Biology subjects

Xu, H.

Publications and source records attributed to Xu, H..

At least 145 records · Page 8Linked to original sources

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

Human myelocyte and metamyelocyte-stage neutrophils suppress tumor immunity and promote cancer progression

Tumor-infiltrating neutrophils (TINs) are highly heterogeneous and mostly immunosuppressive in the tumor immune microenvironment (TIME). Current biomarkers of TINs and treatment strategies targeting TINs have not yielded optimal responses in patients across cancer types. Here, we separated human and mouse neutrophils into three developmental stages, including promyelocyte (PM), myelocyte & metamyelocyte (MC & MM), and banded & segmented (BD & SC) neutrophils. Based on this separation, we observed the predominance of human but not mouse MC & MM-stage neutrophils in bone marrow (BM), which exhibit potent immunosuppressive and tumor-promoting properties. MCs & MMs also occupy the majority of TINs among patients with 17 cancer types. Moreover, through the creation of a NCG-Gfi1-/- human immune system (HIS) mouse model, which supports efficient reconstitution of human TIN, we found a significant increase of BM MCs & MMs in tumor-bearing mice. By comparing the scRNA-seq analysis results of human neutrophils from both BM and tumors, we found CD63 and Galectin-3 distinguish MC & MM from neutrophil populations in cancer patients. Furthermore, we proposed a strategy with Flt3L to specifically induce the trans-differentiation of MCs & MMs into monocytic cells, and trigger tumor control in NCG-Gfi1-/- HIS mice. Thus, our findings establish an essential role of human MC & MM-stage neutrophils in promoting cancer progression, and suggest their potential as targets for developing potential biomarkers and immunotherapies for cancer.

immunology↗

bioRxiv2025

SMART: An Integrated Platform Revolutionizing Cryo-Electron Microscopy Workflow from Data Acquisition to Atomic Modeling

Recent advancements in cryo-electron microscopy (cryo-EM) have revolutionized structural biology, yet persistent workflow fragmentation between data acquisition, reconstruction, and modeling limits its full potential. This paper presents SMART, the first fully integrated cryo-EM platform unifying three critical AI-driven modules: DataSmart (automated data collection), CryoSmart (3D reconstruction), and ModelSmart (end-to-end atomic modeling). The platform embeds more than 10 distinct AI algorithms across its workflow, achieving unprecedented automation levels. These advancements not only lower the expertise barrier for cryo-EM adoption but also establish a new paradigm for AI-empowered structural biology, with broad implications for high-throughput drug discovery. This protocol provides a comprehensive delineation of the operational procedures for the SMART platform. As an example, this protocol enabled the determination of the 2.1 [A] resolution structure of TRPML1, a Ca2+-permeable, nonselective, six-transmembrane tetrameric cation channel found in late endosomes and lysosomes (LELs) of mammalian cells. This integrated workflow enables users, including those with minimal cryo-EM experience, to efficiently complete the entire pipeline from data collection to atomic model building.

bioinformatics↗

bioRxiv2025

TH5487 specifically targets NLRP3 in FCAS patients resistant to MCC950

The NLRP3 inflammasome plays a central role in innate immunity and is activated in response to mitochondrial dysfunction and oxidized DNA. Here, we demonstrate that repurposed small-molecule inhibitors originally developed for DNA glycosylases, TH5487 and SU0268, potently inhibit NLRP3 activation ex vivo in human Peripheral Blood Mononuclear Cells (PBMCs) with IC50 of 1.62 {micro}M and 3.24 {micro}M, respectively. We show that these inhibitors prevent mitochondrial localization of NLRP3 and directly block inflammasome assembly. They also reshape the immune landscape decreasing IL-1{beta}, while increasing IFN-{beta}. Structural and biophysical analyses reveal a two-site DNA binding model in which NLRP3 engages oxidized DNA with a KD1 of 0.268 nM and KD2 3.02 nM. Importantly, these inhibitors block IL-1{beta} secretion in L353P Familial Cold Autoinflammatory Syndrome (FCAS) patient PBMCs where MCC950 fails, demonstrating the therapeutic potential for inflammasome-driven diseases. Together, our findings reveal a novel druggable mechanism of inflammasome inhibition through interference with oxidized DNA sensing and localization, offering new opportunities for treatment of chronic inflammatory disorders.

immunology↗

bioRxiv2025

Fat-rich diet reprograms intrapulmonary neutrophils to boost tissue-specific antitumor immunity

Neutrophils adapt to tissue-specific signals and exert innate defense functions against infections and malignancies. Fat-rich diet (FRD), such as high-fat diet (HFD) and ketogenic diet (KD), has complex impacts on immunity. However, whether and how FRD shapes tissue-specific functions of neutrophils remain unclear. Here we show that both isocaloric HFD- and KD-fed mice demonstrate enhanced neutrophil-mediated pulmonary tumor resistance than chow diet-fed mice. Intrapulmonary but not systemic neutrophils in FRD-fed mice bear enhanced potential of reactive oxygen species (ROS) production and ROS-dependent tumor cytotoxicity. Mechanistically, FRD-induced increased serum saturated fatty acids and cholesterol stimulate lung vascular endothelial cells (LVECs), which reprogram intrapulmonary neutrophils via contact- and intercellular adhesion molecule-1 (ICAM-1)-dependent mechanisms. Analysis on human lung single-cell RNA sequencing data showed that intensified cell adhesion and priming signals from human LVECs are associated with enhanced antitumor functions in intrapulmonary neutrophils. Our findings highlight the roles of dietary fats in shaping neutrophil functions in a tissue-specific manner. Dietary intervention targeting tissue-specific reprogramming of neutrophils therefore represents a potential strategy against malignancies in the lungs.

immunology↗

bioRxiv2025

β-Glucan Modulates Monocyte Plasticity and Differentiation Capacity to Mitigate DSS-Induced Colitis

Trained immunity involves the reprogramming of innate immune cells after an initial exposure, resulting in heightened inflammatory responses to subsequent stimuli and enhanced bactericidal capacity during infection. However, this proinflammatory state could also exacerbate chronic conditions like inflammatory bowel disease (IBD), which is characterized by persistent inflammation and microbial imbalance. It remains unclear how trained immunity influences IBD pathogenesis and whether it can be harnessed therapeutically. In our study, pretreatment with {beta}-glucan reprogrammed bone marrow hematopoietic progenitors and peripheral monocytes, inducing a profound shift in monocyte plasticity and significantly reducing the severity of dextran sulfate sodium (DSS)-induced colitis. Adoptive transfer of bone marrow or peripheral monocytes from {beta}-glucan-trained mice into naive mice conferred robust protection against colitis, demonstrating that this protective effect is transferable. Trained mice also displayed improved clearance of intestinal bacterial infections. Single-cell RNA sequencing revealed an expansion of reparative Cx3cr1 macrophages derived from Ly6Chi monocytes, correlating with accelerated colonic epithelial regeneration. Collectively, these findings reveal how {beta}-glucan-induced trained immunity modulates monocyte differentiation to ameliorate experimental colitis, highlighting the potential of harnessing trained immunity as a therapeutic strategy to recalibrate innate immune responses and restore gut homeostasis in IBD, shedding light for future clinical applications. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=189 SRC="FIGDIR/small/653036v2_ufig1.gif" ALT="Figure 1"> View larger version (56K): org.highwire.dtl.DTLVardef@1b969c2org.highwire.dtl.DTLVardef@1ce603dorg.highwire.dtl.DTLVardef@10582forg.highwire.dtl.DTLVardef@10d7de6_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2025

Targeting USP2 induces degradation of PML-RARα with or without drug-resistant mutations in acute promyelocytic leukemia

Despite the high efficacy of all-trans retinoic acid (ATRA) and arsenic trioxide (ATO) in treating acute promyelocytic leukemia (APL), approximately 10-20% of patients develop drug resistance due to mutations in PML-RAR and other factors. Here, it is demonstrated that inhibiting USP2 with ML364 or silencing USP2 reduces PML-RAR protein levels in both ATRA-sensitive and -resistant APL cells, an effect reversed by proteasome inhibition. Conversely, USP2 overexpression enhances PML-RAR stability. Mechanistically, USP2 interacts with and deubiquitinates PML-RAR, including drug-resistant mutants. Consistent with PML-RAR degradation, ML364 treatment significantly induces apoptosis in APL cell lines and primary leukemia cells. In conclusion, this study identifies USP2 as a novel deubiquitinating enzyme for PML-RAR and highlights USP2 inhibition as a potential therapeutic strategy for APL with PML-RAR mutations.

cell biology↗

bioRxiv2025

Repositioning of polyubiquitin alters the pathologic tau filament structure

Structurally diverse tau filaments form proteinaceous aggregates in a heterogeneous group of neurodegenerative diseases called tauopathies1. The factors extrinsic to the highly ordered core structure that influence tau filament stability are not well understood. Here, we found that polyubiquitinated tau filaments from Alzheimers disease and vacuolar tauopathy human brain tissue exhibit distinct seeding patterns in mice, in association with differences in tau filament ultrastructure determined by cryo-electron microscopy. Interestingly, chemical modulation of the polarity of polyubiquitin adjacent to the tau core with the small molecule ubistatin B resulted in the repositioning of poorly structured densities towards positively charged residues on the highly structured core filament, leading to shifting of the protofilament-protofilament interface of certain vacuolar tauopathy tau filaments. These results suggest that the structure of tau filaments that are associated with different seeding activities in vivo can be influenced by post-translational modifications.

neuroscience↗

bioRxiv2025

Synaptic vesicles that store monoamines and glutamate differ in protein composition.

Neuromodulators such as the monoamines are known to differ from classical neurotransmitters like glutamate in the time scale of signaling due to activation of slower G protein-coupled receptors. Recent work has suggested that the mode of release also differs between classical and modulatory transmitters. Although many components of neurotransmitter release machinery have been identified, we still understand little about the mechanisms responsible for differences in release. In this study, we address the differences between release of dopamine and glutamate by comparing the composition of synaptic vesicles (SVs) that contain the vesicular monoamine transporter 2 (VMAT2) versus vesicular glutamate transporter 2 (VGLUT2). Previous work has shown that these SV populations differ in frequency dependence, recycling kinetics and biogenesis. Taking advantage of a CRISPR-generated knock-in mouse with a cytoplasmic hemagglutinin (HA) tag at the N-terminus of VMAT2 to immunoisolate monoamine SVs, we find differences in the abundance and isoform expression of many SV protein families. Validation in primary neurons and in brain tissue confirms these differences in SV protein abundance between dopamine and glutamate release sites. Functional analysis reveals that the loss of differentially expressed SCAMP5 selectively impairs the recycling of VGLUT2 SVs, sparing VMAT2 vesicles in the same neuronal population. These findings provide new insights into the molecular diversity of SVs and the mechanisms that regulate the release of dopamine and glutamate, with implications for the physiological role of these transmitters and behavior.

neuroscience↗

bioRxiv2025

Replication competition drives the selective mtDNA inheritance in Drosophila ovary

Purifying selection that limits the transmission of harmful mitochondrial DNA (mtDNA) mutations has been observed in both human and animal models. Yet the precise mechanism underlying this process remains undefined. Here, we present a highly specific and efficient in situ imaging method capable of visualizing mtDNA variants that differ by only a few nucleotides at single-molecule resolution in Drosophila ovaries. Using this method, we revealed that selection primarily occurs within a narrow developmental window during germline cysts differentiation. At this stage, the proportion of the deleterious mtDNA variant decreases without a reduction in its absolute copy number. Instead, the healthier mtDNA variant replicates more frequently, thereby outcompeting the co-existing deleterious variant. These findings provide direct evidence that mtDNA selection is driven by replication competition rather than active elimination processes, shedding light on a fundamental yet previously unresolved mechanism governing mitochondrial genome transmission.

genetics↗

bioRxiv2025

NF2 loss malignantly transforms human pancreatic acinar cells and enhances cell fitness under environmental stress

Pancreatic ductal adenocarcinoma (PDAC) occurs as a complex, multifaceted event driven by the interplay of tumor permissive genetic mutations, nature of cellular origin and microenvironmental stress. In this study, using primary human pancreatic acinar 3D organoids, we performed CRISPR knockout screen targeting 199 previously underappreciated potential tumor suppressors curated from clinical PDAC samples. Our data revealed significant enrichment of a list of candidates, with NF2 emerging as the top target. Functional validation confirmed that loss of NF2 promotes the transition of PDAC to an invasive state, potentially through extracellular matrix modulation. NF2 inactivation was found to enhance PDAC cell fitness under nutrient starvation. This adaptation not only reinforces the oncogenic state but also confers therapeutical resistance. Additionally, we found that NF2 loss is associated with the fibroblast heterogeneity and cancer-stroma communications in tumor evolution. These findings establish NF2 as a critical tumor suppressor in PDAC and uncover its role in mediating nutrient adaptation and drug resistance. Importantly, this study provides new insights into drug resistance mechanisms and potential therapeutic targets in PDAC.

cancer biology↗

bioRxiv2025

Systematic evaluation of blood contamination in nanoparticle-based plasma proteomics

Circulating blood proteomics enables minimally invasive biomarker discovery. Nanoparticle (NP)-based circulating plasma proteomics studies have reported varying number of proteins, ranging from ca 2000 to 7000 but its not clear whether higher protein number is more informative. Here, we first develop OmniProt - a silica-NP workflow for plasma proteomics which is optimized through systematic evaluation of NP types and protein corona formation parameters. Next, we present an Astral spectral library for 10,109 protein groups expressed in human plasma. Using Astral with a throughput of 60 sample-per-day, OmniProt identifies ca 3000 to 6000 protein groups from human plasma. Notably, we found that platelet/erythrocyte/coagulation-related contamination artificially elevates protein identifications and compromises quantification accuracy for circulating plasma proteins in NP-enriched samples. Through controlled contamination experiments, we identified and validated protein biomarkers for contaminations of platelet, erythrocyte, and coagulation, respectively, in NP-based plasma proteomics experiments. We further developed an open-access software Baize for accessing plasma proteomics contamination. Finally, we validated our OmniProt workflow and Baize software in a clinical cohort of 193 patients with CT-indistinct benign nodules or early lung cancers. On average, we identified ca 4400 protein groups for each plasma sample without significant contamination. Five samples were flagged out as contaminated. In conclusion, this study reveals that contamination of plasma samples significantly alters the protein identification and quantification in NP-based plasma proteomics experiments, and presents an open-access software Baize to evaluate plasma proteome contamination.

biochemistry↗

bioRxiv2025

Mechanistic Insights into Proteomic Mutation-Phenotype Linkages from Tiling Mutagenesis Screens

High-throughput mutagenesis screens are powerful tools for mapping mutations to phenotypes. However, deciphering the molecular mechanisms that link mutations to phenotypic outcomes remains a significant challenge. Here, we present ProTiler-Mut, a versatile computational framework that harnesses tiling mutagenesis screens, which introduce variants across entire protein sequences, to facilitate investigation of mutation-to-phenotype associations at multiple levels, including individual residues, protein substructures, and protein-protein interactions (PPIs). As demonstrated through our analyses of base editing (BE) screens targeting DNA Damage Response (DDR) proteins and T cell regulators, ProTiler-Mut provides novel insights into the mutation-phenotype linkages, including: i) refined classification of mutation that reveals separation-of-function (SOF) category beyond the conventional binary classification of loss-of-function (LOF) and gain-of-function (GOF); ii) definition of phenotype-associated hotspot substructures that enable the inference of the function of unscreened pathogenic mutations; and iii) identification of phenotype-associated PPIs disrupted by functional mutations. Through ProTiler-Mut analyses, we identified a substructure harboring pathogenic GOF mutations that disrupt interactions between the kinases MAPK1 and RSK1, leading to MAPK1 activation and elevated expression of the immune checkpoint receptor PD-1. Furthermore, we demonstrate the applicability of ProTiler-Mut to various mutagenesis screening platforms, highlighting its broad utility and generalizability.

bioinformatics↗

bioRxiv2025

Prevalent cross-cell type QTL trans-regulatory genetic effects impacting innate lymphoid cells in the small intestine

Tissue function in homeostasis and disease arise from coordinated interactions between different cell types and can vary between individuals in a population, in part due to the impact of genetic variants. In barrier tissues such as the gut, innate lymphoid cells (ILCs) play critical roles in maintaining tissue homeostasis and immunity, but the genetic basis and associated regulatory circuitry affecting tissue ILCs remain largely unknown. Here, we systematically mapped the regulatory functions of genetic variants across 273,370 gut ILCs profiled by single cell RNA-seq from 274 diversity outbred (DO) mice. Computational analysis identified quantitative trait loci (QTL) impacting ILCs at three levels: cell subtype-specific mRNA expression (local and distal), cell subset proportions, and ILC gene expression programs (in trans), of which we experimentally validated a pivotal role for the transcription factor Rbpj in regulating ILC3s. All three classes of ILC QTL display polygenic inheritance signatures and broadly overlap with QTL affecting peripheral cytokine levels that we measured in another 261 DO mice. Strikingly, nearly half of trans-QTL affecting ILC traits did not overlap (within a 50-kb window) genes expressed in ILCs, and such loci were enriched for genes expressed by non-ILCs especially enteric neurons and intestinal glia. This suggests that such loci may act instead across cell type boundaries and allowed us to recover causal trans-cell type regulatory circuits in the tissue, including loci encoding multiple neuron-expressed peptides, epithelial-cell expressed Sox9, and phagocyte-expressed Ccl17. Finally, human orthologs of genes in QTL impacting gene expression and cell proportions are enriched for autoimmune disease risk heritability, suggesting their relevance to human disease. Our findings highlight how the impact of genetic variants may propagate to maintain tissue homeostasis and show a path to understand causality in tissue biology using quantitative genetics.

genetics↗

bioRxiv2025

AutoLEI: An XDS-based Graphical User Interface for Automated Real-time and Offline Batch 3D ED/MicroED Data Processing

Three-dimensional electron diffraction (3D ED), also known as microcrystal electron diffraction (MicroED), is an emerging method for determining structures of submicron-sized crystals. With the development of rapid and convenient data collection protocols, acquiring dozens of datasets in a single MicroED session has become routine. A fast and automated workflow for processing, scaling and merging a large number of MicroED datasets can significantly accelerate the structure determination process. Herein, we present an XDS-based graphical user interface for automated real-time and offline batch 3D ED/MicroED data processing (AutoLEI). We illustrate the functionality of the GUI through four examples, demonstrating both offline and real-time data processing capabilities. These examples include small organic molecules, metal-organic frameworks (MOFs), and proteins, showcasing the versatility and efficiency of the GUI in various applications. SynopsisA graphical user interface for real-time and offline 3D ED/MicroED data processing by XDS was developed. The GUI aims to improve efficiency, minimize redundant data processing work, and provide users with real-time feedback during data collection.

molecular biology↗

bioRxiv2025

Phase separation of Polycomb-like (PCL) proteins drive PRC2 complex condensates to regulate gene expression

Polycomb group (PcG) proteins function as two major multicomponent protein complexes: Polycomb repressive complex 1 (PRC1) and 2 (PRC2) to repress key developmental genes and maintain epigenetic memory of cell identity during development. Phase-separation has long been implicated in PRC1 mediated gene silencing, but it is still unclear whether PRC2 also utilize a similar mechanism to regulate gene expression. Here we report that Polycomb-like (PCL) proteins, PRC2 accessory factors, can phase-separate in vitro and generate dynamic puncta in vivo. Biochemical and cellular analyses reveal that PCL proteins (hereafter referred to as PCLs) have intramolecular interaction between N- and C-terminal domains, which make PCLs into more compact conformations. The intramolecular interaction not only controls the size of the PCLs phase separation droplets, but also affects the chromatin association of PRC2. Finally, we show that CpG islands, key DNA regulatory elements in mammalian promoters, disrupt the N-C intramolecular interaction of PCLs to expose their middle intrinsically disordered regions (IDRs), which in turn trigger PCLs driven PRC2 condensates formation. Together, this study provides a new perspective on the regulation of PRC2 by PCLs, implicating PCLs read CpG islands to fine-tune their oligomerization to overcome the threshold for PRC2 recruitment to corresponding genomic loci via multivalent interaction with CpG islands chromatin.

biochemistry↗

bioRxiv2025

Critical Environmental Limits: Assessing the Limitations of Core Temperature Inflection Point (CTIP) and Biophysical Modeling

The Core Temperature Inflection Point (CTIP) method and biophysical modeling are widely used to determine critical environmental limits (CEL), yet their validity under prolonged heat exposure remains untested. This study evaluated their predictive accuracy by exposing 36 healthy young adults (20 males, 16 females; age: 20.9-22.4 yr) to five counterbalanced 8-hour heat trials in a controlled chamber (36{degrees}C/74.5% RH, 40{degrees}C/55.0% RH, 44{degrees}C/29.2% RH, 47{degrees}C/35.6% RH, 50{degrees}C/24.5% RH). These conditions were selected based on prior CTIP and biophysical model predictions of CELs. Participants engaged in sedentary office tasks (1.29- 1.67 METs), wore standardized summer clothing (0.39-0.40 clo), and had ad libitum access to an electrolyte drink, with a 500-kcal sandwich provided at midday. Rectal temperature (Trec) was continuously monitored. Contrary to CTIP and biophysical model predictions, all five conditions remained compensable (Trec rise rate [≤] 0.1{degrees}C/h), with mean peak Trec staying well below heatstroke thresholds (38.2 {+/-} 0.4{degrees}C). At 44{degrees}C/29.2% RH, females had significantly lower Trec than males (p < 0.05), but steady-state Trec responses were similar between sexes (all p > 0.10). Collectively, CTIP and biophysical models substantially underestimated CELs, leading to overpredicted heat risk across all trials. These findings challenge the reliability of current predictive methods, suggesting human tolerance may exceed existing estimates. Refining these models is essential for improving heat risk assessment during real-world heatwaves and informing public and occupational health guidelines in a warming climate. NEW & NOTEWORTHYThis study demonstrates that healthy young adults maintained heat balance over 8 hours across five extreme heat conditions (36-50{degrees}C), despite the Core Temperature Inflection Point (CTIP) method and biophysical modeling predicting uncompensability. These findings challenge the reliability of these tools for assessing prolonged heat exposure, highlighting the need for refined models to accurately predict critical environmental limits in real-world scenarios, such as extreme heatwaves.

physiology↗

bioRxiv2025

Cryo-EM structures of a pentameric ligand-gated ion channel in liposomes

Detergents and lipid nanodiscs affect the cryo-EM structures of pentameric ligand-gated ion channels (pLGICs) including ELIC. To determine the structure of a pLGIC in a membrane environment that supports ion channel function, we performed single particle cryo-EM of ELIC in liposomes. ELIC activation and desensitization were confirmed in liposomes with a stopped-flow thallium flux assay. Using WT ELIC and a non-desensitizing mutant (ELIC5), we captured resting, activated and desensitized structures at high resolution. In the desensitized structure, the ion conduction pore has a constriction at the 9 leucine of the pore-lining M2 helix, indicating that 9 is the desensitization gate in ELIC. The agonist-bound structures of ELIC in liposomes are distinct from those in nanodiscs. In general, the transmembrane domain is more loosely packed in liposomes compared to nanodiscs. It has been suggested that large nanodiscs are superior for supporting membrane protein function. However, ELIC localizes to the rim of large circularized nanodiscs, and structures of ELIC in large nanodiscs deviate from the liposome structures more than those in small nanodiscs. Using liposomes for cryo-EM structure determination of a pLGIC increases our confidence that the structures are snapshots of functional states.

biophysics↗