Biology subjects

Deng, W.

Publications and source records attributed to Deng, W..

At least 19 recordsLinked to original sources

bioRxiv2026

Dynamic merging of enhancer- and promoter-associated assemblies is coupled to transcriptional bursting

Classical models attribute distal enhancer function to chromatin looping, whereas emerging models propose that spatially organized assemblies of transcriptional regulators also facilitate enhancer-promoter communication. To investigate how these processes relate to transcription, we developed an endogenous-tagging-based four-color live-cell imaging platform and an integrated quantitative analysis pipeline in mouse embryonic stem cells (mESCs), enabling simultaneous visualization of regulatory assembly dynamics, chromatin organization and transcriptional bursting at the endogenous Sox2 locus. We find that the pluripotency transcription factor OCT4 and the coactivator BRD4 form numerous spatially distinct but dynamically interacting assemblies. At non-bursting Sox2 alleles, the promoter and the distal Sox2 enhancer SCR occupy distinct assembly environments, with stronger BRD4 association at the SCR than at the promoter. At bursting Sox2 alleles, transcription is coupled to transient merging and partial overlap of enhancer- and promoter-associated assemblies, with the SCR, promoter and nascent RNA frequently sharing OCT4/BRD4-enriched assembly environments across a broad range of SCR-promoter distances. Pharmacological perturbation of bromodomain-dependent BRD4 function preferentially suppresses long-duration, high-amplitude bursts. Together, our findings support a model in which transcriptional bursting is coupled to the dynamic reorganization and merging of enhancer- and promoter-associated regulatory assemblies, linking chromatin-looping and assembly-based views of enhancer-promoter communication.

molecular biology↗

bioRxiv2026

A light-responsive lipid nanoparticle platform delivering Cas9 RNP for VEGFA editing suppresses choroidal neovascularisation

Choroidal neovascularisation (CNV) is a major cause of irreversible vision loss. Conventional anti-VEGF therapies for CNV often require repeated intravitreal injections, imposing substantial treatment burden and cumulative injection-related risk. Genome editing to disrupt VEGFA could provide durable suppression, potentially from a single administration. However, durability depends on permanently disrupting the target in a well-controlled way, as unrestricted VEGFA loss can cause choriocapillaris atrophy and compromise retinal function. This represents a major safety barrier to clinical translation. In this study, we developed lipid-based nanoparticles to release Cas9/sgRNA ribonucleoprotein (RNP) complexes under light illumination, enabling light-controlled editing of the VEGFA gene. The nanoparticles had a mean hydrodynamic diameter of approximately 130 nm with low polydispersity (0.296) and high sgRNA encapsulation efficiency (90.33%). In human retinal pigment epithelial cells, light exposure increased the extent of VEGFA disruption relative to the non-illuminated condition, as confirmed by targeted next-generation sequencing, with corresponding reductions in VEGFA mRNA and protein expression. Following a single intravitreal injection in a laser-induced rat CNV model, the nanoparticles combined with light illumination significantly reduced vascular leakage and CNV-associated structural abnormalities compared with both vehicle and non-illuminated controls, and ocular safety assessment revealed no apparent nanoparticle-related structural abnormalities in the retina. This study provides a foundation for spatially controlled VEGFA genome editing as a potential therapeutic strategy for VEGFA-driven macular neovascular disease.

bioengineering↗

bioRxiv2026

Lipid nanoparticle-mediated RNAi targeting of KRAS mutants sensitises colorectal cancer cells to chemotherapy

KRAS mutations, including G12D, G12V and G13D, are major oncogenic drivers in colorectal cancer (CRC) and are associated with therapeutic resistance. However, approved KRAS inhibitors remain focused on G12C subtype, leaving these prevalent variants without approved targeted therapies. Here, we developed a lipid nanoparticle (LNP)-based RNA interference (RNAi) strategy to target these three KRAS variants using a single small interfering RNA (siRNA) sequence. The developed siRNA-LNPs reduced KRAS mRNA and protein expression in HCT116 (G13D), SW620 (G12V) and LS180 (G12D) cells, but not in KRAS wild-type HT29 cells, suppressing the cellular proliferation, clonogenic survival and migration. Transcriptomic profiling revealed changes in KRAS associated signalling, including suppression of cell-cycle and DNA repair programmes, providing a rationale for combining siRNA silencing with the fluoropyrimidine chemotherapeutic agent 5-fluorouracil (5-FU). The combination produced synergistic effects across all three KRAS-mutant CRC cell models and enhanced the apoptosis in both monolayer cultures and 3D spheroids. Collectively, these findings provide proof of concept for this combination strategy and support its further investigation as a potential therapeutic approach for KRAS-mutant CRC.

molecular biology↗

bioRxiv2026

Cortex-wide single-cell activity mapping in freely moving mice

How the cortex supports complex, naturalistic behavior can be revealed only in freely moving animals. Although head-mounted microscopes promise access to the neural dynamics underlying such behavior, fundamental trade-offs among field of view, spatial resolution, device weight, and background contamination have prevented cortex-wide single-neuron recording, leaving distributed cortical coordination largely inaccessible. To meet these challenges, we introduce COMET (Cortex-wide Observational Miniature Epifluorescence Technique), a 3.75-g head-mounted mesoscope that integrates an aberration-controlled wide-field optical design, uniform high-efficiency excitation and single-coaxial power-and-data transmission with an end-to-end denoising and signal-extraction pipeline. This enables cortex-wide calcium imaging at single-cell resolution in freely moving mice. COMET captures activity from >15,000 layer 2/3 neurons per 5-min recording over fields of view up to 10 mm in diameter while preserving natural behavior. Benchmarking against benchtop two-photon microscopy demonstrates high spatial concordance of detected neurons and matched event-level calcium dynamics. We apply COMET to social interaction to quantify region-specific engagement and to an auditory trace fear-conditioning task that requires natural movement and behavioral expression, revealing time-ordered changes in regional firing-rate structure and functional connectivity preceding a global conditioned response. COMET provides an open, lightweight hardware-software framework for linking single-neuron activity to cortex-scale network dynamics during freely expressed behaviors under changing internal or external conditions.

neuroscience↗

bioRxiv2026

ERBB2 establishes the timing of progesterone priming required for uterine receptivity

Progesterone is essential for establishing uterine receptivity and sustaining early pregnancy. However, how the uterus acquires the competence to respond to progesterone at the appropriate time remains poorly understood. Here we show that ERBB2 governs the timing of progesterone responsiveness required for uterine receptivity. In endometrial samples from women with recurrent spontaneous abortion (RSA), ERBB2 is downregulated specifically in the stromal compartment, accompanied by a parallel reduction in stromal progesterone receptor (PGR). Uterine deletion of Erbb2 in mice blunts the rise of stromal PGR at the onset of the receptive window, deferring implantation and impairing decidualization despite normal circulating progesterone and estrogen. Epithelial deletion has no such effect, placing the requirement in the stroma. High-dose progesterone fails to rescue the phenotype; advancing progesterone priming by 24 hours restores stromal PGR, HAND2, Bmp2, and on-time implantation. Collectively, the present study provides evidence that ERBB2 directs the temporal window of stromal progesterone priming by setting the threshold for PGR induction, thereby synchronizing the maternal-fetal interface and safeguarding pregnancy.

developmental biology↗

bioRxiv2026

Reprogramming VHL with molecular glues enables selective degradation of caspase-2

Molecular glue degraders (MGDs) reprogram E3 ligases to eliminate neosubstrates, yet their application has largely been confined to CRBN. Here, we identify caspase-2 as a new neosubstrate for von Hippel-Lindau (VHL), expanding the scope of VHL-based MGDs. Guided by a focused VHL ligand library design, we employed TurboID-based proximity labeling to discover stereoisomeric compounds (dCASP2-1 and dCASP2-2) that selectively recruit caspase-2 to VHL and promote its ubiquitin-proteasome system-dependent degradation. Further structure-activity relationship (SAR) studies yielded dCASP2-3 and dCASP2-4, which enhanced degradation potency (by 622-fold relative to dCASP2-1) and abolished enantioselectivity. Mechanistic mapping localized the degrader-induced interface to a two-helix region of the caspase-2 CARD domain, with residues H33, P34, and D100 essential for VHL engagement. Degron-guided computational modeling of the VHL/MGD/caspase-2 ternary complex provided structural insight into neosubstrate recognition. Together, we report the development of VHL molecular glues that selectively and potently degrade caspase-2, offering chemical probes to interrogate its functions in apoptosis and stress responses, while broadening the substrate landscape of VHL-based MGDs.

biochemistry↗

bioRxiv2026

Virtual-cell verification enables self-auditing AI discovery for immune rejuvenation

Artificial-intelligence agents propose drug-discovery hypotheses faster than experiments can test them, yet their conclusions are rarely verified, against the underlying biology, the predicted perturbation, or the agent's own scoring logic. We close this verification gap with an agentic framework built on three verifiers. First, PACE, a phenotype verifier, resolves immune aging into ten directionally scored, cell-type-resolved gene-set modules, selected for cross-cohort stability across four PBMC cohorts, and outperforms five established aging clocks in an independent in-house aging cohort of 434 elderly donors. Second, CellQ, a virtual-cell verifier built with multi-modal LLM, compresses each single-cell transcriptome into eight discrete tokens aligned to a language model's vocabulary through residual vector quantization; it attains state-of-the-art perturbation prediction and uniquely resolves the weak, module-level shifts that differential-expression recovery misses. Third, an Analyzer-Planner-Auditor agent verifies its own scoring logic: screening 110 compounds in primary human PBMCs, it found aged-down modules more reversible than aged-up modules and revised its objective from an equal-weight mean to a balance-constrained minimum, a self-correction that generalized to an independent 13-compound T-cell assay. By verifying its predictions and its own objective against experiment, the framework points beyond hypothesis-generating AI toward self-correcting AI scientists whose objectives could continuously evolve.

bioinformatics↗

bioRxiv2026

New Protein Function Characterization for Human Paralog Discovery, Scraping the Bottom of the Genomics Barrel

Increasing the number of related protein paralogs is important for fully understanding protein relationships, yet it remains challenging for sequences in the twilight zone. Here, we present an integrated homolog detection framework that combines sequence-based (BLASTp, MMseqs2), structure-based (Foldseek), and embedding-distance-based (PROST) similarity metrics to identify additional paralogs. To characterize functionally related protein pairs, we develop protein-family-specific supervised logistic regression models trained on curated functional annotations from MEROPS proteases and KinHub kinases. The resulting model successfully classifies proteins, with ROC-AUC of 0.99 and F1-score of 0.92 for test datasets. Applying this model, we initially identify 686 protease and 298 kinase new candidates. Subsequent structural validation, and previous annotation comparisons yield 7 new protease and 3 new kinase paralogs in the human proteome, mostly lacking prior functional characterization. An additional outcome is structural identification of catalytically important residues for larger numbers of proteases and kinases. Despite the small number of new paralogs for well-studied proteases and kinases, our results demonstrate that integrating orthogonal homolog approaches with family-specific regression models provides a robust, scalable strategy for discovering new functionally related proteins, which is a generalizable approach for novel protein function discovery and can be applied more broadly to under-annotated proteomes.

genomics↗

bioRxiv2026

PABPC1 Modulates Immunoglobulin pre-mRNA Alternative Polyadenylation

Alternative polyadenylation is a mechanism by which cells tune gene expression, and dysregulation can lead to development of disease. PABPC1 has been implicated in poly(A) site selection, but its function in gene regulation remains contradictory and poorly defined. Here, we investigate its role in B cell development, where APA controls immunoglobulin secretion. To define this role, we mapped PABPC1-RNA interactions using CLAP-seq and perturbed PABPC1 expression using a degron based strategy. PABPC1 localizes to the 3UTR in 70% of its gene targets and primarily binds to A-rich regions. Integration with transcriptomic data suggests PABPC1 downregulates 60% of its gene targets. While transcriptome-wide shifts in 3 UTR length were limited, PABPC1 binding was specifically enriched in genes exhibiting significant 3 UTR shortening. Using a foundational genomics model, we find the PAS-proximal region is the most predictive of gene expression within PABPC1 binding sites. Positional analysis revealed PABPC1 localizes closer to the PAS in genes downregulated following depletion. In immunoglobulin transcripts, PABPC1 binds to both secreted and membrane isoforms and is more enriched at the secretory PAS, and depletion modestly alters immunoglobulin expression. Together, our findings demonstrate PABPC1 primarily shortens and downregulates its targets in a context dependent manner. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/720383v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@1a93633org.highwire.dtl.DTLVardef@257f55org.highwire.dtl.DTLVardef@1cad5bcorg.highwire.dtl.DTLVardef@19662fa_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2026

Cell Communication Network factor 4 promotes tumor-induced immunosuppression in breast cancer.

Cell Communication Network factor 4 (CCN4/WISP1) is a matricellular protein secreted by cancer cells that is upregulated in essentially all invasive breast cancers and promotes immunosuppression in melanoma. Recent work suggests that limited anti-tumor immunity also associates with poor patient outcomes in patients with breast cancer. Motivated by increased CCN4 correlating with dampened anti-tumor immunity in primary breast cancer, we test for a direct causal link by knocking out CCN4 (CCN4 KO) in the Py230 and Py8119 mouse breast cancer models. Tumor growth is reduced when CCN4 KO breast cancer cells are implanted in immunocompetent but not in immunodeficient mice. Correspondingly in size-matched tumors, CD4+ and CD8+ T cells are significantly increased in CCN4 KO tumors while the myeloid compartment is shifted from polymorphonuclear- to monocytic-myeloid-derived suppressor cells (MDSC). This shift in the MDSC compartment is also associated with a significant reduction in splenomegaly. Among mechanisms linked to local immunosuppression, CCN4 knockout has a similar impact on the secretome of both breast cancer and melanoma cell lines. Overall, our results suggest that CCN4 promotes tumor-induced immunosuppression in the context of breast cancer and is a potential target for therapeutic combinations with immunotherapies.

cancer biology↗

bioRxiv2025

RAPMS 2.0 Improves Specificity and Throughput for Proteomic Identification of RNA Binding Proteins

RNA-protein complexes are critical factors in development, homeostasis, and disease. RNA proteomics methods are essential for characterizing these complexes but suffer from high levels of background, which hinders identification of ribonucleoprotein (RNP) components. Here, we present RNA Antisense Purification followed by Mass Spectrometry 2.0 (RAP-MS 2.0), an updated version our original RAP-MS protocol with innovations in bead preparation, RNA capture, and peptide purification. RAP-MS 2.0 has lower background than our original protocol, and allows lysate to be reused to capture multiple RNAs. We demonstrate that RAP-MS 2.0 recapitulates known RNPs for 7SL, 7SK, RMRP, U1, U2, U6, U7, and Xist. Additionally, we use RAP-MS 2.0 to identify novel RNA-protein interactions between Xist and TREX components and U1 with FET family transcriptional regulators.

molecular biology↗

bioRxiv2025

VRC01 Selects Rare HIV Escape Mutations After Acquisition in Antibody-Mediated Prevention Trials

Broadly neutralizing antibodies (bnAbs) show promise in HIV prevention, yet viral escape remains a challenge. In the Antibody Mediated Prevention (AMP) trials, the CD4 binding site (CD4bs) bNAb VRC01 blocked acquisition by VRC01-sensitive strains. However, its influence on viral evolution post-acquisition is not fully understood. Here we analyzed >12,000 HIV env sequences from 47 participants from the AMP trials, identifying VRC01-mediated de novo escape mutations in 8 of 26 VRC01-treated participants but none in 21 placebo participants. These mutations were found at very low frequency (<1%) in global viruses. Escape mutations, primarily located in the Loop-D and {beta}23/V5 regions of Env, conferred cross-resistance to several CD4bs bnAbs, while more potent CD4bs bnAbs like N6 and 1-18 largely retained their activity. Our findings demonstrate that prophylactic VRC01 can select for viral escape after infection, underscoring the need for next-generation bnAbs with improved breadth and potency to enhance durability and efficacy of antibody-based HIV prevention.

microbiology↗

bioRxiv2025

Uterine stromal Erbb3-Igf1 signaling is critical to functional gland development conducive to implantation

Preimplantation uterine gland differentiation, involving glandular branching, is a prerequisite for secretion of leukemia inhibitory factor (LIF), thereby establishing uterine receptivity to embryo implantation. This differentiation process is disrupted by the absence of FOXA2, a transcription factor specifically present in uterine glands. It remains unclear whether preimplantation glandular differentiation is regulated exclusively by gland-intrinsic mechanisms or is influenced by the surrounding microenvironment through mesenchymal-epithelial interactions. We show here that uterine deletion of Erbb3 leads to impaired gland branching and FOXA2 expression, as well as reduced female fertility. In contrast, females with uterine epithelial-specific deletion of Erbb3 show normal fertility, suggesting a critical role of stromal ERBB3 signaling. A transcriptomic profiling of day 3 stromal cells identified Igf1 as a candidate paracrine factor mediating the stromal-epithelial interaction. Thus, the uterine deletion of Igf1r leads to a reduced gland branching phenotype similar to that observed in Erbb3d/d uteri. These results demonstrate that ERBB3 regulates glandular branching and embryo implantation via the ERBB3-IGF1-IGF1R signaling pathway.

developmental biology↗

bioRxiv2025

Large Numbers of New Human Paralogs Discovered

The identification of paralogs is critical for understanding protein evolution, function, and for drug design, yet many human proteins remain unannotated and poorly classified. Sequence-based homology detection alone often fails to detect distant paralogs, especially in the "twilight zone" or beyond, regarding sequence identity. Here we present an integrated homolog detection framework that combines results from BLASTp, MMseqs2, Foldseek, and the large protein language model-based tool PROST, followed by validation based on comparison of structures and for enzymes comparison of the specific structures of the catalytic residues. Using all-versus-all exhaustive comparisons across the 20,647 human proteins, we systematically identify novel paralogs and assess their catalytic residues for two serine protease clans. We discovered 14 previously uncharacterized human serine carboxypeptidases, validated against experimentally determined PDB structures, with 11 of these displaying conserved catalytic triads. We further identify 203 new paralogs for human kinases, with 163 of these in the major clusters that represent previously uncharacterized kinase subtypes and 30 putative novel human transcription factors. Across both serine protease subtypes, structural alignments enable the prediction of the previously unknown catalytic residues for those lacking UniProt annotations of active site residues. By integrating sequence, structure, and LPLM embedding-based approaches, the framework enables the discovery of surprisingly large numbers of unknown paralogs, permitting defining catalytic residues, and expands the understanding of protein functional landscapes. These findings provide the foundation for a large number of future functional, evolutionary, and therapeutic investigations.

genomics↗

bioRxiv2025

Long-Read Deep Sequencing Reveals High Rates of Multilineage Transmission and Rapid Viral Population Changes in Acute HIV Infection

Understanding the selective forces acting upon HIV early in infection is crucial to design prevention strategies. By leveraging deep sequencing and the short diagnostic intervals of the FRESH and RV217 cohorts (median 4 days) between the last-negative and first-positive RNA tests, we captured a precise and early snapshot of acute HIV infection. The frequency of multiple transmitted viruses of 38% in these as well as placebo recipients from the AMP trials was higher than previously published, with the true frequency likely to be higher. The relative abundance of lineages fluctuated substantially over time in two-thirds of the multilineage infections, generating uncertainty in identifying the specific viruses that were transmitted and founding the infection. Viral populations exhibited diversity and selection on the Gag and Env proteins at the earliest times examined, with sites inferred to be undergoing negative selection most evident. These data may help explain vaccination failures and provide new targets for prevention.

microbiology↗

bioRxiv2025

Enteropathogenic Escherichia coli manipulates the host exocyst complex to enhance pedestal formation

Enteropathogenic Escherichia coli (EPEC), a major cause of diarrheal disease, produces plasma membrane pedestals that promote colonization of host cells. Critical for pedestal formation is EPECs type III secretion system, which injects [~] 20 effector proteins into human cells. One of these effectors is Tir, which inserts into the host plasma membrane and stimulates the assembly of actin filaments essential for pedestal generation. To date, actin polymerization is the only host process known to contribute to pedestal formation. Here we report that EPEC co-opts the membrane trafficking pathway of polarized exocytosis, which acts together with actin polymerization to allow the efficient production of pedestals. Polarized exocytosis is mediated by the exocyst -- a human octameric complex that uses intracellular vesicles to expand the plasma membrane. We found that EPEC stimulated exocytosis at sites of pedestal formation in a manner dependent on the exocyst. The bacterial effector EspH recruited the exocyst and promoted exocytosis. Genetic inactivation of espH or RNA interference (RNAi)-induced depletion of exocyst components reduced both the frequency and size of pedestals. Additional RNAi experiments indicated that the exocyst is dispensable for actin filament assembly in pedestals. Co-depletion of components of the exocyst and the Arp2/3 complex showed that exocytosis and actin polymerization make additive contributions to pedestal formation. Collectively, these results indicate that exocyst-mediated expansion of the plasma membrane acts together with actin polymerization to optimize the generation of pedestals. IMPORTANCEEnteropathogenic E. coli (EPEC) induces the formation of host cell plasma membrane protrusions called "pedestals" that mediate tight adherence to intestinal epithelial cells. Substantial evidence indicates that the generation of pedestals requires bacterial-induced polymerization of the host cell actin cytoskeleton. However, it remains unknown if EPEC manipulates other human processes to augment the efficiency of pedestal generation. The significance of this research is to demonstrate that EPEC co-opts a second host process called polarized exocytosis to increase the frequency of pedestals and the size of these structures. EPEC induces exocytosis by using its effector protein EspH to recruit the human exocyst complex, which mediates the insertion of membrane at sites of bacterial attachment. Our results demonstrate EPECs ability to coordinate host membrane trafficking with cytoskeletal changes to enhance infection. Remarkably, EPEC shares this strategy with the intracellular bacterial pathogens Listeria monocytogenes and Shigella flexneri.

microbiology↗

bioRxiv2025

Trsp is required by regulatory T cells to prevent lethal autoimmunity in mice

Selenoproteins are involved in immune cell metabolism, yet the roles of these proteins in T cell development and function remain largely unknown. The Trsp gene encodes the selenocysteine tRNA (tRNASec) required for translation of all selenoproteins. In this study, we found that Trsp was required for thymopoiesis, with the majority of tRNASec-deficient T cells not progressing beyond double negative 3 stage, with egressed thymocytes undergoing peripheral homeostatic expansion. Trsp-deficient CD4+ T cells exhibited impairments in TCR and IL-2 signaling and did not cause inflammation in experimental models. On the other hand, Trsp-deficient regulatory T (Treg) cells exhibited defects in suppressive function ex vivo and Treg-specific Trsp deletion using Trspfl/flFoxp3YFP-Cre (Trsp!{iota}Treg) mice caused fatal autoimmunity similar to FOXP3-deficient mice. Reducing oxidative stress via 2-HOBA administration prolonged survival in these Trsp!{iota}Treg mice. These findings indicate that tRNASec is required for T cell homeostasis and may be therapeutic targets in inflammation. One sentence summaryTrsp, a gene required for translation of all selenoproteins, is essential for all T cell development and function, especially regulatory T cells.

immunology↗

bioRxiv2025

CASP3-GSDME-mediated trophoblast pyroptosis contributes to systemic inflammation

Early-onset preeclampsia (EOPE) is associated with excessive apoptosis and inflammation, but the mechanistic link between these processes remains enigma. Here, we report elevated circulating pro-apoptotic proteins in EOPE patients at early pregnancy, along with concurrent CASP3 activation and GSDME cleavage in a subset of EOPE placentas. Using multiple trophoblast cell lines, we demonstrate that trophoblast cells, which highly express GSDME, undergo a shift from apoptosis to CASP3-dependent pyroptosis, driving inflammation. Notably, pyroptotic trophoblasts further induce pro-inflammatory macrophage polarization within placental villi organoids, establishing a feedback loop that amplifies both trophoblast pyroptosis and inflammatory responses in trophoblast organoids-macrophage assembloids. In vivo, CASP3-GSDME-mediated trophoblast pyroptosis contributes to systemic inflammation in wild-type pregnant mice but not in Gsdme-/- mice. Screening of EOPE prevention drugs reveals Vitamin D as a suppressor of GSDME activation and pyroptosis in trophoblast cells. These findings highlight the CASP3-GSDME axis as a promising therapeutic target for preeclampsia prevention.

cell biology↗