The role and molecular mechanism of flgK gene in biological properties, pathogenicity and virulence genes expression of Aeromonas hydrophila.

Aeromonas hydrophila is a highly pathogenic aquatic resident bacterium that can cause co-morbidity in aquatic animals, waterfowl, poultry, and humans. Flagellum is the motility organ of bacteria important for bacterium tissue colonization and invasion. The flgK gene encodes a flagellar hook protein essential for normal flagellar formation. In order to explore the role of flgK in A. hydrophila, a flgK gene mutant strain of A. hydrophila (∆flgK-AH) was constructed using an efficient suicide plasmid-mediated homologous recombination method, and gene sequencing confirmed successful mutation of the flgK gene. The biological properties, pathogenicity and virulence genes expression were compared. The results showed that there was no significant difference in the growth, hemolytic, and swarming abilities, but the swimming and biofilm formation abilities of ∆flgK-AH were significantly reduced and the transmission electron microscope (TEM) results showed that the ∆flgK-AH strain did not have a flagellar structure. The median lethal dose (LD50) value of the ∆flgK-AH in Carassius auratus was 1.47-fold higher than that of the wild-type strain (WT-AH). The quantitative real-time PCR results showed that only the expression level of the lapA gene was up-regulated by 1.47 times compared with the WT-AH, while the expression levels of other genes were significantly down-regulated. In conclusion, flgK gene mutant led to a decline in the pathogenicity possibly by reducing swimming and biofilm formation abilities, these biological properties might result from the down-regulated expression of flagellate and pilus-related genes.

Construction and efficacy of Aeromonas veronii mutant Δhcp as a live attenuated vaccine for the largemouth bass (Micropterus salmoides).

Aeromonas veronii is a human and animal co-pathogenic bacterium that could have a significant negative impact on both human health and aquaculture. In this study, a mutant strain of A. veronii with deletion of the hemolysin co-regulated protein (hcp) gene was constructed (Δhcp-AV). Compared with the wild strain, Δhcp-AV showed significantly reduced growth capacity and biofilm formation ability. Motility tests showed that the hcp gene had no significant effect on the swimming and swarming ability. In addition, the pathogenicity was also reduced. To evaluate the efficacy of Δhcp-AV as a live attenuated vaccine for prevention of Aeromonas veronii infection, we compared the immune response of largemouth bass (Micropterus salmoides) after immunization with 500 µL of 1.47 × 105 CFU/mL of Δhcp-AV and 4 × 108 CFU/mL of inactivated A. veronii. Obvious increases of serum immune related enzyme activity were observed in immunization groups. Expression levels of immune-related genes in Δhcp-AV group were up-regulated, and higher than those in inactivated A. veronii group. After challenging with live A. veronii, the relative percent survival (RPS) was 100% in Δhcp- AV group, whereas the RPS was 76.67% in inactivated A. veronii group. Our data suggest that the live attenuated vaccine Δhcp- AV could elicit a stronger immune response and provide a higher RPS than inactivated A. veronii. These data suggest that hcp gene is an important virulence factor of A. veronii, and the live attenuated vaccine Δhcp-AV is safe and effective for prevention A. veronii infection in M. salmoides farming.

Research on the quality markers of antioxidant activity of Kai-Xin-San based on the spectrum-effect relationship.

Background: Kai-Xin-San (KXS) is one of the classic famous traditional Chinese medicine prescriptions for amnesia, which has been applied for thousands of years. Modern pharmacological research has found that KXS has significant therapeutic efficacy on nervous system diseases, which is related to its antioxidant activity. However, the antioxidant material basis and quality markers (Q-makers) of KXS have not been studied. Objective: The objective of this study is to explore the Q-makers of antioxidant activity of KXS based on spectrum-effect relationship. Methods: Specifically, the metabolites in KXS extracts were identified by UPLC-Q-Exactive Orbitrap MS/MS. The fingerprint profile of KXS extracts were established by high-performance liquid chromatography (HPLC) and seven common peaks were identified. Meanwhile, 2, 2-diphenyl-1-picrylhydrazyl (DPPH) test was used to evaluate the free radical scavenging ability of KXS. The spectrum-effect relationship between its HPLC fingerprint and DPPH free radical scavenging activity was preliminarily examined by the Pearson correlation analysis, grey relation analysis (GRA), and orthogonal partial least squares discrimination analysis (OPLS-DA). Further, the antioxidant effect of KXS and its Q-makers were validated through human neuroblastoma (SH-SY5Y) cells experiment. Results: The results showed that 103 metabolites were identified from KXS, and the similarity values between HPLC fingerprint of twelve batches of KXS were greater than 0.900. At the same time, the results of Pearson correlation analysis showed that the peaks 8, 1, 14, 17, 18, 24, 16, 21, 15, 13, 6, 5, and 3 from KXS were positively correlated with the scavenging activity values of DPPH. Combined with the results of GRA and OPLS-DA, peaks 1, 3, 5 (Sibiricose A6), 6, 13 (Ginsenoside Rg1), 15, and 24 in the fingerprints were screen out as the potential Q-makers of KXS for antioxidant effect. Besides, the results of CCK-8 assay showed that KXS and its Q-makers remarkably reduced the oxidative damage of SH-SY5Y cells caused by H2O2. However, the antioxidant activity of KXS was decreased significantly after Q-makers were knocked out. Conclusion: In conclusion, the metabolites in KXS were successfully identified by UPLC-Q-Exactive Orbitrap MS/MS, and the Q-makers of KXS for antioxidant effect was analyzed based on the spectrum-effect relationship. These results are beneficial to clarify the antioxidant material basis of KXS and provide the quality control standards for new KXS products development.

TSC1/2 mutations-a unique type of mutation suitable for liver transplantation of Hepatocellular carcinoma.

BACKGROUND: This study aimed to investigate the relationship between the prognosis of patients with hepatocellular carcinoma (HCC) after liver transplantation and mammalian target of rapamycin (mTOR) pathway-related genes-TSC1/2. METHODS: We retrospectively analyzed the clinical data of 46 patients who underwent liver transplantation for HCC and performed next generation sequencing to analyze the relationship between the efficacy of sirolimus after liver transplantation for HCC and mutations in mTOR pathway-related genes, especially tuberous sclerosis complex (TSC) mutations. RESULTS: The average age of 46 patients with liver transplantation for HCC was 51±21 years. After surgery, 35 patients received an anti-rejection/anti-tumor regimen that included sirolimus, and 11 patients did not receive sirolimus. There was no significant difference in survival rate between the two groups (P=0.761). The gene sequencing results showed mTOR-related pathway mutations in 10 patients, of whom five (10.9%) had TSC1/2 mutations. Of the 35 patients using sirolimus, those with mTOR-related mutations had significantly better survival rates than patients without mTOR-related mutations (P=0.016). CONCLUSIONS: According to genetic sequencing results, a personalized treatment plan for specific genetic mutations should be selected in patients undergoing liver transplantation for HCC. Patients with mTOR-related gene mutations, especially TSC mutations, can gain significant benefits from the use of mTOR inhibitors such as sirolimus.

Effects of exogenous p53 transfection on the gene expression in the human brain glioma cell line U251.

The p53 gene is activated in response to several malignancy-associated stress signals by transactivation of downstream genes and by transcription-independent mechanisms. In order to identify new p53 downstream genes, we established a new system of p53 gene inducible expression, U251-pTet-p53 cell line, with the Tet-On Gene Expression System, in which exogenous p53 gene could overexpress in doxycycline (Dox) medium but not in the medium without Dox. By comparing their random primer RT-PCR products, it was proved that exogenous p53 gene expression could lead to many genes differential expression, some up-expressed and others down-expressed. All of these differential expressed genes may be p53 downstream genes. We can gain the magnitude of p53 downstream genes, which provides the basis of directly cloning of novel p53 downstream genes and further studying of p53 regulatory network.