Chloromycetin resistance of clinically isolated E coli is conversed by using EGS technique to repress the Chloromycetin acetyl transferase

AIM: To explore the possibility of repression of chloromycetin (Cm) acyl transferase by using external guided sequence (EGS) in order to converse the clinical Ecoli isolates from Cm- resistant to Cm- sensitive.METHODS: EGS directed against chloromycetin acetyl transferase gene (cat) was cloned to vector pEGFP-C1 which contains the kanamycin (Km) resistance gene.The recombinant plasmid pEGFP-C1+EGScat1+cat2 was constructed and the blank vector without EGS fragment was used as control plasmids. By using the CaCl2 transformation method, the recombinant plasmids were introduced into the clinically isolated Cm resistant but Km sensitive E colistrains. Transformants were screened on LB agar plates containing Km. Extraction of plasmids and PCR were applied to identify the positive clones.The growth curve of EGS transformed bacteria cultured in broth with Cm resistance was determined by using spectrophotometer at A600. Drug sensitivity was tested in solid culture containing Cm by using KB method.RESULTS: Transformation studies were carried out on 16 clinically isolated Cm-resistant (250 μg/mLof Cm) E colistrains by using pEGFP-C1-EGScat1cat2 recombinant plasmid. Transformants were screened on LB-agar plates containing Km after the transformation using EGS.Of the 16 tested strains, 4 strains were transformed successfully. Transformants with EGS plasmid showed growth inhibition when grown in liquid broth culture containing 200 μg/mL of Cm. In drug sensitivity test,these strains were sensitive to Cm on LB-agar plates containing 200 μg/mL of Cm. Extraction of plasmids and PCR amplification showed the existence of EGS plasmids in these four transformed strains. These results indicated that the Cat of the four clinical isolates had been suppressed and the four strains were converted to Cm sensitive ones.CONCLUSION: The EGS directed against Cat is able to inhibit the expression of Cat, and hence convert Cmresistant bacteria to Cm-sensitive ones. Thus, the EGS has the capability of converting the phenotype of clinical drug-resistant isolates strains to drug-sensitive ones.

Notch1 Drives the Formation and Proliferation of Intrahepatic Cholangiocarcinoma

The molecular mechanisms underlying the development of intrahepatic cholangiocarcinoma(ICC)are not clear yet.In this study,we investigated the involvement of Notch1 in the development of ICC.The cDNA microarray analysis showed that Notch1 expression was higher in ICC tissues than in normal biliary epithelial cells.Stable transfection of Notchl receptor intracellular domain(NICD1)by hydrodynamic tail vein injection induced ICC formation in mice.Western blotting confirmed that Notchl signaling was activated in human ICC cell lines and mouse ICC tissues.Silencing Notchl with specific short interfering RNA(siRNA)inhibited the proliferation of ICC cells.Flow cytometry and Western blotting indicated that apoptosis was induced in Notchl-silenced ICC cells compared with controls.Additionally,Notchl silencing was associated with the inhibition of hairy and enhancer of split-1(Hes1)and activation of the phosphatase and tensin homolog(PTEN)/p53 pathway.Taken together,these data suggest that Notchl drives ICC formation and proliferation;downregulation of Notchl induces apoptosis in ICC cells;Notchl signaling may serve as a novel therapeutic target for the treatment of ICC.

The Role of CARD9 in Metabolic Diseases

Caspase recruitment domain containing protein 9(CARD9)is an adaptor protein that plays a critical role in pattern recognition receptors(PRRs)-mediated activation of NF-kB and mitogen-activated protein kinase(MAPK).This elicits initiation of the pro・inflammatory cytokines and leads to inflammatory responses,which has been recognized as a critical contributor to chronic inflammation.Current researches demonstrate that CARD9 is strongly associated with metabolic diseases,such as obesity,insulin resistance,atherosclerosis and so on.In this review,we summarize CARD9 signaling pathway and the role of CARD9 in metabolic diseases.

Erratum to:The Role of CARD9 in Metabolic Diseases

The article“The Role of CARD9 in Metabolic Diseases”,written by Cheng TIAN,Ya-li TUO,Yi LU,Chuan-rui XU,Ming XIANG,was originally published electronically on the publisher’s internet portal on May 2020 without open access.With the author(s)’decision to opt for Open Choice,the copyright of the article is changed to©The Author(s)2020 and the article is forthwith distributed under a Creative Commons Attribution 4.0 International License(https://creativecommons.org/licenses/by/4.0/),which permits use,sharing,adaptation,distribution and reproduction in any medium or format,as long as you give appropriate credit to the original author(s)and the source,provide a link to the Creative Commons license,and indicate if changes were made.