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Based on network pharmacology tools and public bioinformatics databases, the pharmacodynamic target and key mechanism of icariin (ICA) in the treatment of ovarian cancer (OC) were identified and experimentally verified. Our previous research showed that TNF, MMP9, STAT3, PIK3CA, ERBB2, MTOR, IL2, PTGS2, KDR and F2 are important targets of ICA in the treatment of OC. TNF, as a hub gene in tumor tissues, was associated with poor prognosis. ICA acted on OC mainly through the biological functions of various kinases, and the pathway with the highest accuracy (p -value) was PI3K. Meanwhile, we observed a close upstream and downstream relationship between NF- κ B and the Pl3K-AKT pathway. This study further verified the mechanism of ICA in promoting apoptosis of SKOV3 cells through the NF- κ B signaling pathway and the tandem relationship between NF- κ B and the Pl3K-AKT pathway. The assay results demonstrated that ICA can promote the apoptosis of SKOV3 cells as indicated by the proapoptotic markers Bax, Bcl-xl and Caspase-3 and the key factors of the NF- κ B signaling pathway (NF- κ Bp65, p-NF- κ Bp65, p-I κ B α and I κ B α). ICA can block the classical NF- κ B pathway by inhibiting I κ B α phosphorylation and consequently blocking the activation of the NF- κ B pathway in SKOV3 cells. ICA can also promote apoptosis by blocking the activation of the NF- κ B pathway in SKOV3 cells via inhibition of NF- κ Bp65 nuclear translocation. After using a PI3K pathway inhibitor, we further discovered that ICA may reduce AKT signal transduction by inhibiting the level of Akt phosphorylation, resulting in a loss of PI3K/Akt-dependent activation of the NF- κ B pathway. [ABSTRACT FROM AUTHOR]

Pathogenic and non-pathogenic Vibrio parahaemolyticus strains were simultaneously detected and quantified using a novel viable multiplex real-time PCR (novel qPCR). We used a new PCR primer and probe, ure R, as a surrogate for detection of the toxin trh gene as the primer was better at identifying variant V. parahaemolyticus trh strains. The specificity of all primers and probes used in this study were validated on three standard strains of V. parahaemolyticus , 42 clinical strains, 12 wild strains, 4 strains of Vibrio spp., and 4 strains of other bacteria. Then, propidium monoazide (PMA) was applied to inhibit DNA of dead cell, and the results of PMA optimized treatments were 15 μM concentration, 5 min incubation periods, 15 min light exposure periods and 30 RPM rotational speed, which resulted in time and cost savings. Pathogenic and non-pathogenic strains were quantified using a two-reaction tube method where the tlh , tdh , and ure R genes were amplified. Additionally, standard curves with a 7-log dynamic range were generated for quantifying viable V. parahaemolyticus and the amplification efficiencies were 108.68, 105.17, and 115.61% for tlh+ , tdh+ , and ure R +. This novel qPCR accurately monitored V. parahaemolyticus contamination rates in shrimps (Penaeus vannamei) and clams (Ruditapes philippinarum) sampled from retail stores located in a major district in Shanghai. In conclusion, our assay can prioritize the detection and quantification of viable pathogenic V. parahaemolyticus and can prove to be a more effective tool for reducing infection risks from consumption of seafood in Shanghai. [ABSTRACT FROM AUTHOR]

Microbial community plays a prominent role in food quality and safety, which was regarded as the major cause for food spoilage and foodborne disease. Pre-PCR propidium monoazide (PMA) treatment is an effective approach to exclude DNA amplification signal from dead cells, and application of this method in the analysis of bacterial community might help us better understand the diversity and distribution of viable bacteria in food. In this study, the effects of PMA treatment on bacterial community characteristics in shrimp were assessed by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) fingerprinting technique. The results showed that the microbial diversity was dramatically influenced by PMA treatment. During storage at 4 °C, Shannon–Wiener index and PCoA analysis indicated there were significant differences between bacterial diversity in samples treated with and without PMA. At the level of species, compared with samples without PMA treatment, Vibrio cholera , Exiguobacterium acetylicum , Aeromonas enteropelogenes , Eubacterium multiforme , Vibrio mimicus in PMA-treated samples disappeared during storage. When samples stored at 25 °C, PMA treatment has little influence on banding patterns in DGGE profiles. In conclusion, this study provides an effective tool for precisely monitoring the diversity of viable bacteria in food samples, especially at low temperature. [ABSTRACT FROM AUTHOR]

A novel TaqMan-based multiplex real-time PCR method combined with propidium monoazide (PMA) treatment was firstly developed for the simultaneous quantification of viable Vibrio parahaemolyticus and Listeria monocytogenes in raw shrimp. The optimization of PMA concentration showed that 100 μM was considered optimal to effectively inhibit 10 CFU/mL dead cells of both bacteria. The high specificity of this method was confirmed on tests using 96 target and non-target strains. The optimized assay could detect as low as 10-10 CFU/g of each strain on the artificially contaminated shrimp, and its amplification efficiencies were up to 100 and 106 % for V. parahaemolyticus and L. monocytogenes, respectively. Furthermore, this assay has been successfully applied to describe the behavior of these two pathogens in raw shrimps stored at 4 °C. In conclusion, this PMA TaqMan-based multiplex real-time PCR technique, where the whole procedure takes less than 5 h, provides an effective and rapid tool for monitoring contamination of viable V. parahaemolyticus and L. monocytogenes in seafood, improving seafood safety and protecting public health. [ABSTRACT FROM AUTHOR]

Listeria monocytogenes is an important foodborne pathogen. To effectively control this pathogen, it is necessary to have a method that can detect and differentiate L. monocytogenes from other Listeria species in food, environmental, and clinical samples. A new multiplex PCR method using new primers targeting the iap gene was developed to detect and differentiate L. monocytogenes and other Listeria species, and the method was applied to examine deli meat samples. Sixteen Listeria strains (8 L. monocytogenes , 2 Listeria innocua , 1 Listeria seeligeri , 3 Listeria welshimeri , 1 Listeria ivanovii , and 1 Listeria grayi ) from the American Type Culture Collection were tested using the new PCR method in comparison to 16S rRNA sequencing assay and a previously described PCR method based on the detection of iap gene with other primers. The new PCR method was able to differentiate L. monocytogenes from the other Listeria species, while the 16S rRNA sequencing assay could not distinguish L. welshimeri from L. monocytogenes and the previously published multiplex PCR assay was not able to distinguish L. monocytogenes from L. innocua or L. ivanovii . Furthermore, 150 deli meat samples were analyzed by the newly developed multiplex PCR in comparison to ISO11290-1 method and 16S rRNA sequencing assay to detect L. monocytogenes and differentiate it from the other 5 species of Listeria . The developed PCR provided the same results as the ISO method. Out of 150 samples, 16 (10.7%) samples and 5 (3.3%) samples were tested positive and confirmed for Listeria spp. and L. monocytogenes , respectively. The multiplex PCR method is capable of discriminating between L. monocytogenes and other Listeria species. The method is simple and specific when combined with simplified enrichment procedures. [ABSTRACT FROM AUTHOR]