DNA topology regulates PAM-Cas9 interaction and DNA unwinding to enable near-PAMless cleavage by thermophilic Cas9.

CRISPR-Cas9-mediated genome editing depends on PAM recognition to initiate DNA unwinding. PAM mutations can abolish Cas9 binding and prohibit editing. Here, we identified a Cas9 from the thermophile Alicyclobacillus tengchongensis for which the PAM interaction can be robustly regulated by DNA topology. AtCas9 has a relaxed PAM of N4CNNN and N4RNNA (R = A/G) and is able to bind but not cleave targets with mutated PAMs. When PAM-mutated DNA was in underwound topology, AtCas9 exhibited enhanced binding affinity and high cleavage activity. Mechanistically, AtCas9 has a unique loop motif, which docked into the DNA major groove, and this interaction can be regulated by DNA topology. More importantly, AtCas9 showed near-PAMless editing of supercoiled plasmid in E. coli. In mammalian cells, AtCas9 exhibited broad PAM preference to edit plasmid with up to 72% efficiency and effective base editing at four endogenous loci, representing a potentially powerful tool for near-PAMless editing.

Network pharmacology analysis combined with experimental validation to explore the therapeutic mechanism of salidroside on intestine ischemia reperfusion.

ETHNOPHARMACOLOGICAL RELEVANCE: Salidroside (SAL), a phenolic natural product present in Rhodiola rosea, are commonly used in the treatment of various ischemic-hypoxic diseases, including intestinal ischemia-reperfusion (IR) injury. However, their efficacy and potential mechanisms in the treatment of intestinal IR injury have not been investigated. OBJECTIVE: The objective of the present study is to investigate the pharmacological mechanism of action of SAL on intestinal IR injury using a network pharmacology approach combined with experimental validation. METHODS: In the present study, we used the Traditional Chinese Medicine Systematic Pharmacology (TCMSP) database and analysis platform and Comparative Toxicogenomics Database (CTD) to predict possible target genes of SAL, collected relevant target genes of intestinal IR injury from GeneCards and DisGenet websites, and collected summary data to screen common target genes. Then, the protein-protein interaction (PPI) target network was constructed and analyzed by STRING database and Cytoscape 3.8.2 with the above intersecting genes. Then, gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed and the component-target-pathway network was constructed, followed by the use of molecular docking and molecular dynamic simulation to verify the possible binding conformation between SAL and candidate targets to further explore the potential targets of SAL in the treatment of intestinal IR injury. Finally, an in vivo model of mouse superior mesenteric artery ligation was established to assess the anti-intestinal IR injury effect of SAL by assessing histopathological changes in mouse small intestine by HE staining, detecting inflammatory factor expression by ELISA kit, and detecting the expression of key protein targets by Western blotting. RESULTS: A total of 166 SAL target genes and 1740 disease-related targets were retrieved, and 88 overlapping proteins were obtained as potential therapeutic targets. The pathway enrichment analysis revealed that the pharmacological effects of SAL on intestinal IR injury were anti-hypoxic, anti-inflammatory and metabolic pathway related, and the molecular docking and molecular dynamic simulation results showed that the core bioactive components had good binding affinity for TXNIP and AMPK, and the immunoblotting results indicated that the expression levels of TXNIP and AMPK in the small intestinal tissues of mice in the drug-treated group compared with the model group were significantly changed. CONCLUSION: SAL may target AMPK and TXNIP domains to act as a therapeutic agent for intestinal IR. These findings comprehensively reveal the potential therapeutic targets for SAL against intestinal IR and provide theoretical basis for the clinical application of SAL in the treatment of intestinal IR.

The crucial roles of long noncoding RNA SNHGs in lung cancer.

Lung cancer is one of the most common malignant tumors with growing morbidity and mortality worldwide. Several treatments are used to manage lung cancer, including surgery, radiotherapy and chemotherapy, as well as molecular-targeted therapy. However, the current measures are still far from satisfactory. Therefore, the current research should focus on exploring the molecular mechanism and then finding an effective treatment. Interestingly, we and others have embarked on a line of investigations focused on the mechanism of lung cancer. Specifically, lncRNA small nucleolar RNA host gene has been shown to be associated with biological characteristics and therapeutic resistance of lung cancer. In addition, small nucleolar RNA host genes may be used as diagnostic biomarker in the future. Herein, we will provide a brief review demonstrating the importance of small nucleolar RNA host genes in lung cancer, especially non-small cell lung cancer. Although lncRNA has shown a crucial role in tumor-related research, a large number of studies are needed to validate its clinical application in the future.

The crucial roles of long noncoding RNA SNHGs in lung cancer

Lung cancer is one of the most common malignant tumors with growing morbidity and mortality worldwide. Several treatments are used to manage lung cancer, including surgery, radiotherapy and chemotherapy, as well as molecular-targeted therapy. However, the current measures are still far from satisfactory. Therefore, the current research should focus on exploring the molecular mechanism and then finding an effective treatment. Interestingly, we and others have embarked on a line of investigations focused on the mechanism of lung cancer. Specifically, lncRNA small nucleolar RNA host gene has been shown to be associated with biological characteristics and therapeutic resistance of lung cancer. In addition, small nucleolar RNA host genes may be used as diagnostic biomarker in the future. Herein, we will provide a brief review demonstrating the importance of small nucleolar RNA host genes in lung cancer, especially non-small cell lung cancer. Although lncRNA has shown a crucial role in tumor-related research, a large number of studies are needed to validate its clinical application in the future (AU)