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Objective:Hyperoxia is a necessary therapy in some neonatal diseases, and long-term therapeutic hyperoxia may induce severe damaging effects on intestinal epithelial cells.The aim of this study was to investigate whether hyperoxia could promote the expression of ASK1 and P38 in intestinal epithelial cells through ROS.Methods:The human colon adenocarcinoma cell line Caco-2 cells were treated with different concentrations of H 2O 2(100 μmol/L, 200 μmol/L and 400 μmol/L)and 85% oxygen in vitro.The expression of ASK1 was detected by immunofluorescence, and the expression of P38 and p-P38 were detected by Western Blot and Real-time PCR. Results:With the increase of H 2O 2 concentration, the fluorescence intensity of ASK1 increased.The fluorescence intensity of ASK1 in the hyperoxia group was significantly stronger than that of the control group and the H 2O 2 groups.With the increase of H 2O 2 concentration(100 μmol/L、200 μmol/L、400 μmol/L), the expression of P38 protein(0.21±0.02, 0.28±0.13, 0.44±0.07)and p-P38 protein(0.09±0.02, 0.19±0.03, 0.37±0.07)gradually increased.The expression of P38 mRNA in 200 μmol/L and 400 μmol/L H 2O 2 groups(4.03±0.68、3.94±0.71)was significantly higher than that in 100 μmol/L H 2O 2 group(3.05±0.47)( P<0.01). The expressions of P38 protein, p-P38 protein and P38 mRNA in the hyperoxia group were significantly higher than those in the H 2O 2 group( P<0.01). Compared with the control group, the expressions of P38 protein, p-P38 protein and p38 mRNA in the hyperoxia group and H 2O 2 groups increased significantly( P<0.01). Conclusion:The expression of ASK1 and P38 in intestinal epithelial cells increased significantly under hyperoxia, which indicated that hyperoxia might activate ASK1 and thereby regulate the expression of downstream P38 through ROS, resulting in intestinal epithelial cells damage.
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Objective To evaluate the analytical performance of a novel HBV DNA assay based on automated DNA extraction and real-time fluorescence quantitative PCR .Methods Analytic verification studies.Accuracy and lower limit of detection were assessed by determining a panel of HBV standard plasma of WHO.HBV standard plasma (genotype A, B, C and D) at 6 different concentrations were measured 18 times to evaluate precision and reproducibility .Pseudo-viral particles at high HBV DNA concentration were serially diluted to assess linear range .One hundred and forty-four clinical specimens were quantified for HBV DNA so as to evaluate the correlation between the new test and COBAS ? system. Results Quantification of HBV standard plasma showed acceptable accuracy , with each deviation between observed and expected values within ±0.35 lg IU/ml (-0.17-0.32 lg IU/ml).Intra-assay coefficients of variation ( CV) for genotype A , B, C and D were 3.87% -6.32%, 0.45% -14.68%, 0.16% -8.36% and 0.64%-13.01%respectively, and the inter-assay CV were 5.67%-9.69%, 1.28%-15.68%, 0.36%-9.05%and 1.69%-13.65%, separately.Linearity assessment exhibited an excellent dynamic range of linear quantification from 20 to 1.0 ×1010 IU/ml ( r =0.998, P <0.001 ) .And the satisfactory results obtained at 3 levels of HBV DNA concentration (10, 20, 50 IU/ml, respectively) confirmed the claimed lower limit of detection with 5/5 detectable rate at 20 IU/ml.Furthermore, good correspondence was observed between the new HBV DNA assay and the COBAS ? system with 100% ( 144/144 ) qualitative coincidence and significant correlation based on 104 positive data ( r=0.984, P<0.000 1).Conclusions The novel fully-automated real-time PCR assay displayed good analytical and clinical performance for highly sensitive detection of HBV DNA.It was well suited for monitoring antiviral responses as well as drug resistance according to current clinical practice guidelines for the management of chronic HBV infection .
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Objective Toinvestigate the molecular characteristics including antibiotic resistance,strain type,serotype,virulence,biofilm formation of Streptococcus pneumoniae isolated from Shanghai adult patients.Methods A total of 37 non-repetitive S.pneumoniae isolates causing community acquired and hospital acquired infections of adults were collected from Shanghai Huashan Hospital from January 2011 to December 2013.The inhibitory zone diameter or minimum inhibitory concentrations (MICs) of 9 antimicrobial agents (penicillin,vancomycin,erythromycin,clindamycin,levofloxacin,cefprozi,ceftriaxone,cefotaxime and linezolid) were determined by Kirby-bauer (K-B) method or Etest method;Serotypes were tested by polymerase chain reaction (PCR) and S.pneumoniae antisera agglutination;Genomic characteristics of different serotype strains were determined by pulsed field gel electrophoresis (PFGE)method;Multilocus sequence types (MLST) was used for strain type;Semi quantitative biofilm formation test was used for the biological membrane formation.Ten main pneumococcal virulence genes (cbpA,pspA,cps2A,lytA,nana,pavA,piaA,ply,psaA and spxB) were detected by PCR and gel electrophoresis.Statistical analysis was performed using Stata software and association statistics were tested using Fisher's exact test.Results The most frequent serotypes were 19F (13.5％),23 F (13.5％),14 (10.8％),19A (10.8％).The penicillin resistance rate was 64.9％.Serotypes 19 F,19A and 23 F were significantly associated with penicillin resistance (x2 =5.89,P =0.015) and the isolates belonged to these serotypes were all multi-drug resistant (MDR).ST81 and ST271 showed high resistance rates to several antibiotics including penicillin (x2 =4.57,P =0.033).Biofilm formation was significantly associated with serotypes 19A (x2 =5.55,P =0.018) and strain type ST320 (x2 =4.33,P =0.037),but not associated with penicillin resistance (x2 =0.16,P =0.686).Virulence gene lytA,pavA,ply,psaA,spxB were found in all isolates.Conclusions Penicillin resistance rate of S.pneumoniae in adult is rising.Specific serotype,epidemic clone and antibiotic resistance are closely related,and can provide the basis for the infection control.The virulence factors such as PspA will be the new targets for vaccine development to reduce S.pneumoniae infection in the future.
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Objective To investigate and compare the molecular epidemiological characteristics of Streptococcus pyogenes isolates from Shanghai adult and pediatric patients in terms of antimicrobial susceptibility ,clone type ,emm type ,biofilm formation and virulence for better infection control and treatment .Methods Thirty‐nine nonduplicate clinical isolates of S . pyogenes from adult and pediatric patients were analyzed by determining the susceptibility to antimicrobial agents by Kirby‐Bauer method;clonal typing by multilocus sequence typing ( MLST ); genotyping by emm gene sequence analysis ,which encoding M protein;genomic characteristics of different emm type strains by pulsed field gel electrophoresis (PFGE );and biofilm formation by semi‐quantitative biofilm formation test . Twenty main virulence genes of S .pyogenes ,including 12 superantigen genes and 8 other key genes were detected by PCR and gel electrophoresis . Results A total of 39 nonduplicate S .pyogenes isolates were analyzed .The most common genotype was emm 12‐ST36 (64 .1% ) and emm 1‐ST28 (17 .9% ) .Isolates from adult and pediatric patients had the same dominant genotype , emm 12‐ST36 . The isolates from children showed significantly higher resistance rate to erythromycin and clindamycin than those from adult patients (P<0 .000 1) .Particular emm type and clone type were frequently identified in the same PFGE cluster .Statistical analysis showed that biofilm formation was significantly associated with emm type 1 (P=0 .005) and erythromycin/clindamycin resistance (P=0 .000 3) .The strains from children showed higher biofilm formation than those from adult patients (P<0 .000 1) .We found that virulence genes speA ,speJ and spd3 were significantly associated with emm type 1 (P<0 .000 1 ,P=0 .005 5 ,P<0 .000 1) ,while speI and sic were significantly associated with emm type 12 (both P<0 .000 1) .We also found that the prevalence of speC ,speH ,ssa , smeZ ,and sdaD genes was significantly different between emm type 12 and emm type 1 (P= 0 .023 8 , P< 0 .000 1 , P<0.0001,P= 0.0003,and P= 0.0068,respectively).TheprevalenceofvirulencegenesspeH,smeZandsdaDwas significantly different between the emm type 12 strains from children and those from adults (all P< 0 .000 1) .Conclusions There is a strong agreement between emm type ,clone type ,virulence genes and the clusters defined by PFGE profiling of S . pyogenes .S .pyogenes isolates from adult and pediatric patients are different in terms of antibiotic resistance and biofilm formation .Certain emm type is significantly associated with antibiotic resistance and virulence ,which is useful for infection control .Dominant virulence genes may be the potential target for developing new vaccine to reduce S .pyogenes infection in the future .
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Objective To investigate the relationship between nuclear factor(NF)-κB activity and lactacystin induced prostate cancer cell apoptosis.Methods Two prostate cancer cell were divided into two groups:blank control group treated with culture solution,lactacystin group treated with different concentration of lactacystin(0.5,1.0,2.0,4.0 μ mol/L),the action time were 8,16 and 24 hours.The cell survival rate was measured by MTT assay.NF-κB DNA binding activity was measured by enzyme-linked immunosorbent assay,the expression of NF-κB P65 nuclear protein was detected by Western blot assay,and caspase-3 activity was analyzed by enzyme analysis assay.Results On basal condition,the NF-κ B DNA binding activity was much higher in DU145 cell than that in LNCaP cell(t=4.728,P=0.001).Compared with blank control group,different concentration of lactacystin groups'NF-κ B DNA binding activity in both the LNCaP and DU145 cell were reduced.The expression of NF-κB p65 nuclear protein decreased along with raising of lactacystin concentration in LNCaP cell,but it did not change in DU145 cell.On basal condition,caspase-3activity in DU145 cell was higher than that in LNCaP cell(t=4.519,P=0.001).After lactacystin acting of 24 hours,caspase-3 activity increased along with raising of lactacystin concentration in both the LNCaP and DU145 cell(2.0 μmol/L lactacystin group compared with 1.0 μmol/L lactacystin group,DU145 cell P=0.000,LNCaP cell P=0.000).Conclusions Lactacystin has different killing effects on prostate cancer cell.The mechanism may be related to inducing the apoptosis by down-regulation of NF-κB activity.There may be additional cell survival/death pathway in androgen-independent prostate cancer cell.