Development and use of a droplet digital PCR (ddPCR) assay to achieve sensitive and fast atypical porcine pestivirus detection.

Atypical porcine pestivirus (APPV) is a recently discovered RNA virus, which mainly caused congenital tremor in piglets. Droplet digital PCR (ddPCR) is an absolute quantitative method that does not rely on the standard curve but has high sensitivity and accuracy. The present study aimed to develop a ddPCR detection assay for APPV. Furthermore, we evaluated the limit of detection, sensitivity, specificity and reproducibility of the ddPCR and real-time quantitative PCR (qPCR) and tested 135 clinical samples to calculate the detection rate of the two methods. The results showed that both methods had a strong linear relationship and quantitative correlation. The ddPCR assay had a minimum detection limit of 0.15 copies/µL for APPV, with a sensitivity 100 times that of qPCR. We tested clinical samples and found that the APPV ddPCR had a 27.4% positive detection rate, noticeably higher than that of the qPCR (14.8%). Additionally, the APPV ddPCR method had excellent repeatability and specificity. In brief, our study provided a novel, feasible and sensitive diagnostic technique to identify and monitor APPV.

Inhibiting 6-phosphogluconate dehydrogenase selectively targets breast cancer through AMPK activation.

PURPOSE: 6-phosphogluconate dehydrogenase (6PGD), a key enzyme of the oxidative pentose phosphate pathway, is involved in tumor growth and metabolism. Although high 6PGD activity has been shown to be associated with poor prognosis, its role and therapeutic value in breast cancer remain unknown. METHODS: The levels and roles of 6PGD were analyzed in breast cancer cells and their normal counterparts. The underlying mechanisms of 6PGD's roles are also analyzed. RESULTS: We found that 6PGD is aberrantly activated in breast cancer as shown by its increased transcriptional and translational levels as well as enzyme activity in breast cancer tissues and cell lines compared to normal counterparts. Although similar degree of enzyme activity inhibition was achieved in both breast cancer and normal breast cells, 6PGD inhibition by siRNA-mediated knockdown or pharmacological inhibitor physcion is more effective in inhibiting growth and survival in breast cancer than normal breast cells. Moreover, inhibiting 6PGD significantly sensitizes breast cancer response to chemotherapeutic agents in in vitro cell culture system and in vivo xenograft breast cancer model. We further show that 6PGD inhibition activates AMPK and its downstream substrate ACC1, leading to reduction of ACC1 activity and lipid biosynthesis. AMPK depletion significantly reverses the inhibitory effects of physcion in breast cancer cells, confirming that 6PGD inhibition targets breast cancer cell via AMPK activation. CONCLUSIONS: Our work provides experimental evidence on the association of 6PGD with poor prognosis in breast cancer and suggests that 6PGD inhibition may represent a potential therapeutic strategy to augment chemotherapy efficacy in breast cancer.