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The activation of the NLRP3 inflammasome is a pivotal step in hyperinflammation in sepsis; however, the regulatory mechanisms underlying its activation are not fully understood. In this study, we found that 14-3-3ε facilitates NLRP3 inflammasome activation by enhancing NLRP3 K63 deubiquitination and promoting its translocation to the mitochondria-associated ER membranes (MAMs) for full activation. Mass spectrometry revealed that 14-3-3ε binds to NLRP3 in macrophages during sepsis. Plasma 14-3-3ε levels were elevated in patients with sepsis and were positively associated with disease severity. 14-3-3ε promoted NLRP3 inflammasome activation by facilitating NLRP3 aggregation and NLRP3-ASC assembly. The interaction between 14-3-3ε and NLRP3 was dependent on phosphorylation at the S194 site of NLRP3 NACHT domain. The NLRP3-14-3-3ε interaction promoted K63 deubiquitination and enhanced the translocation of NLRP3 to MAMs, which is necessary for full activation of NLRP3 inflammasome. Furthermore, macrophage-conditional KO of 14-3-3ε or treatment with BV02, a 14-3-3 inhibitor, improved the survival rate and alleviated organ injuries in septic mice. Taken together, our data indicate that 14-3-3ε functions as a positive regulator of the NLRP3 inflammasome and could be a target for sepsis treatment.

Although well-differentiated thyroid carcinoma (WDTC) is characterized by a robust treatment response, aggressive subtypes, such as anaplastic thyroid carcinoma (ATC), remain highly lethal. To understand thyroid cancer evolution in both children and adults, we analyzed single-cell transcriptomes of 423,733 cells from 81 samples and spatially resolved key tumor and microenvironment populations across 28 tumors with spatial transcriptomics, including rare and unique composite WDTC/ATC tumors and pediatric diffuse sclerosing thyroid carcinomas. Additionally, we identified gene signatures of stromal cell populations in 5 large thyroid cancer bulk RNA-sequencing cohorts. Through this multi-institutional effort, we defined a population of POSTN+ myofibroblast cancer-associated fibroblasts (myCAFs) that are intimately associated with invasive tumor cells and correlate with poor prognosis, lymph node metastasis, and disease progression in thyroid carcinoma. We also revealed a population of inflammatory CAFs that are distant to tumor cells and are found in the inflammatory stromal microenvironment of autoimmune thyroiditis. Together, our study provides spatial profiling of thyroid cancer evolution in samples with mixed WDTC/ATC histopathology and identifies a prognostic myCAF subtype with potential clinical utility in predicting aggressive disease in both children and adults.

Serine-rich splicing factor 3 (SRSF3) is crucial for the metabolic functions of the liver. The genetic deletion of SRSF3 in mouse hepatocytes impairs hepatic lipid and glucose metabolism and leads to fibrosis and formation of hepatocellular adenoma that progresses to hepatocellular carcinoma. SRSF3 protein is proteosomally degraded in metabolic-dysfunction associated fatty liver disease (MAFLD) and metabolic-dysfunction-associated steatohepatitis (MASH). We show here that depleting SRSF3 protein in hepatocytes promoted R-loop accumulation and increased DNA damage in the liver. Prevention of SRSF3 degradation in vivo protected hepatocytes from DNA double-strand breaks in mice with MASH. This protection extended to other DNA-damaging agents such as camptothecin, palmitic acid, or hydrogen peroxide when tested on HepG2 cells in vitro. SRSF3 interacted with TRIM28 and MDC1, which are components of the ATM DNA-damage repair complex, and knockdown of any of these 3 proteins reduced the expression of the other 2 proteins, suggesting they form a functional complex. Lastly, by preventing degradation of SRSF3, we were able to reduce tumors in a diethyl-nitrosamine-induced (DEN-induced) model of cirrhotic HCC. These findings suggest that maintenance of SRSF3 protein stability is crucial for preventing DNA damage and protecting liver from early metabolic liver disease and progression to HCC.

The native human glomerulus features a slit diaphragm resembling a densely interwoven fishnet.

Neutrophils play a pivotal role in the progression of metabolic dysfunction-associated steatohepatitis (MASH) by mediating inflammatory responses. However, the heterogeneity of neutrophil subsets in MASH and their specific contributions to disease progression remain unclear. In this study, analysis of liver biopsies from 265 patients revealed a strong association between elevated neutrophil counts and MASH severity, particularly fibrosis. Five distinct neutrophil subsets were identified in human liver tissue, with PAD4+ neutrophils serving as key drivers in MASH progression. Mechanistically, PAD4+ neutrophils generate neutrophil extracellular traps (NETs) and activate hepatic stellate cells via the TAOK1-dependent MAPK signaling pathway. Inhibition of PAD4+ neutrophils in vivo attenuated the progression of liver fibrosis without exacerbating liver injury. Collectively, these findings elucidate the pivotal involvement of PAD4+ neutrophils in MASH progression and identify them as promising therapeutic targets for mitigating fibrosis and inflammation.

The central nervous system (CNS) parenchyma has conventionally been believed to lack lymphatic vasculature, likely owing to a non-permissive microenvironment that hinders the formation and growth of lymphatic endothelial cells (LECs). Recent findings of ectopic expression of LEC markers including prospero homeobox 1 (PROX1), a master regulator of lymphatic differentiation, and the vascular permeability marker plasmalemma vesicle-associated protein (PLVAP) in certain glioblastomas (GBM) and brain arteriovenous malformations have prompted investigation into their roles in cerebrovascular malformations, tumor environments, and blood-brain barrier (BBB) abnormalities. To explore the relationship between ectopic LEC properties and BBB disruption, we used endothelial cell-specific Prox1 overexpression mutants. When induced during embryonic stages of BBB formation, endothelial Prox1 expression induces hybrid blood-lymphatic phenotypes in the developing CNS vasculature. This effect is not observed when Prox1 is overexpressed during postnatal BBB maturation. Ectopic Prox1 expression leads to significant vascular malformations and enhanced vascular leakage, resulting in BBB disruption when induced during both embryonic and postnatal stages. Mechanistically, PROX1 downregulates critical BBB-associated genes, including β-catenin and claudin-5, which are essential for BBB development and maintenance. These findings suggest that PROX1 compromises BBB integrity by negatively regulating BBB-associated gene expression and Wnt/β-catenin signaling.