Quencher-free fluorescent assays by controlled DNA partitioning in the aqueous two-phase system with crowding-enhanced kinetics.

Fluorescent DNA assays are promising in disease diagnosis, environmental monitoring, and drug screening, encompassing both heterogeneous and homogeneous assay types. Nevertheless, heterogeneous assays suffer from tedious washing steps and slow reaction kinetics, whereas homogenous assays require well-designed fluorophore pairs to modulate signal off/on. Herein, we developed a cost-effective and efficient quencher-free fluorescent DNA assay using an aqueous two-phase system (ATPS). Using a strand-displacement reaction, we showed that similar sensing performance could be achieved at a much lower cost. Furthermore, the unique crowding environment in ATPS accelerated strand-displacement reactions by up to six-fold and reduced DNA amplification time from 120 min to 30 min. Our assay demonstrated robust sensing in serum environments and successful detection of miRNA extracted from cells. This innovative assay format has the potential for biosensor development with both heterogeneous readout and rapid reaction kinetics in various applications.

Identification and analysis of key hypoxia- and immune-related genes in hypertrophic cardiomyopathy.

BACKGROUND: Hypertrophic cardiomyopathy (HCM), an autosomal dominant genetic disease, is the main cause of sudden death in adolescents and athletes globally. Hypoxia and immune factors have been revealed to be related to the pathology of HCM. There is growing evidence of a role for hypoxia and inflammation as triggers and enhancers in the pathology in HCM. However, the role of hypoxia- and immune-related genes in HCM have not been reported. METHODS: Firstly, we obtained four HCM-related datasets from the Gene Expression Omnibus (GEO) database for differential expression analysis. Immune cells significantly expressed in normal samples and HCM were then screened by a microenvironmental cell population counter (MCP-counter) algorithm. Next, hypoxia- and immune-related genes were screened by the LASSO + support vector machine recursive feature elimination (SVM-RFE) and weighted gene co-expression network analysis (WGCNA). Single-gene enrichment analysis and expression validation of key genes were then performed. Finally, we constructed a competing endogenous RNA (ceRNA) network of key genes. RESULTS: In this study, 35 differentially expressed hypoxia genes were found. By using LASSO + SVM-RFE analysis, 10 more targets with differentially expressed hypoxia genes were identified. The MCP-count algorithm yielded five differentially expressed immune cells, and after assessing them for WGCNA characteristics, 612 immune genes were discovered. When hypoxia and immune genes were combined for cross-tabulation analysis, three hypoxia- and immune-related genes (ATP2A2, DDAH1, and OMA1) were identified. CONCLUSION: Based on hypoxia characteristic genes, three key genes were identified. These were also significantly related to immune activation, which proves a theoretical basis and reference value for studying the relationship between HCM and hypoxia and immunity.

Identification and analysis of mitochondria-related key genes of heart failure.

Mitochondria-induced cell death is a vital mechanism of heart failure (HF). Thus, identification of mitochondria-related genes (Mito-RGs) based on transcriptome sequencing data of HF might provide novel diagnostic markers and therapeutic targets for HF. First, bioinformatics analysis was conducted on the GSE57338, GSE76701, GSE136547, and GSE77399 datasets in the Gene Expression Omnibus. Next, we analyzed HF-Mito differentially expressed genes (DEGs) using the protein-protein interaction (PPI) network for obtaining critical genes and exploring their functions. Subsequently, immune cell scores of the HF and normal groups were compared. The potential alteration mechanisms of the key genes were investigated by constructing a competing endogenous RNA network. Finally, we predicted potential therapeutic agents and validated the expression levels of the key genes. Twenty-three HF-Mito DEGs were acquired in the GSE57338 dataset, and the PPI network obtained four key genes, including IFIT3, XAF1, RSAD2, and MX1. According to gene set enrichment analysis, the key genes showed high enrichment in myogenesis and hypoxia. Immune cell analysis demonstrated that aDCs, B cells, and 20 other immune cell types varied between the HF and normal groups. Moreover, we observed that H19 might affect the expression of IFIT3, AXF1, and RSAD2. PCGEM1 might regulate RSAD2 expression. A total of 515 potential therapeutic drugs targeting the key genes, such as tretinoin, silicon dioxide, and bisphenol A, were acquired. Finally, IFIT3, RSAD2, and MX1 expression increased in HF samples compared with normal samples in the GSE76701 dataset, conforming to the GSE57338 dataset analysis. This work screened four key genes, namely, IFIT3, XAF1, RSAD2, and MX1, which can be further explored in subsequent studies for their specific molecular mechanisms in HF.

The Function of FoxK Transcription Factors in Diseases.

Forkhead box (FOX) transcription factors play a crucial role in the regulation of many diseases, being an evolutionarily conserved superfamily of transcription factors. In recent years, FoxK1/2, members of its family, has been the subject of research. Even though FoxK1 and FoxK2 have some functional overlap, increasing evidence indicates that the regulatory functions of FoxK1 and FoxK2 are not the same in various physiological and disease states. It is important to understand the biological function and mechanism of FoxK1/2 for better understanding pathogenesis of diseases, predicting prognosis, and finding new therapeutic targets. There is, however, a lack of comprehensive and systematic analysis of the similarities and differences of FoxK1/2 roles in disease, prompting us to perform a literature review.

Ginsenoside Rg2 Ameliorates Myocardial Ischemia/Reperfusion Injury by Regulating TAK1 to Inhibit Necroptosis.

Necroptosis contribute to the pathogenesis of myocardial ischemia/reperfusion (MI/R) injury. Ginsenoside Rg2 has been reported to have cardioprotective effects against MI/R injury; however, the underlying mechanism remains unclear. This work aimed to investigate the effect of ginsenoside Rg2 on necroptosis induced by MI/R and to explore the mechanism. In this study, hypoxia/reoxygenation (H/R) injury model was established in H9c2 cells. In vivo, male C57/BL6 mice were subjected to myocardial ischemia 30 min/reperfusion 4 h. Rg2 (50 mg/kg) or vehicle was intravenously infused 5 min before reperfusion. Cardiac function and the signaling pathway involved in necroptosis were investigated. Compared with H/R group, Rg2 significantly inhibited H/R-induced cardiomyocyte death. Rg2 treatment effectively inhibited the phosphorylation of RIP1, RIP3, and MLKL in H/R cardiomyocytes, and inhibited RIP1/RIP3 complex (necrosome) formation. In mice, Rg2 treatment manifested significantly lower ischemia/reperfusion (I/R)-induced myocardial necroptosis, as evidenced by decrease in phosphorylation of RIP1, RIP3, and MLKL, inhibited lactate dehydrogenase (LDH) release and Evans blue dye (EBD) penetration. Mechanically, an increased level of tumor necrosis factor α (TNFα), interleukin (IL)-1ß, IL-6, and MCP-1 were found in MI/R hearts, and Rg2 treatment significantly inhibit the expression of these factors. We found that TNFα-induced phosphorylation of RIP1, RIP3, and MLKL was negatively correlated with transforming growth factor-activated kinase 1 (TAK1) phosphorylation, and inhibition of TAK1 phosphorylation led to necroptosis enhancement. More importantly, Rg2 treatment significantly increased TAK1 phosphorylation, enhanced TAK1 binding to RIP1 while inhibiting RIP1/RIP3 complex, ultimately reducing MI/R-induced necroptosis. These findings highlight a new mechanism of Rg2-induced cardioprotection: reducing the formation of RIP1/RIP3 necrosome by regulating TAK1 phosphorylation to block necroptosis induced by MI/R.