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Xu, J.

Publications and source records attributed to Xu, J..

At least 253 records · Page 14Linked to original sources
bioRxiv2025

Plug-and-play assembly of biodegradable ionizable lipids for potent mRNA delivery and gene editing in vivo

mRNA-based gene editing therapeutics offer the potential to permanently cure diseases but are hindered by suboptimal delivery platforms. Here, we devise a robust combinatorial chemistry for plug-and-play assembly of diverse biodegradable ionizable lipids and identify a lead candidate that produces superior lipid nanoparticles for various gene editing tools delivery in vivo. Our study highlights the utility of this synthetic approach and the generality of this platform for potent in vivo gene editing.

bioengineering↗
bioRxiv2025

Cryptochrome 4b protein is likely irrelevant for the radical pair based magnetoreception in the European robin

Avian cryptochrome 4 (Cry4) protein is a putative magnetosensitive molecule facilitating precise long-distance navigation in migratory birds. Two splice variants of Cry4 were reported in European robin (Erithacus rubecula), namely ErCry4a and ErCry4b. It is known that ErCry4a protein exhibits electron transfer between the flavin adenine dinucleotide (FAD) cofactor and tryptophan residues that generates magnetically sensitive radical pairs for magnetoreception. However, little is known about the ErCry4b isoform. We therefore characterized the properties of ErCry4b to see whether it fulfills prerequisites to be a radical pair magnetic sensor molecule. Our results show that ErCry4b protein does not bind FAD in vitro. Computational structure simulations revealed that the FAD non-binding in ErCry4b is likely due to protein structure dynamics. Furthermore, ErCry4b protein abundance in the robin retina, cerebellum and liver is below the detection limit of immunoprecipitation assays coupled with mass spectrometry. Meanwhile, transcript analyses show that ErCRY4b mRNA abundance is 10 times less than ErCRY4b in the retina. In conclusion, ErCry4b does not fulfill the prerequisites to be a radical pair based magnetic sensing molecule due to the lack of FAD binding, and it might not even be expressed as a functional protein in the European robin.

biochemistry↗
bioRxiv2025

Orthosteric interactions with PIP2 activate TMEM16A channels

TMEM16A channels pass Ca2+-activated Cl- currents that drive a plethora of fundamental physiological processes. TMEM16A channels are activated by a rise in intracellular Ca2+ levels but also require interactions with the signaling phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) to gate open. Although PIP2 is essential for the activity of many types of ion channels, its precise binding site and role in channel gating remain poorly understood in most cases, limiting efforts to study channel dynamics and design targeted modulators. In this study, we identify the PIP2 binding interactions that govern TMEM16A gating and permeation. Using a combination of gating molecular dynamics (GMD) simulations and electrophysiological assays, we reveal how the 4 helix of TMEM16A interacts with both the phosphate headgroups and acyl chains of PIP2 to open an electrostatic ring within the channel and stabilize the extracellular opening of the Cl- conduction pathway. These findings provide key insights into the dynamic role of PIP2 in membrane protein function and shed light on the activation mechanism of TMEM16A. This work establishes a framework for rational targeting of TMEM16A in drug development, with potential therapeutic applications in a variety of diseases.

biophysics↗
bioRxiv2025

Unveiling the Complete Spectrum of SARS-CoV-2 Fusion Stages by In Situ Cryo-ET

SARS-CoV-2 entry into host cells is mediated by the spike protein, which drives membrane fusion. While cryo-EM has revealed stable prefusion and postfusion conformations of the spike, the transient intermediate states during the fusion process have remained poorly understood. Here, we designed a near-native viral fusion system that recapitulates SARS- CoV-2 entry and used cryo-electron tomography (cryo-ET) to capture fusion intermediates leading to complete fusion. The spike protein undergoes extensive structural rearrangements, progressing through extended, partially folded, and fully folded intermediates prior to fusion-pore formation, a process that is dependent on protease cleavage and inhibited by the WS6 S2 antibody. Upon interaction with ACE2 receptor dimer, spikes cluster at membrane interfaces and following S2 cleavage concurrently transition to postfusion conformations encircling the hemifusion and pre-fusion pores in a distinct conical arrangement. Subtomogram averaging revealed that the WS6 S2 antibody binds to the spikes stem-helix, crosslinks and clusters prefusion spikes and inhibits refolding of fusion intermediates. These findings elucidate the complete process of spike-mediated fusion and SARS-CoV-2 entry, highlighting the neutralizing mechanism of S2-targeting antibodies.

microbiology↗
bioRxiv2025

Metagenomics reveals potential interactions between CPR bacteria and their phages in groundwater ecosystems

Candidate phyla radiation (CPR) is a vast lineage composed of bacteria with ultra-small size, streamlined genomes, notable defects in core metabolic potential and symbiotic lifestyle, which are widely detected in groundwater ecosystems. Increasing attention has focused on the physiological and ecological significance of CPR bacteria, while the potential interactions between CPR bacteria and their phages still need more exploration. Here, we collected 82 groundwater metagenomic datasets and further derived 1,162 phages with the potential to infect 2,439 groundwater CPR metagenome-assembled genomes (MAGs). Notably, the groundwater CPR MAGs were predominantly infected by temperate phages, and the abundance profiles of phage-CPR interactions in groundwater ecosystems supported the Piggybacking the Winner (PtW) hypothesis, suggesting that lysogenic infection was a strategy co-evolved by CPR bacteria and their phages in groundwater ecosystems. Intriguingly, the groundwater CPR phages encoded various auxiliary metabolic genes (AMGs) that might promote symbiotic lifestyle and metabolic potential of host CPR MAGs. These included AMGs associated with concanavalin A-like lectin/glucanases superfamily and O-Antigen nucleotide sugar biosynthesis, which could enhance surface adhesion of host CPR MAGs. Moreover, AMGs related to ABC transport system and P-type transporter could strengthen metabolic exchange and uptake of essential nutrients from surroundings. Additionally, AMGs involved in various metabolic pathways might alleviate metabolic deficiencies in host CPR MAGs. This study elucidated the mechanisms of phage-CPR interactions and the potential roles of phages in modulating the physiology and ecology of CPR bacteria within groundwater ecosystems.

microbiology↗
bioRxiv2025

Stress drives plasticity in leaf maturation transcriptional dynamics

Leaf development is dynamic, adapting to environmental stress to optimize resource use. For example, in response to drought, Arabidopsis restricts leaf growth to enhance water use efficiency. While this plastic response is well described at the physiological level, the underlying transcriptional changes that facilitate adjustments in leaf development in response to stress remain poorly described. By constructing a [~]1 million single-nuclei transcriptome atlas, we demonstrate that drought stress limits leaf growth by advancing transcriptional responses related to maturation and aging. Notably, we find that these transcriptional changes scale with environmental stress, and help explain how shoot size can decline proportionally with stress intensity. Leveraging these insights, we increased leaf growth under stress by cell-type targeted upregulation of FERRIC REDUCTION OXIDASE 6 in the mesophyll.

plant biology↗
bioRxiv2025

Noncanonical Wnt/Ror2 Signaling Regulates Basal Cell Fidelity and Branching Morphogenesis in the Mammary Gland

The mammary gland epithelium relies on a delicate balance between basal and luminal cell lineages to maintain tissue homeostasis and enable proper development. While the role of canonical Wnt signaling in mammary biology is well-established, the contribution of noncanonical Wnt signaling to lineage identity has remained unclear. Noncanonical Wnt pathways are primarily associated with morphogenesis, cytoskeletal regulation, and cell migration, but whether they are required for maintaining epithelial cell fate remains largely unexplored. Here, we demonstrate that the noncanonical Wnt receptor Ror2 is expressed in both basal and luminal lineages, yet selectively maintained in basal cells throughout development, suggesting a lineage-specific function. Using a p63CreERT2/+ lineage-specific mouse model, we show that Ror2 deletion in basal epithelial cells enhances secondary and tertiary branching while driving a basal-to-luminal fate transition, marked by downregulation of basal markers (K14, K5) and upregulation of luminal markers (K8, K18, ER). Mechanistically, Ror2 loss disrupts RhoA-ROCK1-YAP1 signaling, leading to cytoskeletal reorganization, chromatin remodeling, and increased accessibility at luminal regulatory loci. Notably, ROCK1 inhibition phenocopies Ror2 loss, reinforcing the critical role of the RhoA-ROCK1 axis in basal cell maintenance. These findings provide direct genetic and mechanistic evidence that noncanonical Wnt signaling is essential for maintaining basal lineage fidelity, offering new insights into the mechanisms regulating epithelial plasticity. Given the fundamental importance of lineage stability in epithelial homeostasis, our results suggest that disruptions in Wnt/Ror2 signaling may contribute to aberrant fate transitions relevant to breast cancer progression.

developmental biology↗
bioRxiv2025

Molecular basis of vitamin K-dependent protein γ-glutamyl carboxylation

Gamma-glutamyl carboxylase (GGCX) in human is the sole enzyme responsible for catalyzing the carboxylation of glutamate (Glu) to {gamma}-carboxyglutamate (Gla), a process that relies on the deprotonation of reduced vitamin K. This post-translational modification of vitamin K-dependent proteins (VKDPs) plays a pivotal role in numerous biological processes. Mutations in GGCX are responsible for multiple clinical phenotypes; however, the molecular mechanisms of GGCX activity and its pathology remain poorly understood. In this study, we reported the cryo-electron microscopy (cryo-EM) structures of GGCX bound with the either FIX or FX, two VKDPs and vitamin K, and employed the biochemical and cellular assays as well as molecular dynamics simulations to elucidate its dual-catalytic properties. The enzyme comprises a transmembrane domain that anchors the vitamin K-binding site, along with an ER luminal domain that facilitates binding of coagulation factor IX (FIX) at the exosite. The catalytic center orchestrates the oxidation of vitamin K and the carboxylation of Glu residues through a Cap-H2 coupling mechanism, along where disease-associated mutations in GGCX are predominantly clustered. These findings offer a comprehensive molecular framework for understanding the allosteric regulation of GGCXs dual enzymatic activities and provide valuable insights for developing strategies to monitor and therapeutically target vitamin K metabolism in related diseases.

biochemistry↗
bioRxiv2025

Structural basis for HIV-1 capsid adaption to rescue IP6-packaging deficiency

Inositol hexakisphosphate (IP6) promotes HIV-1 assembly via its interaction with the immature Gag lattice, effectively enriching IP6 within virions. During particle maturation, the HIV-1 protease cleaves the Gag polyproteins comprising the immature Gag lattice, releasing IP6 from its original binding site and liberating the capsid (CA) domain of Gag. IP6 then promotes the assembly of mature CA protein into the capsid shell of the viral core, which is required for infection of new target cells. Recently, we reported HIV-1 Gag mutants that assemble virions independently of IP6. However, these mutants are non-infectious and unable to assemble stable capsids. Here, we identified a mutation in the C-terminus of CA - G225R - that restores capsid formation and infectivity to these IP6-packaging-deficient mutants. Furthermore, we show that G225R facilitates the in vitro assembly of purified CA into capsid-like particles (CLPs) at IP6 concentrations well below those required for WT CLP assembly. Using single-particle cryoEM, we solved structures of CA hexamer and hexameric lattice of mature CLPs harbouring the G225R mutation assembled in low-IP6 conditions. The high-resolution (2.7 [A]) cryoEM structure combined with molecular dynamics simulations of the G225R capsid revealed that the otherwise flexible and disordered C-terminus of CA becomes structured, extending to the pseudo two-fold hexamer-hexamer interface, thereby stabilizing the mature capsid. This work uncovers a structural mechanism by which HIV-1 adapts to a deficiency in IP6 packaging. Furthermore, the ability of G225R to promote mature capsid assembly in low-IP6 conditions provides a valuable tool for capsid-related studies and may indicate a heretofore unknown role for the unstructured C-terminus in HIV-1 capsid assembly.

microbiology↗
bioRxiv2025

IQI: Individualized Quantitative trait loci (QTL) Index

There is currently no standard efficient method to precisely detecting and characterizing the difference of quantitative trait loci (QTL) effect among groups and/or populations at individual level. The main reason is that the traditional global regression based QTL analyses can only derive an overall effect of genetic variants on the quantitative trait in a specific group. It is a big challenge to obtain the individual-level QTL effect, which is necessary for accurate testing and precise characterization. Here, we first proposed an individualized QTL index (IQI) that represents the degree of regulation between genetic variants and the trait for each individual. Based on IQI, Differential QTL (DQ) analysis framework was presented to accurately detect the case-control differences in more detail. In addition, IQI can be a new feature/biomarker applied to various accurate bioinformatics analysis, such as differential analysis, disease prediction, and survival analysis. Overall, IQI affords a new perspective on pathogenic mechanisms and will facilitate the study of genetic regulation more detailedly and precisely.

bioinformatics↗
bioRxiv2025

CDKN2ALow cancer cells outcompete macrophages for microenvironmental zinc to drive immunotherapy resistance

Anti-PD1 therapies are primarily thought to rely on functional T cell responses; yet tumors with limited T cell infiltration can still benefit, suggesting alternative mechanisms contribute to therapeutic efficacy. Indeed, we found that myeloid-rich, T cell-poor tumor models respond to anti-Pd1, and this is dependent on a cancer cell-macrophage crosstalk mediated by cancer cell Cdkn2a expression. Mechanistically, we found that cancer cells with decreased Cdkn2a expression (Cdkn2aLow), which occurs in [~]50% of all human cancers, reorganize zinc compartmentalization by upregulating the zinc importer Slc39a9 at the plasma membrane. Increased cancer cell plasma membrane Slc39a9 leads to intracellular zinc accumulation in cancer cells and depletion of zinc in the tumor microenvironment (TME), resulting in zinc-starved tumor-associated macrophages (TAMs) with reduced phagocytic activity. Restoring zinc availability in TAMs--via dietary supplementation or Slc39a9 knockdown in cancer cells--reprograms TAMs to a pro-phagocytic state and sensitizes Cdkn2aLow tumors to anti-Pd1 therapy. Remarkably, Slc39a9 knockdown tumors respond to anti-Pd1 in Rag1-/- mice, and co-injection of zinc-replete macrophages is sufficient to drive an anti-Pd1 response in immunodeficient mice, demonstrating the T cell-independent nature of this response. Clinically, TAMs from CDKN2ALow cancer patients show reduced zinc and phagocytosis gene signatures. Moreover, patients with lower circulating zinc levels have significantly worse time-to-event outcomes than those with higher levels. Together, these findings uncover a previously unrecognized mechanism by which Cdkn2aLow cancer cells outcompete TAMs for zinc, impairing their function and limiting anti-Pd1 efficacy. They also provide evidence that macrophages alone, without T cells, can enhance anti-PD1 response through zinc-mediated reprogramming of phagocytosis.

cancer biology↗
bioRxiv2025

Microbes under climatic refugia: Self-stabilizing subcommunity rank dynamics in large-river deltaic estuaries

Microorganisms, akin to macroorganisms, can be sheltered by climatic refugia, overlooked for optimism about microbial extinction. By categorizing microbial communities into ecologically distinct subcommunities, we conducted a two-year survey of large-river deltaic estuaries (LDEs) which are regarded as refugial hotspots, and established rank hierarchies to monitor temporal changes. We found that, consistent with macroecology, LDEs harbor distinct microbial community structures, with the abundance distribution of subcommunities exhibiting resilience against environmental perturbations, persisting on an annual cycle. In particular, while the evenness of subcommunity rank abundance curves fluctuates frequently, shifts in rank order are less dynamic but, once initiated, have a more pronounced impact on subsequent rank dynamics. Our results suggest that the microbial subcommunity rank structure in LDEs is both persistent and robust, aligning with classic patterns of species abundance distributions. This study emphasizes that, despite differences in their roles, the stable functioning of microbial rank systems is also fundamental to the refugial capacity of ecosystems and should be given equal importance.

ecology↗
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

SMTdb: A comprehensive spatial meta-transcriptome resource in cancer

Microbes have been found in various tumors and the research of tumor microbiome has been garnering increased attention. However, it remains challenge to investigate the microbiome in cancer at spatial resolution. The rapid advent of spatially resolved transcriptomics techniques has given rise to map transcripts at single-cell resolution in various types of cancer. Here, we constructed a comprehensive spatial meta-transcriptome resource by manually curating 203 fresh frozen (FF) slices from 20 cancers encompassing 334,253 spots and 1,908,646 cells. SMTdb (http://bio-bigdata.hrbmu.edu.cn/SMTdb/) was constructed to provide comprehensive insights into the abundance, distribution and enriched TME regions of 1218 microbiota in spatial tissue slices. SMTdb enables vast interactive data exploration of spatial distribution and expression of microbiota, host gene modules associated with certain microbiota and co-occurrence between microbiota and immune cells within tumor microenvironment. The atlas resource serves as a one-stop and time-effective platform to investigate the interactions among microbial ecosystems and hosts in cancer.

bioinformatics↗
bioRxiv2025

Biosynthetic plasticity enables CD8+ T cell functional resilience under nutrient stress

Summary / AbstractTo maintain lineage-specific functions, cells must acquire and allocate nutrients across diverse cellular processes, even in metabolically-dysregulated environments. The mechanisms allowing CD8+ T cells to maintain immune function in perturbed environments are poorly understood. We find that CD8+ T cells adapt to nutrient stresses over time, reconfiguring gene-regulatory and metabolic networks to license functional recovery. Under acute stress, T cells reorient translational programming, limiting nutrient demand while prioritizing stress-sensitive metabolic and transcriptional responses. Within these responses, the transcription factors ATF4 and CEBPG jointly establish an adaptive metabolic program, promoting amino acid synthesis and uptake while maintaining mitochondrial anaplerosis. Despite diminished energetic capacity under environmental stress, this program prevents failure of central carbon metabolism, mitigating stress amplification and cellular dysfunction to potentiate anti-tumor immunity. Altogether, we demonstrate that biosynthetic plasticity via translational and metabolic reprioritization confers functional resilience to immune cells in unfavorable environments, offering novel strategies to enhance immunotherapies.

immunology↗
bioRxiv2025

The epigenetic factor Zrf1 regulates intestinal stem cell proliferation during midgut regeneration

Stem cells are essential for tissue maintenance and regeneration, balancing self-renewal and differentiation to support homeostasis and repair. Through an RNAi screen in the Drosophila midgut, we identified the epigenetic factor Zrf1 as a critical regulator of intestinal stem cell (ISC) proliferation. Functional analyses reveal that Zrf1 integrates inputs from multiple signaling pathways and interacts with components of the RNA-induced silencing complex (RISC). Zrf1 mutants exhibit elevated expression of transposable elements (TEs) and chromatin disruption, highlighting a broader role in genome stability. Single-nuclei RNA sequencing (snRNA-seq) further demonstrated the influence of Zrf1 on chromatin organization and TE repression, particularly within stem cell progenitors. Our findings suggest that Zrf1 is potentially a key chromatin regulator necessary for maintaining stem cell proliferation and genome integrity, enhancing our understanding of the molecular controls underlying stem cell function and chromatin dynamics.

genetics↗