Objective To investigate acquired carbapenemases and prevalence of carbapenem- resistant gram-negative bacill.Methods The antimicrobial susceptibility was determined by agar dilution method.Metallo-B-lactamase(MBLs)were screened by EDTA-disk synergy tesL The encoding genes of MBLs were amplified by PCR followed by sequencing.Strain homology Was investigated by pulsed-field gel electronphoresis(PFGE).Results In 141 carbapenem-resistant Pseudomonas aeruginosa(P.aeruginosa), there were three resistant patterns which were imipenem(IMP)-resistant+meropenem(MEM)-resistant (66.7%),IMP-resistant+MEM-sensitive(32.6%),and IMP-resistant+MEM-sensitive(0.7%).AⅡthe carbapenem-resistan Acinetobacter baumannii(A.baumannii),Acinetobacter lwoffi(A.lwoffi),Citrobactor freundii(C.freundii),Klebsielta pneumoniac(K.pneumoniac)and Serratia were resistant to imipenem and meropenem.Four strains of 141 P.aeruginosa were positive by EDTA-disk synergy test,and they produced VIM-2-type Metallo-B-1actamase.Of 34 carbapenem-resistan A.baumannii,30 strains produced OXA-tllrpe.Among them, OXA23, OXA24 and OXA66 accounted for 79.4%,38.2% and 67.6%,respectively.And 22 of 34 strains(64.7%)produced multiple OXA-carbapenemases.All 7 strains A.lwoffi produced OXA-23-type carbapenemases.A11 11 strains C.freundii,5 strains k pneumoniac and 1 strains Serratia produced KPC-2-type carbapencmases.And 6 of 11 strains C.freundii produced new subtype IMP-8.Of 15 PFGE type in 34 strains A.baumannii,14 strains belonged to A-type,7 strains belonged to B-type.Seven A.lwoffi strains distilbuted in difierent PFGE type.Four strains of P.aeruginosa producing VIM-2.type Metallo-8-lactamase did not have the same PFGE type.Eleven C.freundii strains had the same PFGE type.Five k pneumoniae strains had the sanle PFGE type.Conclusions Drug resistance to 12 common antibiotics in carbapenem-resistant gram-negative bacill was higher than non-carbapenem-resistant gram-negative.The former produced many kinds of carbaponemases and the strains prducing carbapenemases were prevalent in the C.freundii, A.baumanii, and k pneumoniae.

Tumor morphology from the microscopic appearance has being served as the basis of tumor classification,but has serious 1imitations.Molecular aberrations,including DNA,RNA and protein,ale playing an ever increasing role in guiding classification,prognosis,and therapeutic strategies in cancer patients.A specific molecular profile correlating with important clinical parameters should allow physicians to base management decisions on the molecular characteristics of an individual patient's tumor.The application of high-throughput technologies to tumor,3 should make it possible to generate comprehensive molecular profiles.The integration of clinical,morphological and molecular data could result in a more precise classification of cancers,and lead to better understanding,individualized predicting and treatment,which would give the maximum benefit to the patients.

Objective To evaluate the DLC-l gene promoter methylation in acute lymphoblastic leukemia(ALL)in children by methylation specific-PCR(MSP),explore the its prognostic value.Methods Both pyrosequecing and MSP was used to detect DLC-1 gene methylation in bone marrow samples from 34 children with acute lymphoblastic leukemia,5 normal bone marrow samples and acute leukemia T cell line 6T-CET.DLC-1 mRNA expression in cells with、or without treatment with 5-aza-2'-deoxycytidine wag investigated by real time RT-PCR Results MSP analysis showed that DLC-1 promoter methylation Was identified in 21 of bone marrow samples from ALL patients,but was absent in 5 normal bone marrow specimens.These results were completely agreement with pyrosequencing analysis.In additional,the latter can give the quantitative analysis of methylation status in specific CpG sites.No association Was observed between DLC-1 methylation and patient’S clinical-biologic characteristics including age,gender,WBC count,FAB classification,immunology typing and clinical typing(P>0.05)In 18 ALL patients achieving complete remission(CR)witIl DLC-1 methylation,14 relapsed in 12 monthhs,while only 4 relapsed in 1 patients without DLC-1 gene metllylation.Treatment of the cell line 6T-CEM harboring methylation with 5-aza-2'-deoxycytidine increased DLC-1 expression in dose dependent manner. Conclusions This is the first report of high frequency of promoter methylation of DLC-1 in ALL It shows that DLC-1 methylation iS useful for predicting relapse of ALL.Pyrosequencing assay Call provide a sensitive,easy-to-use method for quantitative assessment of DLC-1 methylation.

Objective To evaluate the matrix effect of processed materials in serum total glycerol measurement and to assess the accuracy of routine test systems.Methods With an isotope dilution liquid chromatography tandem mass spectrometry method as the comparative method，matrix effects of 28 processed materials on 8 routine test systems were evaluated ccording to the NCCLS EP 14 protocol.The processed materials and 20 flesh patient specimens were analyzed with both the comparative method and each of the evaluated methods and results obtained with the two methods were plotted.Two-tailed 95% prediction intervals for the mean of the flesh patient specimen were computed and results on the processed aterials were compared with these intervals for evaluation of matrix effect.Results with the two methods on fresh samples were also compared for assessment of the accuracy of the routine test systems.Results Some of the processed samples showed matrix effects on some of the routine test systems.The observed matrix effects were system-specific and aused either positive or negative biases.Calibration biases were also observed on some test systems.Conclusion Matrix effect and calibration bias have been observed in serum total glycerol measurements.Continued efforts are needed for improving the accuracy of serum total glycerol measurements.

Objective To investigate the significance of human cyomeg Movirus(HCMV)pp65 IgG antibody avidity index(AI)for the clinical diagnosis of HCMV primary infection through the experimental model of HCMV primaly infection in BALB/c mice.Methods 6～8 weeks,female,specific-pathogen-free BALB/c mice were divided into 5 groups.6 mice in each group. And he mice were injected with 2×106 PFU/m1,2×105 PFU/mi,2×104 PFU/ml,2×103 PFU/ml and 2×102 PFU/ml of HCMV intraperitoneally respectively. Another 6 mice were injected intraperitoneally with the maximum dose of HCMV kept at 56℃ for 30 min as inactivated virus group.And HF negative control group was established at same time. All the mice ere sacrificed to obtain brain and lung tissues for the following experiments after 1 montll.(1)Tissue samples obtained from mice were inoculated in human embryo fibroblasts(HF)monolayers after routine treatment for virus isolation.HCMV specific eytopathie effect(CPE) was observed bv inverted phase-contrast microscopy.HCMV UL83 DNA in the ultures as tested by PCR and pp65 antigen was detected by indirect immunofluorescence.(2)Extracted mRNA from tissue samples and HCMV pp67 mRNA were analyzed by reverse transcriptase PCR(RT-PcR).(3)Immunoglobulin M(IgM)antibody and immunoglobulin G(1gG)antibody avidity Was investigated for their usefulness in distinguishing primary genital HCMV infections rom nonprimary infections with ELISA kit using truncated pp65 protein.ResultsHCMV can be isolated in the tissues from the mice injected with 2×106 PFU/ml and 2×105 PFU/m1.RT- PCR and ELISA showed positive results in the same groups.The infective rates were 100%.The analysis of the low doses groups,inactivated group and HF negative ontrol group all showed negative results.Conclusions BALB/c mice can be infected with HCMV and appeared as primary infection after1 month.Determination of HCMV pp65 IgM and HCMV p065 IgG-AI by ELISA incorporated with virus isolation and RT-PCR are helpful for distinguishing primary infections from nonpfimary infections.The detection of HCMV p65 IgM and HCMV pp65 IgG-AI by ELISA utilizing recombinant protein pp5 as antigens can be used for preliminary screening.

Objective To investigate the mechanism that cause allelic dropout of amelogenin gene on X chromosome(Amel-X)when using routine Sullivan106/112 bp primer set in sex identification and discuss its influence on the forensic sex identification and the clinical diagnosis.Methods Amel-X dropout was validated with Sullivan212/218 bp and Haas-Rochholz80/83 bp primer sets.Amplification of amelogenin gene was used to analyze dropout of the Amex-X followed by sequencin.Results Sullivan212/218 bp and Hgas-Rochholz80/83 bp primer sets could be used to identify gender correctly.Three types of point mntation were observed in the forward primer binding region of the Sullivan106/112 bp primer set by sequencing in the lost Amel-X,including single point mutation at 2nd and 13th sites,respectively,and heterozygous multiple point mutations at 2nd and 13th sites.Conclusions Point mutation in the primer binding region may result in a failure to amplify amelogenin allele and thus lead to a null allele.This finding should be mid attention to because it may interfere with the sex identification.

Objective To explore the peptides that can mimic the blood type A antigen and evaluate the anti-A antibody detection value of these peptides.Methods The anti-A monoclonal antibodv (NaM87-1F6)was used to panning the phage clones from a phage display 12-mer peptide library.Positive clones were identified by phage ELISA,phage mieropanning methods.Phage DNA Was sequenced and the corresponding peptide sequences were deduced.Agglutination inhibition test WaS performed to assess the ability of phage clones to inhibit the binding between the type A red blood cell and the anti-A antibody. ABO-ELISA based on the selected peptides was compared with classical haemagglutination test jn the detection of senlm anti-A antibody.Results Seven positive clones were chosen after panning,phage ELISA and phage micropanning.Six clones displayed peptide EYWYCGMNRTGC(C5),the other one displayed peptide QIWYERTLPFTF(C17).The phages displaying the selected peptides could specifically inhibit agglutination of type A red blood cells(RBCs)by anti-A antibodies.In the ABO-ELISA based on C5 and C17,the receiver operating characteristic(ROC)Curve showed that area under curve(AUC)were 0.889 (P=0.000),0.75l(P=0.000)respectively.The Spearman correlation Coeffieient between the ABO-EliSA value and the antibody titer derived from haemagglutination assay were 0.743(P<0.01),0.664(P<0.01)respectively.As for C5,0.300 was the best cut-off for ABO-ELISA with 82.2% sensitivity and 83.3% specificity.As for C17,the sensitivity and specificity of ABO-ELISA was 68.9% and 63.3% respectively when the cut-off value was 0.250.Conclusions The peptides EYWYCGMNRTGC and QIWYERTLPFTF can mimic the blood type A antigenic epitope.ABO-ELISA based on these peptides has the potential for the detection of anti-A antibody.

Objective To establish a rapid method to detect mutations in rpoB genes of rifampin-resistant Mycobacterium tubereulosis in dinical specimens using Real-time fluorescence PCR molecular beacon assay.Methods 174 strains of Mvcobacterium tuberculosis clinical isolates were analyzed using real-time fluorescence PCR molecular beacon assay foilowed with DNA sequencing while 12 strains of NTM and 4 strains of bacteria other than Mycobacterium tuberculosis were used as the contrast.Results Eighty-two 89.1 of 92 rifampin (RIF)-resistant strains and 3 of 82 RIF-sensitive strains were found to harbor mutation in the rpoB gene using real-time fluorescence PCR-molecular beacon assay.The specificity, sensitivity,and accuracy of this assay were 96.3%,89.1%,and 92.5%,respectively-Eithty-three of 92 RIF-resistant strains and 1 of 82 RIF-sensitive strains were found to harbor mutation in the rpoB gene using the direct DNA sequencing.The specificity,sensitivity,and accuracy of the direct DNA sequencing were 98.8,90.2%,and 94.2%,respectively.As compared with real-time PCR molecular beacon assay,171 of 174(98.3%)strains of myeobactefium tuberculosis clinical isolates had the salne results.Conclusion Real-time fluorescence PCR-molecular beacon assay can be used as a rapid screen method to detect RIF-resistant isolates.

Objective To study the molecular genetic background of Bel subtype at ABO blood group.Methods Three samples and fifteen samples were diagnosed as Bel subgroup and normal control samples by serological test,respectively.The extracted DNA was genotyped by sequence specific primer- polymerase chain reaction foilowed by sequencing for Exon6 and exon7 at ABO locus and clones were sequenced.Results A novel Bel variant allele(GenBank EF117687) was identified in a Bel individual.The Bel allele was different from the regular B101 allele by single 952G>A missense mutation in exon7.resulting in an amino acid subsfitution of Val for Met at 318 locus.No mutations were detected in the fifteen control samples and the other two Bel allele samples.Conclusions The mutation position was fimt found to lie on coding region of ABO gene behind nucleotide 930.The mutation of G952A in the al,3 galactosyhransferase gene may be one of the molecular genetic basis of Bel ohenotype.

Objective To assess the impact of pigment epithelium-derived factor(PEDF)on the proliferation and apoptosis of the glioma cells by detecting expression of apoptosis related proteins.Methods U87 cells were treated with PEDF(1000μg/ml,U87PEDF),or without PEDF(U87com0),cell proliferation assays were performed by MTT assay to test the effect of PEDF on proliferation of glioma cells;Apoptosis assays were performed by flow-cytometric analysis;Western-blot Was used for evaluating the expression of p16 protein.Results The induced inhibitony rates of glioma cells by PEDF were(54.29±0.62)% Compaxed with the control(t=2.63,P<0.05).The apoptosis assay showed that(21.84±0.36)% of PI- negative/annexin V-positive Was present in the U87 PEDF cells.The appoptosis was associated with the incteases of p16 protein(0.82±0.09)compared with tlle control(0.43±0.03,P<0.05).Conciusion PEDF may play a significant role in apoptosis regulation and proliferation of glioma cell accompanied with the increase of the p16 protein.