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Selection of Stable Reference Genes for Real-Time Quantitative PCR Analysis in Edwardsiella tarda
( Zhongyang Sun ),( Jia Deng ),( Haizhen Wu ),( Qiyao Wang ),( Yuanxing Zhang ) 한국미생물 · 생명공학회 2017 Journal of microbiology and biotechnology Vol.27 No.1
Edwardsiella tarda is a gram-negative pathogenic bacterium in aquaculture that can cause hemorrhagic septicemia in fish. Many secreted proteins have already been identified as virulent factors of E. tarda. Moreover, since virulent phenotypes are based on the expression regulation of virulent genes, understanding the expression profile of virulent genes is important. A quantitative RT-PCR is one of the preferred methods for determining different gene expressions. However, this requires the selection of a stable reference gene in E. tarda, which has not yet been systematically studied. Accordingly, this study evaluated nine candidate reference genes (recA, uup, rpoB, rho, topA, gyrA, groEL, rpoD, and 16S rRNA) using the Excel-based programs BestKeeper, GeNorm, and NormFinder under different culture conditions. The results showed that 16S rRNA was more stable than the other genes at different culture growth phases. However, at the same culture time, topA was identified as the reference gene under the conditions of different strains, different culture media, and infection, whereas gyrA was identified under the condition of different temperatures. Thus, in experiments, the expression of gapA and fbaA in E. tarda was analyzed by RT-qPCR using 16S rRNA, recA, and uup as the reference genes. The results showed that 16S rRNA was the most suitable reference gene in this analysis, and that using unsuitable reference genes resulted in inaccurate results.
( Xinyue Chang ),( Chengli Teng ),( Haizhen Wu ),( Jiang Ye ),( Qiyao Wang ),( Huizhan Zhang ) 한국미생물생명공학회(구 한국산업미생물학회) 2019 Journal of microbiology and biotechnology Vol.29 No.8
Edwardsiella piscicida is the causative agent of edwardsiellosis, which has caused enormous economic losses worldwide. In our previous research, an attenuated live vaccine known as WED and based on the virulent strain E. piscicida EIB202 can effectively protect turbots against edwardsiellosis via intraperitoneal injection, while vaccination by immersion exhibits a weaker effect. During the development of the immersion vaccine, we surprisingly found the counts of ΔpEIB202/ EIB202 colonized on zebrafish were 100 times lower than those of EIB202. However, pEIB202 carries 53 predicted ORFs and has several copies in E. piscicida EIB202, impeding the study of its function. Thus, the replication region is located at a 1,980 bp fragment (from 18,837 to 20,816 bp), containing a transcriptional repressor and a replication protein. Moreover, the minimal replication plasmid, named pRep-q77, has low copies in both E. coli and E. piscicida, but is more stable in E. piscicida than in E. coli. This work lays a foundation for further examination of the function of the virulence plasmid pEIB202.
Cathepsin K Activity Controls Injury-Related Vascular Repair in Mice
Hu, Lina,Cheng, Xian Wu,Song, Haizhen,Inoue, Aiko,Jiang, Haiying,Li, Xiang,Shi, Guo-Ping,Kozawa, Eiji,Okumura, Kenji,Kuzuya, Masafumi American Heart Association, Inc. 2014 Hypertension Vol.63 No.3
<P>Cathepsin K (CatK) is one of the most potent mammalian collagenases. We showed previously the increased expression of CatK in human and animal atherosclerotic lesions. Here, we hypothesized that ablation of CatK mitigates injury-induced neointimal hyperplasia. Male wild-type (CatK<SUP>+/+</SUP>) and CatK-deficient (CatK<SUP>−/−</SUP>) mice underwent ligation or a combination of ligation and polyethylene cuff-replacement injuries to the right common carotid artery just proximal to its bifurcation, and they were then processed for morphological and biochemical studies at specific time points. On operative day 28, CatK<SUP>−/−</SUP> significantly reduced neointimal formation and neovessel formation in both single- and combination-injured arteries compared with the Cat K<SUP>+/+</SUP> mice. At early time points, CatK<SUP>−/−</SUP> reduced the lesion macrophage contents and medial smooth muscle cell proliferation, the mRNA levels of monocyte chemoattractant protein-1, toll-like receptor-2, toll-like receptor-4, chemokine ligand-12, and the gelatinolytic activity related to matrix metalloproteinase-2/-9. An aorta-explant assay revealed that smooth muscle cell movement was impaired in the CatK<SUP>−/−</SUP> mice compared with the CatK<SUP>+/+</SUP> mice. In addition, the smooth muscle cells and macrophages from CatK<SUP>−/−</SUP> mice had less invasive ability through a reconstituted basement membrane barrier. This vasculoprotective effect was mimicked by Cat inhibition with <I>trans</I>-epoxysuccinyl-L-leucylamido-{4-guanidino} butane (E64<I>d</I>). These results demonstrate an essential role of CatK in neointimal lesion formation in response to injury, possibly via the reduction of toll-like receptor-2/-4–mediated inflammation and smooth muscle cell proliferation, suggesting a novel therapeutic strategy for the control of endovascular treatment–related restenosis by regulating CatK activity.</P>
Preparation and properties of coated silica/aluminum cermet materials via powder metallurgy method
Jieguang Song,Yue Liu,Chunxiao Wu,Xueqing Yang,Huihui Luo,Chunyan He,Chengbin Li,Haizhen Yang 한양대학교 세라믹연구소 2020 Journal of Ceramic Processing Research Vol.21 No.2
Cermet is a combination of metal and ceramic, it exhibits some special properties, so it is one of the most widely usedengineering materials. In this paper, the properties of encapsulated SiO2/Al cermet were prepared by powder metallurgymethod. Results showed that the density of SiO2/Al cermet is increased with an increasing Al content. When the Al contentis high, and the high temperature is sintered, the liquid phase can fully bond the surrounding SiO2 particles to form a relativelycontinuous and dense structure, thereby obtaining a high microstructure density. When the SiO2:Al ratio is increased from 1:1to 1:3, the hardness is gradually increased. The hardness and density of the sample are gradually increased with an increasingmolding pressure. The surface hardness of the cermet is increased first and then decreased with an increasing sinteringtemperature. When the SiO2:Al ratio is 1:3, the holding time is 10 min under molding pressure of 15 MPa, and the sinteringtemperature is 900 °C for 1 h, the surface hardness of the obtained coated SiO2/Al cermet is high, the value is 175.2 HV.
sRNA EsrE Is Transcriptionally Regulated by the Ferric Uptake Regulator Fur in Escherichia coli
Bingbing Hou,Xichen Yang,Hui Xia,Haizhen Wu,Jiang Ye,Huizhan Zhang 한국미생물·생명공학회 2020 Journal of microbiology and biotechnology Vol.30 No.1
Small RNAs (sRNAs) are widespread and play major roles in regulation circuits in bacteria. Previously, we have demonstrated that transcription of esrE is under the control of its own promoter. However, the regulatory elements involved in EsrE sRNA expression are still unknown. In this study, we found that different cis-regulatory elements exist in the promoter region of esrE. We then screened and analyzed seven potential corresponding trans-regulatory elements by using pull-down assays based on DNA affinity chromatography. Among these candidate regulators, we investigated the relationship between the ferric uptake regulator (Fur) and the EsrE sRNA. Electrophoresis mobility shift assays (EMSAs) and β-galactosidase activity assays demonstrated that Fur can bind to the promoter region of esrE, and positively regulate EsrE sRNA expression in the presence of Fe2+.
Zuo, Chaohui,Xia, Man,Liu, Jingshi,Qiu, Xiaoxin,Lei, Xiong,Xu, Ruocai,Liu, Hanchun,Li, Jianliang,Li, Yongguo,Li, Qinglong,Xiao, Hua,Hong, Yuan,Wang, Xiaohong,Zhu, Haizhen,Wu, Qunfeng,Burns, Michael,Li Asian Pacific Journal of Cancer Prevention 2015 Asian Pacific journal of cancer prevention Vol.16 No.1
Objectives: Intrahepatic recurrence is the major cause of death among patients with hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) after curative surgical resection. Several approaches have been reported to decrease the recurrence rate. The objective of our study was to compare the clinical effects of transcatheter arterial chemoembolization (TACE) combined with interferon-alpha (IFN-${\alpha}$) therapy on recurrence after hepatic resection in patients with HBV-related HCC with that of TACE chemotherapy alone. Methods: We retrospectively analyzed the data from 228 patients who were diagnosed with HBV-related HCC and underwent curative resection between January 2001 to December 2008. The patients were divided into TACE (n = 126) and TACE-IFN-${\alpha}$ (n = 102) groups for postoperative chemotherapy. The TACE regimen consisted of 5-fluorouracil (5-FU), cisplatin (DDP), and the emulsion mixed with mitomycin C (MMC) and lipiodol. The recurrence rates, disease-free survival (DFS), overall survival (OS), and risk of recurrence were evaluated. Results: The clinicopathological parameters and adverse effects were similar between the 2 groups (P > 0.05). The median OS for the TACE-IFN-${\alpha}$ group (36.3 months) was significantly longer than that of the TACE group (24.5 months, P < 0.05). The 3-and 5-year OS for the TACE-IFN-${\alpha}$ group were significantly longer than those of the TACE group (P < 0.05) and the recurrence rate was significantly lower (P < 0.05). The TACE and IFN-${\alpha}$ combination therapy, active hepatitis HBV infection, the number of tumor nodules, microvascular invasion, liver cirrhosis, and the BCLC stage were independent predictors of OS and DFS. Conclusions: The use of the TACE and IFN-${\alpha}$ combination chemotherapy after curative hepatic resection safely and effectively improves OS and decreases recurrence in patients with HBV-related HCC who are at high risk. Our findings can serve as a guide for the selection of postoperative adjuvant chemotherapy for patients with HBV-related HCC who are at high risk of recurrence.