Actins ; metabolism ; Animals ; Creatinine ; blood ; Kidney ; pathology ; Kidney Diseases ; metabolism ; pathology ; Male ; Radiation Injuries, Experimental ; metabolism ; pathology ; Rats ; Rats, Wistar ; Vimentin ; metabolism

Objective To explore the molecular types of methicillin-resistant Staphylococcus aureus (MRSA) strains present in major hospitals in Qingdao area, using pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) methods, trying to find out the epidemiological characteristics of these MRSA isolates. Correlation of the PFGE types with microbiological phenotypes and clinical data was also studied. Methods 360 isolates of MRSA were procured during 2003 to 2007 from major hospitals in Qingdao. PFGE technology was applied to comparatively analyze the chromosomal DNA digested with endonuclease Sma Ⅰ . Comparison of DNA fragments patterns from each MRSA strain and cluster analysis were performed with the Bionumericus version ' 2.0' software. A dendogram was generated using PFGE macrorestriction fragments on gel images. Data was used to predict the possibility of each PFGE type via SPSS software version 11.0, using the variables as predictors including groups on patient's age, gender, source and the site where MRSA was isolated. Antibiotic sensitivity patterns of these MRSA isolates were determined by K-B tests, and a correlation between these patterns and PFGE types was investigated. Housekeeping genes were amplified with PCR and sequenced in representative strains of variant PFGE types to identify their allelic profile. Results 5 types of PFGE patterns (M0-M4) were identified with MI being the predominant and M2 next to it which was significantly correlated to the isolates from wounds. M3 type strains were mainly isolated from ICU wards and there were a few cases complied with M4 type with no correlated variant factors found in this study. A unique pattern of MRSA isolates with its M0 distinct from other types had not been reported. No significant association was found between PFGE individual types,gender or age groups. M1 and M2 types were the major proportional PFGE patterns among different hospitals. No vancomycin-resistant isolates were detected among 360 MRSA strains. No significant association was found between individual antibiotic resistance and specific PFGE types. Data from MLST analysis showed that the aUelic profiles of M1 and M3 type strain had the same ST239 linage which was commonly present in China. For M2 and M4 representative strains, the allelic profiles were ST5 and ST240, respectively. ST45 and ST398 were corresponding to two PFGE patterns clustered as M0 type. Conclusion Nosocomial infection due to MRSA was evenly distributed among different age groups and no gender bias was observed. The PFGE types of MRSA strains isolated in major hospitals in Qingdao were highly correlated with the sources of isolates and ST239 isolate seemed the prevalent and widespread one. Strategies should be designed to further monitor and prevent or minimize the spread of ST5 MRSA isolates and the like, in Qingdao area.

OBJECTIVETo establish a new method of multiplex semi-nested polymerase chain reaction (PCR) to detect pathogens in cerebrospinal fluid (CSF).

METHODSAccording to the analysis of the conservative and variable regions in bacterial 16S rRNA genes, we designed universal primers for all bacteria and specific primers for most gram-positive and gram-negative bacteria. All primers were added into the same reaction systems successively of a two-step PCR assay to amplify the different bacterial DNA in CSF, and the results were compared with common culture method with sensitivity and the specificity both detected at the same time.

RESULTSBoth gram-positive and gram-negative bacteria amplified DNA fragment about 1,032 bp after first-step amplification with universal primers. In the second step, specific fragments of 336 bp and 127 bp were amplified in gram-positive and gram-negative bacteria respectively besides fragments of 1,032 bp; The detection limit for E. coli was 8 cfu/ml. The comparison of 62 CSF samples detected by both multiplex semi-PCR and conventional culture method revealed sensitivity, specificity, positive and negative values of 93.8%, 95.7%, 88.2%, and 97.8% respectively for PCR.

CONCLUSIONThe result suggested that the multiplex semi-nested PCR we established was sensitive, specific and rapid method for clinical laboratory to detect pathogens in CSF.

Cerebrospinal Fluid ; microbiology ; DNA Primers ; genetics ; DNA Probes ; genetics ; DNA, Bacterial ; cerebrospinal fluid ; isolation & purification ; Escherichia coli ; genetics ; isolation & purification ; Gram-Negative Bacteria ; genetics ; isolation & purification ; Gram-Positive Bacteria ; genetics ; isolation & purification ; Humans ; Polymerase Chain Reaction ; methods ; RNA, Bacterial ; genetics ; RNA, Ribosomal, 16S ; genetics ; Sensitivity and Specificity ; Staphylococcus aureus ; genetics ; isolation & purification

OBJECTIVETO establish a RP-HPLC method for the determination of calycosin-7-O-beta-D-glucopyranoside in Radix Astragali, and to analyse the calycosin-7-O-beta-D-glucopyranoside content of ten samples of Radix Astragali, collected from different regions.

METHODA Polaris C18(250 mm x 4.6 mm, 5 microns) column was used and a mixture of methanol-water (30:70) was used as the mobile phase at a flow rate of 1.0 mL.min-1. The column temperature was 25 degrees C and the UV detection wavelength was 254 nm.

RESULTThe calibration curve was in good linearity over the range of 0.0106-2.12 micrograms with the regression equation Y = 3035. 97 X - 14.85(r = 0.9999). The average recovery was 95.8% (n = 5, RSD = 1.3%).

CONCLUSIONThe method is simple, quick, sensitive and reproducible. In all of the samples, the calycosin-7-O-beta-D-glucopyranoside contents differ markedly.

Astragalus membranaceus ; chemistry ; classification ; China ; Chromatography, High Pressure Liquid ; Ecosystem ; Glucosides ; analysis ; Isoflavones ; analysis ; Plant Roots ; chemistry ; Plants, Medicinal ; chemistry ; Quality Control ; Species Specificity

OBJECTIVETo study the variation of specific antibody among convalescent of severe acute respiratory syndrome (SARS) patients through a three-year program.

METHODSSera samples were collected from SARS cases in the 5th, 20th and 35th month after onset of the illness. The SARS-CoV specific antibody was detected for all of them by ELISA and neutralized test simultaneously. The titer of neutralizing antibodies was calculated using Reed-Muench method, and the comparison between different time groups was analyzed regarding the variance of data on repeated measures after logarithm conversion.

RESULTS13, 17 and 13 sera samples were collected in the 5th, 20th and 35th month after onset. Results showed that despite the fact that the positive rates of ELISA antibody were 100%, 82.4% and 84.6% respectively,the neutralizing antibody was still positive for all the samples. The average neutralizing antibody titers were 1:43 (1:16-1:203), 1:36 (1:17-1:59) and 1:21 (1:10-1:39) on the 5th, 20th and 35th month after onset, and the differences were statistically significant (F = 60.419, P < 0.001). On the 35th month after the onset, 30.8% (4/13) of the patients were still having the neutralizing antibody level of above 1:36, but the neutralizing antibody level in another 30.8% (4/13) of the patients had decreased to as low as 1:10, when the cut-off level was set as 1:8.

CONCLUSIONResults of the study indicated that the neutralizing antibody of SARS cases could last for at least three years, but the sera specific antibody in SARS cases decreased gradually when time went by. However, neutralizing antibody in some of the cases decreased to a lower level on the 35th month. Further follow-up study was worthwhile to observe the long-lasting profile of antibody existence on SARS cases.

Antibodies, Neutralizing ; analysis ; Enzyme-Linked Immunosorbent Assay ; Follow-Up Studies ; Humans ; Severe Acute Respiratory Syndrome ; immunology

To establish a fluorescent quantitative PCR method (FQ-PCR) with TaqMan probe for simultaneous detection of polyomavirus (BKV) and cytomegalovirus (CMV) and to evaluate its clinical application in the renal transplantation recipients. The conservative sequences of BKV and CMV were targeted and amplified by nested PCR technique. The PCR products were cloned into the plasmids pcDNA3. 1(+). The recombinant plasmid containing target sequences of BKV and CMV were constructed as external standards. The TaqMan-based assay was optimized. For evaluating the assay, the sensitivity was determinated by diluted standard (5 X 103-10icopies/mL), and the specificity was verified by negative control and positive control, and the precision was assessed by intra-assay coefficient of variation (ICV) through detecting standard repeatedly (20 times). A total of 480 blood samples of renal transplantation recipients were used to detect BKV and CMV DNA simultaneously with FQ-PCR, and the concentrations of FK506 were measured by ELISA. The association of DNA copy and concentrations of FK506 was analyzed. The cloned target BKV and CMV DNA was confirmed by sequencing and analysis. The sensitivity of the FQ-PCR assay reached 5 X 103 copies/ml in detecting BKV or CMV DNA. Control DNA verified the assay specifically detecting target DNA. The precision of the assay to quantif target DNA copies was acceptable (Intra-assay CV was 3.44% for BKV and 2.23% for CMV; Inter-assay CV was 4. 98% for BKV and 3.76% for CMV;). Of 480 samples, 130 samples (27. 08%) were CMV DNA positive, significantly higher than the BKV DNA positive (13.33%, 64/480, P<0.05). The positive BKV or CMV DNA was found to be associated with high concentrations of FK506 (P<0. 05). In conclusion, the developed real-time PCR assay for detecting both CMV and BKV DNA simultaneously was s high sensitive, precise and time-effectiveand could be applied in the monitoring of the CMV and BKV infection in the renal transplantation recipients.
Adolescent ; Adult ; Aged ; Conserved Sequence ; Cytomegalovirus ; genetics ; isolation & purification ; Cytomegalovirus Infections ; diagnosis ; virology ; DNA, Viral ; blood ; Female ; Humans ; Immunosuppressive Agents ; blood ; Kidney Transplantation ; adverse effects ; Male ; Middle Aged ; Polyomavirus ; genetics ; isolation & purification ; Polyomavirus Infections ; diagnosis ; virology ; Real-Time Polymerase Chain Reaction ; methods ; Reproducibility of Results ; Sensitivity and Specificity ; Species Specificity ; Tacrolimus ; blood ; Time Factors ; Tumor Virus Infections ; diagnosis ; virology ; Viral Load ; Young Adult

OBJECTIVETo identify differentially expressed genes in recurrent nasopharyngeal carcinoma (rNPC) by DNA microarrays, and analyze chromosomal localizations and molecular function by bioinformatics.

METHODSThe primary nasopharyngeal carcinoma (pNPC) tissue samples and rNPC tissue samples were selected, and Affymetrix Gene1.0 ST gene chips were used to identify differential expressed genes in rNPC, and the bioinformatics was used to analyze their chromosomal localizations as well as molecular functions.

RESULTSA total of 44 genes were identified to be differential expressed in rNPC. Thirty-six genes were down regulated, 8 genes were up regulated. Functional classification of down-regulation genes showed that most genes (10 genes, 27.8%) belonged to the enzyme activity genes, followed by calcium ion binding genes (7 genes, 19.4%), protein binding genes (5 genes, 13.9%), receptor activity genes (4 genes, 11.1%), ATP binding genes (2 genes, 5.6%), transcription factor genes (2 genes, 5.6%), extracellular matrix binding and growth factor binding have 1 gene respectively (each accounted for 2.8%). In addition, the functions of 4 genes (11.1%) were unknown. Functional classification of up-regulation genes showed most genes (3 genes, 37.5%) were unknown, followed enzyme activity genes (2 genes, 25.0%), receptor activity, calcium ion binding and voltage-gated ion channel activity genes have 1 genes respectively (each accounted for 12.5%). These genes were localized randomly on the most the chromosomes, with a majority of them localized on chromosomes 1, 17. Chromosome 1 contained the most differentially expressed genes (10, 22.7%), followed by chromosomes 17 (5, 11.3%).

CONCLUSIONSThe differential expressed genes in rNPC were supposed to be randomly distributed on most chromosomes, but the majorities were found on chromosomes 1, 17. Abnormality in three groups of genes, including in enzyme activity, calcium ion binding and protein binding associate genes, might play important roles in rNPC. Those genes need to be further studied.

Adult ; Aged ; Carcinoma ; Carcinoma, Squamous Cell ; genetics ; pathology ; Female ; Gene Expression Profiling ; Gene Expression Regulation, Neoplastic ; Genes, Tumor Suppressor ; Humans ; Male ; Middle Aged ; Nasopharyngeal Neoplasms ; genetics ; pathology ; Neoplasm Recurrence, Local ; genetics ; Oligonucleotide Array Sequence Analysis

Objective To analyze the etiologic characteristics of Vibrio cholerae in Guangdong province in 2007.Genetic relationship was observed including among predominated biotype isolates from different areas within the province and among same biotypes isolates from cholera cases and regular surveillance.Methods Isolates from cholera cases and through environmental surveillance were typed by sero-and phage-typings.Similarity of molecular fingerprinting was analyzed through comparing the pulsed field gel electrophoresis(PFGE)pattern of predominated biotype isolates,and those of the same biotype isolates from cholera cases and environment surveillance,respectively.In addition,genetic relationship was determined by clustering analysis,using bionumerics software.Results In total,31 isolates from cholera cases were collected and subtyped for 3 serogroups.V.cholerae O1 El Tor Inaba phage 1d was the predominant biotype which causing most of the cases in Guangdong province in 2007.Data from cluster analysis showed that the similarity among Inaba phage 1d strains from different areas were from 94.5% to 100%.However.16 isolates were collected from environment surveillance programs and the predominated biotype could not be found.Additionally,the biotype distribution of cases isolates was not consistent with those isolates through surveillance.High phylogenetic diversity was observed for the same biotypes isolates from cases and surveillance samples.Conclusion Our data showed that V.cholerae O1 El Tor Inaba phage 1d was the predominated biotype with multi-clone coexisting and circulating in Guangdong province in 2007.It also appeared to be the characteristics of cholera in the non-epidemic period,suggesting that it was necessary to enhance the alert surveillance programs for cholera epidemic based on the molecular typing techniques.

Objective To understand the distribution,molecular characteristics and virulence genes of the O1 and O139 Vibrio cholerae isolates from the Pearl River Estuary water.Methods Vibrio cholerae isolates collected from the Pearl River estuary waters from January 2009 to December 2010,were tested by PCR for eight virulence-related genes,including cholera toxin(ctxA),zonula occludens toxin(zot),accessory cholera enterotoxin(ace),hemolysin(hlyA),toxin-coregulated pilus (tcpA),outer membrane protein(ompU),and the regulatory protein genes(tcpⅠ,toxR).Genetic relation was assessed by pulsed-field gel electrophoresis(PFGE)and the patterns were clustered by BioNumerics.Results From 1152 aquatic samples,69 isolates were identified,including 41 Inaba,18 Ogawa and 10 O139.All the isolates showed ctxA negative,while the hlyA and toxR genes were positive in all the isolates.34.15％(14/41)of the Inaba strains were hlyA + toxR + ompU + ace + zot + tcpI+,while 66.67％(12/18)belonged to Ogawa strains and 70％(7/10)of the O139 strains were hlyA + toxR+.Through PFGE analysis,the O1 isolates formed three clusters in this study.The patterns of O1 isolates differed widely,with the similarity as 72.8％-100.0％,while the patterns of O139 isolates having the similarity of 69.9％-95.5％.Conclusion The non-toxigenic O1 and O139 V.cholerae had a wide distribution in the environment of Pearl River estuary water during the nonepidemic period of cholera.All the aquatic isolates presented diversities on the related virulent genes.

Objective To investigate the clinical therapeutic methods and their curative effects in recurrent ischemic angina for internal mammary artery resterosis after coronary artery bypass grafting (CABG).Methods We enrolled the patients who had recurrence of ischemic angina for restenosis of internal mammary artery graft after CABG as research subjects in the Affiliated Hospital of Yan'an University from January 2014 to January 2016.The 42 patients were divided into three groups according to the different treatment approaches for recurrence of ischemic angina:Group A (n=22)who received internal mammary artery interventional therapy;Group B (n=12)who received coronary artery bypass grafting treatment;and Group C (n=8)who received left subclavian artery proximal stent treatment.Then we compared the clinical therapeutic effects in the three groups.Results The success rate in Group C was 100%,which was the highest in the three groups,and the post-operative restenosis rate was 0.The hospitalization time was significantly shorter in Group A than in Group B (P＜0.05).However,the two groups did not significantly differ in mortality,success rate or restenosis rate (P＞0.05).Conclusion We should select the appropriate treatment according to the patient's specific situation for recurrent ischemic angina. Endovascular treatment has evident therapeutic effects,rapid postoperative recovery,and lower treatment risk, making it the preferred treatment when possible.