Biology subjects

Jo, H.

Publications and source records attributed to Jo, H..

At least 19 recordsLinked to original sources

bioRxiv2026

Machine Learning-Based Early Prediction of Neural Microelectrode Lifetime Under Accelerated Aging Conditions

Objective. Chronically implantable medical devices may require functional lifetimes of decades in order to reduce or eliminate the need for their explantation and replacement, which may involve invasive and sometimes risky surgery. However, when devices become exceptionally long-lived, preclinical studies validating their lifetimes become difficult to execute within reasonable timeframes even when accelerated life testing methods are employed. Computational methods capable of accurately and reliably predicting microelectrode lifetime from early-life data could therefore considerably expedite the development of long-lived implantable devices. Approach. Here, inspired by similar studies predicting functional lifetimes of lithium-ion battery cells from early-life data, we developed a machine learning model based on Elastic Net regression and early-life feature engineering to predict impedance-defined microelectrode lifetimes under accelerated aging conditions and evaluated its predictive performance. We also provide a dataset of electrochemical measurements from aging microelectrodes for public use. Main results. Using measurements collected through Day 5, the 10-feature Elastic Net model achieved a root mean squared error of 3.71 accelerated-aging days and a mean absolute percentage error of 13.90% under device-level validation. In an independent fabrication batch, covariance alignment and calibration using five labeled channels from one device in each target group reduced mean absolute percentage errors from 38.1% to 22.5% for Small electrodes and from 56.6% to 18.2% for Medium electrodes. Significance. These results highlight the potential of machine learning-based approaches to accurately predict neural microelectrode lifetimes from early-life data. Such methods may inform the design of future accelerated life studies, providing meaningful longevity estimates from shorter testing periods and potentially accelerating the development of long-lived implantable devices for clinical use.

bioengineering↗

bioRxiv2026

Skin-Related Effects of Corynebacterium amycolatum SB-1 Ferment Filtrate and Functional Characterization of Ellagic Acid

Previous research has established a link between the genus Corynebacterium in the skin microbiota and skin health; however, studies investigating the specific effects of Corynebacterium-derived substances on skin cells remain limited. In this study, we isolated eight strains of Corynebacterium from the facial skin of female participant. To investigate the potential skin improvement effects of these isolates, three cultured skin cell lines (Hs68, HaCaT, and B16-F10) were treated with the supernatants of each strain. We then evaluated and compared the gene expression levels (ELN, COL1A1, IL-1{beta}, and HAS3) alongside their inhibitory effects on melanin biosynthesis. Corynebacterium amycolatum SB-1 was selected as the final candidate for subsequent analysis, primarily based on its rapid growth rate and high cell yield. Using LC-TOF/MS, we screened the postbiotic candidates from the SB-1 strain and identified ellagic acid as a known polyphenolic antioxidant with potent anti-melanogenic effects beneficial for skin improvement. To validate the effects of ellagic acid on skin cells, we assessed FLG gene expression and its inhibition of melanin biosynthesis. Additionally, RNA sequencing was performed to elucidate the underlying molecular mechanisms, revealing that ellagic acid is significantly involved in inhibiting melanogenesis and promoting collagen synthesis. Taken together, our results highlight ellagic acid derived from strain SB-1 as a highly effective bioactive compound for improving skin health, specifically targeting hyperpigmentation and skin aging.

genomics↗

bioRxiv2026

De novo design of ligand binding proteins using large language models alone

Protein design has rapidly advanced with the advent of sequence- and structure-based machine learning models. However, reasoned design, which applies physicochemical principles and rules derived from sequence-structure-function relationships, has not seen the same benefits from generative machine learning models. Here, we test the ability of common large language models (LLMs; e.g. Claude, ChatGPT, and Gemini) to consider design principles to generate de novo proteins that bind metals and lipophilic small molecules without copying existing sequences. Common LLMs alone are able to 1) generate protein sequences to adopt a desired fold and bind the target ligand and 2) explain the principles that motivate the design choices. Following structure prediction and filtering, we selected a small set of designs (6 to 12 designs per query) for experimental validation, affording metal binders in one round of LLM-based design (25% hit rate) and perfluorooctanoic acid binders in two rounds (25% hit rate in the second round of design). Importantly, the LLMs produce detailed justification to accompany the de novo designed sequences, providing a conceptual framework on which designs can be evaluated. While the successful designs have some deviations from the prompted parameters and LLM-articulated design rationale, these campaigns provide a case study that highlights the utility of LLMs in making protein design more comprehensible and accessible to users without sophisticated design expertise.

biochemistry↗

bioRxiv2026

Solanum americanum Bs2 and ZAR1 homologs recognize Xanthomonas euvesicatoria effectors essential for infection.

Multiple recognition events of pathogen-secreted effectors by immune receptors confer robust disease resistance in plants. Understanding the underlying mechanisms facilitates the discovery and deployment of valuable resistance genes for crop protection. Xanthomonas euvesicatoria causes devastating bacterial spot disease in solanaceous crops but cannot infect the wild relative Solanum americanum. Here, we identified X. euvesicatoria type III effectors (T3Es) that induce cell death in S. americanum when transiently expressed in leaf. By quantifying immune responses of the S. americanum SP2273 accession to X. euvesicatoria multiple-T3E knockout mutants, we demonstrated that at least nine T3Es (AvrBs2, XopAP, XopAU, XopE1, XopJ3, XopM, XopN, XopX, and XopZ1) collectively contribute to effector-triggered immunity (ETI). Among these, AvrBs2 and XopJ3 were the primary drivers of ETI, eliciting robust cell death and defense gene expression when naturally delivered into plant cells. We next generated S. americanum lines concomitantly edited at the corresponding immune receptor loci, SaBs2 and SaZAR1 (SP2273-bz). Genetic complementation of SP2273-bz confirmed that AvrBs2 and XopJ3 are specifically recognized by each of the four SaBs2 homologs and by SaZAR1, respectively. Moreover, enhanced growth of X. euvesicatoria on the characterized SP2273-bz line indicated that both effectors are required for bacterial multiplication in S. americanum. Together, our findings provide a framework for understanding the mechanisms of ETI-mediated resistance and establish a genetic foundation for resistance breeding in solanaceous crops.

plant biology↗

bioRxiv2026

Desert hedgehog enhances endothelial resilience and prevents atherosclerosis by mitigating PAI-1 signaling

BACKGROUNDAtherosclerotic cardiovascular disease remains the leading cause of death worldwide. Most current pharmacotherapies target conventional risk factors that promote atherosclerosis (e.g., hyperlipidemia) rather than intrinsic resilience factors that protect against atherosclerosis in the face of risk factors. Here, we investigated the role of desert hedgehog (DHH), a canonical ligand of the hedgehog signaling pathway, as a novel resilience factor that restrains endothelial mesenchymal transition (EndoMT) and protects against atherosclerosis. METHODSSingle-cell RNA sequencing (scRNA-seq) was performed on atheroprone and atheroprotective regions of the ApoE knockout mouse aorta to identify mechanoresponsive genes associated with atherosclerosis. Endothelial cell-specific Dhh knockout mice were subjected to partial carotid ligation and hypercholesterolemic conditions to investigate the role of endothelial Dhh in atherosclerosis progression. scRNA-seq, bulk RNA sequencing, endothelial lineage tracing, immunoprecipitation-coupled mass spectrometry, and surface plasmon resonance were used to in-vestigate the role and mechanism of DHH in EndoMT. Pharmacological interventions and recombinant DHH administration were performed in vivo to evaluate therapeutic potential of DHH targeting. DHH expression was also examined in human atherosclerotic arteries and serum samples from patients with coronary artery disease. RESULTSDHH protein expression was enriched in arterial endothelium from mice, porcine, and humans. However, DHH expression was significantly reduced in atherosclerotic arteries and serum from patients with coronary artery disease. scRNA-seq of atheroprone and atheroresistant region of mouse aorta identified Dhh as a novel mechanoresponsive gene enriched in aortic regions exposed to unidirectional laminar flow. Endothelial cell-specific Dhh knockout (DhhecKO) mice exhibited increased atherosclerotic lesion area, large necrotic cores, and reduced collagen content following partial carotid ligation. Similarly, under hypercholesterolemic conditions, both male and female DhhecKO mice showed aggravated atherosclerosis progression. scRNA-seq of DhhecKO mouse aortas revealed an increased proportion of endothelial cells undergoing mesenchymal transition, indicating enhanced EndoMT. These findings were corroborated by bulk RNA-sequencing of DHH depleted HUVECs and endothelial lineage tracing in inducible DhhecKO mice. Mechanistically, DHH directly interacted with plasminogen activator inhibitor type 1 (PAI-1) and suppressed PAI-1-induced EndoMT. PAI-1 promoted EndoMT in ECs through activation of canonical TGF-{beta} signaling (SMAD2/3) and noncanonical AKT/ERK1/2 signaling via interaction with low-density lipoprotein receptor-related protein (LRP1). Neutralization of PAI-1 or inhibition of LRP1, AKT/ERK1/2, or SMAD3 signaling abolished DHH deficiency-induced EndoMT. DHH competitively inhibited PAI-1 binding to LRP1, thereby attenuating downstream pro-EndoMT signaling. Intriguingly, treatment with PAI-1 inhibitor TM5275 mitigated endothelial Dhh deficiency induced EndoMT in vivo. Of translational relevance, recombinant mouse DHH protein administration reduced atherosclerosis progression, stabilized plaque, and decreased the expression of EndoMT markers in ApoE knockout mice. CONCLUSIONSDesert hedgehog (DHH) is an intrinsic endothelial cell-enriched resilience factor that protects against EndoMT and atherosclerosis by preventing PAI-1 signaling. The present study implicates endothelial DHH as a potential therapeutic target for atherosclerotic cardiovascular disease. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=163 SRC="FIGDIR/small/729715v1_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@194c8a3org.highwire.dtl.DTLVardef@1004d52org.highwire.dtl.DTLVardef@d47c73org.highwire.dtl.DTLVardef@368866_HPS_FORMAT_FIGEXP M_FIG C_FIG Desert hedgehog (DHH) is an intrinsic endothelial cell-enriched resilience factor that protects against EndoMT and atherosclerosis by preventing PAI-1 binding to LRP1 and downstream AKT/ERK, as well as SMAD2/3 signaling. Clinical PerspectiveO_ST_ABSWhat Is New?C_ST_ABSO_LIDHH was identified as a flow-responsive resilience gene that is downregulated by disturbed flow in endothelial cells. C_LIO_LIEndothelial-specific deletion of Dhh promoted endothelial-to-mesenchymal transition (En-doMT) and atherosclerosis. C_LIO_LIDHH directly interacts with PAI-1 and preclude PAI-1 mediated pro-EndoMT signaling. C_LI What Are the Clinical Implications?O_LIDHH maintains endothelial homeostasis during atherosclerosis. C_LIO_LITargeting endothelial DHH-PAI-1 axis may represent a potential therapeutic strategy to reduce EndoMT and limit plaque progression. C_LIO_LILower circulating DHH level may serve as a potential biomarker of endothelial dysfunction and plaque vulnerability in atherosclerotic disease. C_LI

biochemistry↗

bioRxiv2026

Interplay of stability and dynamics in the optimization of a highly proficient de novo enzyme

The development of highly active de novo enzymes that catalyze new-to-nature reactions is becoming increasingly possible. Here, we identify the features underlying the catalytic efficiency of a highly proficient de novo enzyme, from the original computational design to an optimized catalyst obtained through two rounds of directed evolution. Computational, spectroscopic, and biochemical studies reveal successfully designed features, including precise alignment of catalytic residues, transition state stabilization, and environmental tuning. In the most evolved enzyme, binding of a transition state analog also led to widespread increases in backbone conformational stability throughout the protein, except within a helix near the active site entrance, where the introduction of Gly and Pro increased dynamics and catalytic activity. Thus, the entire protein contributes to catalysis in the most optimized enzyme. Also, the initial design considered only the transition state but not substrate binding, leading to a dynamic Michaelis complex prior to optimization. These studies show the multiple features that need to be optimized to achieve high activity in a designed enzyme.

biochemistry↗

bioRxiv2026

Long-Lasting Electrohydrodynamically Printed Transparent Soft Microelectrode for Implantable Biointerfaces

Reliable and scalable soft implantable neural interface fabrication remains a key challenge for chronic bioelectronic applications. Here, we present a transparent soft microelectrode fabricated with electrohydrodynamic (EHD) printing, utilizing the fluorinated polymer poly(vinylidene fluoride-co-hexafluoropropylene) (PVDF-HFP) and poly (3, 4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT: PSS) to form seamless, selectively patterned multilayer structures with low impedance and long-term stability. Controlled in situ curing during printing yields dense, void-free substrate and encapsulation layers, suppressing interfacial defects and ionic pathways, while maintaining high optical transparency (>60%) with PEDOT:PSS. The printed microelectrodes exhibit low impedance, high charge storage and injection capacities, and stable electrochemical behavior under biomimetic conditions. In addition, the devices demonstrate robust mechanical and electromechanical stability under cyclic deformation in both dry and wet environments, as well as under prolonged electrical stimulation. Accelerated aging studies project multi-year operational lifetimes, and in vitro/in vivo biocompatibility assessments confirm excellent tissue integration. These results establish EHD-printed fluorinated polymer-based microelectrodes as a scalable and durable platform for chronic implantable biointerfaces. ToC O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=182 SRC="FIGDIR/small/726391v1_ufig1.gif" ALT="Figure 1"> View larger version (79K): org.highwire.dtl.DTLVardef@f4f717org.highwire.dtl.DTLVardef@8a34a1org.highwire.dtl.DTLVardef@185540dorg.highwire.dtl.DTLVardef@51462a_HPS_FORMAT_FIGEXP M_FIG C_FIG This report presents an electrohydrodynamically printed transparent soft microelectrode for chronic purposes. Electrohydrodynamic printing promotes seamless multilayer structures with selective deposition and long-term mechanical stability. The devices show low impedance, high charge capacity, and robust electrochemical/electromechanical properties. Accelerated aging projects [~]7.2 year lifetimes, and XPS/SEM-EDS confirm strong ion barrier properties and biocompatibility for chronic implantation.

bioengineering↗

bioRxiv2026

Disease context dictates the cellular targets of IL-17 in inflammatory skin disease

Therapeutic blockade of IL-17 and TNF can effectively treat inflammatory skin diseases such as hidradenitis suppurativa and psoriasis, yet the relative importance of the different cell types that respond to IL-17 and TNF remains unresolved. Keratinocytes are viewed as the dominant effector cells, whereas fibroblasts have recently emerged as important contributors. In mice, topical imiquimod induces IL-17- and TNF-dependent skin inflammation and is frequently used to model psoriasis. Here, we demonstrate that intradermal injection of recombinant IL-17 and TNF elicits skin inflammation with features of hidradenitis suppurativa, including a gene expression program that is distinct from psoriasis and imiquimod-induced inflammation. Single-cell transcriptomic network analysis identified dermal fibroblasts as the dominant cell communication hub in hidradenitis suppurativa and in mice injected with IL-17 and TNF. In contrast, fibroblasts and keratinocytes both show strong network involvement in psoriasis and in mice challenged with imiquimod. Cell-type-specific deletion of IL-17 receptor A in mice revealed that imiquimod-induced inflammation depends equally on IL-17 signaling in fibroblasts and keratinocytes, whereas inflammation induced by intradermal IL-17 and TNF only requires fibroblasts to recognize IL-17 and is independent of keratinocyte IL-17 sensing. Single-cell transcriptomic analysis of these conditional knockout mice further demonstrated that keratinocytes and fibroblasts activate divergent and disease-dependent transcriptional programs following activation by IL-17. Together, these findings introduce a new conceptual framework wherein IL-17 signaling is routed through distinct cellular and molecular pathways depending on disease context and establish complementary experimental systems for interrogating type 17 skin inflammation.

immunology↗

bioRxiv2026

Distinct tau filament folds in familial frontotemporal dementia due to the MAPT S305I mutation

Frontotemporal lobar degeneration with tau inclusions (FTLD-tau) comprise a class of fatal heterogeneous neurodegenerative diseases. Approximately 10% arise from pathogenic MAPT mutations and often cause severe, early-onset disease with pathology that is distinct yet partially overlapping with sporadic cases. Here, we evaluated post-mortem tissue from a patient with FTLD-tau due to MAPT S305I showing neuropathology most consistent with argyrophilic grain disease (AGD), a prevalent limbic tauopathy of aging. Structures determined by cryo-electron microscopy reveal tau filament folds that differ from those found in sporadic AGD or other tauopathies and feature a 4-layer architecture stabilized by the Ile substitution within its core. Comparative structural analysis reveals conserved motifs are shared among AGD, corticobasal degeneration, and MAPT P301T. A well-defined density stacks along a cationic cleft, indicative of a bound RNA-like polyanion or small-molecule. In vitro analysis shows the S305I mutation promotes fibrilization relative to normal tau. These results demonstrate that MAPT S305I stabilizes a distinct aggregation-prone tau fold that likely contributes to disease pathology and heterogeneity beyond its known splicing defects, and underscore potential limitations of using the most pathologically similar genetic form as a model for sporadic FTLD-tau.

neuroscience↗

bioRxiv2026

Cross-cohort analysis reveals conserved gut virome signatures and phage-encoded auxiliary functions in ulcerative colitis

While gut bacteriome dysbiosis is a well-established hallmark of ulcerative colitis (UC), the ecological and functional remodeling of the gut virome conserved across diverse populations remains unclear. Here, by constructing a cross-cohort atlas of the UC-associated fecal virome, we show that viral reorganization closely parallels bacterial dysbiosis, with enrichment of phages targeting disease-associated bacterial taxa. We identified a conserved set of UC-associated viral signatures that robustly distinguished UC from healthy controls using machine-learning-based classification across independent cohorts. Functional profiling further revealed that phages enriched in UC carried higher densities of auxiliary metabolic genes (AMGs) related to virulence and antibiotic resistance than phages depleted in UC, with immune evasion and glycopeptide resistance genes particularly overrepresented. Together, our cross-cohort approach highlights the value of the gut virome-based diagnostic framework for UC and suggests that phage-encoded AMGs may contribute to shaping the gut ecosystem under inflammatory conditions.

microbiology↗

bioRxiv2025

Neuronal PARIS-STAT3 axis drives tau pathology and glial activation in Alzheimers disease

PARIS, a substrate of Parkin, accumulates in Parkinsons disease and promotes disease progression. Here, we demonstrate that PARIS also contributes to Alzheimers disease by elevating STAT3 transcriptional activity, thereby inducing tau pathology, hippocampal atrophy, and glial activation. Genetic depletion of Paris reduced tau phosphorylation and cognitive decline in tauopathy mice, whereas neuron-specific PARIS overexpression caused tau accumulation, gliosis, and memory impairment. In contrast, astrocyte-specific overexpression did not induce pathology, indicating that PARIS acts in neurons to drive tau phosphorylation and glial activation. The pathological features induced by neuronal PARIS overexpression were rescued by STAT3 inhibition, demonstrating that PARIS-STAT3 signaling underlies these effects. Moreover, Paris knockout did not alter pathology in the amyloid-driven mouse model, highlighting specificity for tau pathology. Together, these findings reveal PARIS as a neuronal regulator of STAT3 signaling that exacerbates tau-mediated neurodegeneration and identify the PARIS-STAT3 pathway as a potential therapeutic target in Alzheimers disease.

neuroscience↗

bioRxiv2025

A Conserved Stress-Inducible Hub Enhancer Governs Natriuretic Peptide Expression and Undergoes Dynamic and Reversible Activation in Human Heart Failure

BackgroundNatriuretic peptides (NPs), encoded by the NPPA and NPPB genes, serve as both diagnostic biomarkers and cardioprotective hormones in heart failure. Their expression is tightly regulated by mechanical load, yet the upstream enhancer mechanisms translating hemodynamic stress into transcriptional activation remain incompletely understood. MethodsWe investigated the Nppa/Nppb super-enhancer (SE), which resides in a well-insulated topologically associating domain (TAD) and regulates stress-inducible expression of Nppa and Nppb. We applied CRISPR-based enhancer perturbation, chromatin accessibility and histone profiling, chromatin conformation assays, genetic mouse models, human iPSC-derived cardiomyocytes, and paired failing human hearts before and after left ventricular assist device (LVAD) unloading. ResultsThrough integrative epigenomic and genetic analyses, we identified a conserved element, CR9, as a dominant hub enhancer within this SE. In neonatal rat cardiomyocytes, CRISPR-based activation and inhibition established that CR9 was both necessary and sufficient for NP induction, whereas neighboring elements (CR7/CR8) displayed modest activity but synergized with CR9, establishing a hierarchical and cooperative SE architecture. In vivo, CR9 deletion in mice markedly suppressed Nppa/Nppb expression and diminished chromatin accessibility and histone acetylation across adjacent enhancers and promoters, underscoring its structural as well as transcriptional role. Reinsertion of CR9, even in reverse orientation, restored transcription, confirming its orientation-independent activity. CR9 function remained confined within TAD boundaries, supporting a locus-specific regulation. RNAscope revealed spatial proximity between CR9 enhancer RNA and Nppa/Nppb nascent transcripts. Developmental profiling revealed a switch in enhancer usage from CR6/CR7 in the fetal heart to CR9 in adulthood, indicating a transition from developmental to stress-inducible regulation. In human induced pluripotent stem cell-derived cardiomyocytes, deletion of CR9 nearly abolished NPPA/NPPB expression, establishing its essential role in a human cellular context. Most notably, in paired human failing hearts before and after LVAD unloading, chromatin accessibility at CR9 was dynamically reversed, providing the first direct evidence that enhancer states are plastic and therapeutically modifiable in the human myocardium. ConclusionsCR9 functions as a stress-inducible hub enhancer that coordinates NP transcription under pathological load. This enhancer exhibits reversible activation in human hearts, underscoring enhancer plasticity as a modifiable regulatory layer mediating stress-responsive transcriptional adaptation in the diseased myocardium. Clinical PerspectiveO_ST_ABSWhat is new?C_ST_ABSO_LIWe identified CR9 as a stress-inducible hub enhancer within the Nppa/Nppb super-enhancer that is both necessary and sufficient for natriuretic peptide induction, defining a hierarchical and cooperative regulatory architecture controlling Nppa and Nppb expression. C_LIO_LIWe uncovered a developmental switch in enhancer usage from CR6 in the fetal heart to CR9 in adulthood, revealing how stress-inducible enhancer activity emerges as the heart transitions from developmental to adaptive regulation. C_LIO_LIUsing paired failing human hearts before and after LVAD unloading, we provide the first direct evidence that enhancer states are dynamically reversible in the human myocardium. C_LI What are the clinical implications?O_LIThe reversibility of CR9 activity in human heart failure provides a molecular basis for how natriuretic peptide levels decrease when cardiac function improves with effective therapy, reflecting the underlying enhancer plasticity of the diseasesd myocardium. C_LIO_LIEnhancer plasticity is an emerging and targetable mechanism in heart failure, raising the possibility that stress-inducible enhancers like CR9 could be engineered to switch on cardioprotective genes when the heart is under stress, offering a new strategy for precision gene therapy. C_LI

molecular biology↗

bioRxiv2025

Lung Adenocarcinoma Just Desserts: An Expanding Pie of Activating Oncogenes or a Layer Cake of Integrated Alterations

The molecular landscape of lung adenocarcinoma (LUAD) is often summarized as a "pie chart" of driver oncogenes, suggesting identification and targeting of oncogenic drivers is the best clinical approach. However, this model oversimplifies LUAD biology. Patients with identical RAS or RAF mutations exhibit heterogeneous signaling influenced by co-mutations, transcriptional programs, and lineage context. We propose a hierarchical framework integrating oncogenes within cellular context by examining canonical EGFR mutations. We defined an EGFR mutation signature (mSig) by identifying differentially expressed genes in EGFR-mutant LUADs. Semi-supervised clustering and machine learning models were used to test performance and reproducibility across independent datasets. We analyzed molecular subtypes, lineage markers, co-occurring mutations, and candidate drug targets in EGFR-mSig positive (+) versus negative (-) tumors. The EGFR mSig showed robust classification performance across datasets (AUROC = 0.83-0.95; mean NPV = 96.3%). Validated unsupervised gene expression subtypes and lung lineage markers were closely aligned with EGFR mSig status. mSig(+) tumors were identified, even in tumors without EGFR mutations. A subset of canonical RAS mutations mirrored the EGFR mutation pattern. EGFR-mutant/mSig(-) tumors were enriched for non-Bronchioid subtypes and had co-mutations in TP53 or RAS. Coordinated mutations were identified including RAS, KEAP1, STK11, TP53, and CDKN2A, supportive of prior reports. In sum, novel EGFR mSig that captures the transcriptional footprint of EGFR activation, revealed a subset of EGFR wildtype LUADs with "mutant-like" features. Lineage-informed classification highlights subtype-dependent oncogene activity and supports new therapeutic strategies. A context-specific association between RAS mutation and expression of the dual-specificity phosphatase gene DUSP4 may have therapeutic potential.

genomics↗

bioRxiv2025

C1q limits cystoid edema by maintaining basal beta-catenin-dependent signaling and blood-retina barrier function

Macular edema (ME) causes significant vision impairment and occurs in several prevalent retinal diseases including diabetic retinopathy (DR), choroidal neovascularization (CNV), retinal vein occlusion, and uveitis. Retinal edema typically results from dysfunction of the blood-retina barrier (BRB), which is associated with increased retinal expression of complement components. It is unclear whether the classical complement pathway has detrimental or protective roles in the context of BRB dysfunction. Here, we characterize Tspan12 KODBM (Disrupted Barrier Maintenance) mice, a new mouse model of cystoid edema based on genetically and pharmacologically manipulating beta-catenin-dependent norrin/frizzled4 (FZD4) signaling. We assess BRB function, cystoid edema, ERG, and microglia activation outcomes in an aging study with WT, C1qa KO, Tspan12 KODBM, and Tspan12 KODBM; C1qa KO compound mutant mice. Phenotypic analyses and cell-based experiments indicate that C1QA contributes to maintaining basal beta-catenin-dependent signaling and that the absence of C1QA exacerbates BRB dysfunction, cystoid edema, and neuroinflammation in Tspan12 KODBM; C1qa compound mutant mice. Activation of beta-catenin-dependent signaling by a FZD4/LRP5 agonist antibody modality achieves complete resolution of cystoid edema. This study shows that reducing or enhancing norrin/frizzled4 signaling can increase or decrease cystoid edema, respectively, underscoring its potential as a therapeutic target in ME. Furthermore, this study provides novel insights into the contribution of C1QA to BRB maintenance.

neuroscience↗

bioRxiv2025

Immunomodulatory endothelial cells contribute to T cell recruitment and activation through antigen presentation on MHC class II

AimsA subset of endothelial cells referred to as immunomodulatory endothelial cells (IMEC) has been proposed to regulate T cell responses in atherosclerosis, but their phenotype and function remain poorly understood. Here, we characterized the inflammation-induced emergence of IMEC and their crosstalk with T cells. Methods and ResultsAn in vitro model to study IMEC was established and characterized using flow cytometry and proteomics. Single-cell transcriptome data from human atherosclerotic arteries as well as single cell transcriptome and endothelial cell-specific translatome data from a murine atherogenesis model were used to determine pathophysiological relevance. Immunopeptidomics was performed to detect antigen presentation. T cell chemotaxis, adhesion and activation were assessed through flow cytometry and microscopy. IMEC were induced by treating human endothelial cells with interleukin-1{beta}, interferon-{gamma}, and transforming growth factor-{beta}2. These cells expressed lower levels of classical endothelial cell markers but expressed major histocompatibility complex (MHC) class II, proteins involved in antigen processing and presentation (CD83, CD80 and CD86) and pro-inflammatory cytokines as well as chemokines, including CXCL9. An endothelial cell subpopulation with similar immunomodulatory features was identified in a mouse model of accelerated atherogenesis as well as in human atheromas. Conditioned medium from IMEC enhanced the migration of peripheral blood mononuclear cells and induced T cell chemotaxis, the latter being partially inhibited by antagonizing CXCL9. Proteins related to glycosaminoglycan degradation were significantly downregulated in IMEC which was relevant inasmuch as the glycocalyx plays a key role in the establishment of chemokine gradients. Indeed, the accumulation of heparan sulfates in IMEC contributed to the adhesion of T cells. Notably, IMEC that had been exposed to monocyte lysates presented 627 peptide antigens on MHC class II and induced T cell activation. ConclusionOur data demonstrate the role of IMEC as non-professional antigen-presenting cells that potentially contribute to T cell-mediated immune responses in cardiovascular disease. Translational PerspectiveThis study characterizes immunomodulatory endothelial cells (IMEC) as critical mediators of vascular inflammation through their capacity to process and present exogenous antigens and activate T cells. Induced by pro-atherogenic cytokines (IFN-{gamma}, IL-1{beta}, TGF-{beta}2), IMEC upregulate MHC class II and costimulatory molecules, promote leukocyte chemotaxis, and enhance T cell adhesion through surface heparan sulfate. The identification of IMEC-like populations in both murine models and human atherosclerotic plaques indicates a conserved immunological function in atherogenesis. These findings position IMEC as novel, non-professional antigen-presenting cells and potential therapeutic targets to modulate vascular immune responses in atherosclerotic cardiovascular disease.

immunology↗

bioRxiv2025

Flow-sensitive HEG1 controls eNOS activity to prevent endothelial dysfunction, hypertension, and atherosclerosis

Hypertension (HTN), the chronic elevation of blood pressure, accounts for more atherosclerotic cardiovascular disease deaths than any other modifiable risk factor.1 In the arteries, stable blood flow (s-flow) drives healthy, atheroprotective endothelial cell (EC) functions including nitric oxide (NO) production, barrier function, and anti-inflammatory programs via the action of flow-sensitive proteins. We showed that s-flow stimulates Heart-of-Glass 1 (HEG1) protein expression, localization to cell-cell junctions, and secretion from ECs.2 We found that conditional, endothelial cell-specific knockout of (Heg1ECKO) exacerbates atherosclerosis2, however the mechanism was unknown. Here, we report a new role of HEG1 in controlling EC dysfunction, hypertension and atherosclerosis. We discover a novel mechanism: HEG1 regulates NO bioavailability via a flow-dependent HEG1-eNOS interaction (endothelial nitric oxide synthase, NOS3). Heg1ECKO develops spontaneous hypertension and severe atherosclerosis, both of which are effectively treated by Angiotensin-Converting Enzyme inhibition (ACEi). UK BioBank and Swedish cohort studies reveal that plasma HEG1 levels are associated with hypertension and cardiovascular disease risk.3,4 Our findings suggest HEG1 may serve as a biomarker to advance personalized therapies for EC dysfunction, hypertension, and atherosclerosis.

physiology↗

bioRxiv2025

Disturbed Flow Induces Reprogramming of Endothelial Cells to Immune-like and Foam Cells under Hypercholesterolemia during Atherogenesis

BackgroundAtherosclerosis occurs preferentially in the arteries exposed to disturbed flow (d-flow), while the stable flow (s-flow) regions are protected even under hypercholesterolemic conditions. We recently showed that d-flow alone initiates flow-induced reprogramming of endothelial cells (FIRE), including the novel concept of partial endothelial-to-immune-cell-like transition (partial EndIT), but was not validated using a genetic lineage-tracing model. Here, we tested and validated the two-hit hypothesis that d-flow is an initial instigator of partial FIRE but requires hypercholesterolemia to induce a full-blown FIRE and atherosclerotic plaque development. MethodsMice were treated with adeno-associated virus expressing proprotein convertase subtilisin/kexin type 9 and a Western diet to induce hypercholesterolemia and/or partial carotid ligation (PCL) surgery to expose the left common carotid artery (LCA) to d-flow. Single-cell RNA sequencing (scRNA-seq) analysis was performed using cells obtained from the intima and leftover LCAs and the control right common carotid arteries at 2 and 4 weeks post-PCL. Comprehensive immunohistochemical staining was performed on EC-specific confetti mice treated with PCL and hypercholesterolemic conditions at 4 weeks post-PCL to validate endothelial reprogramming. ResultsAtherosclerotic plaques developed by d-flow under hypercholesterolemia at 2 and 4 weeks post-PCL, but not by d-flow or hypercholesterolemia alone, as expected. The scRNA-seq results of 98,553 single cells from 95 mice revealed 25 cell clusters; 5 EC, 3 vascular smooth muscle cell (SMC), 5 macrophage (M{Phi}), and additional fibroblast, T cell, natural killer cell, dendritic cell, neutrophil, and B cell clusters. Our scRNA-seq analyses showed that d-flow under hypercholesterolemia transitioned healthy ECs to full immune-like (EndIT) and, more surprisingly, foam cells (EndFT), in addition to inflammatory and mesenchymal cells (EndMT). Further, EC-derived foam cells shared remarkably similar transcriptomic profiles with foam cells derived from SMCs and M{Phi}s. Comprehensive lineage-tracing studies using immunohistochemical staining of canonical protein and lipid markers in the EC-specific confetti mice clearly demonstrated direct evidence supporting the novel FIRE hypothesis, including EndIT and EndFT, when d-flow was combined with hypercholesterolemia. Further, reanalysis of the publicly available human carotid plaque scRNA-seq and Perturb-seq datasets supported the FIRE hypothesis and a potential mechanistic link between the genes and FIRE. ConclusionWe provide evidence supporting the two-hit hypothesis: ECs in d-flow regions, such as the branching points, are partially reprogrammed, while hypercholesterolemia alone has minimal endothelial reprogramming effects. Under hypercholesterolemia, d-flow fully reprograms arterial ECs, including the novel EndIT and EndFT, in addition to inflammation and EndMT, during atherogenesis. This single-cell atlas provides a crucial roadmap for developing novel mechanistic understanding and therapeutics targeting flow-sensitive genes, proteins, and pathways of atherosclerosis.

cell biology↗

bioRxiv2025

Endothelial Cu Uptake Transporter CTR1 Senses Disturbed Flow to Promote Atherosclerosis through Cuproptosis

Endothelial cells (ECs) lining blood vessels sense disturbed blood flow (D-flow), which drives mitochondrial dysfunction and atherosclerosis. Copper (Cu) is an essential micronutrient, and its disruption of homeostasis has been implicated in atherosclerosis. Cellular Cu levels are tightly controlled by Cu transport proteins including the Cu importer CTR1. Cuproptosis is a recently discovered form of regulated cell death triggered by mitochondrial Cu accumulation, but its endogenous stimulants and role in atherosclerosis remain unknown. Using EC-specific CTR1-deficient mice and cultured ECs, we show that endothelial CTR1 responds to D-flow by increasing mitochondrial Cu levels through its interaction with the mitochondrial Cu transporter SLC25A3 at caveolae/lipid rafts. This leads to the aggregation of lipoylated mitochondrial proteins, mitochondrial dysfunction, and cuproptosis, thereby exacerbating atherosclerosis. Importantly, mitochondria-targeted Cu-chelating nanoparticles effectively mitigate D-flow-induced cuproptosis and atherosclerosis, highlighting the endothelial CTR1-SLC25A3-mitochondrial Cu axis as a potential therapeutic target.

cell biology↗