Biology subjects

Zhao, G.

Publications and source records attributed to Zhao, G..

At least 19 recordsLinked to original sources

bioRxiv2026

Cancer cell adaptations along a proliferation-robustness axis drive TME pro-tumor phenotypes

Tumors are highly complex dynamic ecosystems with causal relationships between cancer cells and their tumor microenvironment (TME). To disentangle these interactions, we performed long-term co-culture experiments with cancer cells evolving alongside continuously replenished fibroblasts or macrophages. This allowed us to distinguish between adaptive and physiological responses in specific cancer-TME interactions, which we then also recapitulated in patient tumors. We found that co-culture with fibroblasts induced the epithelial-mesenchymal transition in cancer cells and resulted in their increased drug tolerance, while co-culture with macrophages promoted stress, hypoxia responses and enhanced tumorigenicity. Adaptations were recurrent and partially reversible, but could not be explained by clonal selection, suggesting a dynamic non-mutational process. Moreover, adapted cancer cells elicited cell state changes in the fibroblasts and macrophages consistent with an extended phenotype mode of cancer evolution. Our findings reframe cancer-TME interactions by a systematic dissection according to sets of specific adaptations and programmed responses.

cancer biology↗

bioRxiv2026

Charting sex divergence in the human brain across the lifespan

Biological sex fundamentally shapes human brain organization, but sex-specific normative neuroanatomical trajectories across the lifespan remain largely uncharted. Here, we constructed independent, sex-specific lifespan brain charts using structural neuroimaging data from 59,915 healthy individuals (29,760 males and 30,155 females) ranging in age from 266 postconception days to 100 years. By examining 296 structural phenotypes across global, cortical, and subcortical measures, these models revealed widespread sex differences in maturational timing, with males reaching peak milestones later than females. These trajectories demonstrate that sex differences evolve dynamically, with phenotype-specific windows of emergence and maximal separation. Compared with conventional sex-pooled references, sex-specific models achieved superior predictive accuracy and reduced misestimation of individual deviations in healthy populations. Across five neuropsychiatric conditions, sex-specific models improved the detection of extreme deviations and revealed both shared and sex-dependent patterns of disorder-related neuroanatomical abnormalities. These sex-specific charts establish tailored normative references for assessing brain development, ageing, and disease.

neuroscience↗

bioRxiv2026

A telomere-to-telomere genome of Holstein cattle resolves chromosome-end architecture and the genetic basis of telomere length

Telomeres safeguard chromosome-end integrity, yet population-scale dissection of telomere-length genetics has been largely restricted to humans. Here, we report a 3.01-Gb telomere-to-telomere (T2T) genome of Holstein cattle that resolves repeat-rich chromosome ends and centromeres and enables telomere-length quantification at single-chromosome-end resolution. Holstein chromosome ends show marked telomere-length asymmetry and end-specific variation in telomere variant repeats, subtelomeric tandem repeats, GC/CpG composition, and DNA methylation. Using sequencing-depth-corrected telomere-length phenotypes from 9,591 cows, teloGWAS identifies major loci encompassing RTEL1, CTC1, and TERT, while multi-tissue eQTL, sQTL, TWAS, and co-expression analyses prioritize CTC1 and implicate DNA replication, DNA repair, and stress-response networks. Together, these findings extend population-scale telomere genetics beyond humans and establish a sequence-resolved framework for chromosome-end biology and telomere-informed livestock genomics.

genomics↗

bioRxiv2026

Structural basis for catalytic and inhibitory divergence between archaeal and bacterial ammonia monooxygenases

Ammonia oxidation initiates nitrification and is closely linked to microbial N2O production. Ammonia monooxygenase (AMO) catalyzes the first and rate-limiting step of nitrification and is widespread across evolutionarily distinct ammonia-oxidizing archaea (AOA) and bacteria (AOB). The ocean is the largest biome for AOA and AOB, which have distinct ecological niches and markedly different sensitivities to nitrification inhibitors. However, the lack of archaeal AMO structures and inhibitor-bound AMO complexes has hindered mechanistic understanding of the architectural, catalytic, and inhibitory divergence between these two enzyme systems. Here, we report high-resolution cryo-electron microscopy (cryo-EM) structures of marine archaeal AMO captured in active and inactivated states within its native membrane environment, together with inhibitor-bound structures of estuarine bacterial AMO. Archaeal AMO forms an unexpected cup-shaped homotrimer composed of eight subunits per protomer and exhibits substantial architectural divergence from bacterial AMO. Integrated structural, biochemical, kinetic, and computational analyses reveal distinct periplasmic architectures, copper-center organization, and hydrophobic channels between archaeal and bacterial AMOs for ammonium acquisition, catalysis and inhibitor response. These findings provide a structural and mechanistic framework for understanding how archaeal and bacterial AMOs have diverged to distinct ammonia-oxidizing strategies and inhibitor susceptibilities across environmentally important ammonia oxidizers.

molecular biology↗

bioRxiv2026

A multi-tissue epigenomic atlas links the sheep non-coding genome to domestication and complex traits

Non-coding regulatory variation drives complex traits, domestication, and evolutionary adaptation, yet the sheep genome lacks high-resolution functional annotation. Here we present SheepEpimap, a multi-tissue regulatory atlas harmonizing 516 CUT&Tag histone modifications, ATAC-seq, and RNA-seq datasets across 43 adult tissues in sheep. We annotated 2.93 million cis-regulatory elements, yielding 557,441 enhancer-gene pairs and 145,407 variants with allele-specific effects. By training a sequence-to-function deep-learning model, we decoded the base-pair syntax of chromatin accessibility, annotated transcription factor motif instances genome-wide, and constructed 12,210 tissue-specific gene regulatory networks (GRNs). Integrating this resource with multi-tissue expression quantitative trait loci, selection sweeps, and genome-wide association studies prioritized non-coding variants driving domestication and complex traits. Finally, cross-species analysis revealed that sequence-conserved, tissue-matched enhancers were significantly enriched in the heritability of complex traits and diseases in humans. In summary, SheepEpimap (https://genome.ucsc.edu/s/mengzhu/SheepEpimap) provides an open-access foundational ecosystem for sheep functional genomics, precision breeding, and comparative biology.

genetics↗

bioRxiv2026

mRNA-based prime-and-pull vaccination combines benefits of subcutaneous and mucosal BCG vaccination

Pulmonary vaccination has been proposed as a strategy to improve protection against tuberculosis, due to the generation of immune cells that more efficiently survey and eliminate infected cells within the lung. However, uncontrolled replication and excessive inflammation associated with mucosal delivery of live-attenuated vaccines highlight the need for alternative strategies that balance efficacy and safety. Here, we describe a prime-and-pull vaccination approach in which systemic immunity is established by subcutaneous BCG vaccination, followed by the induction of local lung immunity through mucosal delivery of lipid nanoparticle-formulated multi-antigen mRNA. Proof-of-concept studies using a model antigen demonstrated the induction of polyfunctional antigen-specific T cells in the lung with minimal inflammatory cell infiltration, compared with mucosal BCG vaccination. Eight Mycobacterium tuberculosis- and BCG-derived proteins were subsequently selected to generate four multi-antigen mRNA constructs. In vivo vaccination and challenge experiments demonstrated that this prime-and-pull strategy is well tolerated and confers protective immunity against tuberculosis in a murine model. Collectively, these data support a modular mRNA-based prime-and-pull vaccination strategy as a translational approach that bridges the improved immunogenicity and efficacy of mucosal BCG vaccination with the safety profile of parenteral BCG administration. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/739464v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@c0635borg.highwire.dtl.DTLVardef@bb0357org.highwire.dtl.DTLVardef@1d87243org.highwire.dtl.DTLVardef@135bcd7_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2026

Pulmonary delivery of antigen-enhanced BCG overcomes safety barriers in immunocompromised hosts and protects against TB in the absence of adaptive immunity.

The low efficacy of Bacille Calmette-Guerin (BCG) in preventing pulmonary tuberculosis (TB) underscores the need for improved TB vaccines. Recombinant BCG (rBCG) strains secreting the virulence-associated effector molecule ESAT-6 from Mycobacterium tuberculosis (Mtb) markedly improve efficacy and immunogenicity in animal models of TB but have been considered unsuitable for clinical translation due to safety concerns identified in intravenous SCID mouse models. Here, we demonstrate that pulmonary delivery fundamentally reshapes the safety and protective efficacy of the ESAT-6-secreting rBCG strains, BCG::RD1 and BCG::ESAT6-PE25SS. In sharp contrast to intravenous delivery, pulmonary administration was markedly better tolerated, improved survival, and reduced systemic dissemination and brain pathology of severely immunocompromised mice. Strikingly, pulmonary rBCG vaccination also conferred superior protection against aerosol Mtb challenge in wild-type, type 2 diabetic, and adaptive immunity-deficient Rag1-/- and Rag2-/-Il2rg-/- mice, with BCG::RD1 showing the strongest adaptive immunity-independent protection. Mechanistically, pulmonary rBCG vaccination promoted lung innate immune activation, expansion of myeloid-biased progenitors in the bone marrow, and enhanced antimycobacterial activity of macrophages, consistent with trained innate immunity. Collectively, these findings reveal that pulmonary vaccination largely overcomes safety concerns of ESAT-6-secreting rBCG strains and provide preclinical evidence for a viable strategy to improve protection against TB in immunocompromised individuals.

immunology↗

bioRxiv2026

The kinetochore proteins Ndc80 and Dsn1 are required for survival of postmitotic neurons

Dsn1 and Ndc80 are essential proteins of the kinetochore complex and required for chromosome segregation in dividing cells and for regulating development and microtubule dynamics in postmitotic neurons. With conditional deletion of floxed alleles, we here show that Dsn1 and Ndc80 are also required for the viability of postmitotic neurons, both in cultures of hippocampal and cortical neurons and in vivo in the retina. Loss of these proteins triggers apoptosis, as indicated by caspase cleavage and an increase in nuclear DNA breakage. The pro-survival function of the kinetochore components is distinct from that which was previously demonstrated for regulation of neuronal synaptogenesis. The microtubule-binding domain of Ndc80 is required for the synaptogenic functions but Ndc80 lacking this domain can nonetheless rescue the viability of neurons from which Ndc80 has been deleted. Similarly, whereas the synaptogenic function involves regulation of microtubules in axons and dendrites, a nucleus-localized Dsn1 is sufficient to rescue the viability of neurons from which Dsn1 has been deleted. Thus, postmitotic neurons retain a nuclear requirement for components of the kinetochore in order to prevent apoptosis.

neuroscience↗

bioRxiv2026

AI-enabled discovery of small molecules targeting complementary pathways for hair follicle rejuvenation

Hair thinning arises from multi-faceted dysfunction within the hair follicle, driven by both intrinsic cellular pathways and pathways responding to extrinsic hormonal and microenvironmental cues. Here, we present an AI-enabled discovery framework to discover small molecules that promote hair follicle rejuvenation. This framework integrates graph neural networks trained on phenotypic screening data with structure-based virtual screening to prioritize compounds that modulate complementary biological pathways. Through AI-enabled screening, hit-to-lead optimization, and medicinal chemistry, we identified four compounds that increase follicle dermal papilla cell viability, stabilize hypoxia signaling by inhibiting prolyl hydroxylase domain protein 2 (PHD2), and suppress androgen-mediated follicular miniaturization by inhibiting 5-reductases (5-ARs). RNA sequencing analyses confirmed pathway engagement, and functional validation across primary cells and a 3D hair follicle organoid model demonstrated high activity and cellular specificity. The lead compounds were incorporated into a water-based formulation, where they demonstrated robust solubility and combinatorial efficacy to increase sprouting length of follicle organoids. These results establish an AI-enabled platform for discovering multi-pathway modulators of hair follicle rejuvenation.

bioengineering↗

bioRxiv2026

Candidozyma auris utilizes transferrin, but not heme-bound iron for in vivo virulence

Candidozyma auris (C. auris) is an emerging multidrug-resistant fungal pathogen, and its dissemination to the bloodstream and deep-seated organs is associated with high mortality. The limited antifungal armory and pipelines against C. auris pose a major challenge in disease management. Addressing this threat requires a deeper understanding of fungal virulence mechanisms that promote persistence and of host factors that drive susceptibility. Previous in vitro studies showed that iron enhances C. auris resistance to azoles and echinocandins, whereas iron chelation mitigates this effect. Here, we demonstrate that C. auris does not utilize cell-free heme or induce hemolysis but instead extracts and uses iron from transferrin to support growth and virulence. Deletion of the SIT1 siderophore transporter in C. auris attenuated fungal growth and reduced renal injury, while increased transferrin-iron saturation worsened disease outcomes in immunocompetent mice, highlighting the importance of transferrin-bound iron uptake. Mechanistically, C. auris exploits transferrin-bound iron to enhance ergosterol biosynthesis and activate antioxidant defenses, promoting resistance to neutrophil- and caspofungin-mediated killing. These findings identify elevated transferrin saturation as a novel host susceptibility risk factor for disseminated C. auris infection and reveal how iron availability reshapes fungal physiology to drive infection persistence.

microbiology↗

bioRxiv2026

Defining the Candidozyma auris pan-genome and essentiality

Candidozyma auris is a multi-drug resistant fungal pathogen that causes hospital outbreaks, but genetic variation and gene function in this organism remain undercharacterized. Here, we performed pangenome analysis on 695 genetically representative isolates of C. auris and found that only 2.6% of gene families varied in their presence, mainly consisting of gene loss events or clade-specific genes, highlighting the clonal nature of these strains. Using microbial genome-wide association, we identified two loci associated with altered keratinocyte adherence, including genes with unknown function. To look broadly at gene function, we developed a genome-scale insertional mutagenesis approach and identified 614 high-confidence essential genes. Nearly one-third of these genes exhibited divergent essentiality compared to the model yeasts Candida albicans and Saccharomyces cerevisiae. Together, this study highlights organism-specific biology and provides a comprehensive resource for identifying species-specific determinants of virulence and targets for antifungal drug development.

microbiology↗

bioRxiv2026

Sex-specific Genetic Regulatory Effects in Chickens

Sexual dimorphism is a defining vertebrate feature, yet its sex-specific molecular architecture remains poorly understood. Here we established a sex-balanced, uniformly reared chicken cohort to map this landscape, integrating individual whole-genome sequencing with 7,969 bulk and 779,380 single nucleus transcriptomes across 32 tissues from 280 birds. We identified 495,098 independent expression quantitative trait loci for 20,194 genes, including 10,937 loci modulated by cell-type composition. Notably, 340 genes were regulated by 449 loci in a sex-dependent manner, significantly enrichment in endocrine tissues like adipose and the adrenal gland. Furthermore, we fine-mapped 1,219 structural variants, demonstrating their unique roles to tissue- and sex-specific expression beyond SNPs. Ultimately, we showed the utility of these regulatory effects in elucidating the molecular basis of metabolism and complex traits in both chickens and humans. This comprehensive atlas of regulatory effects provides profound insights into the genomic and molecular basis of sexual dimorphism in vertebrates.

genomics↗

bioRxiv2026

An Integrated and Configurable End-to-End Pipeline for Longitudinal Cell Painting Analysis

Cell painting assays generate high-dimensional, multi-channel imaging data that enable systematic characterization of cellular phenotypes. Increasingly, such assays are performed in longitudinal settings and under chronic perturbations, introducing additional challenges related to imaging variability, focus-field heterogeneity, and consistency across time points. Existing analysis workflows often require substantial manual adaptation to handle these complexities, limiting scalability and reproducibility. In this paper, we propose SCALE (Stable Cell painting Analysis for Longitudinal Experiments), an integrated, end-to-end analysis pipeline designed for robust longitudinal analysis of cell painting data. The pipeline combines nucleus-centered segmentation, automated quality control, feature extraction, and signal aggregation within a modular and configurable framework. Once assay-specific configurations are specified, the pipeline executes in a fully automated manner from raw images to downstream summary statistics and analysis-ready outputs. We demonstrate the utility of the pipeline using a chronic radiation exposure cell painting dataset, illustrating its ability to support consistent longitudinal comparisons across conditions and time points.

bioinformatics↗

bioRxiv2026

Interpretable transcriptome-to-phenotype modeling of cell-painting nuclear morphology features from RNA-seq under low-dose radiation exposure

With rapid advancements in high-throughput multi-modal profiling techniques across molecular, cellular to tissue scales, translating such multi-modal data into knowledge discovery for foundational understanding of cellular mechanisms is central in modern biomedical sciences. In this study, we focus on understanding how low-dose radiation exposure perturbs cellular morphology by linking high-dimensional transcriptomic responses to quantitative cell-phenotype readouts over time. We present a time-resolved inverse modeling framework that associates gene expression changes via RNA-sequencing with nuclear morphology features obtained from cell-painting imaging. Morphology responses were defined as treated-control differences for multiple nuclear features including size, shape, intensity, and textures, indexed by radiation dose and week. To capture time-dependent associations while maintaining interpretability, both RNA-sequencing and cell-painting data were stratified into four temporal phases (weeks 1-2, 3-4, 5-6, 7-9) and phase-dependent effects are encoded via gene-phase interaction predictors. To reduce confounding by dose trends and to evaluate generalization across time, we used a two-stage leave-one-week-out procedure: (i) a dose-only baseline model produced out-of-week residuals for each morphology feature, and (ii) elastic-net regression on phase-aware predictors modeled residual variation not explained by dose. Hyperparameters were selected via an exhaustive grid search scored by the correlation between observed residuals and out-of-week residual predictions, with additional sparsity diagnostics based on nonzero coefficient counts per fold. Stable predictors were identified by selection frequency and sign consistency across folds, then pruned further for multicollinearity and parsimony. Final reduced models were fit using ordinary least squares with heteroskedasticity-consistent standard errors to report effect estimates robust to non-constant variance. This workflow yields a transparent, time-stratified set of transcriptomic predictors associated with longitudinal nuclear morphology changes and provides a reproducible foundation for downstream biological interpretation and validation.

bioinformatics↗

bioRxiv2026

A micropeptide component of the OGDH complex modulates ATP production in the TCA cycle

Since its elucidation, the tricarboxylic acid (TCA) cycle has been regarded as a fully defined enzymatic pathway. Here, we report the discovery of a previously unrecognized micropeptide component of this central metabolic system. Through analyzing ribosome profiling data from the mouse sORFs.org database, we discovered APPM (ATP production-promoting micropeptide), a 29-amino acid peptide encoded by a non-canonical small open reading frame within the mouse Ctsb gene. APPM localizes to the mitochondrial matrix and inner membrane, where it directly binds to the E1 subunit (OGDH) of the -ketoglutarate dehydrogenase complex (OGDHc). This interaction modulates OGDHc enzymatic activity, thereby regulating NADH and ATP production. We identified tyrosine-13 as the critical residue mediating the APPM-OGDH interaction and functional activation. Exogenous APPM administration increased ATP levels in vivo and, in a mouse model of heart failure, restored cardiac function, outperforming the reference drug trimetazidine. Collectively, our findings identify APPM as the first micropeptide regulator of the OGDH complex, expanding the known repertoire of the TCA cycle and presenting a potential therapeutic strategy for bioenergetic disorders.

molecular biology↗

bioRxiv2025

Multimodal AI for Single cfDNA Profiling and Cancer Screening

Cell-free DNA (cfDNA) serves as a non-invasive biomarker for cancer detection, but conventional methods face challenges due to the ultra-low abundance of tumor-derived cfDNA (ctDNA) among normal cfDNA. Though nucleosome-bound cfDNA harbors rich epigenomic features that could enable ctDNA identification by single-molecule multi-omics cross-validation, this remains unexplored due to methodological limits. Here, we developed a cfDNA sequencing approach integrating methylation, fragmentomics, and histone modifications at the single-molecule level; together with gene semantics and epigenomic annotations, these modalities were vectorized and fused to represent each cfDNA molecule. We trained a Transformer-based model (cfAI) to profile and evaluate ctDNA likelihood at molecule, gene, and sample levels. cfAI achieved [~]10-fold enrichment of cancer-derived signals over noise and reached 72.6% sensitivity at 93.1% specificity for multi-cancer detection. Our study establishes an innovative framework that overcomes the inherent signal-to-noise limitations of conventional assays and reveals biological features at molecular resolution for cancer detection.

bioinformatics↗

bioRxiv2025

A Comprehensive Treatment-Induced Resistance Atlas of Glioblastoma Reveals a Fibrotic Niche Shielding the Tumor from Immunotherapy

Therapeutic resistance in IDH-wildtype glioblastoma (GBM) is driven by an intricate interplay between cellular plasticity and protective microenvironmental niches. By constructing the GRIT-Atlas--a massive transcriptomic compendium encompassing nearly one million high-quality single cells across 17 cohorts--and cross-analyzing it with 48 independent Visium spatial transcriptomics sections, we discover a recurrent "Spatial Resistance Triad" comprising mesenchymal-like (MES-like) malignant cells, myeloid-derived suppressor cells (MDSCs), and collagen-secreting cancer-associated fibroblasts (CAFs). This large-scale spatial integration reveals that the triad physically fortifies microvascular proliferation (MVP) and pseudopalisading necrosis (PAN) niches. To substantiate these findings at true single-cell resolution, we deploy high-plex spatial molecular imaging (CosMx SMI) to map over 400,000 single cells across a clinical validation cohort. This high-resolution architecture firmly validates the triads geography and, critically, provides structural gradient evidence unmasking a spatiotemporal continuum wherein PAN emerges as a direct functional consequence of MVP advancement and subsequent vascular collapse. Mechanistically, stromal CAF-derived COL6A1 engages CD44 receptors on MES-like cells to accelerate a potent neural stemness cascade within these protective domains. Utilizing a blood-brain barrier (BBB)-penetrant multi-library compound screen, we identify Lacidipine as a multi-modal stromal disruptor that successfully silences CAF activation and halts matrix secretion. Incorporating Lacidipine into the standard chemo-immunotherapy backbone (TMZ + anti-CSF1R) completely dismantles the protective desmoplastic matrix niche, forcing a profound collapse of intracranial tumor burden and dramatically extending overall survival in orthotopic models. Collectively, our study leverages unprecedented single-cell and spatial scale to provide a definitive blueprint of GBM therapeutic evasion, establishing matrix-targeted intervention as a mandatory prerequisite for successful glioblastoma eradication.

cancer biology↗

bioRxiv2025

A high-throughput methods SNIPR-Nm reveals small RNA methylation ratio

Small RNAs regulate gene expression and genome stability, and their functions are shaped by 3'-terminal 2'-O-methylation (Nm). Although Nm is universal in plants and mainly marks animal piRNAs, recent evidence reveals Nm on mammalian miRNAs, highlighting broader regulatory roles. However, current sequencing underestimates Nm-modified RNAs due to ligation bias, and existing qPCR-based methods cannot simultaneously quantify abundance and modification, leaving Nm dynamics largely unexplored. We developed a unified enzymatic strategy that integrates poly(A) polymerase and a thermostable ligase to measure both small RNA abundance and 3'-end Nm status. Applied to mouse testis, it enables robust quantification of Nm ratios for piRNAs and miRNAs. Our method offers a versatile tool for dissecting small RNA methylation in development and disease.

genetics↗