Biology subjects

Zhou, H.

Publications and source records attributed to Zhou, H..

At least 37 records · Page 2Linked to original sources

bioRxiv2026

Subcellularly Resolved 3D Translatome in Mouse Oocytes and Early Embryos

Spatial patterning of mRNA translation is a fundamental process in early embryogenesis. Existing RNA translation profiling methods lack subcellular spatial resolution at the single-molecule level, limiting our understanding of spatial RNA biology in embryogenesis. To address this, we profiled the spatial translatome of intact mouse embryos at near-genomic scale by adapting RIBOmap and incorporating multiplexed organelle staining. In oocytes, 2-cell and 4-cell embryos, we systematically analyzed RNA translation across three spatial scales: organelle, subcellular, and intercellular. We found that functionally related genes exhibit spatially and temporally controlled translation patterns near distinct organelles. Using Harmonics, a graph signal processing framework, we demonstrate that embryo asymmetry emerges at the first cell division and is amplified at later stages. This work paves the way for comprehensively investigating the fundamental spatial post-transcriptional regulation at the earliest moments of mammalian life.

developmental biology↗

bioRxiv2026

Discovery of an NRAS isoform and activation-state selective macrocyclic peptide

Macrocyclic peptides have gained increased attention amid claims they are a "Goldilocks" therapeutic modality that can encode the selectivity of a biologic in a footprint close to that of a small molecule. Here we attempt to find a peptide that binds selectively to NRAS, sparing HRAS and KRAS, while accessing the cytosol via passive cell permeability. To do so, we combine subtractive affinity selection with mRNA display to identify Compound 1, an 11mer macrocyclic peptide which binds NRAS at a novel allosteric site between Helix 3 and Helix 4 of the GTPase domain. Compound 1 has total isoform selectivity and can be tuned to achieve activation-state selectivity with a single amino acid change. While it has preferential affinity for oncogenic NRAS-specific mutations, it does not inhibit NRAS function or achieve passive membrane permeability.

cancer biology↗

bioRxiv2026

Maximizing the productivity of co-transcriptional in vitro transcription

mRNA therapeutics have demonstrated transformative potential in infectious disease, oncology and genetic disorders. Parallel to the rapid clinical advancement of mRNA therapies, research into in vitro transcription (IVT), the pivotal process of mRNA manufacturing, has intensified significantly. However, the majority of these studies omitted cap analog, an essential substrate for co-transcriptional capped IVT (co-IVT), likely to simplify experimental systems. Nevertheless, co-IVT remains the dominant industrial standard for the production of mRNA therapeutics. Here, we report an optimized co-IVT process that achieves a yield of more than 26 g/L while maintaining capping efficiency of over 99.5%, and achieving a 30-fold reduction in double-stranded RNA (dsRNA), the most critical process-related impurity. These performance gains substantially improve mRNA production efficiency and enhance mRNA quality, thereby accelerating the transition to commercial-scale manufacturing of mRNA therapeutics. Moreover, the performance was attained using either of two mechanistically distinct engineered T7 RNA polymerase (RNAP) mutants. Both mutants enabled robust mRNA synthesis under process conditions that have not been previously reported. These observations provide mechanistic insights into IVT and advance our fundamental understanding of this critical bio-manufacturing step.

bioengineering↗

bioRxiv2026

Perturb-seq resolves physiologic programs and bidirectional regulation of non-canonical NF-κB signaling in epidermal organoids

Regulated keratinocyte differentiation is required for formation of the stratified epidermis and a functional barrier. Understanding genetic drivers of keratinocyte differentiation is crucial for understanding several skin diseases. Perturb-seq is a single-cell CRISPR screen that measures transcriptomic responses to perturbations. To date, Perturb-seq experiments have principally focused on 2-dimensional cell culture models lacking hallmarks of skin development - physiological desmosome formation and barrier function. Here, we leverage Perturb-seq in an epidermal organoid model that recapitulates physiologically relevant differentiation programs. We demonstrate that our perturbations significantly impact diverse differentiation programs and reveal bidirectional function of non-canonical NF-{kappa}B signaling in late keratinocyte differentiation.

genomics↗

bioRxiv2026

RBM6-mediated alternative splicing facilitates the adaptation of Eurasian avian-like H1N1 swine influenza virus

The Eurasian avian{square}like (EA) H1N1 swine influenza virus (SIV), derived from avian influenza viruses (AIV), poses a serious threat to public health due to its capacity for cross{square}species transmission and pandemic emergence. The molecular determinants underlying its replication advantage over AIV remain poorly defined. Here, we identify RNA{square}binding motif protein 6 (RBM6) as a novel host factor that differentially regulates the replication of EA H1N1 SIV and AIV. Mechanistically, RBM6 binds to the critical M901 site of the viral M segment, thereby modulating RNA splicing. Substitution of M901C with M901T markedly reduced RBM6 binding, impaired M segment splicing, and attenuated viral replication both in vitro and in vivo. Conservation analysis revealed that M901T is common in avian strains, whereas M901C is predominantly maintained in swine strains, underscoring M901C as a determinant of swine adaptation. Complementation experiments further demonstrated that swine RBM6, but not avian RBM6, restored EA H1N1 SIV replication. Taken together, our findings uncover a previously unrecognized role of RBM6 in shaping influenza virus replication and highlight the RBM6-M901C axis may serve as potential targets for controlling influenza virus adaptation and interspecies transmission.

microbiology↗

bioRxiv2026

Selenium-enriched rapeseed extract synergizes with chemotherapy drug cisplatin in inhibiting proliferation and promoting apoptosis of colorectal cancer cells

Chemotherapy treatment of colorectal cancers (CRC) using cisplatin (CDDP) encounters problems of drug resistance by the cancer cells and cytotoxicity to normal cells, highlighting the urgent need for joint therapeutical strategies. Selenium-enriched rapeseed extracts exhibit anti-cancer effects but the bioactive components and mechanisms remain unclear. Here, we applied different solvents to fractionate the extracts from Selenium-enriched rapeseed and found that the water extract (WE) fraction significantly enhanced the cytotoxic effect of CDDP on cancer cells but no damage on normal cells. HPLC-ICP-MS analysis revealed that methylselenocysteine (MSC) and selenocystine (SeCys2) were the main selenium speciation in WE. Through cell biology and integrative multi-omics analysis, we found a synergistic anti-CRC cell effect when combining CDDP with MSC, sulforaphane (SFN), celastrol (Cel), Indole-3-carbinol (I3C), -linolenic acid (ALA) or linoleic acid (LA). We propose that the CDDP-WE combination treatment holds the promise for improving curative efficacy for chemo-refractory CRC patients in the future.

cancer biology↗

bioRxiv2026

Lactylation of Influenza Virus Polymerase Acidic Protein Promotes Viral Replication and Pathogenicity

Influenza virus poses a potential risk of triggering the next global pandemic. In-depth investigation into the mechanisms underlying influenza virus replication and pathogenicity will provide robust support for controlling influenza virus infection. Although post-translational modifications are known to regulate viral infection, the role of lactylation in influenza virus replication remains elusive. In this study, influenza virus ribonucleoprotein complex subunits are found to be lactylated. Specifically, ATAT1 promotes viral polymerase acidic protein (PA) lactylation and enhances viral replication. In contrast, SIRT1 mediates de-lactylation of PA and exerts an inhibitory effect on viral replication. Further investigations reveal lactylation of PA at residues K605 and K609 is essential for viral replication and pathogenicity. Mechanistically, PA K605/609 residues are localized at the interaction interface of the ANP32-mediated polymerase asymmetric dimer; mutation at these residues inhibits polymerase asymmetric dimerization, thereby impairing RNA production during viral genome replication. Collectively, this study uncovers a novel mechanism by which influenza virus hijacks host enzymes to mediate PA lactylation, and expands the molecular regulatory network of influenza virus infection.

microbiology↗

bioRxiv2026

A Variant-Resistant Linear Epitope in the SARS-CoV-2 Nucleocapsid Protein for Antigen Detection

We previously generated a mouse monoclonal antibody, N179, against the SARS-CoV-2 nucleocapsid (N) protein and developed a colloidal gold-based immunochromatographic test strip. This assay achieved a detection limit of 2 ng/mL and displayed 98% concordance with RT-qPCR results. However, the precise epitope recognized by mAb N179 had not been defined. Using a panel of GST-fused N protein truncation fragments, we mapped the linear B-cell epitope recognized by mAb N179 to the flexible C-terminal tail of the N protein by Western blotting and ELISA. The minimal binding motif required for mAb N179 recognition was identified as 390QTVTLL395. Multiple sequence alignment of 11 representative SARS-CoV-2 lineages, including Alpha, Beta, Gamma, Delta, and Omicron subvariants BA.1, BA.2, and BA.3.2, revealed that this epitope was completely conserved across all variants analyzed. Stringent local pairwise alignment analysis using EMBOSS WATER further showed that the 390QTVTLL395 motif achieved a perfect 6/6 match exclusively in SARS-CoV-2; no identical sequence was detected in the seven common human coronaviruses, four influenza viruses, or five bat coronaviruses examined. Structural prediction analyses indicated that this region is surface-exposed and possesses a strong linear B-cell epitope propensity. Together, these findings identify 390QTVTLL395 as a specific molecular signature of SARS-CoV-2 among the viruses analyzed. Our results provide an epitope-level explanation for the sustained diagnostic reliability of the mAb N179-based assay against emerging variants, clarify the molecular basis for its lack of cross-reactivity, and may inform the rational design of SARS-CoV-2 diagnostics targeting conserved, mutation-resistant epitopes. ImportanceWe identified the exact nucleocapsid protein epitope recognized by monoclonal antibody N179, a diagnostic antibody used in a colloidal gold rapid assay. The identified 390QTVTLL395 motif at residues 390 to 395 was conserved among the SARS-CoV-2 variants analyzed and was not present as an identical continuous sequence in the related respiratory viruses examined. This work supports precise epitope mapping as a useful strategy for evaluating and revalidating diagnostic antibodies as respiratory viruses evolve.

microbiology↗

bioRxiv2026

Internalized Components of Membrane Attack Complexes Disrupt Proteostasis and Acquire Alarmin-Like Properties

Immune effects of membrane attack complexes (MAC) have been widely attributed to their abilities to cause cell death. Here, we show that the MAC component, C9, forms non-cytolytic aggregates with pro-inflammatory effects. Intracellular aggregates of C9 are detected within inflamed tissues of patients in association with endothelial cell (EC) activation but not increased cell death. We identify NUMBL as a Rab35 effector that directly binds surface-bound C9 to promote C9 internalization and entry into the endolysosomal pathway. Within acidified endolysosomes, C9 forms insoluble aggregates that are targeted for degradative aggrephagy in a process that activates NF-{kappa}B. For C9 aggrephagy to occur, ZFYVE21, a Rab5 effector, complexes with RNF34 to bridge C9 aggregates to LC3B+ aggresome membranes. We detect C9 aggregates in vivo, and we show that a ZFYVE21-RNF34 signaling axis is required for C9 aggrephagy and NF-{kappa}B -dependent EC activation in three separate mouse models. Mice with conditional loss of ZFYVE21 in ECs show reduced aggregraphy, resulting in attenuated systemic inflammation and reduced tissue injury following skin transplantation. Our data show that the C9 component of MACs forms intracellular aggregates with alarmin-like properties.

biochemistry↗

bioRxiv2026

Super-Resolution Visual ProteomEx for Hard Tissues and Clinical Samples

Correlating nanoscale tissue architecture with unbiased molecular composition within the same specimen remains a fundamental challenge in spatial biology. Current spatial proteomics methods either lack the imaging resolution required to resolve nanoscale tissue architecture or achieve only limited proteome coverage, and untargeted approaches capable of deep protein identification have yet to be integrated with super-resolution imaging within a single workflow. Here, we present microProteomEx, an integrated platform that combines hydrogel-assisted tissue expansion with mass spectrometry-compatible fluorescence imaging, laser capture microdissection, and bottom-up proteomics to achieve simultaneous three-dimensional super-resolution imaging (effective lateral resolution down to [~]47 nm on conventional diffraction-limited microscopes) and spatially resolved proteomics at a scalable lateral resolution of 29-100 {micro}m (0.02-0.28 nL volumetric resolution). By optimizing fixation, protein anchoring, and a secondary re-embedding strategy, we extend the method to mechanically resilient tissues, including mouse kidney and heart, as well as to formalin-fixed, paraffin-embedded clinical specimens. We demonstrate single-glomerulus and single-plaque proteomics (450-1,000 proteins per structure), resolve proteomic differences between malignant melanoma and giant congenital melanocytic nevus in a rare pediatric case, and characterize the morphology-resolved molecular heterogeneity of cored versus diffuse amyloid-beta plaques in a mouse model of Alzheimers disease across two disease stages. microProteomEx establishes a broadly accessible framework for correlating tissue ultrastructure with deep spatial proteomics, with direct implications for disease biology, biomarker discovery, and precision medicine.

biochemistry↗

bioRxiv2026

A G2 Checkpoint Arrests Cryptococcus neoformans Cell Division in response to Hypoxia

Saturated cultures of the pathogenic yeast, Cryptococcus neoformans, arrest as unbudded cells in the G2 phase of the cell cycle. As cells divided and cultures saturated, we found that oxygen levels in the culture medium dropped nearly tenfold. When saturation-arrested cultures were re-oxygenated without adding fresh growth medium, cells immediately formed a bud and then underwent mitosis. Thus, the arrest is due to low oxygen concentration rather than nutrient depletion. Because the G2 arrest was associated with unbudded cells, we asked whether C. neoformans cells have a morphogenesis checkpoint that blocks mitosis until cells can form a bud. Inhibition of budding by treatment with Latrunculin A also led to G2 arrest, and we determined that this arrest is dependent on the CDK inhibitory kinase, Swe1. This finding suggests that C. neoformans possesses a morphogenesis checkpoint analogous to that in the distantly related Saccharomyces cerevisiae. We also demonstrated that Swe1 is required to enforce the hypoxia-induced G2 arrest. We propose that hypoxia inhibits budding in C. neoformans, which in turn triggers a morphogenesis checkpoint to arrest cells in G2 even when nutrients are plentiful.

cell biology↗

bioRxiv2026

Quantitative Assessment of shRNA Loading and Delivery Efficiency of Engineered Extracellular Vesicles

RNA interference (RNAi) therapeutics enable selective silencing of disease-associated genes. Yet, their clinical application remains largely confined to the liver due to extrahepatic delivery constraints of current platforms such as GalNAc conjugates and lipid nanoparticles. Extracellular vesicles (EVs) offer an attractive alternative delivery strategy owing to their biocompatibility, ability to traverse biological barriers, and amenability to engineering. However, EV-mediated RNA delivery is limited by inefficient endogenous RNA loading and poor cytosolic release following uptake. Here, we establish a modular EV-based platform that addresses both challenges by integrating enhanced endogenous shRNA loading with fusogen-mediated cytosolic delivery. Using Argonaute 2 (AGO2)-assisted loading, we substantially increase shRNA copy numbers per vesicle (up to 3.7 copies/EV) and enable quantitative, molecule-resolved assessment of delivery potency. Engineered EVs achieve robust and reproducible shRNA-mediated gene silencing with picomolar IC50 values across multiple cell types and induce significant target knockdown in the mouse brain following intracerebral administration. Together, these findings demonstrate that coordinated engineering of shRNA loading and cytosolic release can overcome key limitations of EV-mediated small RNA delivery.

bioengineering↗

bioRxiv2026

Candida albicans reprograms host inosine metabolism to drive immunosuppressive macrophage polarization and gastric cancer carcinogenesis

Gastric cancer (GC) is strongly associated with changes in the gastric microbiome. However, the direct contribution of individual pathogenic species to tumor initiation and progression remains poorly defined. Here, we identified Candida albicans (C. albicans), a fungus that ranks as the most significantly enriched taxon in GC tissues, can directly promote gastric carcinogenesis. We found that when C. albicans alone was colonized into the gastric mucosa of mice, it induced the complete sequence of GC-associated preneoplastic lesions, including chronic gastritis, mucinous metaplasia, and atypical hyperplasia, with 16% of colonized animals progressing to low-grade intraepithelial dysplasia. In K19-C2mE transgenic mice, a model of spontaneous GC formation, infection with C. albicans significantly accelerated GC formation, with synergistic effects observed upon co-infection with the well-known gastric carcinogen, Helicobacter pylori. Mechanistically, C. albicans upregulated inosine levels in both gastric tissues and circulation. This inosine promoted polarization of macrophages toward an M2-like phenotype by activating the adenosine A2a receptor (A2aR), creats an immunosuppressive microenvironment that protects GC cells from the tumoricidal effects of anti-tumor immunity. Depletion of M2 macrophages or pharmacological inhibition of A2aR attenuated C. albicans-mediated tumorigenesis. Clinically, elevated inosine levels correlated with prior C. albicans exposure and poorer outcomes of GC patients. Our findings establish C. albicans as a direct pro-tumorigenic pathogen in GC and highlight the inosine-A2aR-M2 macrophage axis as a promising target for therapeutic intervention.

cancer biology↗

bioRxiv2026

Objective curriculum-guided design of multi-property proteins

Designing functional proteins that simultaneously possess multiple bio-chemical properties remains a significant challenge, as key protein properties, such as solubility, stability, binding affinity, and chemical resistance, are often interdependent or even conflicting. Current approaches typically attempt to jointly optimize multiple functional objectives in one shot, followed by extensive screening to identify rare feasible designs. Here, we introduce OCDesign, an objective curriculum-guided framework for multi-property protein design. OCDesign is based on the objective curriculum principle, i.e., the order in which objectives are introduced can shape the accessibility of functional solutions. In each design round of OCDesign, candidate sequences are generated in silico, assessed across multiple properties, selected based on Pareto-optimal trade-offs, and experimentally validated, with each experimental stage testing the role of the newly introduced objective within the curriculum. Using antibody-binding protein A as a model system, we show that one-shot optimization fails to yield functional designs, whereas a staged curriculum--progressing from solubility and structural consistency to binding affinity, and then to alkaline resistance--enables the design of proteins possessing multiple desired properties through substantially fewer wet-lab experiments. These results establish OCDesign as a practical computational-experimental strategy for organizing and integrating multiple objectives in protein design, and suggest that objective ordering is a key determinant of accessibility in high-dimensional design spaces.

bioengineering↗

bioRxiv2026

MedMisBench: Measuring Epistemic Resilience of LLMs Under Misleading Medical Context

Large language models (LLMs) now reach expert-level scores on medical licensing exams, encouraging the assumption that high scores imply safe medical judgment while patients increasingly use them for health advice. We show this assumption is fragile: when misleading context is injected into questions that LLMs originally answer correctly, they abandon the correct answer. We call the ability to maintain correct judgment under adversarial context epistemic resilience, and introduce MedMisBench to measure it. MedMisBench contains 10,932 medical question items and 48,889 misleading context-option pairs spanning medical reasoning, agentic capability, and patient-journey evaluation. Across 11 model configurations, mean accuracy falls from 71.1% on original questions to 38.0% under focused misleading context, with 51.5% attack success. The most damaging injections are formal, rule-like fabrications: authority-framed falsehoods reach 69.5% attack success and exception-poisoning claims reach 64.1%. A 14-member clinical panel from 7 countries identified serious potential harm in 38.2% of reviewed cases. MedMisBench exposes a structural blind spot in LLM evaluation in medical settings: existing benchmarks measure what models know, but not whether they preserve correct medical judgment under misleading context.1

bioengineering↗

bioRxiv2026

Visualization of neuronal morphology of the suprachiasmatic nucleus vasopressin neurons by Cre/FLPe-based genetic two-step sparse labelling - GT-SPARCL

Resolving the morphology of individual neurons in densely packed brain regions remains challenging. Sparse labeling is essential for delineating cellular architecture, yet achieving reproducible low-density labeling e.g. < 1% has been a persistent technical hurdle/problem. We present a genetically encoded two-step strategy, GT-SPARCL (Genetic Two-Step Sparse Cre/FLPe Combination Labeling), which leverages two independent stochastic recombination events to deliver stable, tunable low-density labeling in mice. As a proof of concept, we applied this method to visualizing arginine vasopressin (AVP) neurons in the suprachiasmatic nucleus (SCN), the master circadian pacemaker composed of [~]10,000 neurons per side in mice. Using two-photon imaging of whole-mount SCN blocks, we reconstructed individual AVP neurons and uncovered previously under-resolved morphological heterogeneity. Based on axonal trajectories, we distinguished five structural types, including subclasses with commissural projections to the contralateral SCN and others extending projections beyond the nucleus. The majority ([~]70%) exhibited projections both within and outside the ipsilateral SCN, whereas the second most dominant subset ([~]20%) projected exclusively outside the SCN, representing an output-biased type. In contrast, neurons projecting exclusively within the ipsilateral SCN were exceptionally rare, suggesting that "dedicated" local-circuit AVP neurons do not form a major structural subtype. Collectively, our data indicate that AVP neurons are not structurally uniform but instead comprise diverse projection-defined subtypes, implying subtype-depen-dent contributions to intra-SCN communication, bilateral coupling, and circadian output. Beyond the SCN, our GT-SPARCL method may be applicable for achieving low-density labelling of neurons that can be defined by other specific Cre mouse lines.

neuroscience↗

bioRxiv2026

Cholangiocyte RUNX1 Orchestrates Fibrogenic and Inflammatory Signaling to Drive Biliary Fibrosis

IntroductionBiliary fibrosis and inflammation are central to the pathogenesis of cholangiopathies such as primary sclerosing cholangitis (PSC) and primary biliary cholangitis (PBC). Inflammatory and fibrogenic stimuli, such as transforming growth factor-{beta} (TGF{beta}) and lipopolysaccharide (LPS) signaling, drive these processes, but their underlying transcriptional mechanisms in cholangiocytes remain incompletely defined. We investigated the role of Runt-related transcription factor 1 (RUNX1) as a transcriptional co-regulator of fibroinflammatory signaling in cholangiocytes. MethodsHuman PSC-derived cholangiocytes (PSC-Cs) and mouse large biliary epithelial cells (MLEs) were subjected to RUNX1 knockdown or pharmacologic inhibition (Ro5-3335 or AI-10-104). Cytokine secretion was profiled by Luminex multiplexing; RUNX1 genomic binding and protein interactome were assessed by ChIP-qPCR, ChIP-seq, and LC-MS/MS. In vivo, Mdr2-/- mice received Ro5-3335, and cholangiocyte-selective Runx1 knockout mice (Krt19-CreERT) were challenged with a DDC diet, followed by evaluation of fibrosis and inflammation. ResultsRUNX1 expression was significantly increased in cholangiocytes from PSC and PBC patients, and Mdr2-/- mice. RUNX1 knockdown or inhibition reduced IL6, TNF, and other proinflammatory cytokines in PSC-Cs and attenuated TGF{beta}-, LPS-, and TNF-induced Il6 and Ccl2 expression in MLEs. ChIP-qPCR and ChIP-seq revealed TGF{beta}-induced RUNX1 binding to the Il6 promoter and 727 additional genomic sites enriched for fibrosis and inflammatory pathways; predicted upstream regulators included TGF{beta}, TNF, and NF{kappa}B signaling. Proteomic analysis identified TGF{beta}-induced RUNX1 interactions with SMAD2 and NF{kappa}B2. In vivo, Ro5-3335 treatment in Mdr2-/- mice reduced hepatic collagen, ECM gene expression, immune cell infiltration, and serum liver injury markers and bile acids. Similarly, cholangiocyte-specific Runx1 deletion mitigated fibrosis, inflammation, and liver injury in DDC-fed mice. ConclusionRUNX1 is a central transcriptional hub integrating TGF{beta} and inflammatory signals in cholangiocytes. Its inhibition attenuates biliary fibrosis and inflammation in cholestatic models, supporting RUNX1 as a potential therapeutic target in fibroinflammatory cholangiopathies.

physiology↗

bioRxiv2026

GABA antagonises ABA perception and signalling in plants

Abscisic acid (ABA) is a central regulator of plant growth and stress responses. However, it also negatively impact germination, growth, and reproductive performance, highlighting the need for mechanisms that fine-tune its signalling. Here, we identify {gamma}-aminobutyric acid (GABA) as an endogenous antagonist of plant ABA receptor. We show that GABA binds the clade A protein phosphatase-ABI1, disrupting its association with the ABA receptor PYL1, blocking ABA binding, thereby attenuating ABA signalling. GABA modulates ABA-control of stomatal aperture and seed germination, demonstrating its broad physiological influence. Notably, GABA accumulation during stress coincides with the subsequent decline in ABA, consistent with a delayed feedback signal of ABA activity. Together, this work identifies GABA as an antagonist of ABA and provides a mechanism balancing plant stress resilience and growth.

plant biology↗