Multi-omics analysis reveals the unique landscape of DLD in the breast cancer tumor microenvironment and its implications for immune-related prognosis.

Background: Cuproptosis, i.e., copper-induced programmed cell death, has potential implications in cancer therapy. However, the impact of the cuproptosis-related gene (CRG) dihydrolipoyl dehydrogenase (DLD) on breast cancer (BC) prognosis remains underexplored. Methods: We employed real-time quantitative PCR and multiplexed immunostaining techniques to quantify DLD expression in both BC and the adjacent non-cancerous tissues. Immunofluorescence analysis was employed to assess the influence of DLD on immune cells and immunological checkpoints in the BC microenvironment. DLD knockdown experiments were conducted in BC cell lines MDA-MB-468 and SK-BR-3, with knockdown efficiency validated via western blot. Subsequently, we performed the cell counting kit-8 (CCK-8) assay, clone formation assay, Transwell migration assay, and invasion assay. To construct a prognostic model, we employed a Lasso-Cox regression analysis of immune-related genes associated with DLD. Additionally, we established a competing endogenous RNA network based on CRGs to evaluate potential regulatory pathways. Results: Compared to the adjacent tissues, BC tissues exhibited markedly elevated DLD expression levels. In vitro experiments demonstrated that DLD knockdown effectively inhibited BC cell migration, invasion, and proliferation. DLD exhibited positive correlations with CD68+ macrophages and PD-L1 in the tumor, as well as with macrophages and CD4+ T cells in the stroma. Tumor regions with high DLD expression were enriched in PD-L1 and macrophages, while stromal regions with high DLD expression contained CD4+ T cells and macrophages. The AUC values for 1-, 3-, and 5-year overall survival in TCGA-BRCA training set were 0.67, 0.66, and 0.66, respectively. A nomogram with a C-index of 0.715 indicated that risk score, tumor stage, and age could serve as independent prognostic factors for BC. Conclusion: Our findings underscore the significant predictive significance of DLD in BC and its influence on the tumor microenvironment. DLD represents a promising diagnostic and prognostic marker for BC, offering novel avenues for the identification of therapeutic targets and the enhancement of immunotherapy in BC.

Focal adhesion signaling pathway involved in skin immune response of tongue sole Cynoglossus semilaevis to Vibrio vulnificus infection.

Focal adhesion (FA) plays a key role in cell adhesion, migration and antibacterial immune, but it remained unclear in fish. In this study, half-smooth tongue sole Cynoglossus semilaevis were infected with Vibrio vulnificus, and then immune-related protein in the skin, especially for FA signaling pathway were screened and identified by iTRAQ analysis. Results showed that the differentially expressed proteins (DEPs) in skin immune response (eg., ITGA6, FN, COCH, AMBP, COL6A1, COL6A3, COL6A6, LAMB1, LAMC1, FLMNA) were firstly found in FA signaling pathway. Furthermore, the validation analysis of FA-related genes were basically consistent with the iTRAQ data at 36 hpi (r = 0.678, p < 0.01), and their spatio-temporal expressions were confirmed by qPCR analysis. The molecular characterization of vinculin of C. semilaevis was described. This study will provide a new perspective for understanding the molecular mechanism of FA signaling pathway in the skin immune response in marine fish.

Molecular characterization of carp edema virus disease: An emerging threat to koi Cyprinus carpio in China.

Carp edema virus disease (CEVD) has resulted in great economic losses in koi (Cyprinus carpio koi) and common carp (Cyprinus carpio carpio) populations in the world. In this study, the diseased koi were diagnosed as CEV infection based on 5' untranslated region (5'UTR) and 4a protein genes by the conventional PCR, nested PCR and quantitative PCR (qPCR) analyses. Phylogenetic tree analysis showed that the TJ201708 strain was classified into the genogroup IIa. Furthermore, qPCR of 5'UTR gene revealed that the lowest detection limit was 4.0 fg/µL. The pathogenicity of CEV for koi was demonstrated in the infection experiments. Histopathological examination revealed the petechial hemorrhages of liver and spleen, vacuolization of lamina propria of intestine and swelling and necrosis of respiratory epithelial cells of gills. To our knowledge, this is the first report the qPCR of 5'UTR gene in the detection of carp edema virus.

Detection and characterization of Aeromonas salmonicida subsp. salmonicida infection in crucian carp Carassius auratus.

Aeromonas salmonicida is one of the most important pathogens in salmonids and non-salmonids species. Nevertheless, very little was reported in cyprinids about A. salmonicida infection. Hence, a pathogenic A. salmonicida subsp. salmonicida, namely isolate GCA-518, was isolated from diseased crucian carp Carassius auratus. Its optimal growth conditions were at 28 °C, pH 7.0 and 1.5% NaCl. Furthermore, the quantitative real-time PCR (qPCR) targeting serine protease (aspA) gene was established for rapid detection of the lowest limit of 5.6 × 102 copies per reaction. The pathogenicity was confirmed in crucian carp by intraperitoneal infection. Histopathologic examination displayed multifocal necrosis and infiltration of inflammatory cells in gill, liver, kidney and intestine. This is the first report on typical A. salmonicida infection in cultured crucian carp.

Proteomic Profiling of Zebrafish Challenged by Spring Viremia of Carp Virus Provides Insight into Skin Antiviral Response.

Spring viremia of carp virus (SVCV) causes the skin hemorrhagic disease in cyprinid species, but its molecular mechanism of skin immune response remains unclear at the protein level. In the present study, the differential proteomics of the zebrafish (Danio rerio) skin in response to SVCV infection were examined by isobaric tags for relative and absolute quantitation and quantitative polymerase chain reaction (qPCR) assays. A total of 3999 proteins were identified, of which 320 and 181 proteins were differentially expressed at 24 and 96 h postinfection, respectively. The expression levels of 16 selected immune-related differentially expressed proteins (DEPs) were confirmed by qPCR analysis. Furthermore, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses revealed that DEPs were significantly associated with complement, inflammation, and antiviral response. The protein-protein interaction network of cytoskeleton-associated proteins, ATPase-related proteins, and parvalbumins from DEPs was shown to be involved in skin immune response. This is first report on the skin proteome profiling of zebrafish against SVCV infection, which will contribute to understand the molecular mechanism of local mucosal immunity in fish.