Biology subjects

Xu, Q.

Publications and source records attributed to Xu, Q..

At least 19 recordsLinked to original sources

bioRxiv2026

Scalable hardware and software for high-density, multi-regional electrophysiology in non-human primates

Understanding how distributed neural circuits generate complex behaviors requires long-duration recordings from populations of individual neurons across multiple brain regions. High-density Neuropixels probes have revolutionized rodent systems neuroscience. Extending these capabilities to non-human primates (NHPs) for chronic, multi-regional recordings requires compact, implantable microdrive hardware and reliable automated spike-sorting software. NHPs provide access to complex and flexible behaviors that are difficult to study in other model systems. Extending the Neuropixels revolution to NHPs therefore depends on integrating hardware and software for long-duration, multi-regional recordings. Here, we present a scalable hardware-software platform co-designed for high-density neural data acquisition and reliable single-unit identification in NHPs. The Multi-regional Implantable NeuroDrive (MIND) is a lightweight, implantable microdrive that provides stable recordings from multiple independently positioned Neuropixels probes and is designed to support chronic, ambulatory, head-free recordings. We paired MIND with UnitAssay, an automated post-sorting curation framework that identifies and curates characteristic spike-sorting errors with minimal computational cost. Together, MIND and UnitAssay provide an integrated platform for implanted, multi-regional recordings in NHPs performing complex and naturalistic behaviors. We tested MIND and UnitAssay across parietal, prefrontal, and motor cortices in rhesus macaques performing reaching and eye-movement tasks. MIND enabled stable multi-probe recordings across cortical regions and durations. UnitAssay enabled automated curation of large-scale spike-sorting output and improved reliable single-unit identification relative to Kilosort 4 labels and standard SpikeInterface quality control. Together, MIND and UnitAssay provide a scalable solution for high-density, multi-regional, long-duration electrophysiology in NHPs.

neuroscience↗

bioRxiv2026

Liposomal DAGLβ Inhibitor Shows Substantial Antinociceptive Potency Mediated by AMPK in a Neuropathic Pain Mouse Models

Diacylglycerol lipase-beta (DAGL{beta}), a biosynthetic enzyme for the endocannabinoid 2-arachidonoylglycerol (2-AG), is highly expressed in macrophages and serves as a potential contributor to inflammatory and hypernociceptive responses. Moreover, the DAGL{beta} inhibitor KT109 produces antinociceptive effects in various rodent pain models, and its liposome formulation dramatically increases its antinociceptive potency in a lipopolysaccharide mouse model of inflammatory pain compared to free KT109. Here, we further optimized the liposomal KT109 formulation through adjustment of lipid composition and improved processing, achieving 100-fold higher drug encapsulation efficiency than the previous formulation, uniform size distribution, and excellent storage stability. Further, the second generation liposomal KT109 showed remarkable potency (ED50 [95% CL] = 0.226 [0.113-0.454] mg/kg) in a mouse model of paclitaxel-induced neuropathic pain, compared to free KT109 (ED50 [95% CL] = 31 [21-46] mg/kg). Liposomal KT109 (0.4 mg/kg) fully reversed paclitaxel-induced mechanical hypersensitivity for up to 24 hours, and its antinociceptive effects were sustained following a week of daily injections, with no detectable tolerance. In addition, the adenosine monophosphate-activated protein kinase (AMPK) inhibitor, dorsomorphin, prevented the antinociceptive effects of KT109, which is consistent with previous work demonstrating AMPK activation mediates the antinociceptive phenotype of DAGL{beta} knockout mice.

pharmacology and toxicology↗

bioRxiv2026

A genomic foundation model for interpreting human genetic diversity

Interpreting human genomes requires models that recognize shared biological functions while accounting for variation underlying traits, disease susceptibility and adaptation. Here we present hEVO, a genomic foundation model trained on 4.784 billion distinct haplotype sequences reconstructed from whole genome sequencing (WGS) data for 63,809 individuals selected from 550,237 samples across eight cohorts. Pretraining integrates shared sequence features and naturally occurring allelic differences without clinical or functional labels. Against representatives of the Evo 2, NT, GPN and other genomic model families, hEVO ranked first in 18 of 19 primary comparisons spanning functional annotation, variant interpretation and human evolution. Its pretrained allele scores generalized across all six complementary variant benchmarks without task-specific training, achieving the highest performance across clinical pathogenicity, fine-mapped causal variants and experimentally measured regulatory effects. Retrospective ClinVar reclassification further demonstrated improved identification of pathogenic classifications later downgraded, supporting reassessment of earlier clinical interpretations. Predictive strengths extended to ancestry inference, human accelerated sequences and preference for reported selected alleles. This breadth was achieved with substantially fewer parameters than billion-parameter comparators and low inference overhead in the evaluated workloads. These findings establish human haplotype diversity as a source of transferable biological information for interpreting both the disruption of genome function and the variation retained during human evolution.

genomics↗

bioRxiv2026

Central platelet-rich plasma administration promotes weight loss in diet-induced obese mice

Aims: Platelet-rich plasma (PRP) contains diverse bioactive factors, but its potential role in central regulation of energy balance remains unexplored. This study investigated whether central PRP administration alters metabolic homeostasis in diet-induced obese (DIO) mice and examined associated hypothalamic responses under obese and inflammatory conditions. Materials and Methods: PRP was administered intracerebroventricularly in mouse models of DIO and lipopolysaccharide (LPS)-induced systemic inflammation. Body weight, food intake, energy metabolism, and adiposity were assessed alongside hypothalamic microglial morphology and transcriptomic profiling. Results: A single intracerebroventricular PRP injection reduced body weight in DIO mice, with the effect persisting throughout the 7-day observation period. PRP also decreased cumulative food intake, relative gonadal white adipose tissue weight, and fasting glucose, accompanied by increased energy expenditure, oxygen consumption, fat utilization, and physical activity. In both DIO and LPS-treated mice, PRP reduced IBA1-positive area per microglial cell in the arcuate nucleus without altering microglial number. Hypothalamic RNA sequencing revealed changes in immune and metabolic signaling, extracellular matrix organization, and growth-related pathways, suggesting a complex transcriptional response rather than uniform suppression of inflammation. Conclusions: A single central PRP administration induces sustained weight loss and favorable metabolic changes in DIO mice, accompanied by region-specific hypothalamic microglial and transcriptional responses. These findings identify central PRP as a potential modulator of energy balance and provide a basis for investigating its active components and underlying mechanisms.

neuroscience↗

bioRxiv2026

FMR1 Perturbations Disrupt Sensory Corticostriatal Circuits and Mitochondrial Energetics in Fragile X Syndrome

Fragile X syndrome (FXS) features sensory, social, and cognitive dysfunction, but how fragile X messenger ribonucleoprotein 1 (FMR1) deficiency disrupts sensory corticostriatal circuitry and whether distinct FMR1 perturbations converge on shared cellular mechanisms remain incompletely defined. We integrated diffusion magnetic resonance imaging (MRI) in individuals with FXS and Fmr1-knockout (KO) mice with projection-defined calcium recording, bidirectional optogenetic manipulation, primary somatosensory cortex (S1)-targeted expression of three patient-associated FMR1 missense variants, regional transcriptomics, and optical manipulation of mitochondrial energetic state. Individuals with FXS exhibited disrupted sensory corticostriatal structural connectivity, with mouse analyses further localizing abnormalities to S1-striatal pathways. Fmr1 deficiency impaired recruitment of the S1-dorsomedial striatum (DMS) pathway during social interaction, and projection-specific manipulation demonstrated that S1-DMS dysfunction contributes to social and cognitive deficits. Three patient-associated FMR1 variants, I234T, E329G, and R586Q, converged on social behavioral deficits, which were improved by S1-DMS activation. Transcriptomic analyses revealed convergent oxidative phosphorylation (OXPHOS)-related mitochondrial remodeling in S1 and DMS across Fmr1 deficiency and variant models. Systemic treatment with the mitochondria-targeted antioxidant MitoQ attenuated novel-location-recognition, social-interaction, and social-novelty deficits in Fmr1-KO mice. Moreover, mt-EcGAPR-based Mitochondrial OptoEnergetic Activation (Mito-OptoEA) enhanced mitochondrial energetic state in S1 and improved novel-location recognition and sociability in both Fmr1-KO and I234T models. Together, these findings define a sensory corticostriatal circuit-metabolic framework for FXS in which S1-DMS circuit dysfunction and convergent mitochondrial energetic abnormalities contribute to FMR1-related social and cognitive phenotypes.

neuroscience↗

bioRxiv2026

Rank-Preserving Alignment Enables Cross-platform Learning and Phenotyping for Single-Cell and Spatial Proteomics

Decades of antibody-based protein profiling have generated valuable cohorts across evolving cytometry, sequencing and spatial imaging platforms. Differences in marker coverage, signal scale and antibody performance hinder joint analysis and reuse of cohorts with long-term clinical follow-up. Widely used RNA-based integration methods prioritize shared embeddings rather than directly comparable protein measurements. To fill in the gap, we developed RAMP (Rank-preserving Alignment of Multi-platform Proteomics), an unsupervised framework that aligns shared proteins without cell-type annotations. It combines sample-specific anchoring and cross-platform cell matching with bounded monotone transformations. The corrected values retain each protein marker's expression order across cells within a sample. Across seven integration tasks, RAMP achieved the strongest overall balance of batch correction and cell-type preservation. RAMP also improved marker-threshold transfer while preventing expression-order reversals observed with some comparison methods. These reversals made cell types with originally high marker expression appear lower-expressing than other populations. Such overcorrection can mislead cell identification and protein comparisons, even when the integrated embedding shows improved dataset mixing. By preserving each marker's expression order within a sample, RAMP protects the relationships needed to interpret normalized protein measurements. To investigate whether better harmonized inputs improve downstream learning, we used linear, nearest-neighbor and small neural-network models as a controlled, small-scale testbed. This approach tests an input quality question relevant to proteomic foundation models without undertaking large-scale pretraining. Under matched supervision, RAMP improved cross-platform annotation across all three model families and improved missing-marker prediction over unaligned inputs. These gains demonstrate the value of harmonized inputs for predictive learning and motivate evaluating RAMP as preprocessing for proteomic foundation models. In bone marrow, we used high-quality single-cell protein measurements to impute the failed CODEX CD19 channel, recovering B-cell spatial distributions. The completed panel improved B-cell separation from natural killer cells in the joint embedding. In melanoma, a proliferating CD8 T-cell state near tumor cells was associated with longer survival. Transfer such a spatial information to CITE-seq connected tumor proliferation and infiltration state to an adhesion and activation program involving PD-1, LFA-1 and CD2. In lung cancer, we used single-cell CyTOF measurements to predict unmeasured HLA-ABC states in vascular cancer-associated fibroblasts profiled by spatial imaging mass cytometry (IMC). Higher predicted scores were associated with lower immune-cell fractions and worse disease-free survival in the IMC cohort. In head and neck cancer, transferring a fibroblast/macrophage spatial niche score to CITE-seq identified a CD39/TIGIT-associated CD8 T-cell phenotype. Protein and RNA profiles supported checkpoint-associated features, and transfer back to CODEX showed correspondence with measured LAG3. RAMP enables existing single-cell and spatial cohorts to support new discoveries by connecting molecular programs, tissue organization and clinical outcomes across platforms.

bioinformatics↗

bioRxiv2026

Systemic tumor-cell reprogramming converts solid tumors into in situ biofactories for immunotherapy

A central challenge in oncology is delivering potent drugs to tumors without harming healthy tissues. We therefore asked whether tumor cells themselves could be genetically programmed to produce therapeutic molecules directly within the tumor. We present LENTRA (LENtiviral Tumor-Reprogramming Activator), an antigen-targeted lentiviral platform that enables selective transduction and genetic modification of tumor cells in vivo. Following intravenous administration, LENTRA achieves tumor restricted expression of immune checkpoint inhibitors, cytokines, or therapeutic enzymes in multiple murine solid tumor models. Local production of anti CTLA 4 or IL 12 triggered potent antitumor responses while avoiding systemic toxicities. Tumor restricted secretion of sialidase enhanced dendritic cell and T cell infiltration, and reprogrammed tumor associated macrophages toward an immunostimulatory phenotype, resulting in tumor regression and durable immune memory, including in tumors with heterogeneous antigen expression. In patient derived tumor explants, LENTRA mediated efficient transduction, immune remodeling, and reduced tumor viability. By engineering tumors to produce therapeutic proteins, LENTRA converts resistant tumors into autonomous therapeutic sources and active immune hubs, achieving local TME reprogramming and systemic antitumor immunity with an improved safety profile.

cancer biology↗

bioRxiv2026

FFPE-CUTAC: A Single Assay, Multiple Layers

Background: Archival biobanks of formalin-fixed paraffin-embedded (FFPE) specimens represent a vast and underused resource for retrospective molecular studies linked to clinical follow-up. However, fixation-induced chemical modification and nucleic acid fragmentation limit scalable genomic and transcriptomic profiling. FFPE-CUTAC (Cleavage Under Targeted Accessible Chromatin) is an RNA polymerase II-targeted DNA assay specifically designed to profile regulatory activity in FFPE tissue. Here, we assess its archival robustness, information content, compatibility with RNA-seq, and ability to recover copy-number alterations. Results: In meningioma and breast cancer cohorts, we benchmarked FFPE-CUTAC against matched fresh-frozen RNA-seq, FFPE RNA-seq and FFPE whole-genome sequencing. For FFPE specimens spanning up to 25 years, FFPE-CUTAC showed no systematic collection-year-associated decline in library yield or signal-quality metrics. In contrast, FFPE RNA-seq is more affected by the FFPE tissue degradation and specimen age on read composition, gene-body coverage, and gene detection. By measuring RNA polymerase II occupancy on chromatin rather than mature RNA abundance, FFPE-CUTAC is less constrained by transcript half-life, polyadenylation or transcript-capture design. FFPE-CUTAC extends beyond gene regions to non-coding regulatory elements, while the shared gene-level signals supported its integration with existing RNA-seq cohorts for large-scale, long-term clinical association study. FFPE-CUTAC libraries also retain DNA dosage information and can be used to recover chromosome-arm gain, loss and intact states with 96% concordance to matched whole-genome sequencing. Conclusions: By converting routinely preserved pathology sections into integrated regulatory and copy-number profiles, FFPE-CUTAC provides a practical foundation for constructing clinically annotated disease maps to support molecular stratification, prognostic modeling and treatment-association studies.

cancer biology↗

bioRxiv2026

An Oxidative Stress-Associated Seven-Gene Prognostic Signature in Lung Adenocarcinoma: Integrative Transcriptomic Analysis Across Public Cohorts

Lung adenocarcinoma is molecularly heterogeneous, and oxidative-stress programs can support either tumor restraint or tumor adaptation depending on cellular context. This study integrated public lung adenocarcinoma transcriptomic cohorts to identify oxidative-stress-associated expression features and evaluate their prognostic relevance. Expression profiles from The Cancer Genome Atlas, Genotype-Tissue Expression project, and GEO series GSE31210, GSE40791, and GSE30219 were analyzed. Differential expression, weighted gene co-expression network analysis, functional enrichment, univariable Cox regression, and least absolute shrinkage and selection operator Cox modeling were combined to derive a risk signature. Immune-cell enrichment, gene set enrichment analysis, gene set variation analysis, and pan-cancer analyses were used for biological characterization. A total of 1,305 genes differed between tumor and control samples, including 498 upregulated and 807 downregulated genes. Intersection of differentially expressed genes, the oxidative-stress-associated co-expression module, and the oxidative-stress gene set yielded 44 genes enriched in responses to reactive oxygen species and hydrogen peroxide, antioxidant and peroxidase activities, focal adhesion, Rap1 signaling, and PI3K-Akt signaling. A seven-gene signature comprising FBLN5, HBB, FYN, HGF, TFAP2A, PLIN5, and F2RL1 stratified the 523-sample training cohort and the 207-sample internal validation cohort into groups with different overall survival. Time-dependent areas under the receiver operating characteristic curve at 1, 3, and 5 years were 0.677, 0.622, and 0.649 in training and 0.613, 0.691, and 0.706 in internal validation. In the 85-case GSE30219 external cohort, corresponding values were 0.588, 0.661, and 0.631; survival separation followed the expected direction but did not reach statistical significance (log-rank P = 0.100). Seventeen immune-cell signatures differed between risk groups, while high-risk tumors were enriched for cell-cycle, DNA-replication, mismatch-repair, glycolytic, E2F, G2M-checkpoint, MYC-target, and mTORC1-related programs. The signature therefore captures reproducible oxidative-stress-associated transcriptional variation with moderate prognostic discrimination. Its clinical utility requires prospective evaluation, complete clinical adjustment, and experimental validation.

cancer biology↗

bioRxiv2026

BioSecBench-Function: A Verifiable Benchmark for Reasoning about Biological Function from Experimental Data

Inferring biological function from experimental data is central to understanding emerging pathogens and developing effective countermeasures, yet interpreting these data remains slow and expert-intensive. AI agents could help accelerate this process by reasoning across sequence, structural, and biophysical evidence. We present BioSecBench-Function, a verifiable benchmark for recovering biosecurity-relevant function from real biological data. The benchmark comprises 111 evaluations built from published datasets and graded deterministically against ground truth. We organize evaluations along two dimensions: threat axis (spanning seven biosecurity-relevant question types) and biological question (indicating whether the solution depends primarily on sequence, structure, or biophysical assay data). Across 7,326 runs from twenty-two model-harness configurations, Opus 5 under Claude Code led on endpoint pass rate at 50.3%, and Grok 4.6 under Grok Build led on overall pass rate at 44.1% when refusals counted as failures. Performance varied substantially across both model-harness configurations and task categories. Refusal rates differed sharply by provider, and cost was a poor predictor of accuracy: several configurations exceeded 40% pass rate at low cost. BioSecBench-Function provides a standard for measuring whether agents can be trusted to interpret what a new pathogen or variant does when the next outbreak arrives.

biophysics↗

bioRxiv2026

An inflammation-associated five-gene expression signature stratifies survival and immune states in lung adenocarcinoma: an integrative public-cohort analysis

Background: Inflammation and the tumor immune microenvironment contribute to lung adenocarcinoma (LUAD) progression, but the relationship among inflammation-linked transcriptional heterogeneity, patient survival, and immune-state variation remains incompletely defined. Objective: We aimed to identify inflammation-associated LUAD subtypes, derive a parsimonious survival-stratification signature, and characterize its immune and pathway context across public transcriptomic cohorts. Methods: Expression profiles and clinical data were obtained from TCGA-LUAD, GTEx normal lung, and GEO datasets GSE11969, GSE30219, GSE31210, and GSE40791. A curated set of 596 inflammation-related genes was used for consensus clustering. Differential-expression analysis, functional enrichment, univariate Cox regression, and LASSO-Cox modeling were integrated to construct a gene-expression risk score. The prognostic dataset comprised 730 cases and was randomly divided into training (n=502) and internal-validation (n=228) sets; 85 GSE30219 cases formed an external-validation cohort. Immune-cell enrichment, gene set enrichment analysis (GSEA), gene set variation analysis (GSVA), and pan-cancer analyses were used for biological contextualization. Results: The LUAD-versus-control comparison identified 1,305 differentially expressed genes, including 498 upregulated and 807 downregulated genes. Consensus clustering resolved two inflammation-associated subtypes and 67 subtype-associated genes, of which 64 were higher and 3 were lower in Cluster 1 relative to Cluster 2. Thirty-three genes overlapped between the tumor-control and subtype contrasts. LASSO-Cox regression selected CHRDL1, FDCSP, CXCL13, CYP4B1, and S100P. The 1-, 3-, and 5-year areas under the time-dependent receiver operating characteristic curve were 0.6625, 0.6581, and 0.6658 in the training set; 0.7422, 0.6537, and 0.6761 in internal validation; and 0.6560, 0.6387, and 0.6753 in external validation. Risk groups differed across multiple T-cell, B-cell, natural-killer-cell, myeloid, dendritic-cell, macrophage, and granulocyte signatures. Positive GSEA signals included cell cycle (normalized enrichment score [NES]=2.67; adjusted P=1.42 x 10-), DNA replication (NES=2.52; adjusted P=2.52 x 10-), and mismatch repair (NES=2.20; adjusted P=1.77 x 10-). Conclusions: The five-gene expression score separated LUAD survival groups and captured coordinated proliferative and immune transcriptional states. Its moderate discrimination supports further biological and clinical validation rather than immediate clinical application.

bioinformatics↗

bioRxiv2026

Protein Phosphatase 2A Activation Attenuates Acute Myocardial Injury in Takotsubo Syndrome by Modulating Ferroptosis and Mitochondrial Injury in Cardiomyocytes

BackgroundTakotsubo syndrome (TTS) is an acute stress-induced cardiomyopathy characterized by transient left ventricular dysfunction. Despite its reversible nature, TTS is associated with substantial morbidity and mortality in the acute phase, and no specific treatments are available. The precise molecular mechanisms that connect catecholamine stress to reversible myocardial injury are not fully understood. PP2A, a holoenzyme with serine/threonine phosphatase activity, plays a vital role in normal cardiac development, and its dysregulation has been associated with heart disease. Its role in TTS and its potential as a therapeutic target remain completely unknown. MethodsAnalysis of public multi-omics datasets from stress cardiomyopathy (SCM) and experimental models of TTS, along with treatment of cardiomyocytes with human TTS plasma, was used to investigate a potential role for protein phosphatase 2A (PP2A) in stress-induced myocardial injury. To clarify the functional impact of manipulating PP2A activity in TTS, we used a series of disease relevant cell based and in vivo models, leveraging both genetic and pharmacological approaches to modulate PP2A activity in cardiomyocytes and in mice. To gain mechanistic insights into how PP2A influences TTS pathology and downstream signaling pathways, RNA sequencing, stress-responsive iron handling, mitochondrial function, and cardiac phenotypes were thoroughly evaluated in both in vivo and in vitro studies. ResultsPP2A activity was markedly reduced in cardiac tissues from mice with isoprenaline-induced TTS, as well as in isoprenaline-treated cardiomyocytes. Genetic or pharmacological inhibition of PP2A worsened catecholamine-induced cardiac dysfunction and myocardial injury. Most notably, pharmacological activation of PP2A using an orally bioavailable small-molecule activator strongly mitigated myocardial damage and enhanced cardiac function in TTS models. Mechanistically, PP2A inactivation promoted JNK-MAPK signaling and dysregulated stress-responsive iron-handling pathways, leading to ferritinophagy-mediated ferroptosis and mitochondrial dysfunction. Pharmacological JNK inhibition effectively rescued myocardial injury caused by PP2A deficiency in two TTS animal models and in cardiomyocytes. ConclusionsPP2A inactivation is a key molecular event linking catecholamine stress to myocardial injury in TTS. Restoring PP2A activity or inhibiting downstream JNK attenuates ferritinophagy-dependent stress responses and mitochondrial dysfunction, providing a unifying mechanistic framework and highlighting the PP2A-JNK axis as a potential target for short-term intervention during the acute phase of TTS. CLINICAL PERSPECTIVEO_ST_ABSWhat Is New?C_ST_ABS* Protein phosphatase 2A (PP2A) activity is acutely suppressed during the early phase of Takotsubo syndrome (TTS), with preferential involvement of the apical myocardium, closely mirroring the diseases characteristic clinical phenotype. * Downstream of PP2A inactivation, catecholamine stress triggers JNK-MAPK signaling activation and dysregulated stress-responsive iron handling, including ferritinophagy-mediated ferroptosis and mitochondrial dysfunction in cardiomyocytes. * Pharmacological restoration of PP2A activity or downstream inhibition of JNK mitigates acute myocardial injury and preserves cardiac function, thereby establishing the PP2A-JNK axis as a central stress-responsive pathway in TTS. What Are the Clinical Implications?* Identification of PP2A inactivation as a key molecular event highlights a novel opportunity for targeted intervention during the acute phase. * Studies in preclinical animal TTS models provide the first evidence that illuminates the therapeutic potential of a small-molecule PP2A activator as a strategy for acute TTS and may serve as a broad cardioprotective strategy in settings where cardiac dysfunction is implicated. * Targeting downstream JNK signaling, which is activated by PP2A inactivation, emerges as an additional mechanism-based strategy for attenuating acute myocardial injury in TTS.

molecular biology↗

bioRxiv2026

Nutrient flux governs osteogenic fate commitment through the SLC3A1-cystine Axis

Skeletal and mesenchymal cells have limited bone-forming potential due to their rarity, tendency for senescence and/or unstable osteogenic lineage commitment. We previously identified an osteopotent CXCR4 stem cell population with unclear mechanism for its differentiation potential. Here, we found that the cystine transporter SLC3A1 was selectively enriched in CXCR4+ stem cells, uncovering a role for amino acid transport in regulating osteogenic fate commitment. Enforced SLC3A1 expression reprogrammed mesenchymal cells toward a stable osteogenic state while suppressing adipogenic differentiation. Mechanistically, SLC3A1-mediated cystine flux established a glutathione-dependent metabolic program that preserved mitochondrial fitness and restrained stem cell senescence. SLC3A1 also stabilized IGF-1 through suppression of ZMYND8-mediated ubiquitination, uncovering ZMYND8 as a previously unrecognized E3 ligase regulating osteogenic commitment. Cystine supplementation phenocopied the effects of SLC3A1 activation, promoting osteogenic differentiation and skeletal regeneration without genetic manipulation. In vivo, cystine administration accelerated bone regeneration and attenuated ovariectomy-induced bone loss, while analyses of a human osteoporosis cohort, osteoporotic specimens, and single-cell transcriptomic datasets revealed coordinated suppression of the SLC3A1-cystine-IGF-1 pathway in osteoporotic mesenchymal cells. Taken together, these findings establish SLC3A1-mediated cystine transport as a programmable metabolic determinant of osteogenic fate commitment and identify cystine metabolism as a therapeutically targetable axis for skeletal regeneration and osteoporosis.

cell biology↗

bioRxiv2026

VASP Promotes Aortic Valve Calcification by Interacting with FBLIM1 to Activate the Focal Adhesion Pathway

BackgroundCalcific aortic valve disease (CAVD) is the most prevalent valvular heart disease, yet there is no effective pharmacological therapy to halt or reverse its progression. Focal adhesions (FAs) are dynamic structures that connect cells to the extracellular matrix (ECM), serving not only as mechanical anchors but also as critical signalling hubs that regulate cell adhesion, spreading, migration, differentiation, and mechanotransduction. However, their specific role in CAVD pathogenesis remains largely unknown. MethodsTo identify upregulated hub genes, an integrated analysis of proteomics and RNA sequencing was performed on calcified aortic valves. To investigate functional roles in vitro, we utilized genetic knockdown and overexpression of VASP in valvular interstitial cells (VICs), followed by phenotypic evaluations of osteogenic differentiation, calcification, and elastin (ELN) secretion. Downstream mechanisms were explored using RNA-seq in VASP-overexpressing VICs, alongside pharmacological inhibition of the FA pathway. Finally, protein-protein interaction assays were conducted to map and analyze the physical binding between VASP and the structural domains of FBLIM1. ResultsIntegrated omics analysis identified VASP as a significantly upregulated gene in human calcified aortic valve tissues. Functionally, VASP knockdown inhibited, whereas its overexpression promoted, the osteogenic differentiation of VICs. RNA-seq revealed that VASP overexpression activated the FA pathway, and the pharmacological blockade of this pathway successfully suppressed calcification both in vitro and vivo. Mechanistically, we demonstrated a direct physical interaction between VASP and the third LIM zinc-binding domain of FBLIM1, and knockdown of FBLIM1 effectively reversed the pro-calcific effects of VASP. Finally, VASP overexpression was found to promote the secretion of ELN, which subsequently contributed to the calcification process. ConclusionOur study reveals that the interaction between VASP and FBLIM1 drives CAVD progression by activating the FA pathway, which subsequently leads to excessive ELN secretion. These findings reveal a novel mechanistic pathway and may provide a potential therapeutic target for CAVD intervention. What are the Clinical Implications?We have identified the VASP-FBLIM1-FA-ELN axis as a novel molecular pathway involved in CAVD pathogenesis, greatly enriching our understanding of the complex process of aortic valve calcification. This pathway intricately links cytoskeletal dynamics, cell adhesion signalling, and ECM remodelling, providing a new theoretical framework for pathophysiological research in CAVD. In clinical practice, VASP and its downstream pathway components, particularly FBLIM1 and the FA pathway, may serve as potential biomarkers and therapeutic targets for the early diagnosis and treatment of CAVD. For example, the FA pathway inhibitors PF-573228 and Y15 demonstrated significant anti-calcification effects both in vitro and in vivo, suggesting that targeting this pathway may offer a new nonsurgical intervention strategy for patients with CAVD.

cell biology↗

bioRxiv2026

Single-camera, calibration-free gaze estimation using corneal reflections

BackgroundMice make substantial eye movements during head-fixed visual stimulation, and uncorrected gaze shifts corrupt receptive field measurements and confound stimulus-response relationships. Corneal-reflection video oculography in rodents has provided the methodological foundation for calibrated angular gaze tracking since Stahl (2004) but the calibration procedures used by existing methods -- physical camera rotation, motorized stages, behavioral tasks, or precisely co-aligned dual cameras -- have limited their adoption in many mouse neuroscience laboratories. Most studies instead use uncalibrated pupil tracking, deep learning pose estimation that returns pixel coordinates without angular calibration, or learned shifter networks that lack independent validation. New methodWe present an open-source corneal-reflection eye tracking system for head-fixed mice with two methodological contributions. First, a geometric model recovers gaze in calibrated angular units from the pixel displacements of the pupil and corneal reflections, using the known 3D positions of multiple fiducial LEDs as the source of angular scale. The model requires no estimate of Rp, the per-animal eye-geometry parameter that earlier corneal-reflection methods determine through physical calibration. Second, a self-calibration procedure exploits the redundancy of multiple stationary fiducial LEDs: each LED produces an independent gaze estimate from the same geometric model, and a single residual calibration parameter is determined by minimizing the disagreement between per-LED estimates. This replaces the physical camera-rotation calibrations of earlier video oculography (Sakatani and Isa, 2004, 2007; van Alphen et al., 2013; Kretschmer et al., 2017), the motorized stages of Zoccolan et al. (2010), and the precision dual-camera alignment of Payne and Raymond (2017) with a software operation that requires no moving parts, no behavioral task, and no per-animal procedure. The system provides three interactive GUI stages: (1) pupil and LED detection via Difference-of-Gaussians filtering, (2) 3D geometry definition, and (3) gaze angle computation with blink detection, fiducial correction, and manual curation. ResultsValidation against a rotary-encoder-controlled artificial eye demonstrated mean absolute errors below 1{degrees} across all four fiducial LEDs over the {+/-}20{degrees} working range of mouse eye movements, with Pearson correlations exceeding 0.998 between our methods estimation and encoder ground truth. The self-calibration reduced inter-LED disagreement by a factor of 4-6 in mouse recordings. Gaze-corrected stimulus reconstruction applied to Neuropixels recordings from mouse V1 produced qualitatively sharper receptive field estimates with improved signal-to-noise ratios. Comparison with existing methodsOur method is the first multi-LED, single-camera, fully software-calibrated corneal-reflection eye tracker for mice and includes an integrated open-source pipeline for detection, calibration, blink handling, and artifact correction. The multi-LED redundancy doubles as an internal consistency check -- if two LEDs disagree on gaze direction, the calibration is wrong -- providing a guarantee that learned approaches relying on neural-data-derived correction cannot offer. ConclusionsOur method makes calibrated corneal-reflection eye tracking accessible to non-specialist mouse laboratories using consumer-grade hardware ([~] $2,000-2,700 USD), eliminates the per-animal calibration procedures of earlier methods, and is validated by two independent ground truths at both the absolute angular (artificial eye) and functional (V1 receptive fields) levels. HighlightsO_LIOpen-source corneal-reflection eye tracking for head-fixed mice using a single camera and multiple stationary fiducial LEDs. C_LIO_LIGeometric gaze model derives angular scale from LED positions, eliminating per-animal eye-geometry calibration. C_LIO_LISelf-calibration via multi-LED redundancy replaces physical camera rotation, motorized stages, and dual-camera precision alignment. C_LIO_LIValidated to sub-degree accuracy against a rotary-encoder ground truth across the {+/-} 20{degrees} range of mouse eye movements. C_LIO_LIGaze correction produces sharper V1 receptive field estimates in Neuropixels recordings. C_LI

neuroscience↗

bioRxiv2026

Mechano-Initiated PIEZO1-VEGFR2 Interaction Governs CD34+ Cell Differentiation and Repair in Arteriovenous Fistula

BackgroundEndothelial cell (EC) injury induced by disturbed flow drives neointimal hyperplasia in arteriovenous fistulas (AVFs), where CD34+ cell-mediated repair may be involved. PIEZO1 and VEGFR2 are important mechanosensors with critical role in maintaining endothelial function. However, whether PIEZO1 interacts with VEGFR2 during CD34+ cell differentiation to orchestrate the vascular repair remains unknown. MethodsAVF model was established in several mouse strains. Single cell RNA sequencing was performed for human and mouse samples. Cd34-CreERT2; R26-tdTomato; Piezo1flox/flox mice were used to investigate the effect of Piezo1 deletion on endothelial repair in AVFs. CD34-high human umbilical vein ECs (CD34high HUVECs) was sorted and exposed to different flow patterns to determine the role of shear stress in CD34high cell differentiation. Co-immunoprecipitation, proximal ligation assay and complementary approaches were performed to delineate mechanotransduction initiated by PIEZO1-VEGFR2 interaction. ResultsSingle cell RNA sequencing and immunostaining showed abundant CD34high cells in the vessel wall of AVFs in humans and animal models. Exposure of CD34high HUVECs to different flow patterns showed that laminar shear stress downregulated CD34 while upregulating VE-cadherin and claudin-5 expression. In contrast, oscillatory flow produced the opposite effects, indicating impaired endothelial maturation. PIEZO1 knockdown in CD34high HUVECs attenuated shear stress-induced endothelial marker expression. In Cd34 conditional Piezo1 knockout mouse model of AVF, we observed decreased number of CD34-derived cells, more compact cellular arrangement, and attenuated neointimal hyperplasia. Mechanistically, we found PIEZO1 interacts with VEGFR2, thereby mediating the distinct effects of laminar and oscillatory shear stress on AKT-FoxO1 axis, which critically regulates endothelial marker expression. Furthermore, pharmacological activation of AKT signaling in AVF mouse model enhanced CD34+ cell-mediated endothelial repair and attenuated neointimal hyperplasia. ConclusionPIEZO1-VEGFR2 complex-mediated mechanotransduction plays a key role in regulating CD34+ cell-derived endothelial repair in AVFs via AKT-FoxO1 axis. AKT activation enhances endothelial maturation, thereby attenuating neointimal hyperplasia in AVFs. Novelty and SignificanceO_ST_ABSWhat Is Known?C_ST_ABSO_LIIn arteriovenous fistulas (AVFs), abnormal shear stress induces endothelial cell injury, and the resulting neointimal hyperplasia is a major cause of anastomotic stenosis. C_LIO_LICD34 cells actively participate in vascular endothelial repair. C_LIO_LIPIEZO1 is a mechanoreceptor mediating endothelial sensing of hemodynamic shear stress, contributing to the maintenance of atheroprotective endothelial phenotype under laminar shear stress, whereas its activation induces pro-inflammatory effects under disturbed shear stress. C_LI What New Information Does This Article Contribute?O_LICD34 cells participate in repairing endothelial injury induced by abnormal shear stress in AVFs. PIEZO1 knockout in CD34+ cells improve endothelial repair and attenuates neointimal hyperplasia in AVF. C_LIO_LILaminar shear stress induces CD34 downregulation and upregulates VE-cadherin and claudin-5 expression in CD34-high human umbilical vein endothelial cells, whereas oscillatory shear stress upregulates CD34 expression and suppresses VE-cadherin and claudin-5 expression. C_LIO_LIMechano-stimuli lead to PIEZO1-VEGFR2 complex formation regulating CD34 cell-mediated endothelial repair through the downstream AKT-FoxO1 axis. C_LI Abnormal hemodynamic shear stress-induced endothelial injury initiates neointimal hyperplasia in AVFs. The present study identifies PIEZO1 as a key mechanosensor that regulates CD34+ cell-derived endothelial repair in response to distinct blood flow patterns. PIEZO1 promotes CD34+ cell differentiation into mature ECs for endothelial repair under laminar shear stress, whereas it disrupts the differentiation of CD34+ cells into mature endothelium under oscillatory shear stress. Mechanistically, a novel shear stress-sensing complex comprising PIEZO1 and VEGFR2 was identified in regulating flow-induced differentiation of CD34+ cells into mature ECs via the AKT-FoxO1 signaling axis, thereby controlling the expression of endothelial maturation markers VE-Cadherin and Claudin-5. These findings define a novel PIEZO1-VEGFR2 mechanotransduction axis in CD34+ cell-mediated endothelial repair and support AKT pathway activation as a potential therapeutic strategy against neointimal hyperplasia in AVFs.

cell biology↗

bioRxiv2026

Chaperonin recognition of protein dynamics drives drug resistance

The emergence of drug resistance is typically driven by mutations that alter drug-target affinity, yet the role of host cellular machinery regulating these processes remains unclear. Here, we reveal that the chaperonin GroEL/S promotes drug resistance through recognition of protein dynamics. Using directed evolution of E. coli DHFR under antibiotic stress and varying GroEL/S expression, we identify a well-folded resistance variant whose fitness, despite tight inhibitor binding, is critically potentiated by GroEL/S engagement. X-ray crystallography, NMR, molecular dynamics and kinetic modeling reveal that millisecond-timescale flipping of the M20 loop generates steric accessibility and a kinetic window for chaperonin interaction that forcibly displaces tightly bound inhibitors, thereby overriding thermodynamic equilibrium of inhibitor binding to restore the active enzyme pool and preserve metabolic flux. Our findings not only reveal a novel paradigm of "dynamic recognition" where both conformational kinetics and distribution govern chaperonin recognition but also establish chaperonins as "deligandases" that actively modulate in vivo drug binding, suggesting that chaperone surveillance of the cellular proteome extends beyond quality control to govern native protein function. This mechanism defines a previously unrecognized route for the rapid development of drug resistance, with implications for understanding therapeutics of microbial infections and human malignancies.

biophysics↗

bioRxiv2026

UcTCRp: a TCRβ-based framework for quantitative MAIT- and iNKT-associated repertoire-state profiling

MAIT and iNKT cells are conventionally identified using invariant or semi-invariant TCR chains, antigen-loaded tetramers, or transcriptomic phenotypes. These requirements limit their detection in public and clinical immune-repertoire datasets that contain only TCR{beta} sequences. Here we present UcTCRp, a TCR{beta}-only framework for profiling MAIT- and iNKT-associated repertoire states in bulk immune repertoires. UcTCRp integrates V-gene context and CDR3{beta} sequence features using a transformer-based representation pretrained on more than one million TCR{beta} sequences and supervised with curated cross-species MAIT, iNKT and conventional T cell references. The framework defines conserved model-informative TCR{beta} features, uses V-matched negative sampling to reduce germline-segment shortcuts, and generalizes across independent human and mouse datasets. In paired scRNA-seq/scTCR-seq datasets, UcTCRp recovered transcriptome-defined MAIT and iNKT cells and identified additional MAIT-like candidates supported by receptor evidence but missed by expression-only annotation. Bulk calibration against paired single-cell references and synthetic spike-in experiments established operating characteristics for repertoire-level abundance estimation. These results establish unpaired TCR{beta} repertoires as an actionable substrate for reconstructing unconventional T cell-associated immune states, enabling archived repertoire resources to be repurposed for systems-level studies of tissue immunity, disease and therapeutic response.

bioinformatics↗