Objective　To select suitable media for increasing isolation rate of haemophilus.Methods　Haemophilus was inoculated on seven types of media.The average growth index(GI) of Haemophilus was calculated and Haemophilus was isolated from 325 specimens of the respiratory tract and its isolation rate was compared with that media of brchoc and brchoc-V agare.Results　The GI of media with 2% fresh yeast infusion and 50% of meat infusion was higher than that of those with no yeast and meat infusion.The GI of Haemophilus on BRchoc was higher than that of the BSchoc(P<0.001).The isolation rate of Haemophilus from 325 respiratory tract specimens on BRchoc-V was 77.2%,which was significantly higher than that on the medium BRchoc (32.0%).Conclusions　BRchoc-V is a better medium for isolation of Haemophilus.This medium is effective to improve the isolation rate of Haemophilus.

Objective To investigate the localization of Smad2, Smad3, Smad4, and Smad7 proteins and their expression changes in the 5/6 subtotally nephrectomized rat kidney. Methods The rat model of chronic renal failure was established by performing 5/6 subtotally nephrectomy (SNx) and rats in the control group underwent sham-operation. The rats were sacrificed at 4, 8, and 12 week after operation. The sites and levels of expressions of Smad2, Smad3, Smad4, and Smad7 proteins were examined by immunohistochemical staining. Renal fibrosis was assessed by measuring tissue hydroxyproline. Results Immunohistochemical staining indicated that Smad2, Smad3, and Smad4 proteins were mainly expressed in glomeruli and renal tubular cells, while Smad protein 7 was expressed in glomeruli, but rarely in proximal renal tubular cells. Expressions of Smad2, Smad3, and Smad4 proteins in glomeruli were significantly increased during 4-12 weeks after 5/6 nephrectomy, but the expression level of Smad protein 7 was significantly decreased, but accompanied increase of hydroxyproline content in the renal tissues. Conclusion These results indicate that TGF-?/Smad signaling is involved in the progress of chronic glomerulosclerosis. The high level expressions of Smad2, Smad3, and Smad4 proteins and the down-regulation of Smad7 protein may be the major cause of the glomerulosclerosis in this model.

Objectives To evaluate mini/VITAL and BacT/Alert, two fully automated blood culture systems. Methods Standard mini/VITAL AER and ANA bottles (bioM′erieux, Marcy 1′Etoile, France) and BacT/Alert FAN aerobic and FAN anerobic bottles (organon Netherlands) were used. The bottles were analysed over a period of 7 days. A total of 600 samples of blood, 108 samples of body fluid, and 100 strains of imitation were detected by mini/VITAL and BacT/Alert parallelly. The bottles of flagged positive were smeared and Gram stained before being examined under a microscope. Subcultures were performed in parallel on fresh blood agar and chocolate agar incubated in microaerophilic Conditions with 5% CO 2. All the bottles flagged negative were examined in the same conditions as the positive bottles. Results mini/VITAL: The positive rate for aerobic and anaerobic blood cultures was 10% and 5% respectively, for aerobic body fluid culture was 26 9%. No contaminants were detected. 0.5% false positive and 0.8% false negative were detected. 90% of positive sample were detected in 48 h. The shortest time of report of positive bottles was 1.5 h for blood culture. BacT/Alert: The rate of positive of aerobic and anaerobic blood culture was 9.8% and 5% respectively. The positive rate of body fluid culturs was 26.9%. There were used 3 bottles contaminated. The false positive and false negative rate were 0.5% and 0 98% respective. 95.5% of positive bottles were detected in 48 h. The shortest time of flagged positive was 2.5 h. Conclusion mini/VITAL and BacT/Alert were accurate automated blood culture systems for the detection of bacteremia. There was no significant difference on the speed of detection between BacT/Alert FAN bottles and mini/VITAL standard bottles.

OBJECTIVE To compare the BacT/Alert FN with Botai SN anaerobic blood culture bottles for detection of commonly encountered anaerobes.METHODS Using these two types of anaerobic bottles to culture 5 commonly encountered anaerobes in automatic blood culturing system and BacT/Alert system,and then to analyze the results.RESULTS There were all 32 anaerobic bottles reported positive results by BacT/Alert FN bottles,and only 8 positive bottles were reported by Botai SN bottles.CONCLUSIONS The performance of the BacT/Alert FN is much better than Botai SN anaerobic blood culturing bottle when it is used to detect commonly encountered anaerobes.

OBJECTIVE To investigate the variation of in vitro activity,the ?-lactamases,type diversity and the homology of multiple resistances in Acinetobacter isolated.METHODS The multiple resistant Acinetobacter were selected to detect susceptibility test by K-B antimicrobial agents.The resistant rates were analyzed by WHONET 5.4,the isolates ?-lactamases phenotype was detected by three-dimensional test,genomic types were measured by PFGE.The ?-lactamases genotype was determined by PCR assay with specific-primer,and DNA sequencing was also used to analyze resistance-related gene.RESULTS Twenty-eight of 45 strains were OXA producing strains(68.3%),10 strains were IMP producing strains(24.4%),13 strains were TEM producing strains(31.7%),18 strains were CTX-M producing strains(43.9%),6 strains were PER producing strains(14.6%),and 7 strains were AmpC producing strains(17.1%).None produced SHV ?-lactamases.Twenty-four strains were produced 2 or more than 2 kinds of ?-lactamases.CONCLUSIONS The multiple resistance of Acinetobacter can produce kinds of ?-lactamases,but producing ?-lactamases are not the only one mechanism.

Objective To assess the association of epidermal growth factor receptor (EGFR) mutation with histologic subtypes in lung adenocarcinoma .Methods Lung cancer tissues were collected from 3 028 cases of patients with non‐small cell lung cancer , DNA was extracted respectively ,and EGFR gene exons 18 ,19 ,20 and 2l mutations were dectected by ARMS‐PCR amplification . The association of EGFR mutation with histologic subtypes in lung adenocarcinoma was analyzed .Results The mutation rate of EGFR detection was 39 .7% ,most were exon 19 del and exon 21 L858R (proportion 89 .8% );according to the new classification , EGFR gene mutation in infiltrating lesions with micro infiltrating adenocarcinoma and infiltrating adenocarcinoma were different (P<0 .05) .EGFR mutation rates were higher in moderately differentiated lung adenocarcinoma ,degree of differentiation of EGFR mutation rates were statistically different(P<0 .05) .Conclusion The new classification showes a correlation with molecular diag‐nosis ,different subtypes of EGFR mutation rate is different .There is a certain correlation between EGFR gene mutation and the de‐gree of differentiation in adenocarcinoma .

To rapidly select potent anti-VSTM1-v2 scFv (single-chain antibody fragment) by construction and screening of a humanized scFv library in which a murine VH-CDR3 library was grafted onto a human scFv framework. A murine VH-CDR3 library was amplified from anti-VSTM1-v2 murine cDNA and grafted on human scFv (VH3-VK1) framework. Anti-VSTM1-v2 scFv templates were selected and enriched through ribosome display, TA-cloned into expression vector, and transformed into BL21 (DE3) for soluble expression of target scFv. A total of 1000 clones were randomly picked. Positive ones were first identified using colony PCR, indirect ELISA, Western blotting and then verified with sequencing and dose response ELISA. At last an anti-VSTM1-v2 humanized scFv with good binding affinity (EC50 = 21.35 nmol x L(-1)) was selected from the humanized library of 10(12) members generated in this study. This scFv antibody might have potential applications. This study provides a new approach for rapid screening of humanized antibodies.

Objective To investigate mechanisms of carbapenem resistance in Klebsiella pneumoniae. Methods Two carbapenem-non-susceptible Klebsiella pneumoniae Z4 and Z5 isolated from Beijing Hospital in 2008 were investigated. MICs of antibiotics were determined by agar dilution method.Conjugation experiment was carried out in mixed broth cultures. Plasmid DNA preparations were obtained by using an alkalinelysis technique. Elimination of plasmids was performed by repeated SDS treatment. The crude β-lactamase extracts were subjected to IEF. The genotype of β-lactamases were confirmed by PCRs and DNA sequence analysis. Outer membrane proteins (Omps) were isolated and examined by SDS-PAGE.The ompK35 and ompK36 genes were amplified by using PCR and were sequenced. Results MICs of imipenem, meropenem and ertapenem for Z4 and Z5 were 32, 32 and 256 μg/mi, and 1, 1 and 2 μg/ml.Conjugation study with Escherichia coli EC600 resulted in the transfer of significant reduced carbapenem susceptibility from Z4 and Z5 ( MICs increased at least 8-fold). Klebsiella pneumoniae Z4 produced IMP-4 metallo-β-lactamase, TEM-1 and SHV-1 spectrum β-lactamase and Z5 produced IMP-4, TEM-1 and SHV-12 extended-spectrum β-lactamase. E. coli transconjugants of both Z4 and Z5 produced a single IMP-4.Elimination of IMP-4-encoding plasmid from Z5 resulted in carbapenem susceptibility in the isolate,however, Z5 whose IMP-4-encoding plasmid was eliminated exhibited reduced susceptibility to carbapenems ( MICs of imipenem, meropenem and ertapenem were 0. 25 μg/ml,0. 5 μg/ml and 4 μg/ml). Amplification of integron revealed that blaIMP-4 gene of both Z4 and Z5 located within two different class I integrons which were carried on two plasmids with a similar size of approximately 55 000 bp. SDS-PAGE and ompK35/36 genes sequence analysis of Omp indicated that Z4 failed to express OmpK36, because of a nonsense mutation (CAG into TAG) in the ompK36 gene. Conclusion Production of plasmid-mediated metallo-β-lactamase IMP-4 or production of β-lactamase combined with porin OmpK36 deficiency can lead to reduced susceptibility to carbapenems. High-level carbapenem resistance in Z4 is mainly due to production of IMP-4 and the loss of OmpK36.

Objective To understand the distribution of anaerobes in gingivitis infection, and anaerobes sensitivity to ? lastams Methods 44 specimens were taken from the gingival crevicular fluid of gingivitis. The anaerobic bacteria were cultured and identified Minimal inhibitory concentrations (MIC) were measured by using agar dilution method Results Among all samples, the isolation of anaerobes was 100%, the isolation of P.melaninogenica was 86.4%, P.intermedia was 56.8%, B.forsythus was 52.3%, P.gingivalis was 65.9%, F.mucleatum was 63.6%, P.micros was 52.3%, Actinomyces ssp was 38.6% and Capnocytophaga ssp was 9.1% respectively. The anaerobes are sensitive to ? lastams. Conclusion In gingival crevicular fluid, the infection rates of P.melaninogenica, P.intermedia, B.forsythus, P.gingivalis, F.mucleatum, P.micros, and Actinomyces ssp were significantly higher than those of the other anaerobes. ? lastams can be used for the treatment of anaerobes infected gingivitis.

Objective To explore the damage effect of high level of glucose on ECV304 cells and the approaches in which high level of glucose plays its part to provide theoretical bases for the therapy of diabetes.Methods ECV304 cells were divided into four groups:normal control,high glucose group in which glucose was added to the cells with final concentration of 35 mmol?L-1,Radix Astragali(RA) group in which glucose was added to the cells with final concentration of 35 mmol?L-1,as well as RA with final concentration of 500 mg?L-1,mannitol group in which mannitol was added to the cells with final concentration of 25 mmol?L-1.The cells were cultivated for 24 h after the glucose,RA and mannitol were added to the cells and collected for the determination of intracellular Ca2+ concentration,mitochondrial membrane potential and the morphological changes of cells and mitochondria were observed under electronic microscope.Results The intracellular Ca2+ concentration in the cells of high glucose group was significantly higher than those of RA group,mannitol group and normal control group(P