ABSTRACT
Objective To evaluate the capability of four tests for identification of the in vitro suscepti-bility of tigecycline against Acinetobacter and Enterobacteriaceae isolates.Methods Disk diffusion test was per-formed to detect the sensitivity of 158 Acinetobacter and 339 Enterobacteriaceae isolates to tigecycline.The mini-mum inhibitory concentrations ( MICs) of tigecycline for non-sensitive isolates were detected by using broth dilu-tion method ( BDM) , MIC Test Strip ( MTS) and agar dilution method.The differences with antimicrobial sus-ceptibility among the four different methods were evaluated.Results Tigecycline showed good antibacterial ac-tivity against both non-sensitive Acinetobacter and Enterobacteriaceae isolates with most of the MIC50 values in the sensitivity range of (0.5-2) mg/L and all of the MIC90 values of 4 mg/L.The MIC50 and MIC90 values measured by BDM were respectively 1 mg/L and 4 mg/L.The sensitivity rates presented by the results of BDM were re-spectively 87.1%and 70.2%based on the standards made by Food and Drug Administration (FDA) and Euro-pean Committee on Antimicrobial Susceptibility Testing ( EUCAST) .Agar dilution method indicated that most of the MICs of tigecycline to Acinetobacter and Enterobacteriaceae isolates were two dilutions higher than those de-tected by BDM with essential agreement (EA) rate of 56.5%.Both the very major error (VME) and the major error (ME) values were 0 and the categorical agreement (CA) rate was 46.8%according to the FDA standard.The VME and CA values were 0.8% and 24.2% based on EUCAST standard.Compared with agar dilution method, MTS showed better results in determining the susceptibility of Acinetobacter and Enterobacteriaceae iso-lates to tigecycline with MIC50 and MIC90 values of 1.5 mg/L and 4 mg/L, which was similar to the capability of BDM.Referring to the FDA and EUCAST standards, the sensitivity rates were 83.1% and 21.0%, the CA rates was 81.5%and 29.8%, and the EA rate was 71.8%.Most of the results tested by MTS were one dilution higher than those by BDM.FDA standard showed better correlation than EUCAST standard.Disk diffusion method showed the ME, mE, VME and CA values were respectively 19.4%, 71.8%, 0 and 8.9%according to FDA standard.Conclusion Disk diffusion method, MTS and agar dilution method all showed differences with BDM in susceptibility testing.The capability of MTS was similar to that of BDM.The results evaluated by FDA standard were better than those by EUCAST standard.The in vitro susceptibility of bacteria to tigecycline could be tested by disk diffusion method using FDA standard for evaluation, and confirmed with MTS if isolates were resistance or intermediate strains.The BDM could be performed for further confirmation if necessary.
ABSTRACT
Previous study on TNFRl-mediated hepatocyte apoptosis has been implicated in the development of fulminant viral hepatitis.To interfere with the potentially effective target,plasmid named p-mTNFR1shRNA complimentary to the sequence responsible for mTNFR1 was also constructed and further confirmed by sequence analysis.To investigate the effect of mTNFR1shRNA plasmid on mTNFR1 expression in vivo and the disease progress in MHV-3 induced fulminant hepatitis mice model.By hydrodynamic injection of mTNFRlshRNA plasmid,the survival rate of mice,hepatic pathological change were examined and compared between mice with/without mTNFR1 shRNA plasmid intervention.The expression of mTNFR1 was detected by Real-time PCR,immunohistochemistry staining.The mTNFR1 shRNA plasmid significantly reduced mTNFR1 expression in vivo,markedly ameliorates inflammatory infiltration,prolonged the survival time period and elevated the survival rate from 0 up to 13.3% in Balb/cJ mice with MHV-3 induced fulminant hepatitis.This study was designed to explore the opportunity of RNA interference technique in inhibiting TNFR1 expression,which has been reported to be involved in the development of a variety of diseases including fulminant viral hepatitis and severe chronic hepatitis B.
ABSTRACT
Objective To construct the eukaryotic expression plasmids of human Fas and TNFR1 gene(pcDNA3.0-hFas and pcDNA3.0-hTNFR1)and microRNA(miRNA)expression plasmid of hFas and hTNFR1 named p-hFasmiRNA and p-hTNFR1miRNA,and to investigate their inhibitory effects in vitro.Methods The eukaryotic expression plasmids of human Fas and TNFR1 gene were constructed(pcDNA3.0-hFas and pcDNA3.0-hTNFR1)and have been shown successfully to express hFas and hTNFR1 protein.miRNA expression plasmids of hFas and hTNFR1 named p-hFasmiRNA and p-hTNFR1miRNA complimentary to the sequence responsible for hFas and hTNFR1 respective were constructed,meanwhile irrelevant miRNA plasmid was used as a control.By respective co-transfection of p-hFasmiRNA and pcDNA3.0-hFas,p-hTNFR1 miRNA and pcDNA3.0-hTNFR1 expression construct into 293T cells,the inhibition of hFas and hTNFR1 expression was analyzed by real-time PCR and Western blot.Results The experiments showed the significant inhibitory effect of p-hFasmiRNA on hFas and p-hTNFR1miRNA on hTNFR1 expression at 48 h post-transfection both at RNA level and protein level as well in 293T cell lines with the inhibitory efficiency being as high as 87% for hFas and 80% for hTNFR1,respectively.Conclusion The p-hFasmiRNA and p-hTNFR1miRNA were constructed successfully,and it was verified that they could specifically inhibit the hFas and hTNFR1 expression at the cellular level.
ABSTRACT
<p><b>OBJECTIVE</b>Our previous studies have shown that an anti-sense plasmid to mouse fibrinogen like protein 2 (mfgl2) significantly reduced mfgl2 expression in vivo, markedly ameliorated inflammatory infiltration, fibrin deposition and hepatocyte necrosis, prolonged the survival time period and elevated the survival rate in Balb/cJ mice with murine hepatitis virus type 3 (MHV-3) induced fulminant hepatitis. This study was designed to explore the opportunity of RNA interference technique in the inhibitory application of hfgl2 expression, which has been reported to be involved in a variety of disease developments including fulminant viral hepatitis, acute rejection of allo/xeno transplantation and fetal loss syndrome.</p><p><b>METHODS</b>A plasmid named p-hfgl2shRNA complimentary to the sequence responsible for hfgl2 was constructed; meanwhile irrelevant shRNA plasmid with a random combination of the p-hfgl2shRNA sequence was used as a control. A plasmid named pEGFP-hfgl2 expressing hfgl2-EGFP fusion protein was also constructed for the screening of the effect of p-hfgl2shRNA on the hfgl2 expression. By cotransfection of the constructed p-hfgl2shRNA and pEGFP-hfgl2 or pcDNA3.1-hfgl2 expression plasmids into CHO cells, the inhibition of hfgl2 expression by hfgl2shRNA was analyzed by direct observation through fluorescent microscopy, FACS, real time PCR and immunohistochemistry staining.</p><p><b>RESULTS</b>The experiments showed a significant inhibitory effect of p-hfgl2shRNA on hfgl2 expression at 48 h post-cotransfection by observation of green fluorescent cells, FACS, real time PCR and immunohistochemistry staining, with the inhibitory efficiency reaching as high as 85.5%.</p><p><b>CONCLUSION</b>The study demonstrated that p-hfgl2shRNA successfully interfered with hfgl2 expression in vitro. This provides a foundation to further explore its application in vivo.</p>
Subject(s)
Animals , Cricetinae , CHO Cells , Cricetulus , Fibrinogen , Genetics , Plasmids , RNA Interference , RNA, Small Interfering , GeneticsABSTRACT
This study was designed to explore the RNA interference technique in inhibition of the expression of the mouse fibrinogen like protein 2 (mfgl2), which has been reported to be involved in the development a variety of diseases including fulminant viral hepatitis. A plasmid named p-mfgl2shRNA,complementary to the sequence of mfgl2 was constructed, while another short hairpin RNA (shRNA)which was a mutated form of the mfgl2shRNA sequences was used as a control. A plasmid named pEGFP-mfgl2 expressing the mfgl2-EGFP fusion protein was also constructed for the screening of the effect of p-mfgl2shRNA on mfgl2 expression. By cotransfection of p-mfgl2shRNA and pEGFP-mfgl2 or pcDNA3.1-mfgl2 expression construct into CHO cells or HeLa cells, the inhibition of mfgl2 expression by mfgl2shRNA was analyzed by direct observation through fluorescent microscopy, FACS, RT-PCR and immunohistochemistry staining. The experiments showed the significant inhibitory effect of p-mfgl2shRNA on mfgl2 expression at 48h post-transfection in both CHO and Hela cell lines with the inhibitory efficiency as high as 80.1%. The study demonstrated that the construct of p-mfgl2shRNA successfully interfered with the mfgl2 expression in vitro.