Biology subjects

Lee, Y.

Publications and source records attributed to Lee, Y..

At least 73 records · Page 4Linked to original sources

bioRxiv2025

p53 preserves genome stability in dividing cells under hypo-osmotic stress

Cells respond to frequent osmotic stress to maintain structural integrity and function. Whereas acute volume regulation is well-known, the cellular response to prolonged osmotic stress and its long-term consequences remain poorly understood. In this study, we investigate how lasting hypo-osmotic stress influences cell growth and division and the associated cellular response. We observe that cells undergoing their first division under hypo-osmotic stress experience delayed mitotic entry, lengthened mitotic duration, elevated mitotic errors and aneuploidy formation. However, continued cell division is halted through a p53/p21-dependent G1 arrest. p53 activation occurs post-mitotically and is associated with hyper-condensed chromatin and impaired nuclear expansion in daughter cells after mitotic exit. We show that the altered nuclear size dynamics lead to nuclear accumulation of p53 and activation of downstream gene expression. These findings uncover a mechanosensitive, p53-mediated mechanism that safeguards genome integrity by suppressing erroneous mitosis under hypo-osmotic stress.

cell biology↗

bioRxiv2025

RNA-Binding Protein NF90 Mediates Polycomb-Independent Transactivation by EZH2 to Promote Cancer Growth

Increasing evidence suggests critical roles of the polycomb-independent transactivation function of EZH2 in promoting some cancers, such as prostate cancer (PCa), yet the underlying mechanism remains poorly understood. Here, we identify the RNA-binding protein NF90 as a key mediator of this activity. NF90 interacts with EZH2, but not with other core components of the polycomb repressive complex 2 (PRC2), through its RNA-binding modules. Conversely, EZH2 engages NF90 via its intrinsically disordered RNA-binding domain in an RNA-dependent manner. NF90 and EZH2 mutually recruit each other to the AR promoter, where they cooperatively activate AR transcription and enhance downstream AR signaling. This NF90-EZH2 complex is essential for PCa cell growth: depletion of either factor abolishes proliferation, an effect rescued by AR re-expression. Similar to EZH2, NF90 promotes cell-cycle gene expression, is upregulated in advanced PCa, and is associated with poor clinical outcomes. Collectively, our findings uncover RNA-mediated protein interactions as a central mechanism underlying PRC2-independent transcriptional activation by EZH2 and establish NF90 as a major EZH2 coactivator, a master regulator of the cell cycle, and a promising therapeutic target in advanced PCa.

cell biology↗

bioRxiv2025

A genetic screen in enteroendocrine cells reveals mechanisms that control protein sensing and GLP-1 release

Enteroendocrine cells (EECs) are the principal nutrient sensors in the gastrointestinal (GI) tract and release hormones such as glucagon-like-peptide 1 (GLP-1) that modulate GI function and appetite. While some of the molecules involved in nutrient sensing within EECs have been described, there have been no systematic studies to map the relevant genes and pathways. Here, we developed a strategy to perform a high-throughput screen for genes that are required for nutrient-induced activation of EECs, and we applied this to probe mechanisms for sensing dietary protein. We found that all of the genes previously proposed to function as protein sensors in EECs are, collectively, dispensable for protein sensing in an EEC cell line. Instead, a screen of >20,000 sgRNAs identified numerous genes associated with mitochondrial respiration as being necessary for this process. We showed through secondary assays that impairing oxidative phosphorylation (OXPHOS) reduced EEC activation and GLP-1 release in response to nutrients but not in response to a non-nutritive stimulus. On the other hand, boosting OXPHOS increased EEC activation and GLP-1 release. These data reveal that intracellular metabolism within EECs controls the detection of dietary protein, possibly by monitoring the entry of ingested amino acids into the TCA cycle. More broadly, these findings suggest a general strategy to screen for genes and pathways that might be used to boost the nutrient-regulated release of gut peptides such as GLP-1.

physiology↗

bioRxiv2025

Sensitizing tumor response to topoisomerase I antibody drug conjugate by selective CDK7 inhibition

This study investigates the transcriptional impact of Q901, a highly selective CDK7 inhibitor in clinical development. Q901 primarily disrupted MYC and E2F-dependent transcription program, downregulating cell cycle control and DNA damage repair pathways. CDK7 binding at the promoter-proximal regions was dramatically stabilized by Q901, leading to reduced occupancy of MYC, E2F, and RNA Polymerase II (RNAPII). These findings offered a novel therapeutic strategy to enhance cancer susceptibility to TOP1-DNA protein crosslinks (TOP1-DPCs) induced by TOP1 inhibitors. Resistance to TOP1 inhibitors arises through activation of DNA repair pathway when elongating RNAPII encounters TOP1-DPCs. By suppressing RNAPII transition from initiation to elongation and DNA repair pathways, Q901 stabilizes TOP1-DPCs and sensitizes tumor to TOP1 inhibitors. Preclinical studies demonstrated enhanced tumor suppression when combining Q901 with TOP1 inhibitor-based antibody-drug conjugates (TOP1i-ADCs), highlighting its potential as a therapeutic option for cancers resistant to TOP1i-ADC therapy. TeaserOvercoming ADC cross-resistance through combination approach.

cancer biology↗

bioRxiv2025

Systematic Discovery of Pathogen Effector Functions across Human Pathogens and Pathways

Pathogens deploy effector proteins to exploit host cell biology, and most pathogen open reading frames (ORFs) are rapidly evolving and lack functional annotation. We developed the eORFeome, a scalable functional genomics platform encompassing 3,835 effector ORFs from diverse viruses, bacteria, and parasites. High-throughput barcoded screens across NF{kappa}B, apoptosis, p53, cGAS-STING and MHC-I pathways revealed functions for hundreds of uncharacterized eORFs, unexpected new activities for known effectors, and distinct pathway-specific functions encoded by single ORFs. Illustrating the power of the approach, we identify HHV6A U14 as a p53 antagonist, HHV7 U21 as a dual-function STING antagonist and MHC-I antigen display inhibitor, and adenoviral 13.6K/i-leader protein as a de novo evolved TAP inhibitor that suppresses MHC-I display. These results establish a general framework for systematic effector annotation, uncover new mechanisms of host-pathogen interaction across kingdoms, and highlight pathogen effectors as a versatile toolkit for rewiring and probing human cellular pathways.

systems biology↗

bioRxiv2025

Characterizing spatial functional microniches with SpaceTravLR

The advent of spatial omics has revolutionized our understanding of tissue biology; however, these technologies remain largely descriptive and do not capture how changes in gene regulation propagate across spatial neighborhoods. While in-silico perturbation methods and foundation models aim to model the impact of genetic perturbations, these methods are limited to single-cell approaches that lack spatial resolution. Other studies can delineate morphological domains based on transcriptional similarity, but not spatial functional microniches. We address this major unmet need by developing SpaceTravLR (Spatially perturbing Transcription factors, Ligands and Receptors), a novel interpretable machine learning approach that generalizes across tissues and species, uncovering spatial features linked to functional outcomes, thereby capturing functional microniches with spatial resolution. SpaceTravLR infers how single or combinatorial genetic perturbations rewire signals across the tissue neighborhood, by propagating effects through underlying spatially resolved molecular networks, thereby modeling how perturbations can reshape both the targeted cell and its surrounding neighborhood. SpaceTravLR defines novel spatial microniches across a range of tissues at different scales of organization (niches, neighborhoods and tissues), disease and developmental contexts. SpaceTravLRs perturbation predictions are made solely from spatial omics data and closely align with experimental validation or known outcomes based on mechanistic studies. Critically, our approach enables the generation of mechanistic hypotheses underlying identified niches. We show SpaceTravLR discovered a novel mechanism for Ccr4 that drives the spatial location of a pathogenic population of allergen-specific T helper 2 (Th2) cells as they develop in the lymph node, which was experimentally validated in a murine model. Overall, SpaceTravLR provides a novel interpretable and experimentally validated framework for uncovering how genes act individually and combinatorially through cell-intrinsic and cell-extrinsic circuits to shape spatial tissue organization and function.

systems biology↗

bioRxiv2025

Brainstem enkephalinergic neural circuit underlying cold-induced pain relief in mice

Application of cold or cold-mimicking chemicals to injury has long been recognized as an effective means of pain relief and is widely utilized in daily life. However, underlying neural mechanisms remain elusive. Here, we identified a cold-responsive neuronal subset within lateral parabrachial nucleus (lPBN), the thermosensory relay region in the hindbrain, that mediates cold-induced analgesia in mice. Selective activation of these neurons and their projection to ventrolateral periaqueductal gray (vlPAG) increased nociceptive threshold via opioid receptor signaling in vlPAG. Conversely, ablation of these neurons attenuated analgesia induced by cold-mimicking chemicals. We further identified that these neurons express precursor gene of enkephalin, which is released into vlPAG for pain relief. Activation of cold-responsive neurons in descending pain modulation circuitry reduced spinal cord responses to noxious stimuli, suggesting the involvement of top-down pain modulation pathway. These findings propose a central mechanism underlying cold-induced pain relief, which could be a novel therapeutic target. TeaserCold-sensitive lPBN neurons release enkephalin into vlPAG for top-down analgesic action during cold-induced pain relief

neuroscience↗

bioRxiv2025

CRISPR-Induced NUT Suppression Promotes Differentiation and Enhances Trop2-Targeted Immunocytokine Response in NUT Carcinoma

NUT carcinoma (NC) is an aggressive malignancy driven by NUTM1 gene rearrangements with limited therapeutic options. Here, we show that direct suppression of NUTM1 using CRISPR/Cas9 induces squamous-like differentiation and upregulates TROP2 expression in NC cells. Building on this finding, we developed a TROP2-interferon beta (IFN-{beta}) mutein immunocytokine that selectively targets TROP2-expressing tumors. Combined NUTM1 suppression and TROP2-targeted immunotherapy synergistically enhanced cytotoxic and immune-mediated responses in vitro. Transcriptomic and spatial analyses of NC patient tumors revealed that differentiation status correlates with TROP2 expression, upregulated immune pathways, and favorable clinical outcomes. Our results suggest that overcoming differentiation blockade not only alters tumor phenotype but also creates a more immune-permissive microenvironment. These findings highlight the therapeutic potential of sequential tumor reprogramming followed by targeted immunotherapy in treating NC and propose a broader strategy for overcoming differentiation blockade in fusion-driven cancers.

cancer biology↗

bioRxiv2025

New distribution records of Aedes aegypti, Aedes mediovittatus, and Toxorhynchites portoricensis (Diptera: Culicidae) in Puerto Rico and their relevance to Integrated Vector Management

As of October 2025, Puerto Rico has been experiencing an ongoing dengue outbreak that started in March 2024. The latest island-wide mosquito survey conducted in Puerto Rico during 2018- 2019 covered 41 of the 78 municipalities and detected the presence of Aedes aegypti in 27 of the municipalities. Given the prolonged elevated circulation of dengue virus on the island, we carried out an additional Ae. aegypti survey in June 2025 across 44 out of 78 municipalities, including areas with no prior records of the species. Here, we report the occurrence of Ae. aegypti in 43 out of 44 municipalities surveyed, including ten new municipalities where Ae. aegypti has not been reported. These findings provide insight into the dispersal of Ae. aegypti in Puerto Rico, indicating that the species is much more widespread than previously known. We also provide the expanded species occurrence of Aedes mediovittatus and Toxorhynchites portoricensis, which share the same larval habitat as Ae. aegypti. Notably, 96% of cemeteries surveyed across 24 municipalities served as oviposition sites for Ae. aegypti. However, a few cemeteries demonstrated effective preventative practices that minimize mosquito breeding. This effective management, which resulted in no mosquito breeding in some cemeteries and a tire shop, brings hope that communication and education of property/facility managers can reduce the mosquito populations.

ecology↗

bioRxiv2025

Automatic Classification of Circulating Blood Cell Clusters based on Multi-channel Flow Cytometry Imaging

Circulating blood cell clusters (CCCs) containing red blood cells (RBCs), white blood cells (WBCs), and platelets are significant biomarkers linked to conditions like thrombosis, infection, and inflammation. Flow cytometry, paired with fluorescence staining, is commonly used to analyze these cell clusters, revealing cell morphology and protein profiles. While computational approaches based on machine learning have advanced the automatic analysis of single-cell flow cytometry images, there is a lack of effort to build tools to automatically analyze images containing CCCs. Unlike single cells, cell clusters often exhibit irregular shapes and sizes. In addition, these cell clusters often consist of heterogeneous cell types, which require multi-channel staining to identify the specific cell types within the clusters. This study introduces a new computational framework for analyzing CCC images and identifying cell types within clusters. Our framework uses a two-step analysis strategy. First, it categorizes images into cell cluster and non-cluster groups by fine-tuning the You Only Look Once(YOLOv11) model, which outperforms traditional convolutional neural networks (CNNs), Vision Transformers (ViT). Then, it identifies cell types by overlaying cluster contours with regions from multi-channel fluorescence stains, enhancing accuracy despite cell debris and staining artifacts. This approach achieved over 95% accuracy in both cluster classification and phenotype identification. In summary, our automated framework effectively analyzes CCC images from flow cytometry, leveraging both bright-field and fluorescence data. Initially tested on blood cells, it holds potential for broader applications, such as analyzing immune and tumor cell clusters, supporting cellular research across various diseases.

bioinformatics↗

bioRxiv2025

Advancing Protein Ensemble Predictions Across the Order-Disorder Continuum

While deep learning has transformed structure prediction for ordered proteins, intrinsically disordered proteins remain poorly predicted due to systematic underrepresentation in training data, despite constituting approximately 30% of eukaryotic proteomes. We introduce PeptoneBench, the first benchmark to enable systematic assessment of ensemble generators for both ordered and disordered proteins, integrating diverse experimental observables. Our analysis reveals that existing evaluation metrics exhibit systematic bias toward the structured spectrum of the proteome. Assessment of popular predictors (AlphaFold2, ESMFlow, Boltz2) confirms high accuracy on ordered proteins but shows performance degradation with increasing disorder. We further present PepTron, a flow-matching ensemble generator trained on data augmented with synthetic disordered protein ensembles. On our benchmark PepTron matches BioEmu on disordered regions while maintaining competitive accuracy on ordered protein benchmarks. Our data augmentation approach demonstrates that targeted training strategies can approach the performance of computationally expensive simulation-based methods, establishing a generalizable framework applicable to other protein generative models. All datasets, models, and code are openly available.

biophysics↗

bioRxiv2025

ETV4 restrains the formation of CD74+ cytotoxic exhausted CD8+ T cells

Exhausted CD8+ T (Tex) cells comprise heterogeneous subsets with varying cytotoxic and dysfunctional properties, making inhibitory receptor profiles unreliable indicators of function. Therefore, it is essential to define transcriptional regulators or markers that accurately reflect these states. Here, we identified ETS variant 4 (ETV4) as a transcription factor that restricts cytotoxic Tex differentiation within tumors. ETV4 deficiency promotes the emergence of a CD74+ cytotoxic Tex subset while preserving the progenitor Tex (Tpex) pool. CD74 serves as both a marker and regulator, reinforcing Tpex features and enhancing Tex cytotoxicity. Mechanistically, ETV4 loss derepresses T-bet expression, thereby increasing IFN{gamma} production that induces CD74 expression in a paracrine manner. Single-cell analyses across human cancers revealed that CD74+ Tex cells are enriched for proliferative and metabolic programs and correlate with improved survival. Collectively, we establish the ETV4-CD74 axis as a regulator of Tex fate and promising therapeutic target to augment anti-tumor immunity.

immunology↗

bioRxiv2025

Superoxide dismutases shape manganese stoichiometry in Southern Ocean diatoms

Elemental stoichiometry of biomass is a focal point that connects different biogeochemical cycles. Yet, the mechanistic underpinnings of elemental stoichiometry are poorly quantified in many cases. We combined targeted and untargeted metaproteomics, Bayesian statistical modelling, and geochemical measurements to quantify the contribution of specific proteins to metal stoichiometry in natural populations of Southern Ocean diatoms. Our analyses indicate that a substantial amount of non-photosynthetic manganese (Mn) in diatoms in an Antarctic polynya can be attributed to superoxide dismutases ([~]0.7 {micro}mol Mn: mol Carbon; [~]20% of the total cellular Mn quota). We then used cultures and proteomic profiling of the key polar diatom Fragilariopsis cylindrus to identify environmental controls on superoxide dismutases, and discovered that iron concentration has little influence on the abundance of two Mn superoxide dismutases, while Mn limitation induces the depletion of these Mn superoxide dismutases and an accompanying increase of nickel superoxide dismutase. Overall, we combined metaproteomic approaches to quantify proteomic composition and connected these measurements to metal-to-carbon ratios and their responses to metal availability. Because metal quotas are key parameters in some biogeochemical models, our approach provides a direct mechanism for informing ecosystem-scale models with molecular measurements.

microbiology↗

bioRxiv2025

Exploiting TGF-β-mediated Stromal Programming in Homologous Recombination-Deficient Pancreatic Cancer

The tumor microenvironment (TME) actively contributes to pancreatic ductal adenocarcinoma (PDAC) pathogenesis through dynamic bidirectional tumor-stroma interactions. Here, we demonstrate that homologous recombination-defective (HRD) tumor epithelium reprograms the TME in a genotype-specific manner to enhance cancer aggressiveness. Using genetically engineered mouse models, pancreatic stellate cell (PSC) and cancer-associated fibroblast (CAF) co-culture systems, single-nucleus multiomics, and human PDAC models, we show that tumoral loss of ATM serine/threonine kinase drives CAFs toward SMA+ myofibroblastic differentiation, independently of P53 status. These myCAFs, in turn, promote cancer aggressiveness and chemoresistance. Mechanistically, ATM deficiency increases reactive oxygen species and contractility signaling, enhancing TGF-{beta}1 secretion. Pharmacological TGF-{beta} inhibition reverses myCAF differentiation, sensitizes tumors to chemotherapy, and impairs tumor progression in both murine and human ATM-null models. Our findings reveal that ATM-deficient tumors shape a cancer-promoting niche via TGF-{beta} signaling and identify dual targeting of intrinsic and extrinsic vulnerabilities as a promising precision oncology strategy. SIGNIFICANCEHRD pancreatic cancers reprogram the tumor microenvironment in a genotype-specific manner through TGF-{beta}-driven myCAF-enrichment. Targeting this stromal axis alongside platinum-based chemotherapy improves therapeutic efficacy in ATM-deficient models. These findings highlight the need to integrate epithelial genotype and stromal context for truly personalized treatment strategies in PDAC.

cancer biology↗

bioRxiv2025

Site-specific replacement of large-scale DNA fragments in human cells

Despite advances in genome editing1-4, precisely replacing large-scale fragments in human cells remains a significant challenge. Here, we present a site-specific gene replacement tool, named Prime Assembly (PA), which adapts prime editors to produce one or two pairs of 3-flaps on both the genome and donor plasmids. These 3-flaps anneal to each other precisely, similar to "Gibson Assembly" in DNA oligonucleotides5, allowing megabase-scale genomic excision and/or kilobase-scale donor insertion at the gene of interest. The PA system achieves an efficiency of up to 57.8% in replacing endogenous sequences with a 2.9 kbp donor DNA fragment in HEK293T cells, and presents an accuracy of more than 99% for integrated PA fragments. Ultimately, the site-specific replacement of large-scale coding sequences (CDSs) in disease-related genes can restore gene function across numerous patients with different mutations, providing a gene implantation technique for genome editing and a universal therapeutic approach.

molecular biology↗

bioRxiv2025

Polyelectrolyte domains in intrinsically disordered regions differentially regulate intracellular biomolecular condensation

Intracellular condensation is a hallmark of many proteins containing intrinsically disordered regions (IDRs). Decoding the regulatory mechanisms of condensation encoded within the amino acid sequences of condensate-forming proteins is essential for understanding both how intracellular condensates assemble and how they contribute to various disease pathologies. Here, we investigated the budding yeast P-body proteins Nst1 and Edc3, which exhibit pronounced intracellular self-condensation, and identified charged amino acid clusters in the low complexity regions (LCRs) that modulate their phase behavior. Guided by the stickers-and-spacers framework, we hypothesized that clusters of charged residues within IDRs function in promoting intracellular condensation process. Using a sliding-window approach, we designated and dissected the polyelectrolyte (PE) domains of Nst1 and Edc3 and established their essential contributions to condensate coalescence. In Nst1, the polyanionic (PA) subdomain enhances intermolecular attraction and promotes coalescence, whereas the polyampholyte (PAM) subdomain imparts rigidity to the condensate as a distinct modulatory element and functions as a platform in condensation process of other P-body components. PA of Edc3 also regulates coalescence. Extending these findings to the neurodegenerative disease-associated protein TDP-43, we show that its PA regions are critical for coalescence, consistent with the presence of negatively charged amino acid clusters as potent molecular stickers functioning in condensate coalescence in cells. These results highlight charged-residue clusters as important sequence determinants of protein phase behavior and suggest that they represent promising novel regulatory targets for modulating TDP-43 condensation in cells.

cell biology↗

bioRxiv2025

Sirtuin 1 Activation Mitigates Murine Vasculitis Severity by Promoting Autophagy and Mitophagy

BACKGROUNDSirtuin 1 (SIRT1), a NAD+-dependent protein deacetylase, regulates cardiovascular inflammation by modulating cellular stress, inhibiting NLRP3 activation, and promoting the clearance of damaged mitochondria. However, its precise role in the pathogenesis of Kawasaki disease (KD), a pediatric systemic vasculitis and the leading cause of acquired heart disease in children, remains unclear. METHODSUsing the Lactobacillus casei cell wall extract (LCWE) murine model of KD, we evaluated the severity of vasculitis in mice supplemented with NAD+ precursors, as well as transgenic mice overexpressing SIRT1, and mice with specific deletion of Sirt1 in vascular smooth muscle cells (VSMCs) and myeloid cells. Proteomics analysis was performed on the abdominal aortas of WT and SIRT1-overexpressing mice. We performed immunofluorescent staining of cardiovascular tissues to assess the expression of proteins related to the autophagy/mitophagy pathway and the pathogenic switch of VSMCs. Western blot analysis was performed on primary VSMCs and cardiovascular tissues to determine the impact of SIRT1 on autophagic flux. The production of pro-inflammatory cytokines was measured in bone marrow-derived macrophages and peritoneal lavage of transgenic mice using ELISAs. RESULTSSIRT1 expression was downregulated in cardiovascular lesions of LCWE-injected mice, which was associated with a significant reduction of circulating levels of nicotinamide. Supplementation of mice with NAD+ precursors or genetic overexpression of SIRT1 significantly reduced the development of LCWE-induced KD, while the specific deletion of Sirt1 in VSMCs or myeloid cells exacerbated vasculitis. Proteomics analysis indicated impaired mitophagy/autophagy and the pathogenic synthetic switch of VSMCs in LCWE-injected mice, which was rescued with SIRT1 overexpression and associated with reduced production of proinflammatory cytokines. CONCLUSIONSThis study reveals the presence of an impaired NAD+-SIRT1 axis in the pathogenesis of LCWE-induced KD vasculitis and the therapeutic potential of targeting this axis to reduce cardiovascular lesions and inflammation.

immunology↗

bioRxiv2025

Low-intensity focused ultrasound enables temporal modulation of human midbrain organoid differentiation

Controlling the precise timing of biosignaling cues in complex 3D models such as organoids is critical for guiding cellular differentiation and functional maturation. Ultrasound stimulation, a next-generation neuromodulation modality, offers unique advantages due to its target specificity, ability to elicit mechanosensitive responses, and high spatial resolution. However, there is still a lack of precision stimulation platforms and comprehensive biomarker assays for selective control of organoid development. Here, we introduce a modular piezoelectric ultrasound stimulation platform that integrates seamlessly with conventional multi-well plates, enabling selective neuromodulation of midbrain organoids (mBOs). We demonstrate that ultrasound can specifically modulate cellular differentiation by promoting dopaminergic progenitor markers while delaying terminal differentiation. Importantly, ultrasound stimulation did not induce cellular damage, as confirmed by the absence of apoptosis and DNA damage markers. This work demonstrates the potential of focused ultrasound as a safe, non-invasive, and tunable biophysical cue for temporal regulation of organoid differentiation and maturation. Significance StatementOrganoids are three dimensional in vitro models derived from human tissue that recapitulate the complex features of organs. However, precise modulation of organoid differentiation relies on biochemical factors, which are limited in their temporal controllability. In this study, we demonstrate that low-intensity ultrasound stimulation enables temporal modulation of midbrain organoid differentiation using a modular multi-well stimulation platform. In particular, we show that ultrasound promotes the proliferation of dopaminergic progenitor cells. These findings suggest that ultrasound can serve as a supplementary mechanical cue to regulate midbrain organoid development.

developmental biology↗