Multiplex Detection of RNA Viruses Based on Ligation Reaction and Universal PCR Amplification.

To detect several RNA viruses simultaneously, a method based on multiplex ligation reaction combined with multiplex qPCR or multiplex PCR+capillary electrophoresis was established to detect four RNA viruses: human immunodeficiency virus (HIV), hepatitis C (HCV), influenza A virus (IAV) H1N1 and H5N1. The experimental conditions including ligation probe concentration, annealing procedure, ligation temperature and ligase dosage were optimized intensively. We found that the specificity of the ligation reaction was affected by the probe concentration predominantly, high-probe concentration (100 nM) resulted in splint-independent ligation with efficiency comparable to that with RNA splint. The sensitivity of the ligation reaction was affected by the annealing mode apparently as the sensitivity of the step-down annealing mode was 100 times higher than that of the isothermal annealing at 37 °C. Under the optimized condition, this assay could detect virus RNA as low as 16 viral copies per reaction in doubleplex and triplex real-time quantitative PCR detection with satisfactory specificity and precision. By multiplex PCR+capillary electrophoresis, four RNA viruses could be detected in one tube with the sensitivity of 10 copies per reaction.

Deciphering the molecular landscape: evolutionary progression from gynecomastia to aggressive male breast cancer.

BACKGROUND: Gynecomastia denotes the benign proliferation of glandular breast tissue and stands as a recognized risk factor for male breast cancer. Nonetheless, the underlying carcinogenic mechanisms orchestrating the progression from gynecomastia to cancer remain poorly understood. METHODS: This study employed single-cell RNA sequencing (scRNA-seq) to meticulously dissect the cellular landscape of gynecomastia and unravel potential associations with male breast cancer at a single-cell resolution. Pseudotime and evolutionary analyses were executed to delineate the distinct features characterizing gynecomastia and male breast cancer. The TCGA database, along with cell-cell communication analysis and immunohistochemistry staining, was harnessed to validate differential gene expression, specifically focusing on CD13. RESULT: From the copy number variation profiles and evolutionary tree, we inferred shared mutation characteristics (18p+ and 18q+) underpinning both conditions. The developmental trajectory unveiled an intriguing overlap between gynecomastia and malignant epithelial cells. Moreover, the differential gene CD13 emerged as a common denominator in both gynecomastia and male breast cancer when compared with normal mammary tissue. Cell-cell interaction analysis and communication dynamics within the tumor microenvironment spotlighted distinctions between CD13+ and CD13- subsets, with the former exhibiting elevated expression of FGFR1-FGF7. CONCLUSIONS: Our investigation provides novel insights into the evolutionary progression from gynecomastia to male breast cancer, shedding light on the pivotal role of CD13 in driving this transition. The identification of CD13 as a potential therapeutic target suggests the feasibility of CD13-targeted interventions, specifically tailored for male breast cancer treatment.

Klotho improves cardiac fibrosis, inflammatory cytokines, ferroptosis, and oxidative stress in mice with myocardial infarction

The anti-aging protein Klotho has been associated with cardiovascular health protection. Nevertheless, the protective mechanism remains unknown. The present study is aimed at exploring the effect of Klotho on cardiac remodeling and its potential mechanism in mice with myocardial infarction (MI). We used left anterior coronary artery descending ligation to develop an MI model for in vivo analyses. In contrast, H9C2 cells and cardiac fibroblasts were used to establish the oxygen–glucose deprivation (OGD) model in in vitro analyses. In vivo and in vitro models were treated with Klotho. Compound C, an AMPK signaling inhibitor, was used to determine whether Klotho’s effects are mediated through the AMPK/mTOR signaling pathway. Echocardiography, Masson trichrome staining, immunofluorescence, immunohistochemistry, real-time polymerase chain reaction (RT-PCR), and western blot were used to detect the related indicators. The findings of the in vivo model indicate that Klotho treatment improved the mice’s cardiac function, reduced cardiac fibrosis, and attenuated myocardial inflammatory factors, ferroptosis, and oxidative stress. The results of the in vitro model were in line with the findings of in vivo modeling. An AMPK inhibitor, Compound C, reversed all these effects. In conclusion, Klotho potentially improves cardiac remodeling in MI mice by regulating AMPK/mTOR signaling, demonstrating Klotho as an effective MI therapeutic agent. (AU)