Biology subjects

Zhu, X.

Publications and source records attributed to Zhu, X..

At least 109 records · Page 6Linked to original sources

bioRxiv2025

Alcohol Use Disorder Associated Gene FNDC4 Alters Glutamatergic and GABAergic Neurogenesis

Large-cohort genome-wide association studies (GWAS) for alcohol use disorder (AUD) and AUD-related phenotypes have identified more than one hundred genetic loci. Functional study of those GWAS-identified loci might represent an important step toward understanding AUD pathophysiology. We found that genetic loci which are splicing quantitative trait loci (sQTLs) for the fibronectin III domain containing 4 (FNDC4) gene in the brain were identified by GWAS for both AUD drug treatment outcomes and AUD risk. However, FNDC4 function in the brain and how it might contribute to AUD pathophysiology remain unknown. In the present study, we characterized GWAS locus-associated FNDC4 splice isoforms, studies which suggested that FNDC4 alternative splicing results in loss-of-function for FNDC4. We also investigated FNDC4 function using CRISPR/cas9 gene editing, and the creation of human induced pluripotent stem cell (iPSC)-derived neural organoids joined with single-nucleus RNA sequencing. We observed that knock-out (KO) of FNDC4 resulted in a striking shift in the relative proportions of glutamatergic and GABAergic neurons in iPSC-derived neural organoids, suggesting a possible important role for FNDC4 in neurogenesis. We also explored potential mechanism(s) of FNDC4-dependent neurogenesis with results that suggested a role for FNDC4 in mediating neural cell-cell interaction. In summary, this series of experiments indicates that FNDC4 plays a role in regulating cerebral cortical neurogenesis in the brain. This regulation may contribute to the response to AUD pharmacotherapy as well as the effects of alcohol on the brain.

genomics↗

bioRxiv2025

A synergistic generative-ranking framework for tailored design of therapeutic single-domain antibodies

Single-domain antibodies (sdAbs) have emerged as powerful therapeutic agents due to their small size, high stability, and superior tissue penetration. However, unlike conventional monoclonal antibodies (mAbs), sdAbs lack an Fc domain, limiting their functional versatility and manufacturability. To address this challenge, we developed TFDesign-sdAb, a deep learning-based generative-ranking framework that enables rational engineering of sdAbs with tailored functionalities. Our framework integrates a structure-aware diffusion model (IgGM) for large-scale candidate generation and a fine-tuned sorter (A2binder) that evaluates and prioritizes them based on predicted functionality. Unlike traditional CDR-focused approaches, TFDesign-sdAb optimizes both complementarity-determining regions (CDRs) and framework regions (FRs), allowing sdAbs to acquire new functional properties while maintaining antigen specificity. We validated our approach by conferring Protein A binding to human VHs and nanobodies that originally lacked this feature, achieving high expression rates, strong binding affinities, and successful purification via industry-standard Protein A affinity chromatography. High-resolution structural characterization (1.49 [A] and 2.0 [A]) of the redesigned sdAb-Protein A complexes revealed conserved FR-mediated binding motifs that recapitulate natural Fc-Protein A interactions, validating the accuracy of our model. Furthermore, our pipeline streamlined the antibody engineering process, achieving a 100% success rate in generating Protein A-binding sdAbs while maintaining their original antigen-binding affinity. This work demonstrates the power of AI-driven design in overcoming long-standing limitations in antibody engineering and presents a scalable, generalizable solution for enhancing sdAb functionality.

bioinformatics↗

bioRxiv2025

Differential Regulation of Hepatic Macrophage Fate by Chi3l1 in MASLD

Metabolic dysfunction-associated steatotic liver disease (MASLD) progression involves the replacement of protective embryo-derived Kupffer cells (KCs) by inflammatory monocyte-derived macrophages (MoMFs), yet the regulatory mechanisms remain unclear. Here, we identify chitinase 3-like 1 (Chi3l1/YKL-40) as a critical metabolic regulator of hepatic macrophage fate. We observed high expression of Chi3l1 in both KCs and MoMFs during MASLD development. Genetic deletion of Chi3l1 specifically in KCs significantly exacerbated MASLD severity and metabolic dysfunction, whereas MoMF-specific Chi3l1 deletion showed minimal metabolic effects. Mechanistic studies revealed that this cell type-specific regulation arises from differential metabolic requirements: KCs display elevated glucose metabolism compared to MoMFs. Chi3l1 directly interacts with glucose to inhibit its cellular uptake, thereby selectively protecting glucose-dependent KCs from metabolic stress-induced cell death while having negligible effects on less glucose-dependent MoMFs. These findings uncover a novel Chi3l1-mediated metabolic checkpoint that preferentially maintains KCs populations through glucose metabolism modulation, providing important new insights into the pathogenesis of MASLD and potential therapeutic strategies targeting macrophage-specific metabolic pathways.

molecular biology↗

bioRxiv2025

Comparative phenotypic, genomic and transcriptomic characterisation of two Salmonella Typhimurium strains for a first-in-human challenge model

BackgroundInvasive non-typhoidal Salmonella (iNTS) disease remains a major public health challenge in sub-Saharan Africa. Salmonella Typhimurium is responsible for the majority of cases, with specific lineages being associated with increased risk of bloodstream infection. We have recently developed a Salmonella Typhimurium controlled human infection model (CHIM) to better understand disease pathogenesis and to provide a platform to test candidate vaccines. Selecting appropriate challenge strainsis a central design consideration in developing challenge model protocols. We describe the rationale, manufacture, and detailed characterisation of the two Salmonella Typhimurium strains used in the first-in-human NTS CHIM. MethodsTwo Salmonella Typhimurium strains, 4/74 (ST19) and D23580 (ST313), were selected from the UK Health Security Agency National Collection of Type Cultures and manufactured under Good Manufacturing Practice (GMP) conditions. Challenge agent stocks underwent microbial limits testing, viability and stability assessments, and phenotypic characterisation including growth kinetics, motility, acid sensitivity, and antibiotic susceptibility. Whole-genome sequencing was performed to confirm genetic stability post-manufacture. The effect of pre-challenge handling conditions was assessed in saline and sodium bicarbonate buffers, and bacterial survival was evaluated under simulated gastric and intestinal conditions using a modified Rossett-Rice model. Transcriptomic profiling was undertaken to determine whether sodium bicarbonate exposure altered expression of key virulence genes. ResultsBoth strains retained their expected phenotypic characteristics, including reduced motility and melibiose utilisation in D23580. GMP stocks remained pure, viable, and stable post-manufacture, with no unexpected genomic mutations detected. Both strains were susceptible to clinically relevant antibiotics used in the study. Survival was maintained in neutral and mildly acidic conditions, with significant reduction below pH 3.5. Stability was preserved for up to one hour in saline buffer and during simulated gastric transit in the Rossett-Rice model. Transcriptomic analysis showed no significant changes in Salmonella pathogenicity island-1 or 2, or flagellar gene expression following sodium bicarbonate exposure ConclusionsThese findings confirm that the Salmonella Typhimurium 4/74 and D23580 strains retained genomic and phenotypic integrity post-manufacture. The resulting challenge stocks provide the foundation for an ongoing NTS CHIM that aims to advance understanding of NTS and iNTS pathogenesis and support candidate vaccine testing.

microbiology↗

bioRxiv2025

The receptor binding properties of H5Nx influenza A viruses have evolved to promiscuously bind to avian-type mucin-like O-glycans

Highly pathogenic H5Nx influenza A viruses are causing unprecedented, season-independent outbreaks across avian and mammalian species, including dairy cattle, a novel reservoir. The sialoside-binding properties of influenza A hemagglutinin (HA) are strongly related to its ability to infect and transmit between hosts. Mucin-like O-glycans, omnipresent in respiratory tracts, have been understudied as viral receptors due to their complexity. To address this, we synthesized 25 O-linked glycans with diverse sialosides, including modifications by fucosides and sulfates. Our findings reveal that H5Nx 2.3.4.4b viruses uniquely bind core 3 sialyl-Lewisx and Sia-Gal-{beta}3GalNAc, glycans not recognized by classical H5 or other avian viruses. By determining its crystal structure, we resolved the structural features of both structures in an H5 hemagglutinin (HA) from a 2016 2.3.4.4b virus. While these viruses do not bind human-type receptors, their promiscuous receptor specificity enhances binding to human tracheal tissues, suggesting that O-glycan recognition contributes to their zoonotic potential.

microbiology↗

bioRxiv2025

SND1 Impairs Calcium Homeostasis in Right Ventricular Failure by Binding and Destabilizing SERCA2a in Cardiomyocytes

BackgroundRight ventricular failure (RVF) is a major contributor to a poor prognosis in patients with pulmonary arterial hypertension (PAH); however, its underlying molecular mechanisms remain incompletely understood. We identified a significant upregulation of the cardiac fetal gene SND1 in the right ventricular myocardium of RVF rats. This upregulation may be a key component of cardiac fetal reprogramming and play a critical role in RVF progression. However, the precise molecular mechanisms by which SND1 contributes to RVF remain unelucidated. MethodsRat models of PAH were established by intraperitoneal injection of monocrotaline (MCT). The pulmonary artery pressure and right ventricular function of rats were assessed using transthoracic echocardiography combined with right heart catheterization. Weighted gene co-expression network analysis (WGCNA) of proteomic data identified a significant association between SND1 upregulation and RVF progression. Calcium transient and contractility measurements in single cardiomyocytes were performed to evaluate the effect of SND1 on sarcoplasmic reticulum (SR) function. Liquid chromatography with mass spectrometry/mass spectrometry analysis combined with co-immunoprecipitation (Co-IP) was used to identify sarcoplasmic/endoplasmic reticulum Ca2+-ATPase 2A (SERCA2a) as the protein interacting with SND1. The effect of SND1 on the progression of RVF was validated using cardiomyocyte-specific SND1 conditional knockout rats. ResultsProteomic and molecular analyses revealed that the cardiac fetal gene SND1 is reactivated in RVF and may contribute to disease progression. In vitro, SND1 knockdown in neonatal rat ventricular myocytes (NRVMs) enhanced contractility, improved calcium transient amplitude and velocity, and increased cell survival under calcium overload. Mechanistically, SND1 interacts with SERCA2a and promotes its proteasomal degradation, impairing SR calcium reuptake. SND1 knockdown restored SERCA2a stability. In vivo, cardiomyocyte-specific SND1 knockout improved right ventricular function, reduced SERCA2a degradation and apoptosis, and increased survival in RVF rats. ConclusionOur study revealed that SND1 reactivation (a fetal gene) contributes to the progression of RVF by promoting SERCA2a degradation and impairing calcium handling. Targeted suppression of SND1 enhances cardiomyocyte contractility and survival, highlighting SND1 as a potential therapeutic target for improving right ventricular function in PAH. Clinical perspectiveO_ST_ABSWhat Is New?C_ST_ABSO_LIThis study identifies SND1, a reactivated fetal gene, as a key contributor to right ventricular failure (RVF) progression in pulmonary arterial hypertension (PAH). C_LIO_LISND1 interacts with SERCA2a, promoting its proteasomal degradation, which disrupts sarcoplasmic reticulum calcium reuptake and weakens cardiomyocyte contractility. C_LIO_LICardiomyocyte-specific SND1 deletion restores SERCA2a levels, improves calcium handling, enhances right ventricular function, and increases survival in RVF rats. C_LI What Are the Clinical Implications?O_LIThe reactivation of fetal genes such as SND1 is not just a marker but a pathogenic driver in maladaptive right ventricular remodeling. C_LIO_LITargeting SND1 represents a novel therapeutic approach to improve right ventricular contractility and prevent heart failure progression in patients with PAH. C_LIO_LIThese findings may inform the development of gene- or protein-targeted therapies aimed at stabilizing SERCA2a and preserving right heart function in high-risk populations. C_LI

cell biology↗

bioRxiv2025

Glial betaPix is essential for blood vessel integrity in the zebrafish brain

The formation of blood-brain barrier and vascular integrity depends on the coordinative development of different cell types in the brain. Previous studies have shown that zebrafish bubblehead (bbh) mutant, which has mutation in the betaPix locus, develops spontaneous intracerebral hemorrhage during early development. However, it remains unclear in which brain cells betaPix may function. Here, we established a highly efficient conditional knockout method in zebrafish by using homology-directed repair (HDR)-mediated knockin and knockout technology, and generated betaPix conditional trap (betaPixct) allele in zebrafish. We found that betaPix in glia, but neither neurons, endothelial cells, nor pericytes, was critical for glial and vascular development and integrity, thus contributing to the formation of blood-brain barrier. Single-cell transcriptome profiling revealed that microtubule aggregation signaling stathmins and pro-angiogenic transcription factors Zfhx3/4 were down-regulated in glial and neuronal progenitors, and further genetic analysis suggested that betaPix may act upstream on the PAK1-Stathmin and Zfhx3/4-Vegfaa signaling to regulate glia migration and angiogenesis. Therefore, this work reveals that glial betaPix plays an important role in brain vascular development in zebrafish embryos and possibly human cells.

developmental biology↗

bioRxiv2025

Decoding the Moving Mind: Multi-Subject fMRI-to-Video Retrieval with MLLM Semantic Grounding

Decoding dynamic visual information from brain activity remains challenging due to inter-subject neural heterogeneity, limited per-subject data availability, and the substantial temporal resolution gap between fMRI signals (0.5 Hz) and video dynamics (30 Hz). Current approaches face persistent challenges in addressing these temporal mismatches, demonstrate limited capacity to integrate subject-specific neural patterns with shared representational frameworks, and lack adequate semantic granularity for aligning neural responses with visual content. To bridge these gaps, we propose a framework addressing these limitations through three innovations: (1) a Dynamic Temporal Alignment module that resolves temporal mismatches via exponentially weighted multi-frame fusion with adaptive decay coefficients; (2) a Brain Mixture-of-Experts architecture that combines subject-specific extractors with shared expert layers through parameter-efficient tri-modal contrastive learning; and (3) a Multi perspective Semantic Hyper-Anchoring module that resolves cross-subject attention bias via multi-dimensional semantic decomposition, leveraging multimodal LLMs for fine-grained video semantic extraction--enabling the model to match individual attention patterns as different subjects naturally focus on distinct aspects of the same visual stimulus. This module boosts Top-10/Top-100 retrieval by 17.7%/6.6%. Experiments on two video-fMRI datasets demonstrate state-of-the-art performance, with 39%/30% improvements in Top-10/Top-100 accuracy over single-subject baselines and 27% gains against multi-subject models. The framework exhibits remarkable few-shot adaptability, retaining 97% performance when using only 10% training data for new subjects. Visualization analysis confirms this generalization capability stems from effective disentanglement of subject-specific and shared neural representations.

neuroscience↗

bioRxiv2025

Transcriptomic splicing analysis reveals a gene-independent form of TE reactivation in tumors representing unrecognized source of oncogenes and neoantigens

Transposable elements (TEs) are reactivated in tumors and serve as significant contributors to tumor transcriptomic complexity and heterogeneity. However, their repetitive nature and diverse transcriptional forms have hindered efforts to characterize TE-derived transcripts independent of host gene contexts. Here, using our self-developed splicing-junction analysis tool ASJA, we systematically interrogated TE transcription across 32 cancer types, identifying transcripts autonomously initiated from and exclusively spliced among TEs with high TE content, termed TEtrans. Our analysis revealed 5,361 TEtrans exhibiting pan-cancer prevalence, tumor-specific expression and features of mature transcripts, including canonical splicing, 5 caps and polyA tails. Mostly unannotated and enriched in intergenic regions, TEtrans are predominantly derived from primate-specific classes with conserved splice pattern. TEtrans burden demonstrates heterogeneous associations with prognosis and immune activity across cancers, while their expression can be epigenetically dual-regulated by stemness- and inflammation-associated transcription factors. Functional studies uncovered that TEtrans could act as oncogenes, exemplified by tsTE1, a HERVH-derived transcript that promotes colorectal cancer proliferation by enhancing TOP1-mediated DNA supercoil relaxation. Remarkably, [~]7.2% of TEtrans are identified to encode tumor neoantigens, including viral proteins and unannotated peptides, which are shared among patients and validated by proteogenomic analysis. These TEtrans-derived neoantigens are immunogenic both in vitro and in vivo, and exceed neoepitopes from genomic alterations in abundance per tumor, particularly in cancers with low mutational burden. Collectively, as a gene-independent form of TE-derived transcripts, TEtrans represents a unique source of oncogenes and tumor neoantigens, expanding the functional repertoire of TEs in cancer biology and offering new avenues for therapeutic targeting. HighlightsO_LITEtrans are tumor-specific transcripts with conserved splicing patterns, predominantly derived from primate TEs in intergenic regions. C_LIO_LITEtrans expression is epigenetically activated and dually regulated by stemness/inflammation transcription factors. C_LIO_LITEtrans function as oncogenes, exemplified by tsTE1 enhancing TOP1-mediated DNA relaxation. C_LIO_LITEtrans encode tumor neoantigens that elicit CD8+ T cell responses, providing targets for immunotherapy, especially in low-mutation tumors. C_LI Abstract figure O_FIG O_LINKSMALLFIG WIDTH=198 HEIGHT=200 SRC="FIGDIR/small/640928v1_ufig1.gif" ALT="Figure 1"> View larger version (78K): org.highwire.dtl.DTLVardef@1d2d4cforg.highwire.dtl.DTLVardef@918f14org.highwire.dtl.DTLVardef@489465org.highwire.dtl.DTLVardef@1069d17_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2025

Targeting CD38 effectively prevents myelofibrosis in myeloproliferative neoplasms

Proinflammatory signaling is a hallmark of myeloproliferative neoplasms (MPNs). Several studies have shown that monocytes are a major source of proinflammatory cytokines and monocyte-derived fibrocytes play a pivotal role in the pathogenesis of myelofibrosis (MF). To further explore the role of monocytes in MF, we generated inducible NrasG12D/+Jak2V617F/+(NJ) mice. Recipients transplanted with NJ bone marrow cells developed MF with an early onset of anemia and monocytosis. In vitro, NJ recipients bone marrow nucleated cells exhibited increased quantity of CD45+CollagenI+ fibrocytes, which were mainly derived from the Ly6chigh monocytes. RNA sequencing identified a significant elevated expression of CD38 (a nicotinamide adenine dinucleotide (NAD)+ hydrolase) in Ly6chigh monocytes from NJ mice, which results in pronounced lower level of NAD+. In humans, CD14+ monocytes from MF patients showed significantly higher expression of CD38 than controls and monocytes from polycythemia vera (PV) patients with grade 1 fibrosis had higher CD38 expression than those without fibrosis. Finally, we tested that boosting NAD+ via pharmacological CD38 targeting or NAD+ precursor supplementation inhibited the differentiation of fibrocytes in vitro and observed that targeting CD38 can effectively prevent the onset of fibrosis in vivo. Collectively, our findings shed light on the role of CD38 in monocytes and suggest potential clinical applications such as use of CD38 as a biomarker of fibrotic progression and potential clinical utility of CD38 inhibition in patients with MF. Key PointsO_LICD38-overexpressing monocytes are increased in MF murine models and MPN patients progressing to fibrotic-phase disease. C_LIO_LIRestoring intracellular NAD+ levels using the CD38 inhibitor 78c prevented the development of fibrotic-phase disease in MPN murine models. C_LI

cancer biology↗

bioRxiv2025

Ethylene modulates cell wall mechanics for root responses to compaction

Soil stresses impact crop yields, presenting global agricultural challenges. Soil compaction triggers root length reduction and radial expansion driven by the plant hormone ethylene. We report how ethylene controls cell wall properties to promote root radial expansion. We demonstrate how soil compaction stress, via ethylene, upregulates Auxin Response Factor1 in the root cortex, which represses Cellulose Synthase (CESA) genes. CESA repression drives radial expansion of root cortical cells by modifying the thickness and mechanics of their cell walls, which result in a "stiff epidermis-soft cortex" contrast. Our research thus connects ethylene signaling with root mechanics via cell wall strength, and reveals how dynamic regulation of cellulose synthesis crucially controls root growth in compacted soil.

plant biology↗

bioRxiv2025

The fetal hydrops-associated single-residue mutation L322P abolishes mechanical but not chemical activation of the PIEZO1 ion channel

The mechanically activated PIEZO1 ion channel is genetically linked to numerous physiological and pathophysiological processes. For example, deleting PIEZO1 in mice leads to defective lymphatic vessel development, while nonsense mutations in humans are associated with autosomal recessive generalized lymphatic dysplasia (GLD) and non-immune hydrops fetalis. However, it remains unclear whether PIEZO1-dependent biological processes are directly mediated by its intrinsic mechanosensitivity. Here we identified a human fetal hydrops-associated single-residue mutation, L322P (corresponding to L329P in mouse PIEZO1), which completely abolishes PIEZO1s response to mechanical stimuli while maintaining normal plasma membrane expression and responsiveness to its chemical activators, Yoda1 and Jedi1. Remarkably, the mechanical response of the mutant can be restored by Yoda1. These findings demonstrate a direct link between the loss of PIEZO1s mechanosensitivity and the pathophysiological phenotype of fetal hydrops, and raise the therapeutic potential of using PIEZO1 chemical activators to restore the mechanosensitivity of PIEZO1 missense mutants that are associated with genetic diseases such as GLD and hydrops fetalis. Significance StatementGenetic deletion studies have revealed the diverse cellular, physiological, and pathophysiological roles of the mechanically activated PIEZO1 ion channel. However, whether these functions are directly mediated by its mechanosensitivity has remained experimentally unaddressed. In this study, we identified a novel fetal hydrops-associated single-residue mutation, hPIEZO1-L322P, which specifically abolishes PIEZO1s mechanical response while retaining its chemical activation. Remarkably, the mechanical response of the mutant can be fully restored by the PIEZO1 chemical activator Yoda1. These findings address the long-standing question of whether PIEZO1 relies on its mechanosensitivity to mediate in vivo functions, provide critical insights into the mechanosensing mechanism of PIEZO1, and highlight the therapeutic potential of using PIEZO1 chemical activators to rescue the loss of mechanosensitivity caused by missense mutations.

physiology↗

bioRxiv2025

Cloche/Npas4l is a pro-regenerative platelet factor during zebrafish heart regeneration

Zebrafish has full capacity of heart regeneration, but little is known about how blood cells, especially platelets, are involved in this regenerative process. Here we report that cloche/npas4l is a pro-regenerative platelet factor for heart regeneration. We found that haploinsufficiency of npas4l disrupted cardiomyocyte (CM) and endothelial cell (EC) proliferation and heart regeneration after injury. A single-cell transcriptomic atlas revealed that npas4l was dynamically expressed in platelets after heart injury and regulated robust interactions between platelets-CMs or -ECs. Decreasing platelets by either nitroreductase-mediated platelet depletion or platelet-deficient mutant mpl impaired CM and EC proliferation, and over-expression of npas4l in platelets sufficiently induced CM and EC proliferation in uninjured hearts, as well as rescued CM and EC proliferation defects in cloche mutants. Mechanistically, Npas4l positively regulated a panel of ligands expression including bmp6 in platelets to fine-tune CM proliferation and heart regeneration. Therefore, this work unveils a novel platelet Npas4l signaling and presents mechanisms on how platelets modulate CM/EC proliferation via ligand-receptor network during zebrafish heart regeneration.

cell biology↗

bioRxiv2025

Avoidance of fratricide and improved efficacy of CD33-MSLN CAR-iNK cells in treating acute myeloid leukemia

Acute myeloid leukemia (AML) patients are often older, which brings challenges of endurance and persistent efficacy of autologous CAR-T cell therapies. Allogenic CAR-NK cell therapies may offer reduced toxicities and enhanced anti-leukemic potential against AML. In this study, we designed a novel CD33-mesothelin loop CAR (Loop CAR) and evaluated its anti-tumor efficacy in human umbilical cord blood-derived NK (UCB-NK) cells and human pluripotent stem cell-derived NK (hPSC-iNK) cells. The Loop CAR exhibited superior cytotoxicity against dual-antigen-positive tumor cell lines and primary AML cells. To further avoid fratricide caused by endogenous CD33 expression in NK cells, we established a hPSC-derived cell line via knockout of CD33 gene (CD33KO) and engineered Loop CAR. Rather than enforced expression of exogenous CD16, we generated abundant mature CD33KO-Loop CAR-iNK cells highly expressing endogenous CD16 via an organoid induction approach. This innovative strategy effectively mitigated NK cell fratricide and significantly enhanced CD33 and mesothelin-mediated specific cytotoxicity. Moreover, the CD33KO-Loop CAR-iNK cells demonstrated superior tumor-killing activity in AML xenograft mice and significantly prolonged survival. hPSC-derived CD33KO-Loop CAR-iNK cells possess unique advantages and translational potential for treating AML.

cancer biology↗

bioRxiv2025

Engineered CRO-CD7 CAR-NK cells derived from pluripotent stem cells avoid fratricide and efficiently suppress human T-cell malignancies

BackgroundT cell malignancies are highly aggressive hematological tumors with limited effective treatment options. CAR-NK cell therapy targeting CD7 has emerged as a promising approach for treating T-cell malignancies. However, conventional CAR-NK cell therapy faces the challenges of cell fratricide due to CD7 expression on both malignant cells and normal NK cells. Additionally, engineering CARs into human tissue-derived NK cells demonstrates heterogeneity, low transduction efficiency, and high manufacturing costs. MethodsThe human pluripotent stem cells (hPSCs) were genetically modified by knocking out the CD7 gene and introducing the CD7 CAR expression cassette to generate CD7 KO-CD7 CAR-hPSCs. These modified hPSCs were subsequently differentiated into CD7 KO-CD7 CAR-iNK cells using an efficient organoid induction method. The cytotoxicity of CD7 KO-CD7 CAR-iNK cells against CD7+ tumor cells was evaluated. Furthermore, we overexpressed the CXCR4 gene in CD7 KO-CD7 CAR-hPSCs and derived CXCR4-expressing CD7 KO-CD7 CAR-iNK (CRO-CD7 CAR-iNK) cells. The dynamics of CRO-CD7 CAR-iNK cells in vivo were tracked, and their therapeutic efficacy was assessed using human T-cell acute lymphoblastic leukemia (T-ALL) xenograft models. ResultsThe CD7 KO-CD7 CAR-iNK cells derived from CD7 KO-CD7 CAR-hPSCs effectively avoided fratricide, demonstrated normal expansion, and exhibited potent and specific anti-tumor activity against CD7+ T-cell tumor cell lines and primary T-ALL cells. CXCR4 overexpression in CRO-CD7 CAR-iNK cells improved their homing capacity and extended their persistence in vivo. The CRO-CD7 CAR iNK cells significantly suppressed tumor growth and prolonged the survival of T-ALL tumor-bearing mice. ConclusionsOur study provides a reliable strategy for the large-scale generation of fratricide-resistant CD7 CAR-iNK cells with robust anti-tumor effects from hPSCs, offering a promising cell product to treat T-cell malignancy.

cancer biology↗

bioRxiv2025

The Q226L mutation can convert a highly pathogenic H5 2.3.4.4e virus to bind human-type receptors

AbstractH5Nx viruses continue to wreak havoc in avian and mammalian species worldwide. The virus distinguishes itself by the ability to replicate to high titers and transmit efficiently in a wide variety of hosts in diverse climatic environments. Fortunately, transmission to and between humans is scarce. Yet, if such an event were to occur, it could spark a pandemic as humans are immunologically naive to H5 viruses. A significant determinant of transmission to and between humans is the ability of the influenza A virus hemagglutinin (HA) protein to shift from an avian-type to a human-type receptor specificity. Here, we demonstrate that a 2016 2.3.4.4e virus HA can convert to human-type receptor binding via a single Q226L mutation, in contrast to a cleavage-modified 2016 2.3.4.4b virus HA. Using glycan arrays, x-ray structural analyses, tissue- and direct glycan binding, we show that L133a{Delta} and 227Q are vital for this phenotype. Thus, whereas the 2.3.4.4e virus HA only needs a single amino acid mutation, the modified 2.3.4.4b HA was not easily converted to human-type receptor specificity.

microbiology↗

bioRxiv2025

AncestryHub: A web server for local ancestry analysis

SummaryAncestryHub is a web version of local ancestry analysis software. It contains four built-in software and a set of built-in population reference panels. AncestryHub will significantly reduce the efforts and requirements for the users on their personal computational skills and computational hardware environment for ancestry analysis. Availabilityhttps://www.ancestryhub.4dgenome.com Contact: lima@4dgenome.com

bioinformatics↗

bioRxiv2024

Cytosolic Carboxypeptidase 5 maintains mammalian ependymal multicilia to ensure proper homeostasis and functions of the brain

Ependymal multicilia position at one-side on the cell surface and beat synchronously across tissue to propel the flow of cerebrospinal fluid. Loss of ependymal cilia often causes hydrocephalus. However, molecules contributing to their maintenance remain yet fully revealed. Cytosolic carboxypeptidase (CCP) family are erasers of polyglutamylation, a conserved posttranslational modification of ciliary-axoneme microtubules. CCPs possess a unique domain (N-domain) N-terminal to their carboxypeptidase (CP) domain with unclear function. Here, we show that a novel mutant mouse of Agbl5, the gene encoding CCP5, with deletion of its N-terminus and partial CP domain (designated Agbl5M1/M1), developed lethal hydrocephalus due to degeneration of ependymal multicilia. Interestingly, multiciliogenesis was not impaired in Agbl5M1/M1 ependyma, but the initially formed multicilia beat in intercellularly diverse directions, indicative of aberrant tissue-level coordination. Moreover, actin networks are severely disrupted and basal body patches are improperly displaced in mutant cells, suggesting impaired cell level polarity. In contrast, Agbl5 mutants with disruption solely in the CP domain of CCP5 (Agbl5M2/M2) do not develop hydrocephalus despite increased glutamylation levels in ependymal cilia as similarly seen in Agbl5M1/M1. This study revealed an unappreciated role of CCP5, particularly its N-domain, in ependymal multicilia stability associated with their polarization and coordination.

cell biology↗