Background: Thyroid collision tumors (TCTs) are rare thyroid malignancies characterized by the coexistence of distinct tumor types. We investigated the histopathology, immunohistochemistry, and gene mutations to comprehensively characterize the heterogeneity of TCTs. Methods: Immunohistochemistry (IHC), hematoxylin and eosin (HE) staining, and Congo red staining were performed to characterize tumor markers. DNA concentrations of all samples were measured using the Qubit DNA Assay Kit in the Qubit 2.0 fluorometer. Somatic mutations were analyzed using the Genomic Identification of Significant Targets in Cancer (GISTIC) and the NCBI-ClinVar, dbSNP, COSMIC, and HGMD databases. Bioinformatic enrichments were constructed using the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway databases, the OncodriveCLUST software, Cytoscape, and Circos. Results: Immunohistochemistry staining of papillary thyroid carcinoma (PTC) and medullary thyroid carcinoma (MTC) samples showed several distinct biomarkers. According to whole-exome sequencing, a total of 76 single-nucleotide variants (SNVs), 12 insertion–deletion variants (INDELs), and 99 copy-number variations (CNVs) were identified in the TCTs. Moreover, an OTU deubiquitinase, ubiquitin aldehyde–binding protein 2 (OTUB2), with a deletion mutation enhanced the proliferation of thyroid cancer cells in both in vitro and in vivo experiments. Conclusion: HRas proto-oncogene (HRAS) and STAG2 cohesin complex component (STAG2) were synchronously identified as the driver genes, while the OTUB2 deletion mutation may contribute to tumor proliferation and disease progression in TCTs.
Background: Atherosclerosis is a chronic inflammatory disease characterized by lipid-driven immune dysregulation. Argininosuccinate synthase 1 (ASS1) has been implicated in macrophage inflammation, yet its precise mechanistic role in foam cell-mediated vascular injury during atherosclerosis remains unclear. This study investigates whether ASS1 promotes disease progression via the NLRP3/IL-33/ST2 axis. Methods: An in vitro foam cell model was established using phorbol 12-myristate 13-acetate (PMA)-differentiated U937 macrophages treated with oxidized low-density lipoprotein (ox-LDL). The role of ASS1 was assessed via knockdown (si-ASS1) and overexpression (ASS1 overexpression) plasmids. Co-culture systems with human umbilical vein endothelial cells (HUVECs) and human aortic vascular smooth muscle cells (HAVSMCs) were used to evaluate endothelial apoptosis and VSMC proliferation/migration. In vivo, atherosclerosis was induced in apolipoprotein E‑deficient (ApoE)-deficient mice via a 12-week high-fat diet, and ASS1 expression was modulated using AAV9 vectors. Molecular analyses included ROS detection, enzyme-linked immunosorbent assay (ELISA), qPCR, western blot, and immunofluorescence. Plaque burden was assessed via Oil Red O staining. Results: Ox-LDL treatment significantly upregulated ASS1 expression in U937-derived foam cells. ASS1 overexpression enhanced intracellular ROS production, NLRP3 inflammasome activation, STAT3 phosphorylation, and IL-33 secretion. These effects were reversed by ASS1 knockdown. Rescue experiments demonstrated that STAT3 is required for ASS1-mediated NLRP3 activation and IL-33 upregulation. ASS1 altered IL-33 receptor ST2 signaling by increasing the soluble decoy isoform (sST2) and decreasing the membrane-bound signaling isoform (ST2L). In co-culture, ASS1-overexpressing foam cells promoted HUVEC apoptosis (via mitochondrial pathway) and HAVSMC proliferation, migration, and dedifferentiation. NLRP3 overexpression alone mimicked the pro-inflammatory effects of ASS1 and reversed the anti-inflammatory effects of ASS1 knockdown. In vivo, ASS1 knockdown in ApoE-/- mice reduced plaque lipid deposition, serum levels of IL-33 and IL-1β, and vascular expression of NLRP3 and p-STAT3, while ASS1 overexpression exacerbated these parameters. Conclusions: ASS1 drives atherosclerosis by activating the STAT3/NLRP3 inflammasome axis, shifting the IL-33/ST2 balance toward a pro-inflammatory state, and amplifying foam cell–mediated endothelial injury and smooth muscle cell dysfunction. Targeting ASS1 may offer a novel therapeutic strategy for inflammatory vascular disease.
Background: Accurate biodosimetry is essential for effective radiological triage, precise clinical monitoring, and assessment of risks from diagnostic exposures. Immunofluorescent detection of phosphorylated histone H2AX (γH2AX) and p53-binding protein 1 (53BP1) DNA double-strand break repair foci provides high sensitivity for radiation dose assessment within the first hours after exposure. However, inter-laboratory reproducibility of γH2AX/53BP1-based biodosimetry remains limited, and the contribution of pharmacological modifiers is unresolved. Methods: Here, we conducted an inter-laboratory comparison of in vitro radiation dose–response relationships of foci yields measured in cryopreserved umbilical cord blood lymphocytes (UCBLs) and freshly isolated peripheral blood lymphocytes (PBLs) from healthy donors using fluorescent microscopy with emphasis on workflow harmonization and reproducibility across laboratories. Results: Under low-dose γ irradiation, both UCBLs and PBLs exhibited a strong linear, dose-dependent induction of γH2AX, 53BP1, and co-localized foci, with co-localization emerging as the most sensitive endpoint. γH2AX pan-nuclear staining was observed exclusively in UCBLs and functioned as a distinct endpoint under the examined conditions. Under harmonized low-dose conditions, calyculin A at a non-toxic concentration of 1 nM did not provide measurable stabilization or enhancement of ionizing radiation-induced foci (IRIF) yields. Although IRIF yields differed between the two laboratories, dose–response slopes were highly concordant, demonstrating reproducibility under harmonized experimental conditions. Conclusions: These findings demonstrate that inter-laboratory reproducibility of γH2AX/53BP1-based biodosimetry is achieved primarily through disciplined workflow harmonization rather than through pharmacological enhancement. By reinforcing assay reproducibility and biological consistency, this work supports the translational applicability of IRIF-based biodosimetry for broader application in radiation exposure assessment.
Background: Recent studies have identified impaired renal gluconeogenesis as a hallmark of chronic kidney disease. Triptolide is a natural compound widely used in China for the treatment of renal diseases. This study investigated whether triptolide mitigates renal fibrosis by promoting renal gluconeogenesis. Methods: Renal fibrosis was induced in vivo by unilateral ureteral obstruction (UUO) surgery in mice. Transforming growth factor-β (TGF-β)-stimulated human kidney-2 (HK-2) cells were used as an in vitro model to investigate renal fibrosis. Metabolomics, western blotting, immunohistochemistry (IHC), and metabolic assays were performed to investigate the underlying mechanisms. Results: Triptolide reduced the expression of several fibrotic markers in the kidneys of UUO mice. Metabolomic analysis revealed enhanced renal gluconeogenesis following treatment with triptolide, which was confirmed by analyzing gluconeogenic enzyme expression and lactate concentration in UUO kidneys. The pro-gluconeogenic effect of triptolide was further confirmed in TGF-β-stimulated HK2 cells. Inhibition of phosphoenolpyruvate carboxykinase 1 (PCK1) reversed the anti-fibrotic and pro-gluconeogenic effects of triptolide in TGF-β-stimulated HK2 cells. We further demonstrated that peroxisome proliferator-activated receptor-gamma co-activator 1 alpha (PGC1α) expression was downregulated in TGF-β-stimulated HK2 cells and UUO kidneys, and that triptolide reversed this downregulation. Moreover, the PGC1α inhibitor reversed the effect of triptolide on PCK1 expression and glucose metabolism. Finally, IHC analysis revealed that triptolide inhibited histone lactylation in UUO kidneys, which was associated with a decreased production of inflammatory factors and reduced macrophage infiltration. Conclusions: Triptolide may inhibit renal fibrosis by increasing the PGC1α/PCK1 axis, thereby promoting renal gluconeogenesis. This cascade may reduce histone lactylation and renal inflammation, providing a mechanistic pathway for its anti-fibrotic effect.
Background: Alzheimer’s disease (AD) involves a progressive deterioration of cognitive abilities, memory loss, and persistent brain inflammation. Emerging evidence indicates that pyroptosis mediated by the NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome, contributes significantly to AD development. Docosahexaenoic acid (DHA) has demonstrated neuroprotective properties; however, the precise mechanisms by which it modulates pyroptosis in AD have yet to remained incompletely elucidated. Objective: To explore the role of DHA in modulating microglial pyroptosis via the HOXA9-NLRP3 pathway in an AD model. Methods: Effects of DHA on Aβ25–35-induced pyroptosis were assessed in human microglial clone 3 (HMC3) human microglial cells using CCK-8, western blotting, immunofluorescence, and Enzyme-linked Immunosorbent Assay (ELISA) assays. The role of homeobox A9 (HOXA9) in pyroptosis regulation was evaluated through overexpression and knockdown experiments. Dual-luciferase reporter assays together with chromatin immunoprecipitation (ChIP) were used to verify the interaction of HOXA9 to NLRP3 promoter. Amyloid precursor protein / Presenilin-1 double-transgenic (APP/PS1) transgenic AD mice underwent DHA treatment in vivo, and cognitive performance was assessed using the Morris water maze paradigm. Expression of HOXA9, NLRP3, and pyroptosis-related proteins were analyzed by Quantitative Real-time Reverse Transcription PCR (qRT-PCR), Western blotting, and immunofluorescence. Results: DHA treatment significantly reduced Aβ25–35-induced microglial pyroptosis, as indicated by decreased levels of p30-Gasdermin D (GSDMD), cleaved-caspase-1, IL-1β, and IL-18. HOXA9 overexpression reversed the protective effects of DHA, whereas NLRP3 inhibition by MCC950 enhanced DHA inhibition of pyroptosis. Dual-luciferase and ChIP assays confirmed that HOXA9 directly regulates NLRP3 transcription. In APP/PS1 mice, DHA administration enhanced cognitive performance while simultaneously decreasing the expression of pyroptosis-related markers and inflammatory mediators in brain. Inhibition of NLRP3 signaling by MCC950 further strengthened the neuroprotective actions of DHA. Conclusion: DHA ameliorates AD-related cognitive decline and reduces microglial pyroptosis through suppressing the HOXA9-NLRP3 axis. These results offer novel insights into the molecular basis of DHA-mediated neuroprotection and highlight potential therapeutic targets for AD.
