In mammalian cells, long noncoding RNAs (lncRNAs) form complexes with proteins to execute various biological functions such as gene transcription, RNA processing and other signaling activities. However, methods to track endogenous lncRNA dynamics in live cells and screen for lncRNA interacting proteins are limited. Here, we report the development of CERTIS (CRISPR-mediated Endogenous lncRNA Tracking and Immunoprecipitation System) to visualize and isolate endogenous lncRNA, by precisely inserting a 24-repeat MS2 tag into the distal end of lncRNA locus through the CRISPR/Cas9 technology. In this study, we show that CERTIS effectively labeled the paraspeckle lncRNA NEAT1 without disturbing its physiological properties and could monitor the endogenous expression variation of NEAT1. In addition, CERTIS displayed superior performance on both short-and long-term tracking of NEAT1 dynamics in live cells. We found that NEAT1 and paraspeckles were sensitive to topoisomerase I specific inhibitors. Moreover, RNA Immunoprecipitation (RIP) of the MS2-tagged NEAT1 lncRNA successfully revealed several new protein components of paraspeckle. Our results support CERTIS as a tool suitable to track both spatial and temporal lncRNA regulation in live cells as well as study the lncRNA-protein interactomes.

Myocardial ischemic injury is among the top 10 leading causes of death from cardiovascular diseases worldwide. Myocardial ischemia is caused mainly by coronary artery occlusion or obstruction. It usually occurs when the heart is insufficiently perfused, oxygen supply to the myocardium is reduced, and energy metabolism in the myocardium is abnormal. Pathologically, myocardial ischemic injury generates a large number of inflammatory cells, thus inducing a state of oxidative stress. This sharp reduction in the number of normal cells as a result of apoptosis leads to organ and tissue damage, which can be life-threatening. Therefore, effective methods for the treatment of myocardial ischemic injury and clarification of the underlying mechanisms are urgently required. Gaseous signaling molecules, such as NO, H 2 S, H 2 , and combined gas donors, have gradually become a focus of research. Gaseous signaling molecules have shown anti-apoptotic, anti-oxidative and anti-inflammatory effects as potential therapeutic agents for myocardial ischemic injury in a large number of studies. In this review, we summarize and discuss the mechanism underlying the protective effect of gaseous signaling molecules on myocardial ischemic injury.Keywords: myocardial ischemia, NO, H 2 S, H 2 , gas co-dornor, protecting mechanisms SOURCE OF GASEOUS SIGNALING MOLECULESCurrent studies have shown that gas signaling molecules mediate certain inhibitory effects on oxidative stress (25), apoptosis (26), inflammation (27), and autophagy (28), and have protective

Pachymic acid (PA), a bioactive ingredient isolated from Poria cocos Wolf, is reported with potential benefits of anti‐inflammatory, anti‐oxidative actions. It is reasoned that PA may play the potential benefits against cystitis glandularis (CG), an inflammation of the bladder tissue. In this study, we aimed to apply the network pharmacology and molecular docking analyses to reveal concrete anti‐CG targets and mechanisms of PA, and then the bioinformatic findings were verified by using clinical and animal samples. The methodological data from network pharmacology approach showed that 303 and 243 reporting targets of CG and PA, and other 31 shared targets of CG and PA were identified. Subsequently, all top targets of PA against CG were screened out, including cyclooxygenase‐2, epidermal growth factor receptor, tumor antigen p53 (TP53), tumor necrosis factor‐alpha (TNF), interleukin‐1 (IL‐1) beta, proto‐oncogene c‐jun. Molecular docking data demonstrated that PA exerted potent bonding capacities with TNF, TP53 proteins in CG. In human study, the findings suggested that overactivated TNF‐α expression and suppressed TP53 activation were detected in CG samples. In animal study, PA‐treated mice showed reduced intravesical IL‐1, IL‐6 levels, and lactate dehydrogenase content, downregulated TNF‐α and upregulated TP53 proteins in bladder samples. Taken together, our bioinformatics and experimental findings identify the key anti‐CG biotargets and mechanisms of PA. More markedly, these pivotal pharmacological targets of PA against CG have been screened out and verified by using computational and experimental analyses.

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