to immunofluorescence. Rescue-IVM was also performed with half of the human oocyte cohort to obtain MII oocytes. Main results and the role of chanceProteomic profiling identified around 1600 proteins in mouse oocytes and 3100 in mouse cumulus cells across all three treatment groups (at least 2 peptides per protein). Differential expression analysis and pattern analysis collectively revealed a signature of proteins that were consistently differentially expressed between in vivo and in vitro oocyte maturation systems (log2FC of {+/-} 1 and a p-value [≤] 0.05). These subsets of proteins were mapped to biological processes including eukaryotic translation, autophagy and endocytosis pathways within oocytes. Orthogonal validation of clathrin, ribosomal protein L24 and eukaryotic initiation factor 2A supported the proteomic findings and expression was conserved in human oocytes. Changes in reactive oxygen species detoxification and serine biosynthesis were observed in mouse cumulus cells, with fluorescence intensity changes in ferredoxin-1 and phosphoglycerate dehydrogenase supporting the dysregulation of cumulus cell processes during in vitro maturation. Large scale dataThe mass spectrometry data are available via ProteomeXchange with identifier PXD073269. Limitations, reasons for cautionThe foundational mechanisms of oocyte developmental competence remain elusive, particularly in humans where MII oocytes are heterogenous in quality within the same stimulation cycle and patient. In this study, C57Bl6/J mice were used as the model species, allowing precise control over differing models of oocyte quality and capacity to analyse large numbers of oocytes. However, care is required when interpreting the significance of these findings in mice to mechanisms regulating human oocyte quality. Nonetheless, the in vivo stimulation and both IVM protocols used in this study are clinically relevant and developmentally matched. This study has also not addressed oocyte developmental competence in gonadotropin-free IVM oocytes, which is now a clinical reality. Wider implications of the findingsThis study confirms that mouse oocytes, matured in vitro in two clinically relevant systems, show reduced developmental competence when compared to in vivo matured oocytes. Through examination of the global proteome in oocytes, molecular pathways including eukaryotic translation, autophagy and endocytosis were dysregulated in in vitro oocytes. Recent findings have revealed the critical role of these pathways to developmental competence in the context of in vivo development. In cumulus cells, changes in reactive oxygen species detoxification and serine biosynthesis were observed, adding to the extensive knowledge around metabolic activity in cumulus cells as a critical facet of oocyte quality. Combined, this data suggests that the necessary processes of protein storage and degradation in oocytes and metabolism in cumulus cells constitute important components of oocyte quality. These processes appear suboptimal in current IVM systems, providing a future research direction to optimise IVM protocols with consideration to these protein pathways. Study funding/competing interestsThis study was funded by a National Health and Medical Research Council Investigator Fellowship (APP1023210) awarded to R.B.G. and by a gift from Open Philanthropy. The following competing interests are declared: R.B.G.is a consultant to Dioseve Inc.. L.E.W is a co-founder, shareholder, director and advisor of Jumpstart Fertility Inc.. L.E.W. is also an advisor and shareholder in EdenRoc Sciences, the parent company of Metro Biotech NSW and Metro Biotech, and in Life Biosciences LLC and its daughter companies. His UNSW Industry Scientia position is partly funded by Proto Axiom. All other authors have no competing interests to disclose.

cell biology↗

bioRxiv2026

A hypothalamo-septo-hippocampal circuit for REM sleep-dependent consolidation of social memory

REM sleep constitutes a critical window for memory consolidation, yet the brain circuits orchestrating this process remain incompletely defined. Here, we identify a lateral supramammillary nucleus (SuM)-medial septum (MS) projection as a REM sleep-specialized pathway essential for hippocampal memory consolidation. Fiber photometry and optrode recordings revealed that lateral SuM-MS projecting neurons were selectively active during REM sleep. REM-specific optogenetic silencing of this projection impaired consolidation of both social and contextual fear memories. Crucially, silencing of its downstream target, the MS-CA2 pathway, during REM sleep selectively disrupted social memory while sparing contextual fear memory. This functional dissection establishes a hypothalamo-septo-hippocampal circuit (lateral SuM-MS-CA2) dedicated to social memory processing, in parallel to the recently-described direct SuM-CA2 pathway. These results also position the SuM as a REM sleep-hub that routes information via parallel septal pathways to consolidate distinct memory modalities.

neuroscience↗

bioRxiv2025

Efficient Identification of Short Tandem Repeats via Context-Aware Motif Discovery and Ultra-Fast Sequence Alignment

Tandem repeats (TRs) are highly polymorphic genomic elements, associated with diverse molecular traits and implicated in numerous human diseases. However, large-scale analysis of TRs has been limited by computational challenges, including motif recognition, detection in complex regions, and excessive computational cost. Here we present FastSTR, a computationally efficient tool for precise detection and characterization of TRs. FastSTR integrates a context-aware N-gram motif model with a segmented global alignment algorithm to enable accurate motif identification and boundary definition, even for repeat units up to 8 bp. Across 13 species, FastSTR achieved >90% recall and 99% precision, running several times faster than existing methods white outperforming them in both sensitivity and accuracy. Applied to the human genome, FastSTR uncovered previously unannotated HSATII elements, resolved population-specific TR demonstrate, and identified recurrent STR alterations in lung cancer. These results demonstrate FastSTR as a versatile framework for TR annotation and discovery, advancing studies of genome evolution, genetic diversity, and disease.

bioinformatics↗

bioRxiv2025

CircRNA-based, non-integrated, in vivo panCAR-mediated B cell immune resetting in mouse models and non-human primates

B cell immune resetting, defined as the comprehensive depletion and subsequent reconstitution of B cells, represents a pivotal therapeutic approach for B cell-associated disorders. Recently, chimeric antigen receptor T cell (CAR-T) therapy has demonstrated substantial clinical efficacy in treating B cell malignancies and autoimmune diseases. However, this approach remains constrained by its reliance on lymphodepleting conditioning regimens, high invasiveness, high costs, and the risk of severe cytokine release syndrome (CRS). Antibody-based therapies, including monoclonal antibodies and T cell-engager (TCE) bispecific antibodies, suffer from suboptimal tissue penetration, resulting in inadequate B cell depletion in lymphoid and peripheral tissues, and subsequent rapid resurgence of pathogenic B cells. Here, we engineered highly stable circular RNAs (circRNAs) encoding anti-CD19 CAR (circRNA-CAR), which demonstrated prolonged and enhanced CAR protein expression compared to conventional linear mRNAs. By employing immune cell-tropic lipid nanoparticles (LNPs) for in vivo delivery, circRNA-CAR facilitated the generation of panCAR immune cells (including CAR-T, CAR-NK, and CAR-macrophages) in both murine models and cynomolgus monkeys. Compared to rituximab or teclistamab, in vivo panCAR induced more extensive B cell depletion across multiple tissue compartments in mice. Moreover, in vivo panCAR mediated robust B cell depletion, eliminated autoreactive antibodies, and restored renal function by alleviating proteinuria in a systemic lupus erythematosus (SLE) murine model. It also effectively reduced IgE antibody levels, decreased eosinophil activity, and improved lung pathology in an asthma murine model. Notably, in vivo panCAR-mediated B cell depletion significantly attenuated aging-associated phenotypes in 17- month-old aging mice, remarkably outperforming both rituximab and teclistamab. In cynomolgus monkeys, in vivo panCAR achieved sustained B cell depletion lasting approximately 40 to 50 days, followed by reconstitution of a naive B cell repertoire. Collectively, circRNA-based in vivo panCAR enables complete B cell immune resetting, offering a versatile and promising therapeutic platform for a broad spectrum of B cell- and antibody-mediated disorders.

immunology↗

bioRxiv2025

Allostery is a widespread cause of loss-of-function variant pathogenicity

Allosteric communication between non-contacting sites in proteins plays a fundamental role in biological regulation and drug action. While allosteric gain-of-function variants are known drivers of oncogene activation, the broader importance of allostery in genetic disease and protein evolution is less clear. Here, we integrate large-scale experimental measurements and neural network models to provide evidence that allostery is a widespread cause of loss-of-function variant pathogenicity in human genetic diseases. In addition, our analyses reveal a conserved distance-dependent decay of allosteric mutational effects outside of protein active sites. As an important mechanism of pathogenicity, allostery needs to be better mapped, understood, and predicted across the human proteome.

genomics↗

bioRxiv2025

KDM5A enhances MHC-I antigen presentation and potentiates antitumor immunity unleashed by immune checkpoint blockade

While some studies have implicated the histone 3 lysine 4 demethylase KDM5A in tumorigenesis and promotion of metastasis, KDM5A has also been shown to boost immunotherapy responses in melanoma. The distinct functional role of KDM5A in the context of immunotherapy and the underlying mechanisms remain largely unknown. Here, we report that higher KDM5A expression strongly correlates with higher MHC-I expression, CD8+ T cell infiltration, and prolonged survival in colorectal cancer and gastric cancer patients receiving anti- PD-1 therapy. KDM5A promotes MHC-I-mediated antigen presentation and CD8+ T cell-mediated tumor killing across multiple murine cancer models. Mechanistically, KDM5A upregulates MHC-I expression through the SOCS1/IFN-{gamma}/STAT1 signaling and improves tumor cell-intrinsic antigen presentation capacity by inhibiting lysosomal proteases in a demethylase-dependent manner. Moreover, KDM5A directly represses lysosomal cathepsins in dendritic cells and promotes the cross-priming activity. Through a high-throughput chemical compound library-based screen, we identified Clomiphene, an FDA-approved small molecule, which significantly elevates KDM5A expression, and enhances antitumor immunity during anti-PD-1 immunotherapy in mouse models of melanoma and colon cancer. Our findings that KDM5A functions in MHC-I-mediated immune activation during anti-PD-1 therapy present an opportunity for developing KDM5A-enhancing therapies to increase tumor immunogenicity, sensitize solid tumors to immunotherapy and exclude immune evasion.

immunology↗

bioRxiv2025

Probing the CaV1.2 interactome in heart failure identifies a positive modulator of inotropy and lusitropy

BackgroundMaladaptive changes in the function, expression and localization of proteins involved in calcium-handling worsen the impaired contractility of systolic heart failure (HF). Standard proteomics techniques require cell lysis and so are unable to characterize changes specific to the critical sub-cellular domain bounded by the T-tubule and the sarcoplasmic reticulum, known as the cardiac dyad. Traditional approaches are also less likely to capture low-affinity protein-protein interactions on lipid membranes. To improve our understanding of heart failure pathophysiology, we applied proximity proteomics to the cardiac dyad of mice with ischemic cardiomyopathy. MethodsUsing two lines of transgenic mice expressing fusion proteins of the engineered ascorbate peroxidase APEX2 with subunits of the cardiac voltage gated calcium channel, CaV1.2, we labeled the dyad proteome of live, intact myocytes from healthy mice (N=6) and mice with coronary artery ligation HF (N=5) by peroxidase-catalyzed biotinylation. Quantitative mass spectrometry with isobaric tandem mass tags (TMT) was used to assess alterations in the local dyad proteome in myocytes from mice with chronic, remodeled HF. We subsequently generated a mouse with inducible cardiac overexpression of Galectin-1 to examine the effects of this protein on cardiac function and more specifically on the calcium-handling properties of mature myocytes, using cellular electrophysiology, calcium imaging, and echocardiography. ResultsFrom mice with HF, we found significant enrichment of 43 proteins defined by their abundance and proximity to transgenic CaV1.2 1C channels, and a significant reduction in 22 proteins, out a of a total of 2326 proteins quantified. We also significantly enriched 286 proteins, and saw a reduction in 13 proteins, defined by proximity and abundance to CaV1.2 {beta}2B-subunits, out of a total 2236 proteins identified. Pathway analysis revealed HF is associated with increased abundance of components of the 26s proteasome and microtubules in the dyad, as well as the dimerizing, carbohydrate binding protein Galectin-1. Cardiac specific overexpression of Galectin-1 in healthy mice increases activation of CaV1.2 and the Ryanodine receptor and accelerates myocyte relaxation through phosphorylation of Phospholamban. ConclusionsUsing proximity proteomics to examine the effects of HF in vivo, we find increased localization of Galectin-1 to the cardiac dyad, and that overexpression of Galectin-1 accelerates calcium kinetics in the heart.

cell biology↗

bioRxiv2025

APOE genotype confers context dependent neurovascular vulnerability in immune-vascularized human forebrain organoids

The APOE gene is a major genetic determinant of neurovascular and immune function, yet the mechanisms by which its isoforms modulate brain vulnerability to pathogenic stress remain incompletely understood. Here, we employ isogenic human iPSC-derived immune-vascularized--Forebrain Organoid-based Multicellular Assembled Cerebral Organoids (FORMA-COs)--to dissect isoform-specific responses to a clinically relevant viral challenge. We find that APOE2/2 and APOE4/4 FORMA-COs exhibit heightened viral RNA burden and distinct neuroinflammatory profiles compared to APOE3/3. Specifically, APOE4/4 promotes IL-1 and VEGFA induction, whereas APOE2/2 leads to elevated TNF-{beta} and VEGFA protein accumulation, indicating divergent pathways of injury. Integrated transcriptomic analyses, combined with known and predicted APOE protein-protein interaction networks, reveal genotype-dependent enrichment of cytokine signaling, angiogenic remodeling, and immune dysregulation. In vivo validation using humanized mouse models corroborates APOE genotype- specific vascular remodeling, microglial activation, and oligodendrocyte perturbation. These findings demonstrate that APOE genotype confers context-specific susceptibility to neuroimmune and vascular injury, providing insight into genetic risk mechanisms underlying infection-related and neurodegenerative brain disorders.

neuroscience↗

bioRxiv2025

Enhance Memory CD8+ T Cell Formation via Downregulation of Hematopoietic Progenitor Kinase 1 and Sustained Mitochondrial Fitness

Memory CD8+ T cells are pivotal for long-term anti-tumor immunity due to their longevity and rapid response upon encountering tumor cells. Hematopoietic progenitor kinase 1 (HPK1), a member of the Ste20 family of kinases, restricts proximal T cell receptor (TCR) signaling and plays critical roles in T cell priming and activation. However, the impact of HPK1 on CD8+ T cell differentiation is not well understood. Here, we demonstrate that chimeric antigen receptor (CAR) T cells derived from patients lacking HPK1 exhibit a stronger memory phenotype. HPK1 deletion promotes the formation of precursor and central memory CD8+ T cells, resulting in superior and long-lasting antitumor activity in murine cancer models compared to WT mice. Additionally, HPK1 knockout induces metabolic reprogramming by enhancing oxidative phosphorylation (OXPHOS) and mitochondrial respiration complex activity. Mechanistically, HPK1 deletion downregulates mTOR signaling by reducing the phosphorylation of mTOR and S6. Increased mitophagy activity is observed in HPK1-depleted cells, with upregulation of key mitophagy regulators, including Pink1 and Bnip3l, maintaining mitochondrial fitness. Our study reveals that HPK1 regulates CD8+ T cell metabolic reprogramming, guiding cell differentiation and antitumor responses.

immunology↗

bioRxiv2025

SynBioGPT: A Retrieval-Augmented Large Language Model Platform for AI-Guided Microbial Strain Development

Synthetic biology seeks to engineer microbial cell factories for sustainable bioproduction, yet the optimization of these systems is impeded by the complexity of metabolic engineering and the protracted timelines of iterative design-build-test-learn (DBTL) cycles. Traditional computational approaches, such as constraint-based modeling, provide valuable insights but demand extensive manual curation. Large language models (LLMs) hold promises for automating knowledge extraction and strain design, yet conventional models like GPT-4 suffer from outdated corpora and hallucination errors in domain-specific tasks. SynBioGPT v1.0, a Retrieval-Augmented Generation (RAG)-enhanced LLM, enhanced knowledge retrieval using vector search but often retrieved semantically similar yet contextually irrelevant documents. Here, we introduce SynBioGPT v2.0 (https://synbiogpt.biodesign.ac.cn), which mitigates these limitations by decomposing queries into sub-questions and employing keyword-based searches. Tested on a 100-question synthetic biology benchmark, SynBioGPT v2.0 achieved 98% accuracy with the Claude-3.7-sonnet backend, a 10% improvement over v1.0s 88% with Llama3-8B-Instruct. This advance highlight the efficacy of query decomposition and precise retrieval in enhancing LLM utility for synthetic biology.

synthetic biology↗

bioRxiv2025

A Myosin Nanomotor Essential for Stereocilia Maintenance Expands the Etiology of Hereditary Hearing Loss DFNB3

Cochlear hair cells transduce sound using stereocilia, and disruption to these delicate mechanosensors is a significant cause of hearing loss. Stereocilia architecture is dependent upon the nanomotor myosin 15. A short isoform (MYO15A-2) drives stereocilia development by delivering an elongation-promoting complex (EC) to stereocilia tips, and an alternatively spliced long isoform (MYO15A-1) tunes postnatal size in shorter stereocilia, which possess mechanosensitive ion channels. Disruption of these functions causes two distinct stereocilia pathologies, which underly human autosomal recessive non-syndromic hearing loss DFNB3. Here, we characterize a new isoform, MYO15A-3, that increases expression in postnatal hair cells as the developmental MYO15A-2 isoform wanes reciprocally. We show the critical EC complex is initially delivered by MYO15A-2, followed by a postnatal handover to MYO15A-3, which continues to deliver the EC. In a Myo15a-3 mutant mouse, stereocilia develop normally with correct EC targeting, but lack the EC postnatally and do not maintain their adult architecture, leading to progressive hearing loss. We conclude MYO15A-3 delivers the EC in postnatal hair cells and that the EC and MYO15A-3 are both required to maintain stereocilia integrity. Our results add to the spectrum of stereocilia pathology underlying DFNB3 hearing loss and reveal new molecular mechanisms necessary for resilient hearing during adult life.

neuroscience↗