OUHP: an optimized universal hairpin primer system for cost-effective and high-throughput RT-qPCR-based quantification of microRNA (miRNA) expression.

MicroRNAs (miRNAs or miRs) are single-stranded, ∼22-nucleotide noncoding RNAs that regulate many cellular processes. While numerous miRNA quantification technologies are available, a recent analysis of 12 commercial platforms revealed high variations in reproducibility, sensitivity, accuracy, specificity and concordance within and/or between platforms. Here, we developed a universal hairpin primer (UHP) system that negates the use of miRNA-specific hairpin primers (MsHPs) for quantitative reverse transcription PCR (RT-qPCR)-based miRNA quantification. Specifically, we analyzed four UHPs that share the same hairpin structure but are anchored with two, three, four and six degenerate nucleotides at 3'-ends (namely UHP2, UHP3, UHP4 and UHP6), and found that the four UHPs yielded robust RT products and quantified miRNAs with high efficiency. UHP-based RT-qPCR miRNA quantification was not affected by long transcripts. By analyzing 14 miRNAs, we demonstrated that UHP4 closely mimicked MsHPs in miRNA quantification. Fine-tuning experiments identified an optimized UHP (OUHP) mix with a molar composition of UHP2:UHP4:UHP6 = 8:1:1, which closely recapitulated MsHPs in miRNA quantification. Using synthetic LET7 isomiRs, we demonstrated that the OUHP-based qPCR system exhibited high specificity and sensitivity. Collectively, our results demonstrate that the OUHP system can serve as a reliable and cost-effective surrogate of MsHPs for RT-qPCR-based miRNA quantification for basic research and precision medicine.

Composition and risk assessment of perioperative patient safety incidents reported by anesthesiologists from 2009 to 2019: a single-center retrospective cohort study.

BACKGROUND: Patient safety incident (PSI) reporting has been an important means of improving patient safety and enhancing organizational quality control. Reports of anesthesia-related incidents are of great value for analysis to improve perioperative patient safety. However, the utilization of incident data is far from sufficient, especially in developing countries such as China. METHODS: All PSIs reported by anesthesiologists in a Chinese academic hospital between September 2009 and August 2019 were collected from the incident reporting system. We reviewed the freeform text reports, supplemented with information from the patient medical record system. Composition analysis and risk assessment were performed. RESULTS: In total, 847 PSIs were voluntarily reported by anesthesiologists during the study period among 452,974 anesthetic procedures, with a reported incidence of 0.17%. Patients with a worse ASA physical status were more likely to be involved in a PSI. The most common type of incident was related to the airway (N = 208, 27%), followed by the heart, brain and vascular system (N = 99, 13%) and pharmacological incidents (N = 79, 10%). Those preventable incidents with extreme or high risk were identified through risk assessment to serve as a reference for the implementation of more standard operating procedures by the department. CONCLUSIONS: This study describes the characteristics of 847 PSIs voluntarily reported by anesthesiologists within eleven years in a Chinese academic hospital. Airway incidents constitute the majority of incidents reported by anesthesiologists. Underreporting is common in China, and the importance of summarizing and utilizing anesthesia incident data should be scrutinized.

Assessment of Itch and Pain in Animal Models and Human Subjects.

For the past century, scientists have developed a variety of methods to evaluate itch and pain in both animal models and human subjects to throw light on some of the most important pathways mediating these unpleasant sensations. Discoveries in the mechanisms underlying itch and pain in both physiological and pathological conditions relied greatly upon these studies and may eventually lead to the discovery of new therapeutics. However, it was a much more complicated job to access itch and pain in animal models than in human subjects due to the subjective nature of these sensations. The results could be contradictory or even misleading when applying different methodologies in animal models, especially under pathological conditions with a mixed sensation of itch and pain. This chapter introduces and evaluates some of the classical and newly designed methodologies to access the sensation of itch and pain in animal models as well as human subjects.