Biology subjects

Zhou, P.

Publications and source records attributed to Zhou, P..

At least 37 records · Page 2Linked to original sources

bioRxiv2026

B Lymphocyte Protein Factories produced by Hematopoietic Stem Cell Gene Editing

Long-term in vivo production of therapeutic proteins and development of vaccines that elicit protective levels of broadly neutralizing antibodies (bNAbs) against major pathogens face challenges. Here we report on an alternative gene-editing approach using small numbers of hematopoietic stem and progenitor cells (HSPCs) to direct long-term, high-level expression of antibodies or cargo proteins. Edited B lymphocyte offspring can be activated by cognate antigen to undergo clonal expansion and develop into specific antibody or cargo protein-synthesizing plasma cells. These cells produce long-lasting, therapeutic levels of serum antibody against HIV-1 or malaria and an anti-influenza virus bNAb that mediated universal protection from heterologous lethal challenge. Our data provide a paradigm for cell therapy approaches to prevent or treat disease using self-amplifying B cell protein factories.

immunology↗

bioRxiv2026

Multi-Omics Reveals Activated Fibroblasts with Dual Functional Roles in Repair and Negative Barrier in the CD8+ T Cell Cytotoxic Niche for Lung Cancer Neoadjuvant Therapy

A major challenge in elucidating immune activation and tolerance is that single-omics technologies are inherently limited. Since single-cell transcriptomics lacks spatial information and spatial transcriptomics lacks resolution or depth, neither can adequately model the multidimensional tumor immune microenvironment (TiME) features. We present NICHE (Niche Integrated Cellular Heterogeneity Elucidator), an integrative multi-omics framework. By aligning physical cell-cell interactions with single-cell ligand-receptor (L-R) expression, NICHE leverages single-cell transcriptomics, spatial proteomics, and AI modeling to systematically decode the composition, spatial interactions, and communications within immune functional niches. To validate NICHEs capabilities, we applied it to human tonsil tissue. The framework successfully identified key structural and functional units and deeply elucidated the cell-cell interactions and molecular crosstalk within them. Furthermore, these results were validated through orthogonal methods, including single-cell spatial transcriptomics and multiplexed immunofluorescence, which confirmed the accuracy and reproducibility of our analyses. In non-small cell lung cancer (NSCLC) patients undergoing neoadjuvant immunotherapy, CD8+ T cell cytotoxic niches mediate tumor clearance with variable outcomes, to define the cellular and molecular drivers behind this functional spectrum, we utilized NICHE to analyze the TiME from patients with complete versus partial pathological response, both pre- and post-treatment. We found that neoadjuvant immunotherapy may induce the activation of CXCL12+, NECTIN2+, POSTN+, and COL6A1+ fibroblasts through the activation of cytotoxic T cells. Paradoxically, these fibroblasts support tissue repair but also establish an immunosuppressive niche that protects residual tumor cells, ultimately attenuating treatment efficacy. Our work establishes a multidimensional framework for dissecting dynamic immune activity, with direct implications for understanding tumor immunology and identifying novel therapeutic targets.

immunology↗

bioRxiv2026

Therapeutic interleukin-2 rewires skeletal-immune circuits to reverse postmenopausal bone loss

Postmenopausal osteoporosis results from excessive osteoclast activity, but the immune circuits that limit osteoclastogenesis in the bone marrow remain poorly understood. Here, we tested a clinically accessible immunotherapy low-dose interleukin-2 (IL-2) as a therapeutic intervention in mice with established ovariectomy-induced bone loss, integrating microcomputed tomography imaging, single-cell transcriptomics, genetic perturbation, and human monocyte-osteoclast assays. Therapeutic administration of IL-2 reversed trabecular bone loss, expanded CXCR3 regulatory T cells in bone marrow, and promoted IL-10- and CGRP-producing ILC2s that were required for protection. IL-2 also acted directly on the osteoclast lineage, suppressing mouse and human osteoclastogenesis through activation of a STAT1-IRF1-dependent IFN program in RANKL-stimulated monocytes. Together, these data define an IL-2-responsive skeletal-immune network that can be therapeutically engaged to suppress osteoclastogenesis, which opens new avenues for cytokine-based immunotherapies in postmenopausal osteoporosis and related bone disorders.

immunology↗

bioRxiv2026

NORAD orchestrates KLC1-mediated SFPQ transport via liquid-liquid phase separation

Neuronal function requires precise long-distance axonal transport mediated by molecular motors and RNA-binding proteins like SFPQ, though regulatory mechanisms remain poorly defined. We identify long non-coding RNA NORAD as a master regulator of this process through liquid-liquid phase separation (LLPS). While SFPQ and kinesin-1 mediate cargo delivery, the specific RNA coordinating their interaction was unknown. We demonstrate NORAD directly binds kinesin light chain 1 (KLC1) and promotes SFPQ condensation into dynamic LLPS droplets, enabling efficient transport. CRISPR-assisted mapping and functional assays show NORAD depletion disrupts granule dynamics, impairs neuroprotective mRNA localization, and induces axonal degeneration. In vitro reconstitution confirms NORAD-KLC1 synergy enhances SFPQ phase separation, and neuron-specific knockout mice exhibit motor deficits with reduced neuronal density. These findings establish the first evidence of lncRNA NORAD-mediated LLPS in axonal transport, revealing a new paradigm for RNA-guided neuronal maintenance.

molecular biology↗

bioRxiv2025

AAV vectors with enhancer-controlled ITR promoters

Adeno-associated virus (AAV) is limited by its packaging capacity and the unwanted promoter activity of inverted terminal repeats (ITRs). Here we utilized mini-enhancers to regulate ITR promoters and drive AAV cargo expression in the absence of the canonical promoters, which not only solved the ITR issue but also released more payload for the cargo. As an example, this new design successfully enabled robust, tissue-specific SpCas9 gene editing via a single AAV, which otherwise requires a dual-AAV system due to the large size of SpCas9.

bioengineering↗

bioRxiv2025

B lymphocytes that enter the germinal center late preferentially differentiate into memory cells that recognize subdominant epitopes

Immune responses to pathogens and effective vaccines elicit germinal center (GC) responses wherein B cells undergo affinity maturation and develop into plasma cells (PCs) and memory B cells (MBCs). The GC reaction is initially seeded by a limited group of founder B cells, and subsequently further diversified by continual entry of naive B cells that compete with GC founder cells for antigen and T cell help. Whether these later-arriving invaders contribute to the development of PCs or MBCs is not known. To investigate the fate of GC invaders we developed a dual-recombinase reporter approach that enables pre- and post-GC B cell lineage tracing and used it to examine immune responses to vaccination and influenza infection. Notably, fate-mapped invaders preferentially give rise to MBCs as opposed to PCs. Moreover, antibodies expressed by invader-derived MBCs harbor fewer somatic mutations, exhibit lower affinity, and their antibodies bind to subdominant antigenic epitopes relative to founder MBCs. Our findings indicate that invader GC B cells are an important source of humoral immune memory diversification after infection or vaccination.

immunology↗

bioRxiv2025

Targeting the host factor HGS-viral membrane protein interaction in coronavirus infection

While current antivirals primarily target viral proteins, host-directed strategies remain underexplored. Here, we performed a genome-wide CRISPRi screening to identify the host protein, Hepatocyte Growth Factor-Regulated Tyrosine Kinase Substrate (HGS), as essential for the pan-coronaviruses infection both in vitro and in vivo. Mechanistically, HGS directly interacts with the viral membrane (M) protein, facilitating its trafficking to the ER-Golgi intermediate compartment (ERGIC) for virion assembly. Conversely, HGS deficiency caused M retention in the ER, blocking assembly. Leveraging this interaction, we designed M-derived peptides and screened over 5,000 FDA-approved drugs, identifying riboflavin tetrabutyrate (RTB). Both the peptides and RTB bind HGS and disrupt its interaction with the M protein, leading to M retention in the ER and subsequent blockade of virion assembly. These agents demonstrated broad anti-pan-coronavirus activity in vitro and in vivo. Collectively, our findings establish HGS as a druggable host target and identify RTB as a promising broad-spectrum antiviral candidate.

microbiology↗

bioRxiv2025

Directed evolution of a stem-helix targeting antibody enables MERS-CoV cross-neutralization through enhanced binding affinity

Broadly neutralizing antibodies (bnAbs) targeting conserved regions of the betacoronavirus spike are important for pan-betacoronavirus protection and pandemic preparedness. Here, we report on the isolation of a human monoclonal antibody, CC65.1, from a SARS-CoV-2 convalescent donor that targets the conserved S2 stem helix region. CC65.1 neutralizes various sarbecoviruses, including SARS-CoV-2, and binds the MERS-CoV spike but lacks MERS-CoV-neutralizing activity due to insufficient binding affinity. We utilized directed evolution to enhance the binding affinity of CC65.1 for the MERS-CoV S2 stem helix, yielding engineered antibody variants with newly acquired MERS-CoV-neutralizing activity. High-resolution structural analysis reveals key paratope mutations that optimize binding and stabilize epitope engagement. Our findings demonstrate the potential of rational antibody engineering to expand bnAb breadth across divergent betacoronaviruses. This work supports the development of engineered bnAbs and S2-targeted vaccines for broad betacoronavirus countermeasures and highlights strategies to achieve cross-lineage immunity for future pandemic threats. Author SummaryThe persistent emergence of new SARS-CoV-2 variants of concern that evade neutralizing antibody responses and other zoonotic betacoronaviruses with pandemic potential have provided strong motivation to develop broadly neutralizing antibodies (bnAbs) that target more conserved regions of the spike protein in sarbecoviruses and other betacoronaviruses. Here, we employed a directed evolution strategy to engineer the sarbecovirus-neutralizing antibody CC65.1, which targets the conserved S2 stem helix, to enhance its binding affinity with the MERS-CoV stem helix region, thereby conferring MERS-CoV neutralization. High-resolution structural studies of engineered CC65.1 revealed that key mutations reshape the paratope to better accommodate and stabilize the MERS-CoV S2 stem helix, resulting in increased binding affinity and neutralization potency. This study emphasizes the critical role of affinity maturation in expanding neutralization breadth and provides valuable insights for design of bnAbs to prevent and treat pandemic threats by betacoronaviruses.

immunology↗

bioRxiv2025

REV1 inhibition enhances trinucleotide repeat mutagenesis

Trinucleotide repeat (TNR) instability has been implicated in the pathogenesis of numerous neurodegenerative disorders. Because TNR instability causes mutagenesis of the underlying gene, we refer to the repeat instability phenomenon as TNR mutagenesis in this study. While germline expansions destabilize TNR to cause disease anticipation, somatic cell TNR instability drives earlier onset of symptoms and further disease progression. However, the drivers behind these repeat length changes remain unclear. Current models suggest that DNA replication slippage events and the action of genome instability pathways, such as DNA repair, cause TNR mutagenesis. Whether mutagenic polymerases from the translesion synthesis (TLS) pathway result in TNR instability is unclear. TLS polymerases are best at bypassing difficult-to-replicate DNA regions due to bulky lesions or gaps in DNA. While some effects of TLS polymerases on TNR instability have been explored in lower organisms, evidence in human cells is lacking. Using a quantitative GFP reporter with expanded CAG repeats, we show that inhibition of the TLS polymerase REV1 by its inhibitor, JH-RE-06, or siRNA knockdown increases TNR instability and the underlying mutability. These results suggest that REV1 protects Trinucleotide repeat length mutagenesis through potential continuous DNA synthesis when replicative polymerases stall ahead of repeat secondary structures. Collectively, we present evidence of the role of the TLS pathway in TNR instability, with potential implications for understanding mutability mechanisms, disease biology, and therapeutic targeting.

genetics↗

bioRxiv2025

Early-onset β-amyloidosis in human brains with hematological malignances and cardiovascular diseases: Revisiting injury/stress induced axonal pathology

{beta}-Amyloid (A{beta}) and tau pathologies are hallmarks of Alzheimers disease (AD) and they develop in human brain following differential spatiotemporal trajectories. As such, young/adult-onset tau-independent {beta}-amyloidosis is rare. We encountered four such cases among 397 banked brains, with the donors died of hematological malignances (blood cancers) or cardiovascular diseases. To explore the pathological implications, we examined 17 brains (10-87 year-old, y) from blood cancer patients and three (52-82 y) with cardiovascular diseases, focusing on vascular injury, axonal pathology and A{beta} formation. A{beta} plaques occurred in two adult brains (31 y, 63 y) with blood cancers and two (52 y, 65 y) with cardiovascular diseases in the absence of tau. In the blood cancer brains, 17/17 had vascular injuries seen in hematoxylin-eosin stained sections, 13/17 had iron leakage, and 13/17 had axonal pathology. Malignant cell infiltration was found in 5/14 brains with myeloid, lymphocytic and lymphoma malignances, with light chain infiltration in 3/3 brains with multiple myeloma. In the cardiovascular disease brains, A{beta} deposition primarily as diffuse plaques occurred in the cerebral cortex, with vascular and axonal pathologies in the white matter, striatum and internal capsule. Using a multi-labeling approach, the injury/stress induced axonal pathology was found to concur with {beta}-amyloid processor protein elevation and enhanced {beta}-secretase 1 processing but not intraneuronal A{beta} accumulation. The current findings suggest that hematological malignances and cardiovascular diseases are risk conditions for early-onset cerebral {beta}-amyloidosis, potentially attributable to vascular injury.

neuroscience↗

bioRxiv2025

Agropyron cristatum genome provides a new insight for wheat improvement with wild relatives

Exploring novel genetic variation for target traits is crucial for advancing wheat breeding and genetic improvement. Since 1990, we have been transferring genes from Agropyron cristatum, a promising wild relative genetic resource featuring robust spike morphology and stress tolerance, into common wheat by distant hybridization. To efficiently harness A. cristatum genes, we de novo assembled a 25.62-Gb high-quality genome of an autotetraploid accession Z559 with 28 chromosomes, and uncovered its specific genome features and an extensive repertoire of genes associated with yield and resistance to abiotic and biotic stresses. We systematically characterized A. cristatum genome introgressions by resequencing 431 wheat-A. cristatum derivatives and cloned the gene AcGNS1, which functions in the regulation of grain number per spike in wheat. Subsequently, we developed an optimized genome-selection model for wheat-A. cristatum breeding derivatives, enabling the rapid selection of new potential varieties. These findings demonstrate a genomic breeding strategy for leveraging novel genetic resources of wild relatives and advancing wheat improvement.

genomics↗

bioRxiv2025

Rescuing Ischemic Brain Injury by Rewiring Mitochondrial Electron Flow

Mitochondrial metabolic flux alterations are critical drivers of acute ischemia-reperfusion (IR) brain injury. Reverse electron transfer (RET), defined as the upstream flow of electrons from the quinone pool to complex I, is a major source of pathological reactive oxygen species (ROS) under stress conditions. In an in vivo brain IR model, oxygen deprivation induces the buildup of RET-supporting substrates, with glycerol 3-phosphate identified as the dominant contributor in addition to succinate. Rapid oxidation of these substrates by brain mitochondria upon reoxygenation drives massive ROS production, while also leading to over-reduction and dissociation of the complex I flavin mononucleotide (FMN) cofactor. The resulting FMN-deficient complex I becomes catalytically impaired, unable to oxidize NADH or to produce ROS. To mitigate RET and preserve complex I function, we used transgenic mice xenotopically expressing alternative oxidase (AOX). This enzyme bypasses complexes III and IV by directly oxidizing the reduced quinone pool and passing electrons onto molecular oxygen. AOX expression did not alter complex I abundance, supercomplexes assembly, or basal respiration rates, but effectively diverted electrons from the quinone pool, decreasing RET flux via complex I and limiting ROS generation during IR. For the first time we showed that AOX expression and attenuation of RET preserved complex I FMN binding, suppressed oxidative stress, and conferred neuroprotection in vivo. Our findings reveal a novel strategy for rewiring mitochondrial electron flux to mitigate initial IR brain injury, highlighting modulation of the quinone pool by AOX as a potential therapeutic strategy for IR.

biochemistry↗

bioRxiv2025

Targeting Regnase-1 unleashes CAR T cell antitumor activity for osteosarcoma and creates a proinflammatory tumor microenvironment

Negative regulators of T cell function represent promising targets to enhance the intrinsic antitumor activity of CAR T cells against solid tumors. However, the endogenous immune ecosystem in solid tumors often represents an immunosuppressive therapeutic barrier to CAR T cell therapy, and it is currently unknown whether deletion of negative regulators in CAR T cells reshapes the endogenous immune landscape. To address this knowledge gap, we developed CAR T cells targeting B7-H3 in immune-competent osteosarcoma models and evaluated the intrinsic and extrinsic effects of deleting a potent negative regulator called Regnase-1 (Reg-1). Deletion of Reg-1 not only improved the effector function of B7-H3-CAR T cells but also endowed them with the ability to create a proinflammatory landscape characterized by an influx of IFN{gamma}-producing endogenous T cells and NK cells and a reduction of inhibitory myeloid cells, including M2 macrophages. Thus, deleting negative regulators in CAR T cells enforces a non-cell-autonomous state by creating a proinflammatory tumor microenvironment.

immunology↗

bioRxiv2025

Highly heterozygous Citrus changshan-huyou Y. B. Chang originated from ancient hybridization between mandarin and pummelo and displayed distinct tissue-specific allelic imbalance

The genus Citrus is characterized by a reticulate evolutionary history with frequent hybridization, making it an intriguing subject for genome evolution investigation. Citrus changshan Y. B. Chang (Huyou) is a unique landrace first discovered in Zhejiang Province, China with premium fruit quality. The evolutionary origin of Huyou has puzzled local botanists and growers. Here, we sequenced a 120-years-old "ancestral tree" of Huyou using PacBio long read and Hi-C sequencing and assembled 2 high-quality haplotype-resolved genomes HY1 and HY2. Huyou displayed a genome heterozygosity level at 3.07%, among the highest in published citrus genomes. Using a k-mer-based tracing approach, we explicitly resolved that HY1 genome contained 87.8% mandarin, 7.3% pummelo, 0.2% citron origin, whereas HY2 had 85.0% pummelo, 2.9% mandarin, 0.3% citron, implying a hybridization event between mandarin and pummelo. Phylogeny dating showed that HY1 (2.0 Mya) and HY2 (2.18 Mya) had diverged earlier than the split of Citrus clementina and Citrus reticulata, and the split of Citrus grandis and Citrus maxima, respectively. We observed clear chromosomal recombination on chr8 and chr9 in HY1, which may have occurred after the ancestral hybridization. Further transcriptome analyses in 6 tissues revealed a strong allelic dominance of HY2 over HY1 in root tissue and moderately in stem, leaf, flower, and fruits. KEGG enrichment analyses revealed that genes related to antioxidants biosynthesis and lipid metabolisms were most significantly affected by allelic imbalance. This first report of allelic imbalance in citrus species support Huyou as an interesting model to investigate genome evolution following distant hybridization.

evolutionary biology↗

bioRxiv2025

DiNovo: high-coverage, high-confidence de novo peptide sequencing using mirror proteases and deep learning

Despite the recent advancements driven by deep learning, de novo peptide sequencing is still constrained by incomplete peptide fragmentation and insufficient protein digestion in current single protease-based proteomic experiments. Here, we present a software system, named DiNovo, for high-coverage and confidence de novo peptide sequencing by leveraging the complementarity of mirror proteases. DiNovo is empowered by several innovative algorithms, including a mirror-spectra recognition algorithm independent of pre-sequencing, two sequencing algorithms based on deep learning and graph theory, respectively, and target-decoy mapping, a method for sequencing result evaluation free of prior peptide identification. Compared with the trypsin protease used alone, DiNovo using two pairs of mirror proteases led to two to three times high-confidence amino acids sequenced. Compared with previous single-protease de novo sequencing algorithms, DiNovo achieved much higher sequence coverages. DiNovo also showed great potential as a powerful complement or alternative to database search for peptide identification with quality control.

bioinformatics↗

bioRxiv2025

Has AlphaFold 3 Solved the Protein Folding Problem for D-Peptides?

Due to their favorable therapeutic properties, including improved stability, bioavailability, and membrane permeability, D-peptides that bind biological L-proteins represent an important class of systems in computational drug design. A reliable in silico workflow for these systems must correctly preserve stereochemistry while predicting fold and binding pose. The AlphaFold 3 (AF3) model reported by Abramson et al. (2024) enforces a strict chirality violation penalty to maintain chiral centers from model inputs and is reported to have a low chirality violation rate of only 4.4% on a PoseBusters benchmark containing diverse chiral molecules. Herein, we report the results of 3,255 black-box experiments with AF3 to evaluate its ability to predict the fold, chirality, and binding pose of D-peptides in heterochiral complexes. Despite inputs specifying explicit D-stereocenters, we report that the AF3 chirality violation rate for D-peptide binders is much higher at 51% across all evaluated predictions; on average the model is as accurate as chance (random chirality choice, L or D, for each peptide residue). Increasing the number of seeds failed to improve this violation rate. The AF3 predictions exhibit incorrect folds and binding poses, with D-peptides commonly oriented incorrectly in the L-protein binding interface. Confidence metrics returned by AF3 also fail to distinguish predictions with low chirality violation and correct docking vs. predictions with high chirality violation and incorrect docking. We conclude that AF3 is a poor predictor of D-peptide chirality, fold, and binding pose and propose solutions to address these limitations. Significance StatementAlphaFold 3 (AF3) is a model trained to predict protein interactions. This algorithm is tuned to respect chiral centers (L and D). Changing the chirality of even one protein residue can significantly alter chemical properties such as binding and stability. Therefore, an algorithm should exhibit a chiral center error rate of 0%. Although the original AF3 authors reported a 4.4% chirality violation rate, we have found that the rate for D-peptides is much higher at[~] 50%. Our data reveal a crucial structural prediction error in AF3 and demonstrate that this widely used model is as accurate on average as chance (random chirality choice, L or D, for each peptide residue). These results indicate structure prediction of D-peptides is an outstanding problem.

bioinformatics↗

bioRxiv2025

Red Light-Activated Reversible Inhibition of Protein Functions by Assembled Trap

Red light offers strong tissue penetration and low phototoxicity, making it an attractive option for optogenetic applications. However, the available red light-inducible optogenetic tools are rather limited. Here, we present a novel red light-induced protein clustering system, namely R-LARIAT (Red Light-Activated Reversible Inhibition by Assembled Trap), that controls protein functions in a spatiotemporal manner. Our system capitalizes on the rapid and reversible binding of LDBs (nanobodies-based dimerization binders) to a bacterial phytochrome DrBphP, which uses a mammalian endogenous biliverdin chromophore to absorb red light. An anti-GFP nanobody fused with LDBs allows this method to quickly trap a wide variety of GFP-tagged proteins into light-induced protein clusters. Strikingly, our system exhibits an excellent performance in clustering efficiency with high light sensitivity and stability, it can function even when shielded by multiple glass plates. By utilizing the R-LARIAT system to trap and sequester tubulin, cell cycle progression can be blocked in HeLa cells. Therefore, the R-LARIAT system takes advantage of red light with greater tissue penetration and holds the potential to precisely control protein functions in living organisms.

cell biology↗

bioRxiv2025

A monophyletic origin of domesticated barley

The origin of domesticated barley has been debated extensively over the past century. Early botanical and comparative morphological research and recent genetic research supported a polyphyletic origin. A monophyletic origin was proposed after the discovery that a single genetic locus controls the presence of hull-less grains in all domesticated barley. Interpreting the origin of domesticated barley is further complicated by the archaeological record showing that the first domesticates had two rows of grain on the inflorescence, as does its wild progenitor. However, these two-rowed types effectively disappeared from the record for thousands of years, and they were replaced by derived six-rowed types that dominated barleys cultivation history and only reappeared in the record around one thousand years ago. Here, we used two independent datasets with large sample sizes and genome-wide genetic markers to re-evaluate barley cultivation history. We unequivocally demonstrate that modern domesticated barley has a monophyletic origin. Phylogenetic reconstruction and examination of current archaeological records suggests that the west Fertile Crescent was most likely where barley was first domesticated. Modern two-rowed types were likely derived from six-rowed domesticated types not earlier than 3,500 years ago in modern Turkiye and Iran. We conclude that the two distinct non-brittle rachis haplotypes, coinciding with distinct row types in cultivated barley, are likely the results of two chronological events along the same evolutionary line. Our results provide a reconcilable framework for explaining genetic and phytogeographic patterns and pave the way for a consensus on the origin of barley domestication.

evolutionary biology↗