Scuticociliatosis is an invasive external or systemic infection caused by ciliated protozoa, mainly those within the subclass Scuticociliatia (scuticociliates). Many scuticociliates are fish pathogens, including Miamiensis avidus, Philasterides dicentrarchi, Pseudocohnilembus persalinus, and Uronema marinum. Our previous study showed that hemolysis-related genes derived from bacteria through horizontal gene transfer (HGT) may contribute to virulence in P. persalinus. Hemorrhagic lesions are a common feature of scuticociliatosis, but it is not known whether other scuticociliates also have bacteria-derived hemolysis-related genes. In this study, we constructed a high-quality macronuclear genome of another typical pathogenic scuticociliate, U. marinum. A total of 105 HGT genes were identified in this species, of which 35 were homologs of hemolysis-related genes (including hemolysin-like genes) that had previously been identified in P. persalinus. Sequencing of an additional five species from four scuticociliate families showed that bacteria-derived hemolysis-related genes (especially hemolysin-like genes) are widely distributed in scuticociliates. Based on these findings, we suggest that hemolysin-like genes may have originated before the divergence of scuticociliates.

Balantioides coli plays an important role in the diarrhea of weaned piglets, but its pathogenic potential and interaction with gut microbes remain unclear. To investigate the impact of B. coli colonization on the gut bacterial structure and function of weaned piglets, a metagenomic analysis based on shotgun sequencing was performed on fresh fecal samples collected from ten B. coli-colonized piglets and eight B. coli-free ones in this study. The results showed that decreasing diversity and shifted composition and function of the bacterial community were detected in the weaned piglets infected by B. coli. In contrast to the B. coli-negative group, the relative abundances of some members of the Firmicutes phylum including Clostridium, Ruminococcus species, and Intestinimonas butyriciproducens, which produce short-chain fatty acids, were significantly reduced in the B. coli-positive group. Notably, some species of the Prevotella genus (such as Prevotella sp. CAG:604 and Prevotella stercorea) were significantly increased in abundance in the B. coli-positive piglets. A functional analysis of the gut microbiota demonstrated that the differential gene sets for the metabolism of carbohydrates and amino acids were abundant in both groups, and the more enriched pathways in B. coli-infected piglets were associated with the sugar-specific phosphotransferase system (PTS) and the two-component regulatory system, as well as lipopolysaccharide (LPS) biosynthesis. Furthermore, several species of Prevotella were significantly positively correlated to the synthesis of lipid A, leading to the exporting of endotoxins and, thereby, inducing inflammation in the intestines of weaned piglets. Taken together, these findings revealed that colonization by B. coli was distinctly associated with the dysbiosis of gut bacterial structure and function in weaned piglets. Lower relative abundances of Clostridiaceae and Ruminococcaceae and higher abundances of Prevotella species were biomarkers of B. coli infection in weaned piglets.

Microglia function as a vital constituent in the maintenance of brain homeostasis. Aberrant microglial activation, however, may contribute to neurodegenerative diseases. Lithospermic acid (LA) is a plant-derived polycyclic phenolic carboxylic acid isolated from Salvia miltiorrhiza. The present study investigated the potential effects of lithospermic acid on LPS-induced neuroinflammation in BV2 microglial cells and determined the mechanism of action of this compound. Cells were pre-treated with lithospermic acid for 1 h and incubated with LPS for 24 h. qPCR, immunofluorescence, and immunoblot assays were used to determine the expression of iNOS, COX2, NF-κB p65, and HSP90 expression. ELISA was employed to measure the production of pro-inflammatory cytokines. Lithospermic acid dramatically reduced LPS-stimulated cell migration and decreased NF-κB p65 nuclear translocation. Furthermore, lithospermic acid also markedly decreased the production of pro-inflammatory cytokines, including IL-6, IL-1β, and TNF-α in a dose-dependent manner. Additionally, lithospermic acid inhibited NO and PGE2 production in response to LPS, and it also inhibited the expression of iNOS and COX2 in a dose-dependent manner. Molecular docking and experimental verification have demonstrated that lithospermic acid inhibits the activity and expression of HSP90. Small interfering RNA knockdown of HSP90 expression, which abrogated LPS-induced inflammation. These findings suggest that the lithospermic acid targeting HSP90 attenuates LPS-induced inflammatory response via the NF-κB signalling pathway in BV2 microglial cells. Collectively, lithospermic acid may offer therapeutic benefits for neurodegenerative disorders associated with microglial activation and could serve as a potential inhibitor/agent for the treatment of neuroinflammation.

Pseudocohnilembus persalinus, a unicellular ciliated protozoan, is one of commonest facultative pathogens. We sequenced the macronuclear genome of P. persalinus in 2015, which provided new insights into its pathogenicity. Here, we present the P. persalinus genome database (PPGD) ( http://ciliates.ihb.ac.cn/database/home/#pp ), the first genome database for the scuticociliatosis pathogens. PPGD integrates P. persalinus macronuclear genomic and transcriptomic data, including genome sequence, transcript, gene expression data, and gene annotation, as well as relevant information on its biology, morphology and taxonomy. The database also provides functions for visualizing, analyzing, and downloading the data. PPGD is a useful resource for studying scuticociliates or scuticociliatosis. We will continue to update the PPGD by integrating more data and aim to integrate the PPGD with other ciliate databases to build a comprehensive ciliate genome database.

Dysregulation of intracellular pH (pHi) is a hallmark biological feature of cancer cells, which hijack pHi homeostasis to sustain malignant phenotypes including uncontrolled proliferation, invasion, and metabolic reprogramming. Meanwhile, alkaliptosis, a newly defined pH-dependent form of regulated cell death specifically triggered by the small-molecule compound JTC-801, has emerged as a promising therapeutic target for cancer intervention. However, reliable tools for monitoring dynamic pHi changes during alkaliptosis remain insufficient. RpHluorin2 is an optimized ratiometric pH-sensitive green fluorescent protein variant with enhanced fluorescence intensity and stability. Herein, we established a stable RpHluorin2-expressing cell line and further developed a multi-dimensional fluorescence detection workflow, which encompasses a microplate reader, fluorescence microscopy, flow cytometry, a small animal imaging system (IVIS Spectrum), and a protein dot blot assay coupled with IVIS. Our results demonstrated a strong linear correlation between RpHluorin2 fluorescence intensity and cell number, with a coefficient of determination ( R ² ) of 0.9998. Furthermore, across all employed detection methods, JTC-801 treatment induced dose-dependent and time-dependent reductions in RpHluorin2 fluorescence, an observation that is consistent with the elevation of pHi during alkaliptosis. This platform enables high-throughput screening, single-cell analysis, and biochemical validation, providing a robust tool for mechanistic research on alkaliptosis and the development of pH-targeted anticancer therapies.