Biology subjects

Ouyang, K.

Publications and source records attributed to Ouyang, K..

5 recordsLinked to original sources

bioRxiv2026

Single-cell profiling resolves gain- and loss-of-function mechanisms in CASR to advance mechanism-aware variant classification

Multiplexed assays of variant effect (MAVEs) and computational methods have advanced variant classification but have struggled to distinguish the molecular mechanism of variants with sufficient accuracy, limiting utility for multi-mechanism genes. Here, we coupled single-cell RNA-seq profiling with supervised machine learning to predict the mechanism of variants in CASR, a gene in which gain of function (GOF) variants cause hypocalcemia and loss of function (LOF) variants cause hypercalcemia. We engineered endogenous CASR-depleted HEK293 cells to express a single exogenous variant copy, measured their transcriptional profiles at single-cell resolution, trained a multi-class machine learning model on 96 expert-annotated variants (25 benign, 35 LOF, 36 GOF), and predicted mechanism for 157 variants of uncertain significance, of which 16 were predicted GOF and 54 LOF. The model showed high performance (macro F1 0.98, overall accuracy 0.98), and its predictions were corroborated by clinical phenotype and serum calcium measurements across more than 300,000 patients in a commercial genetic testing database. These results highlight the effectiveness of using molecular mechanism annotations with scRNA-seq to train models that distinguish molecular mechanisms of pathogenicity. The approach has immediate relevance for clinical variant classification and establishes a foundation for mechanism-guided clinical management in disorders with mechanism-dependent interventions, with potential application to other multi-mechanism genes.

genetics↗

bioRxiv2026

Kindlin-2-Moesin interaction orchestrates sprouting angiogenesis via modulating endothelial membrane mechanics and VEGF signaling

Membrane mechanics play a crucial role in cellular signaling and fate determination, yet their impact on angiogenesis remains poorly understood. Here, we identify Kindlin-2 as a key regulator of sprouting angiogenesis via regulating endothelial membrane tension through its interaction with Moesin, a crucial linker protein between the cell membrane and the actin cortex. Mechanistically, Kindlin-2 binds to the N62 residue of Moesin, limiting its overactivation and maintaining proper membrane tension to facilitate VEGFR2 endocytosis and downstream signaling. Using both developmental and pathological models, we demonstrate that the interaction of Kindlin-2 and Moesin is enhanced in high angiogenic conditions, and endothelial Kindlin-2 deletion reduces angiogenesis. Furthermore, mutation of Moesin at N62 phenocopies the effects of Kindlin-2 loss. Together, these findings uncover a previously unrecognized mechanism linking membrane tension regulation to angiogenesis and provide new insights into targeting the Kindlin-2-Moesin axis for therapeutic intervention in neovascular diseases.

developmental biology↗

bioRxiv2026

Oxidative stress induces E. coli aryl polyene expression, sensitizing the bacterial stress response and modulating the redox environment of innate immune cells

Aryl polyenes (APEs) are specialized polyunsaturated outer membrane lipids that protect their producers from oxidative stress and contribute to biofilm formation. APEs are produced by an abundant biosynthetic gene cluster (BGC) family conserved across Gram-negative bacterial clades. The APE biosynthesis pathway involves 11 different enzymes and cumulates in the attachment of APEs to an anchor molecule in the Gram-negative outer membrane. Unlike most other small molecule BGCs, the APE BGC does not contain a dedicated regulatory gene that controls production of its metabolically costly compounds. Building from our prior observations of APEs role in acute oxidative stress protection, we here use a uropathogenic Escherichia coli (UPEC) strain to show that APE expression conveys a potential competitive advantage characterized by increased early-stage growth, sensitization of the bacterial oxidative stress response, and dampening of the redox stress of innate immune cells after in vitro infection. Our data indicate that APEs could act as a UPEC fitness factor, and in future work we aim to study their contribution to overall bacterial pathogenicity and survival, as well as how APEs could facilitate the transition from an oxygen poor environment such as the gut to the oxygen rich environment of the urinary tract. ImportanceBacterial pathogens use various mechanisms to achieve a competitive advantage under harsh conditions such as upon interaction with their host. We studied the function of aryl polyenes (APEs), specialized polyunsaturated fatty acids in the outer membrane, in the context of a uropathogenic E. coli strain. APE expression is induced by an oxidative environment and contributes to early-stage growth and sensitization of the oxidative stress response. Furthermore, APE-expressing E. coli dampens the intracellular oxidative milieu of target host phagocytes. These findings suggest a role for APEs as a fitness factor, and create opportunities to study their in vivo function, and explore them as a potential drug target.

microbiology↗

bioRxiv2024

A single-cell 3D spatiotemporal multi-omics atlas from Drosophila embryogenesis to metamorphosis

The development of a multicellular organism is a highly intricate process tightly regulated by numerous genes and pathways in both spatial and temporal manners. Here, we present Flysta3D, a comprehensive multi-omics atlas of the model organism Drosophila, spanning its developmental lifespan from embryo to pupa. Our datasets encompass 3D single-cell spatial transcriptomic, single-cell transcriptomic, and single-cell chromatin accessibility information. By integrating these multi-dimensional data, we constructed cell state trajectories that uncover the detailed profiles of tissue development. With a focus on the central nervous system (CNS) and midgut, we dissected the spatiotemporal dynamics of gene regulatory networks, cell type diversity, and morphological changes from a multi-omics perspective. This extensive atlas provides an unprecedentedly rich resource and serves as a systematic platform for studying Drosophila development with integrated single-cell data at an ultra-high spatiotemporal resolution.

developmental biology↗

bioRxiv2022

FH variant pathogenicity promotes purine salvage pathway dependence in kidney cancer

The tricarboxylic citric acid cycle enzyme fumarate hydratase (FH) is a tumor suppressor. When lost in cells, its substrate fumarate accumulates to mM levels and drives oncogenic signaling and transformation. Germline alterations lead to an autosomal dominant condition known as hereditary leiomyomatosis and renal cell cancer (HLRCC) where patients are predisposed to various benign tumors and an aggressive form of kidney cancer. FH alterations of unclear significance are frequently observed with germline testing; thus, there is an unmet need to classify FH variants by their cancer-associated risk, allowing for screening, early diagnosis and treatment. Here we quantify catalytic efficiency of 74 FH variants of uncertain significance. Over half were enzymatically inactive which is strong evidence of pathogenicity. We generated a panel of HLRCC cell lines expressing FH variants with a range of catalytic activities, then correlated fumarate levels with metabolic features. We found that fumarate accumulation blocks purine biosynthesis, rendering FH-deficient cells reliant on purine salvage to maintain purine nucleotide pools. Genetic or pharmacologic inhibition of the purine salvage pathway reduced HLRCC tumor growth in vivo. Together, these findings suggest pathogenicity of many patient-associated FH variants and reveal purine salvage as a targetable vulnerability in FH-deficient tumors. Statement of SignificanceThis study functionally characterizes patient-associated FH variants with unknown significance for pathogenicity. This study also reveals nucleotide salvage pathways as a targetable feature of FH-deficient cancers, which are shown to be sensitive to the purine salvage pathway inhibitor 6-mercaptopurine. This presents a new rapidly translatable treatment strategy for FH-deficient cancers.

cancer biology↗