Search Results (25)

X-linked dystrophin-deficient muscular dystrophy (DD-MD) is an uncommon neuromuscular disorder in cats. We described an adult male cat with chronic tongue protrusion, dysphagia, muscle hypertrophy, and a history of rhabdomyolysis associated with anesthesia. Clinical pathology revealed markedly increased CK activity, muscle histopathology demonstrated a dystrophic phenotype, and an absence of dystrophin protein was confirmed by immunofluorescent staining. Whole genome sequencing identified two potential disease-causing variants, including a new missense variant in the DMD gene (c.2207T>C; p.Gln736Arg), which was considered causative of the clinical phenotype. A second variant in the CLIC2 gene was also detected but was considered unlikely to cause myopathic signs. The clinical course remained stable over 1.5 years with supportive management and dietary modification, and no further episodes of rhabdomyolysis occurred. This case expands the known spectrum of feline DMD variants and highlights the value of genetic testing combined with muscle histopathology for diagnosing chronic presentations of MD. Avoidance of inhalant anesthetics may be important in managing affected cats due to the risk of acute muscle injury. Full article

Colorectal cancer (CRC) remains a leading cause of cancer mortality, yet no systematic effort has linked druggable CRC driver genes to downstream ion channel effectors. We integrated differential expression analysis, weighted gene co-expression network analysis (WGCNA), and protein–protein interaction (PPI) network pharmacology to identify CRC hub genes and their ion channel connections, validated by dual single-cell perturbation approaches: variational graph autoencoder-based virtual knockout (VGAE-KO) and experimental HCT116 CRISPRi Perturb-seq (6 genes, 8445 cells). WGCNA identified 100 hub genes spanning three functional programs. Ribosomal proteins link to K⁺ channels (RPS21 → KCNQ2, targetable by EMA-approved ataluren, passed dual validation at 97.8th–98.7th percentile). RNA processing genes connect to Cl⁻ channels (LSM7 → CLIC1, strongest signal at 99.8th–99.4th percentile). Immune checkpoint receptors (LAG3, CD27) connect via PPI intermediates to ${\mathrm{Ca}}^{2+}$ and K⁺ channels, targetable by relatlimab (FDA-approved) and varlilumab (Phase 2). This work maps previously unknown links between CRC driver genes and ion channel regulation, with the ataluren-RPS21-KCNQ2 axis ready for pharmacological testing. Full article

Background: Exercise is a promising non-pharmacological intervention for cocaine addiction but molecular mechanisms of exercise-related genes in addiction remain unclear. This study aimed to identify exercise-related signature genes for cocaine addiction and to assess the potential causal relationship between exercise and cocaine addiction using two-sample Mendelian randomization (MR) analysis. Methods: Midbrain transcriptomic data were analyzed for differentially expressed genes (DEGs) and intersected with exercise-related genes. Functional enrichment, protein-protein interaction (PPI) and immune infiltration analyses explored their roles while signature genes were screened via LASSO/Random Forest and validated by ROC curves. GSEA explored pathways and MR confirmed exercise’s causal effect. Results: A total of 244 DEGs were identified, including 27 exercise-related, and six signature genes (CALM3, CCL2, CD44, CLIC1, JUN, VCAM1) showed AUC values between 0.714 and 0.868 in distinguishing cocaine-addicted individuals from controls. Functional analyses revealed enrichment in immune-inflammatory pathways, metabolic processes and neuro-immune interactions and immune infiltration analysis showed cocaine addicts had elevated pro-inflammatory cells, reduced regulatory cells and signature genes correlated with immune dysregulations. MR analysis suggested a statistically significant protective association between genetically proxied higher levels of exercise and cocaine addiction risk (p < 0.05). Conclusions: These six genes may be potential biomarkers and therapeutic targets, and exercise may protect against cocaine addiction by regulating immune-inflammatory responses, metabolic pathways and neuroplasticity, although further validation in larger, independent cohorts and experimental models is required. Full article

Increasing evidence points to members of the chloride intracellular ion channel (CLIC) protein family performing a variety of functions within cells—classifying them as moonlighting proteins—and serving as natural cellular antioxidant protective agents. Apart from their role as membrane-inserting ion channels, members of the CLIC family also possess enzymatic oxidoreduction activity in their soluble form. The current study is the first to specifically examine the S-glutathionylation catalytic activity of several purified recombinant CLIC protein members (rCLIC1, rCLIC3, and rCLIC4) by directly measuring their ability to deglutathionylate and glutathionylate a synthetic model peptide via an in vitro tryptophan fluorescence quenching assay. Effects of pH and temperature on this activity were also assessed. Our findings provide insights into a likely previously uncharacterised mechanism by which CLIC proteins serve as cellular antioxidant protective enzymes via their S-glutathionylation capabilities. Full article

The breeding of disease-resistant pigs has consistently been a topic of significant interest and concern within the pig farming industry. The study of pig blood indicators has the potential to confer economic benefits upon the pig farming industry, whilst simultaneously providing valuable insights that can inform the study of human diseases. In this study, an F2 resource population of 489 individuals was generated through the intercrossing of Large White boars and Min pig sows. A total of 17 haematological parameters and T lymphocyte subpopulations were measured, including white blood cell count (WBC), lymphocyte count (LYM), lymphocyte count percentage (LYM%), monocyte count (MID), monocyte count percentage (MID%), neutrophilic granulocyte count (GRN), percentage of neutrophils (GRN%), mean platelet volume (MPV), platelet distribution width (PDW), platelet count (PLT), CD4+/CD8+, CD4+CD8+CD3+, CD4+CD8−CD3+, CD4−CD8+CD3+, CD4−CD8−CD3+, and CD3+. The Illumina PorcineSNP60 Genotyping BeadChip was obtained for all of the F2 animals. Subsequently, a genome-wide association study (GWAS) was conducted using the TASSEL 5.0 software to identify associated variants and candidate genes for the 17 traits. Significant association signals were identified for PCT and PLT on SSC7, with 1 and 11 significant SNP loci, respectively. A single nucleotide polymorphism (SNP) on SSC12 was identified as a significant predictor of the white blood cell (WBC) trait. Significant association signals were detected for the T lymphocyte subpopulations, namely CD4+/CD8+, CD4+CD8+CD3+, CD4+CD8−CD3+, and CD4−CD8+CD3+, with the majority of these signals observed on SSC7. The genes CLIC5, TRIM15, and SLC17A4 were identified as potential candidates for influencing CD4+/CD8+ and CD4−CD8+CD3+. A missense variant, c.2707 G>A, in the SLC17A4 gene has been demonstrated to be significantly associated with the CD4+/CD8+ and CD4-CD8+CD3+ traits. Three missense variants (c.425 A>C, c.500 C>T, and c.733 A>G) have been identified in the TRIM15 gene as being linked to the CD4+/CD8+ trait. Nevertheless, only c.425 A>C has been demonstrated to be significantly associated with CD4-CD8+CD3+. In the CLIC5 gene, one missense variant (c.957 T>C) has been identified as being associated with the CD4+/CD8+ and CD4-CD8+CD3+ traits. Additionally, significant association signals were observed for CD4+CD8+CD3+ and CD4+CD8−CD3+ on SSC2 and 5, respectively. Subsequently, a gene ontology (GO) enrichment analysis was conducted on all genes within the quantitative trait loci (QTL) intervals of platelet count, CD4+/CD8+, and CD4−CD8+CD3+. The MHC class II protein complex binding pathway was identified as the most significant pathway among the three immune traits. These results provide guidance for further research in the field of breeding disease-resistant pigs. Full article

(1) Background: Influenza A Virus (IAV) uses host cellular proteins during replication in host cells. IAV infection causes elevated expression of chloride intracellular channel protein 1 (CLIC1) in lung epithelial cells, but the importance of this protein in IAV replication is unknown. (2) In this study, we determined the role of CLIC1 in IAV replication by investigating the effects of CLIC1 knockdown (KD) on IAV viral protein translation, genomic RNA transcription, and host cellular proteome dysregulation. (3) Results: CLIC1 KD in A549 human lung epithelial cells resulted in a significant decrease in progeny supernatant IAV, but virus protein expression was unaffected. However, a significantly larger number of viral RNAs accumulated in CLIC1 KD cells. Treatment with a CLIC1 inhibitor also caused a significant reduction in IAV replication, suggesting that CLIC1 is an important host factor in IAV replication. SomaScan^®, which measures 1322 proteins, identified IAV-induced dysregulated proteins in wild-type cells and in CLIC1 KD cells. The expression of 116 and 149 proteins was significantly altered in wild-type and in CLIC1 KD cells, respectively. A large number of the dysregulated proteins in CLIC1 KD cells were associated with cellular transcription and predicted to be inhibited during IAV replication. (4) Conclusions: This study suggests that CLIC1 is involved in later stages of IAV replication. Further investigation should clarify mechanism(s) for the development of anti-IAV drugs targeting CLIC1 protein. Full article

Chloride intracellular ion channel (CLIC) proteins exist as both soluble and integral membrane proteins, with CLIC1 capable of shifting between two distinct structural conformations. New evidence has emerged indicating that members of the CLIC family act as moonlighting proteins, referring to the ability of a single protein to carry out multiple functions. In addition to their ion channel activity, CLIC family members possess oxidoreductase enzymatic activity and share significant structural and sequence homology, along with varying overlaps in their tissue distribution and cellular localization. In this study, the 2-hydroxyethyl disulfide (HEDS) assay system was used to characterize kinetic properties, as well as the temperature and pH profiles of three CLIC protein family members (CLIC1, CLIC3, CLIC4). We also assessed the effects of the drugs rapamycin and amphotericin B, on the three CLIC proteins’ enzymatic activity in the HEDS assay. Our results demonstrate CLIC1 to be highly heat-sensitive, with optimal enzymatic activity observed at neutral pH7 and at a temperature of 37 °C, while CLIC3 had higher oxidoreductase activity in more acidic pH5 and was found to be relatively heat stable. CLIC4, like CLIC1, was temperature sensitive with optimal enzymatic activity observed at 37 °C; however, it showed optimal activity in more alkaline conditions of pH8. Our current study demonstrates individual differences in the enzymatic activity between the three CLIC proteins, suggesting each CLIC protein is likely regulated in discrete ways, involving changes in the subcellular milieu and microenvironment. Full article

The choroid plexus (ChP) is a complex structure in the human brain that is responsible for the secretion of cerebrospinal fluid (CSF) and forming the blood–CSF barrier (B-CSF-B). Human-induced pluripotent stem cells (hiPSCs) have shown promising results in the formation of brain organoids in vitro; however, very few studies to date have generated ChP organoids. In particular, no study has assessed the inflammatory response and the extracellular vesicle (EV) biogenesis of hiPSC-derived ChP organoids. In this study, the impacts of Wnt signaling on the inflammatory response and EV biogenesis of ChP organoids derived from hiPSCs was investigated. During days 10–15, bone morphogenetic protein 4 was added along with (+/−) CHIR99021 (CHIR, a small molecule GSK-3β inhibitor that acts as a Wnt agonist). At day 30, the ChP organoids were characterized by immunocytochemistry and flow cytometry for TTR (~72%) and CLIC6 (~20%) expression. Compared to the −CHIR group, the +CHIR group showed an upregulation of 6 out of 10 tested ChP genes, including CLIC6 (2-fold), PLEC (4-fold), PLTP (2–4-fold), DCN (~7-fold), DLK1 (2–4-fold), and AQP1 (1.4-fold), and a downregulation of TTR (0.1-fold), IGFBP7 (0.8-fold), MSX1 (0.4-fold), and LUM (0.2–0.4-fold). When exposed to amyloid beta 42 oligomers, the +CHIR group had a more sensitive response as evidenced by the upregulation of inflammation-related genes such as TNFα, IL-6, and MMP2/9 when compared to the −CHIR group. Developmentally, the EV biogenesis markers of ChP organoids showed an increase over time from day 19 to day 38. This study is significant in that it provides a model of the human B-CSF-B and ChP tissue for the purpose of drug screening and designing drug delivery systems to treat neurological disorders such as Alzheimer’s disease and ischemic stroke. Full article

Activated microglia potentially cause neurodegeneration in Parkinson’s disease (PD). Matrix metalloproteinase (MMP)-9 plays a crucial role in the pathogenesis of PD, but the modulator of microglial release of MMP-9 remains obscure. Given the modulatory effect of chloride intracellular channel protein 2 (CLIC2) on MMPs, we aimed to determine the role of CLIC2 in regulating microglial MMP expression and activation. We found that CLIC2 is expressed in microglia and neurons in rat brain tissue and focused on the function of CLIC2 in primary cultured microglia. Exposure to recombinant CLIC2 protein enhanced microglial invasion activity, and its knockdown abolished this activity. Moreover, increased activation of MMP-9 was confirmed by the addition of the CLIC2 protein, and CLIC2 knockdown eliminated this activation. Additionally, increased expression of CLIC2 was observed in PD-modeled tissue. In conclusion, CLIC2 increases MMP-9 activity in the microglia, which are involved in PD pathogenesis. Full article

Background: Overexpression of chloride intracellular channel protein 1 (CLIC1) in tumor cells has been confirmed, but it has received less attention in the tumor blood vessel endothelium. Aim: The assessment of CLIC1 expression in ccRCC tumor blood vessels and its relationship with TNM parameters and tumor cell CLIC1 expression. Methods: CLIC1 immunostaining in ccRCC was evaluated in 50 cases in both malignant cells and tumor blood vessels (CLIC1 microvessel density-CLIC1-MVD) and was correlated with TNM staging parameters. Results: CLIC1-MVD was observed in approximately 65% of cases, and CLIC1 co-localization in both tumor and endothelial cells was observed in 59% of cases. ccRCC was classified into four groups (Classes 0–3) based on the percentage of positive tumor cells, with each group including sub-groups defined by CLIC1 expression in the endothelium. Class 3 (60–100% positive tumor cells) had the highest CLIC1-MVD, with an impact on T and M parameters (p value = 0.007 for T, and p value = 0.006 for M). For cases with CLIC1 intracellular translocation, there was a strong correlation between CLIC1-MVD and M (p value < 0.001). Conclusions: Co-expression of ccRCC tumor and endothelial cells promotes tumor progression and metastasis and should be investigated further as a potential therapeutic target for ccRCC and other human malignancies. Full article

Background: Hypoxia is recognized as a key feature of cancer growth and is involved in various cellular processes, including proliferation, angiogenesis, and immune surveillance. Besides hypoxia-inducible factor 1-alpha (HIF-1α), which is the main mediator of hypoxia effects and can also be activated under normoxic conditions, little is known about its counterpart, HIF-2. This study focused on investigating the clinical and molecular landscape of HIF-2-altered urothelial carcinoma (UC). Methods: Publicly available next-generation sequencing (NGS) data from muscle-invasive UC cell lines and patient tumor samples from the MSK/TCGA 2020 cohort (n = 476) were interrogated for the level of expression (mRNA, protein) and presence of mutations, copy number variations, structural variants in the EPAS1 gene encoding HIF-2, and findings among various clinical (stage, grade, progression-free and overall survival) and molecular (tumor mutational burden, enriched gene expression) parameters were compared between altered and unaltered tumors. Results: 19% (7/37) of UC cell lines and 7% (27/380) of patients with muscle-invasive UC display high EPAS1 mRNA and protein expression or/and EPAS1 alterations. EPAS1-altered tumors are associated with higher stage, grade, and lymph node metastasis as well as with shorter PFS (14 vs. 51 months, q = 0.01) and OS (15 vs. 55 months, q = 0.01). EPAS1 mRNA expression is directly correlated with that of its target-genes, including VEGF, FLT1, KDR, DLL4, CDH5, ANGPT1 (q < 0.001). While there is a slightly higher tumor mutational burden in EPAS1-altered tumors (9.9 vs. 4.9 mut/Mb), they are enriched in and associated with genes promoting immune evasion, including ARID5B, SPINT1, AAK1, CLIC3, SORT1, SASH1, and FGFR3, respectively (q < 0.001). Conclusions: HIF-2-altered UC has an aggressive clinical and a distinct genomic and immunogenomic profile enriched in angiogenesis- and immune evasion-promoting genes. Full article

Background: As microRNA-142 (miR-142) is the only human microRNA gene where mutations have consistently been found in about 20% of all cases of diffuse large B-cell lymphoma (DLBCL), we wanted to determine the impact of miR-142 inactivation on protein expression of DLBCL cell lines. Methods: miR-142 was deleted by CRISPR/Cas9 knockout in cell lines from DLBCL. Results: By proteome analyses, miR-142 knockout resulted in a consistent up-regulation of 52 but also down-regulation of 41 proteins in GC-DLBCL lines BJAB and SUDHL4. Various mitochondrial ribosomal proteins were up-regulated in line with their pro-tumorigenic properties, while proteins necessary for MHC-I presentation were down-regulated in accordance with the finding that miR-142 knockout mice have a defective immune response. CFL2, CLIC4, STAU1, and TWF1 are known targets of miR-142, and we could additionally confirm AKT1S1, CCNB1, LIMA1, and TFRC as new targets of miR-142-3p or -5p. Conclusions: Seed-sequence mutants of miR-142 confirmed potential targets and novel targets of miRNAs can be identified in miRNA knockout cell lines. Due to the complex contribution of miRNAs within cellular regulatory networks, in particular when miRNAs highly present in RISC complexes are replaced by other miRNAs, primary effects on gene expression may be covered by secondary layers of regulation. Full article

CLICs are the dimorphic protein present in both soluble and membrane fractions. As an integral membrane protein, CLICs potentially possess ion channel activity. However, it is not fully clarified what kinds of roles CLICs play in physiological and pathological conditions. In vertebrates, CLICs are classified into six classes: CLIC1, 2, 3, 4, 5, and 6. Recently, in silico analyses have revealed that the expression level of CLICs may have prognostic significance in cancer. In this review, we focus on CLIC2, which has received less attention than other CLICs, and discuss its role in the metastasis and invasion of malignant tumor cells. CLIC2 is expressed at higher levels in benign tumors than in malignant ones, most likely preventing tumor cell invasion into surrounding tissues. CLIC2 is also expressed in the vascular endothelial cells of normal tissues and maintains their intercellular adhesive junctions, presumably suppressing the hematogenous metastasis of malignant tumor cells. Surprisingly, CLIC2 is localized in secretory granules and secreted into the extracellular milieu. Secreted CLIC2 binds to MMP14 and inhibits its activity, leading to suppressed MMP2 activity. CLIC4, on the other hand, promotes MMP14 activity. These findings challenge the assumption that CLICs are ion channels, implying that they could be potential new targets for the treatment of malignant tumors. Full article

Transcriptional regulation is a major mechanism by which organisms integrate gene x environment interactions. It can be achieved by coordinated interplay between cis-regulatory elements (CREs) and transcription factors (TFs). Euryhaline tilapia (Oreochromis mossambicus) tolerate a wide range of salinity and thus are an appropriate model to examine transcriptional regulatory mechanisms during salinity stress in fish. Quantitative proteomics in combination with the transcription inhibitor actinomycin D revealed 19 proteins that are transcriptionally upregulated by hyperosmolality in tilapia brain (OmB) cells. We searched the extended proximal promoter up to intron1 of each corresponding gene for common motifs using motif discovery tools. The top-ranked motif identified (STREME1) represents a binding site for the Forkhead box TF L1 (FoxL1). STREME1 function during hyperosmolality was experimentally validated by choosing two of the 19 genes, chloride intracellular channel 2 (clic2) and uridine phosphorylase 1 (upp1), that are enriched in STREME1 in their extended promoters. Transcriptional induction of these genes during hyperosmolality requires STREME1, as evidenced by motif mutagenesis. We conclude that STREME1 represents a new functional CRE that contributes to gene x environment interactions during salinity stress in tilapia. Moreover, our results indicate that FoxL1 family TFs are contribute to hyperosmotic induction of genes in euryhaline fish. Full article

We recently identified a deviant bovine immune phenotype characterized by hyperproliferation of lymphocytes after polyclonal stimulation. This phenotype was first discovered in dams that responded to PregSure BVD vaccination by producing pathological antibodies, triggering the fatal disease “bovine neonatal pancytopenia” in calves. The aim of the study was to gain deeper insights into molecular processes occurring in lymphocytes of immune phenotypes and the effect on their secretome after immune stimulation. Two discovery proteomic experiments were performed with unstimulated and Pokeweed Mitogen (PWM) stimulated lymphocytes, using label-free LC-MS/MS. In lymphocytes, 2447 proteins were quantified, and 1204 proteins were quantified in the secretome. Quantitative proteome analysis of immune deviant and control samples after PWM stimulation revealed clear differences. The increase in abundance of IL17A, IL17F, IL8, CCL5, LRRC59, and CLIC4 was higher in controls through mitogenic stimulation. In contrast, the abundance of IFNγ, IL2, IL2RA, CD83, and CD200 increased significantly more in immune deviant lymphocytes. Additional pathway enrichment analysis of differentially secreted proteins also yielded fundamental differences between the immune phenotypes. Our study provides a comprehensive dataset, which gives novel insights into proteome changes of lymphocytes from different bovine immune phenotypes. These differences point to the development of diverse immune responses of bovine immune phenotypes after immune stimulation. Full article