Macrophage adaptation to oxidative stress may influence the progression of acetaminophen (APAP)-induced liver injury, but the underlying mechanisms remain incompletely understood. In this study, we combined hepatic transcriptomic and proteomic data with untargeted metabolomics to prioritize candidates for functional investigation. Joint pathway analysis highlighted arachidonic acid metabolism, and arachidonate lipoxygenase 3 (ALOXE3) was prioritized for functional investigation based on its concordant upregulation at the mRNA and protein levels and its previously characterized hydroperoxide isomerase activity. We then combined spatial and single-cell transcriptomics with macrophage-directed ALOXE3 manipulation to examine its distribution and function. Spatial and single-cell analyses provided cellular context for Aloxe3 expression, including its detection in macrophage-associated populations. Single-cell analysis showed higher scores for a glutathione (GSH) metabolism-related gene set in Aloxe3-positive than in Aloxe3-negative macrophages. In vivo, macrophage-directed ALOXE3 overexpression reduced serum ALT and AST activities and hepatic necrosis, whereas knockdown produced the opposite effects. In bone marrow-derived macrophages exposed to conditioned medium from APAP-treated hepatocytes, ALOXE3 knockdown increased 4-hydroxynonenal signals and reactive oxygen species-associated fluorescence and reduced GSH-associated fluorescence. Overexpression produced the opposite changes. These findings support a role for macrophage ALOXE3 in redox responses during APAP-induced liver injury.
Mutations in thyroid hormone receptor (TR) are etiologically linked to severe neurodevelopmental disorders, highlighting its critical role in the development and function of the nervous system. However, since neural progenitor cell (NPC) emergence precedes endogenous thyroid hormone synthesis, the ligand-independent functions of TR during early neurogenesis remain elusive. We employed hESC-derived NPCs in combination with TR knockdown to investigate the functions of unliganded TR (uTR). By integrating multi-omics approaches, including RNA sequencing, co-immunoprecipitation coupled with mass spectrometry, ChIP-seq profiling of NuRD components and histone modifications, and high-throughput chromatin conformation capture techniques, we elucidated the mechanisms by which uTR regulates neurogenesis and differentiation. Depletion of uTR triggered extensive transcriptional reprogramming, significantly disrupting key neurodevelopmental pathways. Mechanistically, we demonstrate that uTR physically interacts with the Nucleosome Remodeling and Deacetylase (NuRD) complex. Notably, TR knockdown predominantly compromised the protein stability and genomic recruitment of the NuRD subunit MTA1. The loss of uTR was accompanied by altered NuRD-mediated repression and a marked increase in histone H3 lysine 9 acetylation (H3K9ac), while H3K27ac profiles and the higher-order 3D chromatin architecture were largely maintained. Our findings collectively establish uTR as a critical epigenetic modulator that fine-tunes the neurodevelopmental program, potentially through the uTR-NuRD-H3K9ac signaling axis during the key early window of neurogenesis. This study highlights the importance of ligand-independent signaling in maintaining chromatin homeostasis during early neural development and provides novel insights into the pathogenesis of TR-related disorders.
Cardiomyopathies are major causes of heart failure and sudden cardiac death, yet the functional consequences of many disease-associated genes and variants remain poorly defined. Here, we establish a scalable, segment-based in vivo platform in Drosophila for quantitative cardiac phenotyping and precision modeling of cardiomyopathy subtypes. By integrating established cardiac imaging approaches into a standardized framework, termed Segmental Cardiac Output and Performance Evaluation (SCOPE), we systematically compared cardiac function across abdominal heart segments. This analysis identified segment A3 as the dominant pumping region, exhibiting the largest systolic and diastolic diameters, highest fractional shortening, and greatest cardiac output, thereby providing an optimal and reproducible site for functional assessment. Leveraging this platform, we generated genetically defined models of dilated and hypertrophic cardiomyopathy through heart-specific knockdown of LamC and bt, homologs of human LMNA and MYBPC3, respectively. LamC silencing produced hallmark features of dilated cardiomyopathy, including chamber dilation, cardiomyocyte loss, reduced contractility, and decreased cardiac output. In contrast, bt silencing induced hypertrophic remodeling characterized by increased heart wall thickness and impaired cardiac performance. These models recapitulate key structural and functional characteristics of human cardiomyopathy subtypes. Together, this work establishes a scalable in vivo platform for segment-resolved cardiac phenotyping that enables precise modeling of cardiomyopathy, systematic functional validation of candidate genes and variants, and future discovery of genetic and pharmacologic modifiers.
Peripheral neuropathies arise from diverse genetic and acquired insults, and Schwann cell dysfunction is central to demyelination, impaired axon-glia support, and failed regeneration. Human pluripotent stem cell (hPSC)-derived Schwann cells have emerged as a scalable and genetically defined platform for modeling these disorders in a human context. This Review summarizes current hPSC-to-Schwann cell differentiation strategies and examines how these systems have been applied to model peripheral neuropathies, including Charcot-Marie-Tooth disease, neurofibromatosis type 1, diabetic peripheral neuropathy, and peripheral nerve injury. It further positions hPSC-derived Schwann cells relative to primary human Schwann cells, adult stem cell-derived Schwann-like cells, and non-human models, and discusses how emerging three-dimensional organoid, bioprinted, and microphysiological platforms address multicellular complexity and translational bottlenecks. hPSC-derived Schwann cell models are becoming an important bridge between reductionist mechanistic studies and more physiologically complex peripheral nerve platforms. Their continued value will depend on improving maturation, functional validation, multicellular integration, and translational reproducibility.
VCX3A, a member of the VCX/VCY gene family, is predominantly expressed in spermatogonia of the male testis. Emerging evidence implicates that mutations or duplication of VCX3A may contribute to nonobstructive azoospermia. Although VCX3A has been characterized as a nuclear RNA-binding protein, its functional role in spermatogonia remains poorly defined. Among the members of the VCX gene cluster, VCX3A showed the highest expression in human testicular tissue. Functionally, exogenous VCX3A promoted the proliferation and migration of the mouse spermatogonial cell line GC-1, whereas knockdown of endogenous VCX3A in NCCIT cells, a human embryonal carcinoma line, suppressed both behaviors. Biophysical analyses revealed VCX3A to be an intrinsically disordered protein that undergoes liquid–liquid phase separation in vitro as well as within the nuclei of GC-1 and NCCIT cells. irCLIP-seq profiling demonstrated that VCX3A binds preferentially to the intronic regions of protein-coding genes, with marked enrichment of an RRDGG motif. IP–MS and co-immunoprecipitation confirmed that VCX3A interacts with the splicing regulator KHDRBS1 and associates with pre-mRNAs transcribed from genes governing cell motility and proliferation, including Lpp and Frmd5. VCX3A modulated the alternative splicing of these pre-mRNAs, thereby increasing the abundance of specific transcript isoforms. Notably, either deletion of the RGG-like domain of VCX3A or knockdown of Khdrbs1 disrupted VCX3A-mediated splicing regulation and abolished its stimulatory effects on GC-1 cell proliferation and migration. VCX3A is an RNA-binding protein that cooperates with KHDRBS1 in GC-1 cells to coordinately regulate the alternative splicing of pre-mRNAs derived from motility- and proliferation-related genes such as Lpp and Frmd5, thereby enhancing the proliferative and migratory capacity of GC-1 cells. Our findings provide the direct mechanistic evidence linking VCX3A protein to the post-transcriptional control of spermatogonial cell behavior.
