Absolute quantification of viable Vibrio cholerae in seawater samples using multiplex droplet digital PCR combined with propidium monoazide.

Introduction: Toxigenic Vibrio cholerae serogroup O1 and O139 are the pathogens responsible for the global cholera epidemic. V. cholerae can settle in the water and spread via the fecal-oral route. Rapid and accurate monitoring of live V. cholerae in environmental water has become an important strategy to prevent and control cholera transmission. Conventional plate counting is widely used to detect viable bacteria but requires time and effort. Methods: This study aims to develop a new assay that combines triplex droplet digital PCR (ddPCR) with propidium monoazide (PMA) treatment for quantitatively detecting live V. cholerae O1/O139 and cholera enterotoxin. Specific primers and probes were designed according to the conserved regions of gene rfb O1, rfb O139, and ctxA. The amplification procedures and PMA treatment conditions were optimized. The specificity, sensitivity, and ability of PMA-ddPCR to detect viable bacteria-derived DNA were evaluated in simulated seawater samples. Results and Discussion: The results revealed that the optimal primer concentrations of rfb O1, rfb O139, and ctxA were 1 µM, while the concentrations of the three probes were 0.25, 0.25, and 0.4 µM, respectively. The best annealing temperature was 58°C to obtain the most accurate results. The optimal strategy for distinguishing dead and live bacteria from PMA treatment was incubation at the concentration of 20 µM for 15 min, followed by exposure to a 650-W halogen lamp for 20 min. In pure culture solutions, the limit of detection (LODs) of V. cholerae O1 and O139, and ctxA were 127.91, 120.23 CFU/mL, and 1.5 copies/reaction in PMA-triplex ddPCR, respectively, while the LODs of the three targets were 150.66, 147.57 CFU/mL, and 2 copies/reaction in seawater samples. The PMA-ddPCR sensitivity was about 10 times higher than that of PMA-qPCR. When detecting spiked seawater samples with live bacterial concentrations of 1.53 × 102 and 1.53 × 105 CFU/mL, the assay presented a higher sensitivity (100%, 16/16) than qPCR (50.00%, 8/16) and a perfect specificity (100%, 9/9). These results indicate that the developed PMA-triplex ddPCR is superior to the qPCR regarding sensitivity and specificity and can be used to rapidly detect viable toxigenic V. cholerae O1 and O139 in suspicious seawater samples.

Benefits of early administration of Sacubitril/Valsartan in patients with ST-elevation myocardial infarction after primary percutaneous coronary intervention.

OBJECTIVE: To evaluate the effects of early administration of Sacubitril/Valsartan (Sac/Val) in patients with ST-elevation myocardial infarction after primary percutaneous coronary intervention (pPCI). METHODS: This prospective, controlled, single-center study randomized 186 ST-segment elevation myocardial infarction patients to one of the following two groups: Sac/Val group: early administration of Sac/Val within 24 hours after pPCI; control group: conventional angiotensin-converting enzyme inhibitors (ACEI) application. The creatine Kinase (CK) peak after the surgery, the incidence of acute heart failure during hospitalization, level of NT-proBNP and left ventricular ejection fraction (LVEF) measured by ultrasound before discharge and soluble suppression of tumorigenicity2 (sST2), LVEF, infarct size determined by single photon emission computed tomography (SPECT), readmission rate within 6 months were recorded and compared between two groups. RESULTS: Compared to the control group, Sac/Val could decrease the CK peak and the incidence of acute heart failure after pPCI; the level of NT-proBNP was lower and LVEF was higher before discharge in the Sac/Val group. After 6 months, the patients who had taken Sac/Val had a higher LVEF, a smaller infarct size determined by SPECT, lower sST2 and readmission rate. CONCLUSION: Patients with ST-elevation myocardial infarction after primary percutaneous coronary intervention could benefit from early administration of Sacubitril/Valsartan, the effect was superior to conventional ACEI.

Effects of MgSO4 and magnesium transporters on 6-hydroxydopamine-induced SH-SY5Y cells.

AIMS: The magnesium ion (Mg2+) fulfils several important functions for living organisms. We investigated whether there is a protective effect of MgSO4 on 6-OHDA-induced neurotoxicity in SH-SY5Y cells, and gained insight into the effects of cellular mRNA and protein expression of the magnesium transporters SLC41A1, NIPA1, MagT1 and CNNM2 on 6-OHDA-induced neurotoxicity. MAIN METHODS: The effect of MgSO4 on cell viability in 6-OHDA-treated SH-SY5Y cells was measured using a CCK-8 kit. The mRNA and protein expression of SLC41A1, NIPA1, MagT1, and CNNM2 were detected using reverse transcription-qPCR and Western blot. KEY FINDINGS: The results showed that SH-SY5Y cells treated with 25-50µM 6-OHDA for 24h significantly decreased cell viability, while if pre-incubated with 0.125-1mM MgSO4 for 1h before adding 6-OHDA it partially prevented the cell damage. There was a significant decrease in cellular mRNA and protein expression of SLC41A1, NIPA1, MagT1 and CNNM2 in 6-OHDA treated SH-SY5Y cells, and MgSO4 can reverse its decline. SIGNIFICANCE: Our results suggest that MgSO4 may protect SH-SY5Y cells against 6-OHDA-induced cell injury and that gene expression of SLC41A1, NIPA1, MagT1, and CNNM2 might be involved in dopaminergic neurons.