Biology subjects

Shen, J.

Publications and source records attributed to Shen, J..

At least 55 records · Page 3Linked to original sources

bioRxiv2026

OSTM1 is a ubiquitin E3 ligase that suppresses B-cell malignancy by activating the cAMP/PKA/CREB pathway

Osteoclastogenesis-associated transmembrane protein 1 (OSTM1) is a glycosylated, membrane-integral protein that regulates lysosomal homeostasis, with loss-of-function mutations causing autosomal recessive osteopetrosis. Through a whole-genome CRISPR/Cas9 screen, we identified OSTM1 as a previously unrecognized tumor suppressor in B-cell malignancies. Consistent with this role, OSTM1 is frequently deleted or downregulated across a broad spectrum of human B-cell cancers. In mice, B-cell-specific monoallelic or biallelic ablation of Ostm1 cooperates with Cdkn2a loss to drive lymphomagenesis with near-complete penetrance. Mechanistically, we uncover a cytosolic, non-glycosylated fraction of OSTM1 that functions as a ubiquitin E3 ligase to promote proteasomal degradation of phosphodiesterase 3B (PDE3B). As PDE3B hydrolyzes cAMP and suppresses the tumor-protective PKA/CREB/CREBBP signaling axis, loss of OSTM1 leads to PDE3B stabilization, attenuation of cAMP signaling, and enhanced oncogenic transformation. Together, our findings establish OSTM1 as a critical suppressor of B-cell lymphomagenesis through regulation of the cAMP/PKA/CREB pathway.

cancer biology↗

bioRxiv2026

Development of Integrin α5β1-targeted PET/NIR imaging probes for glioblastoma intraoperative navigation and intracavity targeted radionuclide therapy

Incomplete extent of tumor resection is the major factor of glioblastoma recurrence, leading to its poor prognosis. Developing therapies that enable the minimal tumor cell remnant during surgical resection is a major challenge to combat this aggressive cancer. This study develops a glioblastoma integrin 5{beta}1-selective peptide binder through series strategy screening and labeling it with 68Ga, indocyanine green (ICG), or 177Lu for efficacious multimodal treatment. This strategy combines positron emission tomography (PET) imaging for preoperative diagnosis, intraoperative NIR-II fluorescence-guided surgery for maximal tumor resection, and followed by intracavity targeted radionuclide therapy for elimination of residual tumor cells. We identified GR, with five arginine mutation, exhibiting superior integrin 5{beta}1 binding affinity and brain penetration. [68Ga]GR demonstrated substantial tumor uptake and prolonged retention time in both mouse models and tissues from glioblastoma patients. The guidance of ICG-GR achieved accurately intraoperative tumor imaging and maximal tumor resection in orthotopic mouse glioblastoma models. Notably, combining intracavity administration of [177Lu]GR following ICG-GR guiding resection markedly inhibited tumor growth and reduced possibility of tumor recurrence compared with surgery alone (unguided or ICG-GR-guided) or unguided surgery followed by [177Lu]GR. Therefore, we have reported the integrin 5{beta}1-targeted radionuclide and optical agents for maximal elimination of tumor cells during surgical resection in glioblastoma patients. HIGHLIGHTSO_LIGR is a glioblastoma-specific peptide with both favorable binding affinity superior brain penetration. C_LIO_LIICG-GR is a glioblastoma-targeted NIR probe enabling maximal surgical resection. C_LIO_LIICG-GR-guided surgery with [177Lu]GR-based adjuvant therapy allows robust elimination of tumor cells. C_LI

cancer biology↗

bioRxiv2026

AutoPELSA: an automated sample preparation system for proteome-wide identification of target proteins of diverse ligands

Protein-ligand interactions are fundamental to cellular function and drug discovery, and ligand-modification-free strategies have emerged as powerful tools for proteome-wide interrogation of these interactions. Among these, the peptide-centric local stability assay (PELSA) stands out for its high sensitivity. It uses a single digestion step to detect ligand-induced local stability shifts, enabling precise binding region localization and affinity estimation. However, manual PELSA workflows suffer from multiple labor-intensive steps that introduce variability and constrain throughput, limiting large-scale applications. To address these limitations, we developed AutoPELSA, an automated platform that streamlines the PELSA workflow including the limited proteolysis step and the following peptide separation step. AutoPELSA completes 96 samples in [~]4 h, enabling high-throughput analysis under native conditions. AutoPELSA reliably detected targets of both strong-affinity ligands (e.g., staurosporine, identifying 114 kinase targets) and low-affinity ligands, including -ketoglutarate (30 known targets). Furthermore, a mixed-ligand dose-response strategy enabled simultaneous determination of binding affinities for multiple ligand-protein interaction regions in a single experiment. Overall, AutoPELSA provides a scalable and modification-free platform for proteome-wide identification and affinity profiling of ligand-protein interactions.

molecular biology↗

bioRxiv2026

Leveraging Human Pangenome for Improved Somatic Variant Detection

Somatic variant detection is technically challenging due to low variant allele fractions, the confounding presence of germline variation, and reference bias. Linear references such as GRCh38 miss sample-specific variation, causing misalignments and incorrect variant calls. Although telomere-to-telomere donor-specific assemblies (DSAs) accurately represent individual genomes, their application is limited by cost and technical barriers. Alternatively, the graph-based human pangenome provides a scalable framework to improve read alignment and perform genome inference. Here, we benchmarked somatic variant detection using GRCh38, graph-based pangenomes, and pangenome-inferred DSAs with a HapMap mixture dataset and the COLO829 melanoma cell line. Pangenome-guided alignment improves read mapping and somatic variant calling accuracy. Furthermore, personalized pangenomes partially reconstruct donor-specific genomic content, improving accuracy, reducing germline contamination, and enabling detection of events in loci absent or poorly represented in GRCh38. These findings demonstrate that graph-based and personalized pangenomes are effective strategies for enhancing somatic variant detection compared with GRCh38.

genomics↗

bioRxiv2025

In situ chromatin dynamics and HIV-1 nuclear trafficking

The organization and dynamics of chromatin within intact nuclei remain poorly defined, limiting understanding of how nuclear architecture influences macromolecular transport. HIV-1 provides a striking example of a large complex that must traverse this dynamic landscape to access integration sites within transcriptionally active regions. Here, we combine a functional nuclear import system with correlative cryo-electron tomography to visualize chromatin architecture and HIV-1 cores in situ during nuclear penetration. Native nucleosomes resolved at 5.6 [A] reveal four structural classes, from compact conformations at the nuclear periphery to open, flexible forms in the interior, defining a spatially heterogeneous and dynamic chromatin landscape. Within this environment, HIV-1 cores decorated with nuclear factors exclude nearby nucleosomes and preferentially associate with open chromatin. Disruption of CPSF6-capsid interactions abolishes these associations, confining viral cores to peripheral chromatin. These findings establish an in situ framework linking chromatin structure and dynamics to HIV-1 nuclear trafficking and provide a structural basis for viral navigation within the dynamic nuclear landscape.

microbiology↗

bioRxiv2025

Insights into the AAV packaging mechanism: Cryo-EM Structure of the AAV2 Rep-Capsid Packaging Complex

The Adeno-associated virus (AAV) has become the most used viral vector for gene therapy applications to treat monogenic diseases, with over 250 clinical trials and six FDA-approved biologics. AAV has a single-stranded DNA genome encapsulated in a 60-subunit icosahedral protein shell. Assembly of empty capsids occurs in the nucleus, and in a subsequent step, the genome is packaged by the motor activity of the AAV Rep proteins. The translocation of ssDNA has been suggested to occur through one of the twelve channels at the fivefold symmetry axis. While there is substantial evidence that Rep proteins directly interact with the capsid, the specific molecular determinants, stoichiometry, and translocation mechanism remain unknown. To understand how Rep proteins assemble on the capsid, we examined Rep-capsid complexes using cryo-electron microscopy with single-particle reconstruction. Our results show that Rep proteins can assemble into the capsid fivefold pore as either pentameric or hexameric rings. The different ring complexes dock into the pore similarly, using the post-sensor1 {beta}-hairpin motif (pos1{beta}h2) as a capsid interaction module. This interaction induces the folding of a segment in the capsid HI loop, resulting in an expansion of the pos1{beta}h2 {beta}-sheet. Our structures show that any of the AAV Rep proteins can interact with the capsid, and this mode of interaction may be a conserved mechanism across all parvoviruses. Collectively, our results provide insights into the mechanism of AAV genome encapsidation and could inform strategies to improve recombinant AAV packaging efficiency.

biochemistry↗

bioRxiv2025

GatorSC: Multi-Scale Cell and Gene Graphs with Mixture-of-Experts Fusion for Single-Cell Transcriptomics

Single-cell RNA sequencing (scRNA-seq) enables high-resolution characterization of cellular heterogeneity, but its rich, complementary structure across cells and genes remains underexploited, especially in the presence of technical noise and sparsity. Effectively leveraging this multi-scale structure is essentially an information fusion problem that requires integrating heterogeneous graph-based views of cells and genes into robust low-dimensional representations. In this paper, we introduce GatorSC, a unified representation learning framework that models scRNA-seq data through multi-scale cell and gene graphs and fuses them with a Mixture-of-Experts architecture. GatorSC constructs a global cell-cell graph, a global gene-gene graph, and a local gene-gene graph derived from neighborhood-specific subgraphs, and learns graph neural network embeddings that are adaptively fused by a gating network. To learn noise-robust and structure-preserving embeddings without labels, we couple graph reconstruction and graph contrastive learning in a unified self-supervised objective applied to both cell- and gene-level graphs. We evaluate GatorSC on 19 publicly available scRNA-seq datasets covering diverse tissues, species, and sequencing platforms. Experiments showed that GatorSC consistently outperforms state-of-the-art deep generative, graph-based, and contrastive methods for cell clustering, gene expression imputation, and cell-type annotation. The learned embeddings are used for accurate trajectory inference, recovery of canonical marker gene programs, and cell-type-specific pathway signatures in an Alzheimers disease single-nucleus dataset. GatorSC provides a flexible foundation for comprehensive single-cell transcriptomic analysis and can be readily extended to multi-omic and spatial modalities.

bioinformatics↗

bioRxiv2025

Colorectal cancer relies on an immunosuppressive cellular topography and genomic adaptations for establishing brain metastases

Colorectal cancer (CRC) brain metastases have a poor prognosis and limited treatment options, including resistance to radiation therapy. Little is known about the molecular and cellular mechanisms that enable CRC tumor cells to adapt to the brain and establish a supportive tumor microenvironment. To address this gap we used spatial transcriptomics to analyze 51 CRC brain metastases. A subset had matched primary colon tumors and longitudinally paired metastatic resections before and after radiation treatment. We identified the critical spatial cellular features of the tumor epithelium and the surrounding tumor microenvironment that support metastatic growth in the brain. CRC brain metastases developed a stromal microenvironment with abundant fibroblasts and tumor-associated macrophages. A fibroblast-macrophage cellular neighborhood promoted angiogenesis, extracellular matrix remodeling, and immune suppression. Tumor cells showed local adaptations. In endothelial-rich regions, they were proliferative whereas in macrophage-rich regions, they were more differentiated and immune evasive. Compared with paired primary tumors, CRC brain metastases showed increased chromosomal instability, with activation of RNA-processing, stress response, and junctional remodeling pathways. After radiation treatment, resistant clones had increased epithelial-mesenchymal transition, while the immunosuppressive stroma remained intact. We identified tumor-derived MIF, GDF15, PRSS3 and SEMA3C ligands and macrophage-derived SPP1 that have the potential to affect multiple cell types in the metastatic niche. These ligand-receptor interactions drive angiogenesis, stromal activation and immune suppression. In a macrophage-tumor-fibroblast co-culture model, knockout of SPP1 in macrophages led to reduced expression of lipid-metabolism related genes and disrupted tumor-promoting interactions. Together, these results indicate that CRC growth in the brain is sustained by a specific cellular organization with immunosuppressive multicellular interactions.

cancer biology↗

bioRxiv2025

Live visualization of extracellular matrix dynamics during development and regeneration in zebrafish

Extracellular matrix (ECM) plays fundamental roles in animal development, regeneration, and disease. The difficulty of tagging endogenous matrix proteins in vertebrates has limited the understanding of ECM composition and dynamics in complex tissues. To visualize vertebrate ECM components, we tagged zebrafish Laminin, gamma 1 (Lamc1), Collagen, type I, alpha 2 (Col1a2), and Transforming growth factor, beta-induced (Tgfbi) using C-terminus in-fusion genome editing. Analysis of these knock-in lines revealed distinct expression of each protein in various tissues during development and regeneration. Fluorescent recover after photobleaching (FRAP) analysis further indicated that Lamc1 is stable in fin fold matrix but more dynamic in myoseptal matrix of developing zebrafish, while Col1a2 and Tgfbi are stable matrix components in myosepta. Strikingly, we found that Col1a2-mScarlet protein accumulates at the amputation plane during tailfin regeneration, where it remains concentrated for several days and distant from the regeneration blastema. This "foundation" region also displayed a distinct transcriptome suggesting active and dedicated events at the base of the regenerating appendage. Our resource enables live capture of ECM dynamics that can identify new events in developing and regenerating zebrafish. Summary statementExtracellular matrix resources for zebrafish development and tissue regeneration.

developmental biology↗

bioRxiv2025

A biased allosteric modulator functions as a molecular glue to induce β2AR dimerization

Family A G-protein coupled receptors (GPCRs) are typically described as monomers, yet growing evidence suggests they can form dimers with distinct signaling properties1-3. The mechanisms and therapeutic potential of such dimerization, however, remain poorly understood. Here, we show that AP-7-168, an optimized derivative of a {beta}-arrestin-biased negative allosteric modulator of the {beta}2-adrenergic receptor ({beta}2AR) that sustains bronchorelaxation in cell and tissue models4, functions as a molecular glue to promote {beta}2AR homodimerization. Cryo-EM structures reveal a unique binding mode in which two AP-7-168 molecules pack within a pocket formed by transmembrane helices 3, 4, and 5 of two protomers, stabilizing a dimeric conformation that selectively prevents {beta}-arrestin coupling. In cells, AP-7-168 robustly induces {beta}2AR dimerization and drives enlarged nanocluster formation. Combined with extensive functional studies, our findings unveil a novel allosteric mechanism by which a small molecule biases {beta}2AR signaling through dimerization, highlighting ligand-induced dimerization as a strategy for GPCR modulation.

biochemistry↗

bioRxiv2025

Mass spectrometry-based proteomic and glycoproteomic drafts of fifteen human body fluids

The study of proteins and N-glycosylation in human body fluids (HBFs) is crucial for understanding both physiological and pathological states, offering valuable insights for disease diagnostics and monitoring. In this study, we present a comprehensive proteomics and glycoproteomics dataset featuring 15 types of precious HBFs (plasma (P), urine (U), cerebrospinal fluid (CSF), pleural fluid (PF), ascitic fluid (AF), synovial fluid (SF), pericardial fluid (PCF), peritoneal dialysis effluent (PDE), bile (B), gastric juice (GJ), seminal plasma (SP), saliva (SA), pancreatic juice (PJ), sweat (SW), and tear (T)). We also provide detailed protocols for protein processing specific to each type of HBF. The resulting peptides and enriched intact N-glycopeptides were analyzed via liquid chromatography-tandem mass spectrometry (LC-MS/MS) in data independent acquisition (DIA) and combined electron transfer/higher-energy collisional dissociation and stepped collision energy/higher-energy collisional dissociation (EThcD-sceHCD) mode, respectively. The resulting spectra and searched files have been uploaded to the online repository iProX (www.iprox.cn) and made easily accessible (PXD068799) to facilitate further research into HBF proteomics and glycoproteomics.

systems biology↗

bioRxiv2025

ALM enables contextual decision-making via dynamic reconfiguration of local circuits

Cognitive operations often require flexible implementation of stimulus-response contingencies, depending on context. We developed an olfactory task in which mice learned to associate a test odor with a directional lick response, conditional on a preceding context odor drawn from a different odor set. Two-photon imaging revealed that anterior lateral motor cortex (ALM) contains distinct populations encoding context, test odors, and choice. Optogenetic silencing during the context and delay periods impaired performance, suggesting that ALM contributes to configuring the appropriate contingency. Although context odors that instructed the same mapping were represented by separate populations, their influence converged at the level of choice-selective neurons. A subpopulation of these neurons exhibited dual selectivity for context and choice, forming what we term "contingency neurons." These findings suggest that ALM supports flexible behavior not by abstracting over context cues, but by dynamically reconfiguring local circuits to route sensory input to the appropriate motor output.

neuroscience↗

bioRxiv2025

Polyclonal origins of human premalignant colorectal lesions

Cancer is generally thought to be caused by expansion of a single mutant cell1. However, analyses of early colorectal cancer lesions suggest that tumors may instead originate from multiple, genetically distinct cell populations2,3. Detecting polyclonal tumor initiation is challenging in patients, as it requires profiling early-stage lesions before clonal sweeps obscure diversity. To investigate this, we analyzed normal colorectal mucosa, benign and dysplastic premalignant polyps, and malignant adenocarcinomas (123 samples) from six individuals with familial adenomatous polyposis (FAP). Individuals with FAP have a germline heterozygous APC mutation, predisposing them to colorectal cancer and numerous premalignant polyps by early adulthood4. Whole-genome and/or whole-exome sequencing revealed that many premalignant polyps--40% with benign histology and 28% with dysplasia--were composed of multiple genetic lineages that diverged early, consistent with polyclonal origins. This conclusion was reinforced by whole-genome sequencing of single crypts from multiple polyps in additional patients which showed limited sharing of mutations among crypts within the same lesion. In some cases, multiple distinct APC mutations co-existed in different lineages of a single polyp, consistent with polyclonality. These findings reshape our understanding of early neoplastic events, demonstrating that tumor initiation can arise from the convergence of diverse mutant clones. They also suggest that cell-intrinsic growth advantages alone may not fully explain tumor initiation, highlighting the importance of microenvironmental and tissue-level factors in early cancer evolution.

cancer biology↗

bioRxiv2025

Illuminating the Druggable Proteome with an AI Protein Profiling Platform

Most human proteins lack chemical probes or pharmaceutical modulators, leaving much of the proteome unexplored. 1,2 Activity-based protein profiling has enabled proteome-scale discovery of protein-ligand interactions and covalent inhibitors, 3-5 but remains limited by probe chemistry, protein abundance, and discordant ligandability assignments across studies. 6-9 Machine-learning (ML) models can in principle generate proteomewide ligandability maps, but the predictive utility of current models is limited by the requirement for structures and the use of incomplete and weak training labels. 9-12 Here we developed an artificial intelligence protein profiling (AiPP), a sequence-based multitask platform built on the ESMC protein language model, 13,14 to accelerate proteomewide therapeutic discovery and target identification. While its primary task is identification of covalently ligandable cysteines, seven additional task heads and two external modules provide broader context by annotating reversible ligand-binding residues, disordered molecular recognition features, cysteine functional context, and reactivities. Central to the development is the LatentLift clustering approach, which leverages latent space similarities to reconcile conflicting experimental labels and facilitate model training. Applied to the human proteome, AiPP generated a cysteine-directed ligandability atlas that overcomes the limitations of current chemo-proteomic maps. As a proof of concept, we demonstrate that AiPP can guide the discovery of covalent allosteric inhibitors targeting the previously undruggable protein tyrosine phosphatase PTPN6. By linking sequence, ligandability and biological context, AiPP provides an open resource for therapeutic discovery, while LatentLift offers a general strategy for harmonizing proteomics data for ML applications.

biophysics↗

bioRxiv2025

Dual DNA-binding capability of Cdc13 coordinates with Ku to safeguard telomere integrity

In eukaryotes, telomeres, nucleoprotein complexes assembled on telomeric 3-overhang DNA, protect linear chromosome ends from degradation and inappropriate DNA repair. In budding yeast, Saccharomyces cerevisiae, telomere assembly involves the Cdc13-Stn1-Ten1 (CST) complex, the Ku70-Ku80 (Ku) complex, and the Rap1-Rif1-Rif2 complex. Among these, the CST complex, centered on the binding of the G-rich single-stranded region by the Cdc13 subunit, is essential for telomere maintenance and protection. Here, we show that Cdc13 also interacts with the duplex DNA adjoining its bound single strand. This junction binding enables Cdc13 to reposition the Ku complex, potentially suppressing Ku-mediated end joining, while allowing the Cdc13 and Ku complexes to synergize in protecting telomeric DNA ends, a function that can be disrupted by mutation of a conserved residue (K504E). cdc13-K504E cells remain viable but are hypersensitive to Exo1 overexpression, particularly when combined with ku80{Delta}. Surprisingly, cdc13-K504E and other telomere protection mutants are prone to adaptive metabolic reprogramming during stationary-phase growth, leading to increased fitness. This reprogramming is inheritable and centers on elevated expression of thiamine biosynthesis, salvage, and intake pathways. Our findings reveal a stress-induced adaptive response associated with telomere erosion, indicating that telomere erosion may serve as a timer for its activation.

biochemistry↗

bioRxiv2025

Core gastric microbiota linked to pathogenesis and preserved across age stratified cohorts

Mounting evidences reveal a complex microbial niche in gastric environment. Nevertheless, the phylogenetic stability of gastric microbiota remains undetermined. Here, using full-length 16S rRNA and ITS amplicon sequencing applied to gastric brushes from a pediatric endoscopic cohort (n=243), we identified a core microbiota comprising 11 genera, which clustered into two antagonistic communities (CST1A and CST1B). CST1A was enriched in healthy subjects, while CST1B correlated with gastroduodenal disease. Notably, this bipartite architecture arose through bacterial interactions, with fungal taxa demonstrating minimal integration. Further stratified analyses revealed that H. pylori infection preferentially depleted CST1A taxa. The subclusters exhibited divergent metabolic profiles, with CST1A specializing in motility processes and CST1B in nucleotide/peptidoglycan synthesis. Cross-cohort validation of six datasets (n=1,183) confirmed the persistent relevance of the CST1A-CST1B structure across disease stages. Collectively, our findings define a core gastric microbiota established in early life, linked to gastric disease risk, and persisting into adulthood. HighlightsO_LIIdentification of a gastric bacterial network established in early life. C_LIO_LIDysbiosis in two subclusters within the core microbiota is associated with pediatric peptic ulcers. C_LIO_LIHelicobacter pylori infection reduces the abundance of the core microbiota, with a particularly greater inhibitory effect on CST1A. C_LIO_LIThe core microbiota is preserved across age stratified cohorts and is associated with gastric cancer. C_LI

microbiology↗

bioRxiv2025

Metabolic engineering of Barbarea vulgaris identifies β-amyrin synthase LUP5 as a determinant of saponin-mediated insect resistance

Plant-insect coevolution has been a major driver of specialized metabolite diversification, yet the genetic basis of natural variation in defensive chemistry remains poorly understood. The wild crucifer Barbarea vulgaris comprises two ecotypes, an insect-resistant G-type and a susceptible P-type, characterized by distinct triterpenoid saponin profiles. To investigate the causal relationship between saponin composition and insect resistance, we established a stable transformation system for B. vulgaris. Expression of the G-type {beta}-amyrin synthase gene LUP5 in the susceptible P-type conferred up to a 95% reduction in Plutella xylostella feeding, accompanied by increased accumulation of three hederagenin-derived monodesmosidic saponins. Comparison of LUP5 expression driven by its native promoter and by the constitutive 35S promoter revealed that the native promoter leads to increased hederagenin accumulation and is activated later in development, which may prevent early metabolic stress and allow coordinated expression of downstream pathway genes. In contrast, silencing of CYP72A552 by RNAi decreased total hederagenin levels by approximately 40% without affecting resistance, indicating threshold-dependent defense. Our results provide direct in planta evidence that LUP5 is a key determinant of natural variation in insect resistance in B. vulgaris, underscoring the pivotal role of the saponin backbone in herbivore deterrence. By linking promoter activity to metabolite structural diversity, this work provides mechanistic and conceptual insight into how plants coordinate specialized metabolism and defense.

plant biology↗

bioRxiv2025

DAXX governs the silencing of LINE1 during spermatogenesis in mice

The suppression of transposable elements in germline is critical for safeguarding fertility. Here we identify that the DAXX, a potent transcription repressor and an H3.3 chaperone, is indispensable for transposable elements silencing during spermatogenesis. Male mice with conditional knockout of Daxx in germ cells mediated by Ddx4-cre display delayed meiotic progression, deformed and reduced production of sperms, and an age-dependent decline in fertility. The ablation of DAXX results in activation of evolutionarily young LINE1 and ERVs subfamilies. Mechanistically, DAXX interacts with DMAP1 to maintain DNA methylation of LINE1 in meiotic cells. In summary, this study identifies DAXX as a previously unknown regulator of spermatogenesis and demonstrates that it functions as an epigenetic regulator to maintain the silencing the young LINE1 by DNA methylation.

developmental biology↗