Biology subjects

Lee, J.

Publications and source records attributed to Lee, J..

At least 235 records · Page 13Linked to original sources

bioRxiv2025

De Novo design of a potent Wnt Surrogate specific for the frizzled7 subtype members

In humans, 19 Wnt ligands interact with 10 Frizzled (Fzd) receptors and the co-receptors LRP5/6 to initiate signaling. Wnts and Fzds are highly promiscuous, making it challenging to dissect the specific outcomes of individual Wnt-Fzd interactions. Developing Wnt surrogates with specificity for individual Fzd subtypes could be pivotal. We present a modular, potent, and Fzd7-specific Wnt surrogate that consists of three de novo designed modules, a Fzd7 binder, an LRP6 binder and a homodimeric protein. The Fzd7-specific module was designed by targeting two less conserved surface patches on the cysteine-rich domain (CRD) of Fzds to achieve both selectivity and affinity. It exhibits a strong binding affinity (KD < 2.3 nM) for the very closely related Fzd7 subtype members (Fzd7, Fzd1, Fzd2) with no measurable binding to the CRDs of the other seven Fzd receptors. This Wnt surrogate induced spheroid organoid formation from intestinal stem cells at subnanomolar concentration, and promoted full hair follicle regeneration and robust hair growth in mice. These results suggest that our strategy could be extended to design modular Wnt surrogates capable of selectively activating individual Fzd receptors, providing a valuable tool kit for development and differentiation, organoid cultures and targeted regeneration.

bioengineering↗

bioRxiv2025

Inertial sensing of water content in tumor spheroids

Cellular water content governs the concentration of all biomolecules inside a cell, thereby influencing the physical and functional properties of the cell. However, measurements of water content in physiologically relevant cell culture models remain largely unavailable, particularly in 3D models such as tumor spheroids and organoids. Here, we achieve such measurements using a commercially available, industrial-grade, steel tube. The steel tube functions as a mechanical resonator that inertially senses the buoyant mass of particles. For microgram-scale particles [≥]400 {micro}m in diameter, we achieve <1% precision error in buoyant mass with a 5-minute acquisition interval. By sequentially measuring the buoyant mass of individual, glioblastoma patient-derived tumor spheroids in media of different densities and cell permeabilities, we determine the absolute and fractional (v/v) water content of the spheroids, along with their dry mass, volume, and density properties. We achieve ~0.4% precision error in fractional water content with a throughput of 3 spheroids per hour. This enables us to detect both inter-spheroid variability in fractional water content and acute responses to kinase inhibition. Overall, we establish a simple and accessible technique for quantifying water content in living 3D cell culture models, opening new avenues for studying biophysical regulation in multicellular systems.

bioengineering↗

bioRxiv2025

Computational design of a single-domain antibody that specifically recognizes WT1 peptide-loaded class I MHC

T cell receptor (TCR)-like antibodies that recognize peptide-loaded class I MHC (pMHC) complexes can enable precise targeting of cancer cells, but developing single-domain binders with high specificity and affinity is challenging. Here, we report the computational design and experimental validation of a TCR-like single-domain antibody (sdAb) that specifically recognizes the WT1-derived peptide RMFPNAPYL presented by HLA-A*02:01. Starting from the crystal structure of a Fab antibody bound to RMF/HLA- A02:01, we repurposed the VH domain into a stable, soluble Trastuzumab-derived VH scaffold. The resulting sdAb, RMFsdAb, spans all nine peptide residues and shows no binding to a control pMHC with a different peptide. Its biophysical properties were improved by fusion to human serum albumin domain III (HSA D3), yielding a monodisperse HSA D3-RMFsdAb with 81 nM affinity and specificity for RMF/HLA- A*02:01. We further engineered a bivalent format (RMFsdAb-HSA D3-RMFsdAb), which dramatically increased apparent binding affinity to 0.4 nM. When expressed on a CAR T cell, HSA D3-RMFsdAb functioned as the antigen-recognition domain to selectively activate T cells in the presence of RMF/HLA-A*02:01-positive cells. Our results demonstrate a viable strategy to develop high-specificity, peptide-focused TCR-like sdAbs for pMHC-targeted therapeutics.

biochemistry↗

bioRxiv2025

High-throughput single-cell isolation of Bifidobacterium strains from the gut microbiome

While metagenomic studies can highlight strain-level diversity within microbial communities, the diversity obtained is often incomplete. Moreover, their utility for phenotypic characterizations remains hampered without the subjacent, systematic isolation procedures required with traditional culturomics. In this work, we examined the capabilities of a commercially available high-throughput single-cell dispensing solution to selectively target and isolate diverse strains of a genus of interest, Bifidobacterium, from fecal samples. The general performance of the single-cell dispenser was first assessed, revealing a low doublet frequency of 11.5% and an ability to preserve global genus diversity when a mixed culture of Bifidobacterium was dispensed. Culturing-related factors including the use of an effective selection medium, such as the Bifidus Selective Medium supplemented with mupirocin (BSM-MUP), and the length of incubation were found to be critical in determining isolation success. Leveraging these results, we obtained a total of 622 viable isolates from five Singaporean fecal samples, among which [~]98.7% were bifidobacteria. Whole-genome sequencing of 96 isolates revealed six different Bifidobacterium species with both inter- and intra-subject lineage diversity, and the majority of the assemblies were not previously captured using metagenomic sequencing. Our findings validate the ability of high-throughput culturomics to recover diverse, novel bacterial strains and open up the possibility to robustly interrogate their functional characteristics, advancing our understanding of important microbiomes. IMPORTANCEThe field of microbial culturomics is still in its early stages. Enhancing our ability to isolate and phenotypically test bacterial strains from their multicellular environment is crucial for advancing microbiome research and healthcare development. Given the time- and cost-inefficiencies of traditional culturing methods, a more efficient, high-throughput approach to obtain isolates is needed. In the present study, we assessed a single-cell dispensing platform and developed a workflow to isolate diverse Bifidobacterium strains from fecal samples. We demonstrated here the capability of this novel technology to efficiently obtain hundreds of isolates of a targeted organism, covering both species and strain diversities. This generalizable and scalable method allows for the high-throughput recovery of microbes with little optimization needed for novel targets, providing a fundamental step in improving the culturomic framework to complement metagenomic approaches and enable isolate-level functional studies of important microbiomes.

microbiology↗

bioRxiv2025

A naturalistic reinforcement learning task uncovers historical neural representations

Exploration is essential for reinforcement learning (RL) in human development, facilitating cognitive and behavioral adaptation. However, conventional RL paradigms in neuroimaging studies often rely on highly constrained search spaces and artificial, non-naturalistic scenarios, limiting their capacity to reflect the complexity of real-world human exploration and decision- making. To address this gap, we developed a novel, naturalistic RL paradigm called Photographer, which involves a photograph-taking task set in a virtual street-view environment. Participants navigated city scenes and attempted to infer the paradigms covert goal solely from feedback, defined as the conceptual similarity between their photograph and a hidden target sentence. Representational similarity analysis revealed that feedback history, an encoding of recent trial attempts, was robustly represented in the middle orbital gyrus and inferior frontal gyrus. These neural representations were consistently observed across two independent participant groups. Moreover, although the two regions coactivated during exploration, each was associated with a distinct functional network.

neuroscience↗

bioRxiv2025

Membrane Tethering of Honeybee Antimicrobial Peptides in Drosophila Enhances Pathogen Defense at the Cost of Stress-Induced Host Vulnerability

Antimicrobial peptides (AMPs) represent a promising alternative to conventional antibiotics in combating multidrug-resistant pathogens, yet their clinical translation is hindered by proteolytic instability, cytotoxicity, and poor bioavailability. Here, we demonstrate that glycosylphosphatidylinositol (GPI)-mediated membrane tethering of honeybee defensin1 (Def1) in Drosophila melanogaster enhances its antimicrobial efficacy by [~]100-fold compared to secreted or untethered forms, while preserving physiological and behavioral integrity under baseline conditions. Using a genetically engineered Drosophila model, we expressed three Def1 variants: native (Def1), secreted (s-Def1), and membrane-tethered (t-Def1). Flies expressing t-Def1 exhibited superior bacterial clearance of Pseudomonas aeruginosa and improved survival post-infection, with no adverse effects on locomotion, courtship, or sleep architecture. However, under stress paradigms--including sleep deprivation and dextran sulfate sodium (DSS)-induced gut injury--t-Def1 exacerbated intestinal barrier dysfunction, as evidenced by elevated Smurf phenotype incidence, highlighting a trade-off between antimicrobial potency and epithelial vulnerability. Our work establishes Drosophila as a powerful platform for dissecting AMP mechanisms and engineering spatially targeted therapies, offering translational insights for pollinator health and human infectious disease management. These results advocate for iterative refinement of membrane-anchoring strategies to balance therapeutic efficacy with host safety, advancing the development of next-generation AMPs with minimized off-target effects.

biochemistry↗

bioRxiv2025

From 2D to 4D: a Containerized Workflow and Browser to Explore Dynamic Chromatin Architecture

BackgroundCharacterizing the physical organization of the genome is essential for understanding long-range gene reg- ulation, chromatin compartmentalization, and epigenetic accessibility. Hi-C experiments generate two- dimensional (2D) genome-wide contact maps of chromatin interactions by capturing the spatial proximity between genomic loci, which reveal interaction frequencies but lack the spatial resolution needed to interpret the three-dimensional (3D) genome structure(s). Emerging evidence suggests that epigenetic regulation is closely linked to 3D genome architecture, and that structural changes over time (4D) drive key biological pro- cesses in development, disease, and environmental response. Thus, integrating 3D structure with functional data is critical for a more complete understanding of genome regulation. Previous work, most notably the 4DHiC chromosome modeling framework, has shown that physical multi-dimensional modeling approaches rooted in polymer physics and molecular dynamics can resolve these structures at biologically meaningful resolutions by integrating temporal Hi-C data with physical constraints to uncover dynamic chromosome reorganization. Thus, molecular dynamics simulations, constrained by Hi-C contact matrices, can resolve fine-scale structural changes and reveal functionally significant transitions in chromatin conformation. ResultsHerein, we present the 4D Genome Browser Workflow (4DGBWorkflow) and the 4D Genome Browser (4DGB). The algorithm is based on the 4DHiC method and the containerized tool is an end-to-end workflow that can transform, filter, and view 4D epigenomics and chromatin datasets, allowing non-specialists to apply three-dimensional modeling principles to diverse datasets and experimental conditions. The software executes on a laptop running macOS, Linux or Windows. From input Hi-C files (.hic), the 4DGBWorkflow produces 3D reconstructions of chromosomes, integrates the reconstruction with track data (e.g., epigenetic marks, transcriptome profiles), and provides comparative visualization of the results in a single workflow. ConclusionsThe 4DGBWorkflow and 4D Genome Browser are open-source tools for comparative analysis and visual- ization of 4D chromosome datasets, including chromatin architecture and epigenomic signals. Automatic integration of Hi-C data with molecular dynamics democratizes the construction of time resolved 3D genome structures, simplifying complex simulations and data integration schemes.

bioinformatics↗

bioRxiv2025

Benchmarking the Impact of Anatomical Segmentation on In Vivo Magnetic Resonance Spectroscopy

PurposeEstimation of metabolite concentrations in brain magnetic resonance spectroscopy (MRS) requires correction for differences in tissue water content, relaxation properties, and the proportions of gray matter (GM), white matter (WM), and cerebrospinal fluid (CSF). Accurate knowledge of the relative proportions of these tissue classes within the volume of interest is therefore essential for reliable quantification. Commonly used brain segmentation tools differ in their algorithms, priors, and implementation, potentially introducing variability in MRS-derived concentration estimates. This study investigates the impact of segmentation software on estimated absolute concentrations. MethodsThree segmentation software tools, ANTs, FSL, and SPM, were evaluated. Segmentations were applied to an in vivo test-retest MR dataset to assess (1) differences in estimated tissue fractions, and (2) how these differences propagate into tissue-corrected metabolite concentrations. As an additional validity check and biological benchmark of segmentation performance, age-related associations with GM and total creatine (tCr) were examined. ResultsSignificant differences (p < 0.0001) were observed in tissue fraction estimates between segmentation tools, leading to differences in metabolite concentration estimates of up to 9% under identical acquisition and modeling conditions. Although the strength of the correlation varied between segmentation methods, no statistically significant differences were found. ConclusionThe choice of segmentation methodology contributed substantially to variability in MRS "absolute" metabolite concentration estimates. These results underscore the need for transparent segmentation reporting to ensure reproducibility and cross-study comparability in MRS research. Quantifying the segmentation-driven variability allows researchers to contextualize cross-study differences, helping determine whether observed effects are methodological or biologically meaningful.

neuroscience↗

bioRxiv2025

Tumor-initiating genetics and therapy drive divergent molecular evolution in IDH-mutant gliomas

Astrocytomas and oligodendrogliomas are slow-growing and treatment-sensitive IDH-mutant gliomas diagnosed at ages 30-50. Local tumor regrowth and treatment resistance is inevitable resulting in 3-10 year astrocytoma and up to >20 years oligodendroglioma survival. We sought to identify genetic changes associated with tumor evolution in response to therapy through multi-timepoint whole-genome/whole-exome sequencing of 206 IDH-mutant glioma patient samples collected through the Glioma Longitudinal Analysis (GLASS) Consortium. We validated known genomic markers of tumor progression, including hypermutation and CDKN2A homozygous deletion, and discovered novel genetic alterations that distinguish the response to treatment in astrocytomas compared to oligodendrogliomas. Point mutations in PIK3CA, PIK3R1, and NOTCH1 were newly acquired in recurrent oligodendrogliomas and associated with increased mutation rates. Focal oncogene amplifications, together with CDKN2A homozygous deletions, were associated with an increase in recurrence-specific chromosomal imbalances in astrocytomas. Mutational signature analysis revealed additional differences and detected enrichment for the SBS11, and SBS119 mutational signatures after temozolomide treatment in both IDH-glioma subtypes, whereas astrocytomas showed increased ID8 signatures after radiotherapy. These signatures suggest that the genomes of oligodendroglioma and astrocytoma adapt to the selective pressures of tumor progression and treatment in different ways. However, in both IDH-mutant glioma subtypes we observed a convergence of acquired driver gene alterations with genome-wide changes and worse patient outcomes, signaling selection of treatment-refractory clones. By identifying new prognostic markers and delineating the genomic divergence of oligodendrogliomas and astrocytomas after diagnosis, our results suggest that different DNA damage response mechanisms are engaged following chemo- and radiation therapy.

cancer biology↗

bioRxiv2025

smDeepFLUOR: Single-Molecule Deep Learning Fluorescence Classification

Fluorescence intensity variation has long served as a primary readout for monitoring biological events. However, single-fluorophore signals arising from distinct molecular events often exhibit similar intensity profiles, making further classification challenging using conventional methods. In this study, we introduce smDeepFLUOR, a deep learning-based framework that resolves seemingly homogeneous spatiotemporal fluorescence signals by uncovering latent features imperceptible to conventional analyses. By leveraging a three-dimensional convolutional neural network trained on image sequences captured over 7 x 7 x 10 voxel windows, smDeepFLUOR reliably distinguishes specific from nonspecific protein binding, even across different experimental days, with an accuracy of up to 97%. Remarkably, smDeepFLUOR also captures real-time DNA synthesis kinetics by identifying subtle changes in the spatial distance between the fluorophore and the 3 end of nascent DNA, a feature undetectable by traditional methods. These classifications were achieved without incorporating explicit physical rules or engineered features, implying the presence of intrinsic, previously unrecognized differences in emission patterns. This approach significantly extends the analytical capabilities of single-molecule fluorescence imaging and opens new avenues for minimally labeled and label-free protein activities.

biophysics↗

bioRxiv2025

Convergent DNA Methylation Abnormalities at Bivalent Chromatin in Human Growth Disorders

Loss-of-function mutations in DNMT3A, a DNA methyltransferase, or NSD1, a histone methyltransferase, cause overgrowth syndromes. Conversely, disruption of the DNMT3A domain that binds NSD1-deposited H3K36 dimethylation (H3K36me2) results in growth restriction. To investigate the molecular basis of these opposing growth outcomes, we generated isogenic human embryonic stem cells carrying growth syndrome-associated mutations in DNMT3A and NSD1. Unexpectedly, both overgrowth- and growth restriction- associated DNMT3A mutations led to DNA hypomethylation in a shared subset of active enhancers, implicating H3K36me2 in directing enhancer methylation maintenance. In contrast, bivalent chromatin--marked by both active and repressive chromatin modifications--showed divergent DNA methylation changes: hypermethylation in growth restriction-associated DNMT3A mutants and hypomethylation in overgrowth-associated DNMT3A or NSD1 loss-of-function mutants. These findings identify locus-specific DNA methylation defects as a common molecular feature and nominate dysregulated DNA methylation at bivalent chromatin as a potential driver of abnormal growth phenotypes.

genomics↗

bioRxiv2025

DIA-PASEF Proteomic Profiling Reveals MpkA-Dependent Iron Stress Responses and Siderophore Biosynthesis in Aspergillus nidulans

Filamentous faungi play essential roles in biotechnology as producers of valuable bioproducts and, conversely, as opportunistic pathogens. Aspergillus nidulans is a widely used model organism for fungal genetics and cell biology; however, comprehensive proteomic references for this species remain limited. In this study, we applied a data-independent acquisition-parallel accumulation serial fragmentation (DIA-PASEF) approach to enable efficient and in-depth proteome profiling of A. nidulans. Leveraging ion mobility-based ion cloud information from DDA-PASEF experiments, we developed DIA-PASEF methods that identified 3,904 proteins across biological triplicates grown in rich medium. Compared to prior studies, this represents an increase in protein identifications of more than 140% and was achieved with more than five-fold reduction in analysis time. We employed this newly developed DIA-PASEF methodology to conduct both proteomic and phosphoproteomic analyses under iron-depleted conditions in an MpkA protein-kinase deficient mutant ({Delta}mpkA). The {Delta}mpkA strain exhibited expression of approximately 500 additional proteins and occupancy of over 1,800 additional phosphosites relative to a control. Differentially expressed and phosphorylated proteins increased by more than an order of magnitude in the {Delta}mpkA mutant across both iron-replete and iron-deplete conditions. Gene Ontology (GO) enrichment analysis revealed broader and distinct biological processes under iron-depleted conditions, highlighting adaptive responses specific to iron limitation and MAPK pathway disruption. This work establishes a high-coverage proteomic resource for A. nidulans and provides novel insights into fungal stress responses and signaling network perturbation. Importantly, high-throughput proteomic profiling reveals that limited iron availability and MAPK pathway disruption increases siderophore biosynthesis.

systems biology↗

bioRxiv2025

CloneCoordinate: Open-source software for collaborative DNA construction

Custom DNA constructs have never been more common or important in the life sciences. Many researchers therefore devote substantial time and effort to molecular cloning, aided by abundant computer-aided design tools. However, support for managing and documenting the construction process, and for effectively handling and reducing the frequency of setbacks, is lacking. To address this need, we developed CloneCoordinate, a free, open-source electronic laboratory notebook specifically designed for cloning and fully implemented in Google Sheets. By maintaining a real-time, automatically prioritized task list, a uniform physical sample inventory, and standardized data structures, CloneCoordinate enables productive, collaborative cloning for individuals or teams. We demonstrate how the information captured by CloneCoordinate can be leveraged to troubleshoot assembly problems and provide data-driven insights into cloning efficiency. CloneCoordinate offers a new and uniquely accessible model for how to carry out, and iteratively improve on, real-world DNA assembly.

synthetic biology↗

bioRxiv2025

A reference panel for linkage disequilibrium and genotype imputation using whole-genome sequencing data from 2,680 participants across India

India is the most populous country globally, yet genetic studies involving Indian individuals remain limited. The Indian population is composed of many founder groups and has a mixed genetic ancestry, including an ancestral component not observed anywhere outside of India. This presents a unique opportunity to uncover novel disease variants and develop more tailored medical interventions. To facilitate genetic research in India, a crucial first step is to create a foundational resource that serves as a benchmark for future population studies and methods development. To this end, we have constructed the largest and most nationally representative linkage disequilibrium and genotype imputation reference panels in India to date, using high-coverage whole-genome sequencing data of 2,680 Indian participants from the Longitudinal Aging Study in India-Harmonized Diagnostic Assessment of Dementia (LASI-DAD). As an LD reference panel, LASI-DAD includes 69.5 million variants, representing 170% and 213% increases relative to the 1000 Genomes Project (1000G) and TOP-LD panels, respectively. Besides serving as an LD lookup panel, LASI-DAD facilitates various statistical analyses that rely on precise LD estimates. In a polygenic risk score (PRS) analysis, LASI-DAD improved the predictive performance of PRS by 2.1% to 35.1% across traits and studies. As an imputation reference panel, LASI-DAD improved the imputation accuracy by 3% to 101% (mean = 38%) compared to the TOPMed panel (Version R3) and by 3% to 73% (mean = 27%) compared to the Genome Asia Pilot (GAsP) panel across different minor allele frequencies. The LASI-DAD reference panel is publicly available to benefit future studies.

genetics↗

bioRxiv2025

Novel mouse model of cerebral microbleeds created by Crispr/Cas9-mediated Col4a1 deletion in adult brain microvessels

Cerebral small vessel disease is a leading cause of cognitive decline and stroke in the elderly, with cerebral microbleeds (CMBs) as one of the key imaging biomarkers. Our understanding of its pathophysiology remains limited due to the lack of appropriate animal models. We report a novel mouse CMB model created by disrupting collagen IV, a core component of the vascular basement membrane (BM), specifically within brain microvessels. Targeted deletion of Col4a1 was achieved in adult mice using brain endothelial-specific AAV vectors with CRISPR/Cas9. MRI revealed numerous CMBs with distributions similar to those of human CMBs. CMB burden increased progressively over six months following Col4a1 deletion in a dose-dependent manner, accompanied by cognitive decline and motor incoordination. Histological examination revealed hemosiderin deposits corresponding to MRI-detected CMBs without evidence of macroscopic hemorrhage or white matter lesions, while ultrastructural analysis demonstrated significant BM thinning in Col4a1-depleted microvessels. Analysis of human MRI and genomic data identified significant associations between CMB susceptibility and genetic variants in TIMP2, an endogenous inhibitor of the matrix-degrading enzyme MMP2, underscoring the clinical relevance of our model. These findings establish a direct causal relationship between microvessel COL4A1 and CMB, suggesting that dysregulated collagen IV homeostasis in BM underlies CMB development.

neuroscience↗

bioRxiv2025

In vivo cartography of state-dependent signal flow hierarchy in the human cerebral cortex

Understanding the principle of information flow across distributed brain networks is of paramount importance in neuroscience. Here, we introduce a novel neuroimaging framework, leveraging integrated effective connectivity (iEC) and unconstrained signal flow mapping for data-driven discovery of the human cerebral functional hierarchy. Simulation and empirical validation demonstrated the high fidelity of iEC in recovering connectome directionality and its potential relationship with histologically defined feedforward and feedback pathways. Notably, the iEC-derived hierarchy revealed a monotonically increasing level along the axis where the sensorimotor, association, and paralimbic areas are sequentially ordered - a pattern supported by the Structural Model of laminar connectivity. This hierarchy was further demonstrated to flexibly reorganize across brain states: flattening during an externally oriented condition, evidenced by a reduced slope in the hierarchy, and steepening during an internally focused condition, reflecting heightened engagement of interoceptive regions. Our study highlights the unique role of macroscale directed functional connectivity in uncovering a biologically interpretable state-dependent signal flow hierarchy.

neuroscience↗

bioRxiv2025

Downregulation of LATS1/2 Drives Endothelial Senescence-Associated Stemness (SAS) and Atherothrombotic Lesion Formation

BackgroundAtherothrombosis, the main event leading to acute coronary syndrome (ACS), is strongly linked to disturbed blood flow (d-flow) regions. Although the involvement of the Hippo pathway and its kinases Large Tumor Suppressor Kinase 1and 2 (LATS 1 and 2) in mechanical stress responses is known, the mechanisms by which d-flow simultaneously induces senescence, proliferation, and atherothrombosis remain unclear. MethodsThe role of endothelial cells (EC)-specific LATS1/2 was examined using EC specific knock-out (EKO) mice in a partial left carotid ligation (PLCL) model. Plaque spatial multi-omics analysis was performed by integrating imaging mass cytometry, sequential immunofluorescence (COMET), and spatial metabolomics at the single-cell level in human and mouse atherosclerotic plaques. ResultsIn tamoxifen-inducible Lats1homo(-/-) /Lats2 homo(-/-) EC-specific knockout (EKO) mice, deletion of LATS1/2 induced by tamoxifen led to fatal outcomes, characterized by severe systemic edema and markedly increased vascular permeability. In contrast, Lats1het(+/-)/Lats2 homo(-/-)-EKO mice survived and developed atherothrombotic plaques exhibiting neovascularization even without further additional dietary or genetic intervention. Spatial proteomics analysis revealed that LATS1/2 depletion in ECs triggered a senescence-associated stemness (SAS) phenotype, primarily driven by CD38 upregulation. Complementary spatial metabolomics profiling demonstrated a significant increase in sulfite and taurine within LATS1/2-deficient plaques, indicating lowered sulfite oxidase (SUOX) activity. Mechanistically, CD38 upregulation was found to suppress SUOX expression, induce the reverse mode of mitochondrial complex V, and increase succinate dehydrogenase (SDH) activity along with ATP consumption. Paradoxically, despite ATP depletion, this metabolic disturbance enhanced glutamate metabolism and the tricarboxylic acid (TCA) cycle, sustaining EC proliferation under energetically stressed conditions. The combined effect of LATS1/2 deletion and CD38 activation established a unique EC phenotype defined by increased SAS, leading to proliferation, senescence, and eventual cell death. These pathological processes culminated in the formation of atherothrombotic plaques, which were attenuated by inhibition of CD38. Notably, a similar phenotype--marked by metabolically active ECs--was observed in human atherothrombotic plaques, suggesting translational relevance. ConclusionLoss of LATS1/2 in ECs induces SAS state that promotes excessive EC proliferation, senescent cell accumulation, and the development of structurally fragile, leaky neo vessels--hallmarks of atherothrombotic lesions. CD38-mediated SUOX deficiency further amplifies this pathological process by inducing mitochondrial dysfunction, depleting ATP, and triggering compensatory upregulation of glutamate and TCA cycle metabolism. These findings identify a novel LATS1/2-CD38-SUOX axis in ECs that orchestrates SAS-driven atherothrombosis. Targeting CD38 may represent a promising therapeutic strategy to mitigate vascular dysfunction and plaque instability in high-risk ACS patients. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=191 HEIGHT=200 SRC="FIGDIR/small/660635v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@149d313org.highwire.dtl.DTLVardef@1c0988borg.highwire.dtl.DTLVardef@15efec9org.highwire.dtl.DTLVardef@1a96e03_HPS_FORMAT_FIGEXP M_FIG C_FIG Under normal physiological conditions, LATS1/2 and Lamin A work together to suppress CD38 expression. Lamin A binds directly to the CD38 promoter to repress transcription, and LATS1/2 interact with Lamin A to reinforce this suppression. This collaboration helps maintain low CD38 activity and preserves cellular NAD levels. However, under disturbed flow (d-flow), both LATS1/2 and Lamin A are downregulated. The loss of this dual repression leads to increased CD38 NADase expression. Elevated CD38 accelerates NAD consumption, causing NAD depletion--a hallmark of cellular senescence. Reduced NAD disrupts key metabolic and stress-response pathways, contributing to the onset of the senescent state. At the same time, CD38 suppresses sulfite oxidase (SUOX), leading to sulfite accumulation and mitochondrial redox imbalance. This shift activates the reverse mode of mitochondrial Complex V, which decreases ATP production and increases mitochondrial ROS, intensifying metabolic and oxidative stress in endothelial cells (ECs). In response, ECs compensate by upregulating succinate dehydrogenase (SDH), enhancing TCA cycle activity and glutamate metabolism. This metabolic adaptation provides the biosynthetic building blocks needed for cell growth and proliferation. As a result, ECs adopt a paradoxical phenotype: they show classical features of stress-induced senescence (such as NAD depletion, oxidative stress, and cell cycle arrest signals), while simultaneously undergoing metabolic activation and proliferation, also mediated by YAP. This defines a non-canonical endothelial program known as senescence-associated stemness (SAS), characterized by the formation of abnormal, proliferative, yet fragile neovessels. These dysfunctional vessels contribute to atherothrombosis, setting this process apart from the more stable lesions typical of conventional atherosclerosis.

molecular biology↗

bioRxiv2025

A Structural Atlas of TAP Inhibition by Herpesviruses and Poxviruses

In the host-pathogen arms race, herpesviruses and poxviruses encode proteins that sabotage the transporter associated with antigen processing (TAP), thereby suppressing MHC-I antigen presentation and enabling lifelong infection. Of the five known viral TAP inhibitors, only the herpes simplex virus protein ICP47 has been structurally resolved. We now report cryo-electron microscopy structures of TAP in complex with the remaining four: BNLF2a (Epstein-Barr virus), hUS6 (human cytomegalovirus), bUL49.5 (bovine herpesvirus 1), and CPXV012 (cowpox virus), assembling a structural atlas of viral TAP evasion. Employing divergent sequences, folds and conformational targets, these viral inhibitors converge on a common strategy: they stall TAP from the alternating access cycle, precluding peptide entry into the ER and shielding infected cells from cytotoxic T-cell surveillance. These findings reveal striking functional convergence and provide a structural framework for rational antiviral design.

biophysics↗