OBJECTIVETo clone pIA and pIB genes of Neisseria gonorrhoeae,and to construct pIA-pIB fusion gene and its prokaryotic expression system, and to establish enzyme linked immunosorbent assay (ELISA) based on rPIA-PIB for detecting serum and pus samples from gonorrhea patients and to evaluate the sensitivity and specificity of the ELISA.

METHODSpIA-pIB fusion gene was constructed by polymerase chain reaction (PCR) using linking primers and a prokaryotic expression system of the fusion gene was constructed by using routine molecular biological methods. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) plus BioRad Gel Image Analyzer was used to measure the expression of the target recombinant protein rPIA-PIB. Ni-NTA affinity chromatography was performed to extract and purify rPIA-PIB. An ELISA by using rPIA-PIB as the coated antigen for detecting the specific IgG against rPIA and/or rPIB in gonorrhea patients' sera as well as another ELISA by using rPIA-PIB antiserum as the first antibody for detecting the rPIA and/or rPIB in gonorrhea patients' pus samples were established. In these experiments, ELISAs associated with rPIA, rPIB and their antisera were applied as the controls.

RESULTS100% similarities of the nucleotide and putative amino acid sequences of the pIA-pIB fusion gene were confirmed when compared with the original sequences. The output of rPIA-PIB was 29.8% of the total bacterial proteins. The purified rPIA-PIB only showed a single target protein segment in gel after SDS-PAGE. Using a positive rate (98.3%) of rPIA-PIB-IgG-ELISA to detect 119 cases of gonorrhea patients' serum samples was remarkably higher than that of rPIA-IgG-ELISA (30.3%) or rPIB-IgG-ELISA (66.4%) (P<0.01). The positive rate (91.6%) of rPIA-PIB-ELISA to detect 119 cases of gonorrhea patients' pus samples was also significantly higher than that of rPIA-IgG-ELISA (27.7%) or rPIB-IgG- ELISA (62.2%) (P<0.01).

CONCLUSIONIn this study we successfully constructed pIA-pIB fusion gene of N. gonorrhoeae and its prokaryotic expression system while rPIA-PIB showed obvious superiority used as the antigen in gonorrhea associated detection kits compared to both the rPIA and rPIB.

Antigens, Bacterial ; immunology ; Bacterial Outer Membrane Proteins ; genetics ; immunology ; metabolism ; Base Sequence ; Enzyme-Linked Immunosorbent Assay ; methods ; Gene Expression Regulation, Bacterial ; Humans ; Neisseria gonorrhoeae ; genetics ; immunology ; metabolism ; Recombinant Fusion Proteins ; genetics ; immunology ; metabolism

OBJECTIVETo assess the clinical efficacy of acupuncture and moxibustion on depression in view of the outcome indicators of the patient subjective reports.

METHODSOne hundred and sixty-three cases of depression being in compliance with the inclusive standards were randomized into a soothing-liver and regulating-mind group, an acupoint-shallow-puncturing group and a non-acupoint-shallow-puncturing group. In the soothing-liver and regulating-mind group, the conventional acupuncture was applied to the four-gate points [Hegu (LI 4) and Taichong (LR 3)], Baihui (GV 20) and Yintang (EX-HN 3), the direct moxibustion with moxa cone was applied to the four-flower points [Geshu (BL 17), Danshu (BL 19)]. Finally, the intradermal needling was used at Xinshu (BL 15) and Ganshu (BL 18). In the acupoint-shallow-puncturing group, the acupoints selected were same as those in the soothing-liver and regulating-mind group. But the needle insertion was shallower and the time of moxibustion was shorter. In the non-acupoint-shallow-puncturing group, the spots that were 10 mm lateral to those acupoints in the soothing-liver and regulating-mind group were selected. The operation was same as that in the acupoint-shallow-puncturing group. The treatment was given twice a week in three groups. Totally, 12 weeks of treatment were required. The score of symptom checklist 90 (SCL-90), the self-report symptom inventory was observed before treatment, 1 month and 3 months after treatment separately so as to assess the corresponding short-term, mid-term and long-term efficacies of the program of acupuncture and moxibustion for soothing the liver and regulating the mind.

RESULTSIn each time-point after treatment, for the scores of somatization, obsessive-compulsive symptom, interpersonal sensitivity, depression, anxiety, hostility, paranoid ideation, psychoticism and the other 8 dimensionalities, in comparison between the soothing-liver and regulating-mind group and the non-acupoint-shallow-puncturing group, the differences were significant statistically (all P < 0.05). For the scores of depression, anxiety and hostility, in comparison between the soothing-liver and regulating-mind group and the acupoint-shallow-puncturing group, the differences were significant statistically (all P < 0.05).

CONCLUSIONAcupuncture and moxibustion can improve the scores of SCL-90 scale for the patients with depression. The outcome indicators of the patient subjective reports can accurately assess the clinical efficacy.

Acupuncture Therapy ; Adolescent ; Adult ; Aged ; Depression ; psychology ; therapy ; Female ; Humans ; Male ; Middle Aged ; Moxibustion ; Self-Assessment ; Treatment Outcome ; Young Adult

AIMTo establish a solid phase extraction-high performance liquid chromatographic (SPE-HPLC) method for determining plasma scutellarin concentration, and study its pharmacokinetics after i.v. breviscapine in rabbits.

METHODSMethanol-water-phosphoric acid (50:50:0.5) mixture was used as mobile phase, Nucleosil C18 column (250 mm x 4.6 mm ID) was selected. The wavelength of UV detection was 335 nm. Fifteen rabbits, randomized into 3 groups, were given breviscapine i.v. at the dose of 10, 20 and 40 mg.kg-1. Scutellarin in plasma was determined by SPE-HPLC method.

RESULTSLinearity was obtained over the range of 0.02-10.0 mg.L-1 of scutellarin. The method recovery was 96.15%-99.31%; the within-day and between-day RSDs were all below 10%. After i.v. 10, 20 and 40 mg.kg-1 of breviscapine to rabbits, the concentration-time curve of scutellarin fitted to a three compartment model. The main pharmacokinetic parameters showed no significant difference between low and medium doses, but the difference was significant between high dose and other doses.

CONCLUSIONThis assay method was accurate, sensitive, simple and suitable for the measurement of plasma scutellarin concentration. The pharmacokinetic characteristics were found to fit a three-compartment model following i.v. injection of breviscapine to rabbits. The changes of drug concentration in vivo exhibited linear kinetics ove the dosage range of 10-20 mg.kg-1, but when the dosage was 40 mg.kg-1, the linear kinetic properties disappeared.

Animals ; Apigenin ; Area Under Curve ; Chromatography, High Pressure Liquid ; methods ; Flavonoids ; blood ; pharmacokinetics ; Male ; Rabbits

Objective To investigate the clinical effects and treatment of thyroid dysfunction induced by PegIFNα-2a therapy in patients with chronic hepatitis B.Methods 95 patients with chronic hepatitis B received PegIFNα-2a from 2007-2011were selected. Analyzed the clinical manifestations,laboratory results,treatment and clinical outcome of the patients who had thyroid dysfunction induced by PegIFNα-2a therapy.Results During the treatment of PegIFNα-2a,10 patients ( 10/95,10.53％ ) had thyroid dysfunction,in which 4 were of hyperthyroidism and 6 were of hypothyroidism.After appropriate treatment,all the patients who had thyroid dysfunction had satisfactory outcome.Conclusions PegIFNα-2a therapy may induce thyroid dysfunction.Monitor thyroid function regular and have active treatment ensure the interferon therapy to continue.With the end of the interferon therapy,thyroid function can be back to normal.

OBJECTIVETo clone tpn17 and tpn47 genes of Treponema pallidum and then construct their prokaryotic expression systems,to establish ELISAs based on rTpN17 and rTpN47 as antigens and to evaluate the sensitivity and specificity of the ELISAs for detection of serological diagnosis of syphilis.

METHODSThe whole length of tpn17 and tpn47 genes was amplified by PCR and then their prokaryotic expression systems were constructed. SDS-PAGE was used to measure the expression of the target recombinant proteins rTpN17 and rTpN47. Ni-NTA affinity chromatography was applied to extract rTpN17 and rTpN47, while Western blot was performed to determine the specific immunoreactivity of rTpN17 and rTpN47. By using rTpN17 and rTpN47 as the coated antigen, respectively, ELISAs (rTpN17-ELISA and rTpN47-ELISA) were established to detect serum samples from 200 healthy individuals, 25 RA patients, 17 SLE patients and 211 syphilis patients. The detection effects of the ELISAs were compared to those of TRUST and TPHA.

RESULTThe sequence similarity of the cloned tpn17 and tpn47 genes was 100 % compared with the corresponding sequences in GenBank. The expression outputs of rTpN17 and rTpN47 were approximately 37.2 % and 26.8 % of the total bacterial proteins, respectively. Both the extracted rTpN17 and rTpN47 could take place remarkable conjugation reactions to the sera with positive antibody against Treponema pallidum.The positive detection rate of TPHA (99.1%) was the highest (P<0.001). The positive detection rates of rTpN17-ELISA (85.3 %) and rTpN47-ELISA (84.3 %) were similar (P>0.05). The positive detection rates of TRUST (72.5 %) was lower than that of rTpN17-ELISA (P=0.001) but similar to that of rTpN47-ELISA (P=0.014). The detection results of all the serum samples from healthy individuals, RA patients and SLE patients were negative, whereas 7.1 % (3/42) of the samples from RA or SLE patients were positive.

CONCLUSIONrTpN17 and rTpN47 are still maintaining their original immunoreactivity. The ELISAs using rTpN17 or rTpN47 as the antigen are rapid, simple and convenient, higher sensitivity and specificity methods for serological screening and detection of syphilis.

Antibodies, Bacterial ; Antigens, Bacterial ; Blotting, Western ; Enzyme-Linked Immunosorbent Assay ; methods ; Female ; Humans ; Male ; Syphilis ; diagnosis ; Syphilis Serodiagnosis ; Treponema pallidum ; chemistry ; immunology ; isolation & purification

This paper presents a novel monitor which uses ARM controller AT91SAM7S64 as its main processor, LCM (Liquid Crystal Display Module) for displaying ECG waves, SD (Secure Digital memory) card for data storage and RF module PTR8000 for radio data transmission. This portable monitor boasts alarm function for abnormality and can provide dynamic ECG monitoring for patients.
Computer Communication Networks ; Electrocardiography ; instrumentation ; Humans ; Monitoring, Physiologic ; instrumentation ; Signal Processing, Computer-Assisted ; instrumentation ; Telemetry ; instrumentation ; methods

Objective To retrospectively explore the prevalence of cardiovascular disease (CVD) in inpatients with chronic obstructive pulmonary disease(COPD)in Beijing.Methods The COPD patients who were discharged from the General Hospital of the Liberation Army,Peking Union medical college Hospital and Beijing Hospital between January 1st,2000 to March 20th,2010,were investigated.The prevalence of CVD were calculated.The tendency of the prevalence of CVD by age or discharge year and the difference of the prevalence of CVD between male and female were estimated by using chi-square analysis.Results There were 4960 COPD patients who were in accordance with the inclusion criteria with 3570 males and 1390 females.The mean age was 72.2±10.4 years.Of the COPD patients,48.8% were diagnosed as cardiovascular diseases.The age-adjusted over-all prevalence of CVD was 26.4%.Chronic pulmonary heart disease and other disease of pulmonary artery(15.8%)was the most frequent diseases,followed by heart failure(13.6%),ischemic heart disease (10.6%).In COPD patients,male was more likely to have angina,pulmonary heart disease and other disease of pulmonary artery and acute kidney failure (P＜0.05),while less likely to get arrhythmia (atrial fibrillation/atrial flutter),heart failure,pulmonary embolism,hypertension and diabetes mellitus (P＜0.05).The prevalence of arrhythmia increased with age,however,the ischemic heart disease and heart failure decreased.The proportion of CVD decreased in male patients while increased in females.Conclusion The overall prevalence of CVD comorbidities was 48.8% in 4960 patients with COPD who were older than 40 years in Beijing.There were differences among the groups of various age and sex in the distribution of CVD comorbidities frequencies year by year.

Objective To determine the distribution and sequence conservation of outer membrane protein X (ompX) gene in Salmonella paratyphi A isolates as well as the immunogenicity and irnmono-protection of ompX gene products.Methods OmpX gene in Salmonella paratyphi A isolates was detected by PCR and the amplification products were sequenced after the T-A cloning process.OmpX gene product was expressed with E.coli expression system and the expressed rOmpX was extracted by Ni-NTA affinity chromatography.SDS-PAGE and Bio-Rad Gel Image Analyzer were applied to examine the expression and yield of rOmpX.Both antigenicity and immune-reactivity of rOmpX were detected by immune-diffusion test,ELISA and Western blot assay.The immuneprotective effect of rOmpX against infection of Salmonella paratyphi in mice was determined and the agglutinative titers of sera from rOmpX-immunized mice was measured by micro-Widal' s test.Results All the tested Salmonella paratyphi A isolates had ompX gene with high nucleotide or amino acid sequence identity (99.2％-100.0％ or 98.4％-100.0％).When rOmpX was induced to rabbits to produce high level antibody and combined with antiserum against whole cell of Salmonella paratyphi A,the results displayed a positive Western hybridization signal.Results from ELISA demonstrated that 95.6％ (65/68) of the serum samples from paratyphoid-A patients were positive on rOmpX antibody.Mice that were immunized with 100 μg or 200 μg rOmpX displayed an immune-protective rate of 93.3％ (14/15) or 100.0％ (15/15).Sera from those rOmpX-immunized mice provided 1 ∶ 10-1 ∶ 40 agglutination titers in both H antigens of Salmonella paratyphi A and Salmonella typhi.Conclusion The recombinant expression product of ompX gene could be used as a candidate antigen for developing genetic engineering vaccines against Salmonella paratyphi A infection.

OBJECTIVETo construct prokaryotic expression systems of TCS genes comD/comE/comC of Streptococcus pneumoniae, and to determine the correlation of ComD and ComC with the drug resistance.

METHODSThe entire comD, comE and comC genes were amplified by PCR and their prokaryotic expression systems were established by routine genetic engineering technique. SDS-PAGE and Bio-Rad Agarose Image Analyzor was applied to measure the outputs of target recombinant proteins rComD, rComE and rComC. Rabbits were immunized with these recombinant proteins to prepare antisera. The resistance of S.pneumoniae strains to penicillin and cefotaxime was examined after ComD and ComC were blocked by antisera.

RESULTCompared with the reported sequences, similarities of nucleotide and amino acid sequences of the cloned comD, comE and comC genes were 98.4% approximately 99.3% and 99.1% approximately 100%, respectively. The constructed engineering bacteria E.coli BL21DE3(pET42a-comD), E.coli BL21DE3(pET42a-comE) and E.coli BL21DE3(pET42a-comC) were able to efficiently express the target recombinant proteins and the outputs of rComD, rComE and rComC were 28%, 25% and 35% of the total bacterial proteins, respectively. The double immunodiffusion titers of rabbit antisera against rComD, rComE or rComC were 1:4, 1:4 and 1:8, respectively. After the ComD and/or ComC were blocked by the antisera, the cefotaxime-sensitive S. pneumoniae strains became to resistant to antibiotics but there were no changes for cefotaxime-resistant strains and resistance to penicillin for all tested strains.

CONCLUSIONThe prokaryotic expression systems of S.pneumoniae comD/come/comC genes have been successfully constructed, and the study also indicates that both the ComD and ComC are involved in the drug resistance of S. pneumoniae to cefotaxime.

Animals ; Bacterial Proteins ; biosynthesis ; genetics ; Cefotaxime ; pharmacology ; Escherichia coli ; genetics ; metabolism ; Rabbits ; Recombinant Proteins ; biosynthesis ; genetics ; Recombination, Genetic ; Signal Transduction ; Streptococcus pneumoniae ; drug effects ; genetics ; beta-Lactam Resistance ; genetics

OBJECTIVETo construct prokaryotic expression systems of Streptococcus pneumoniae ciaH and ciaR genes,and to determine their correlation with drug resistance.

METHODSThe total length of ciaH and ciaR genes was amplified by PCR and their prokaryotic expression systems were established by using routine genetic engineering technique. SDS-PAGE was applied to measure the outputs of target recombinant proteins rCiaH and rCiaR. Rabbits antisera and IgGs against rCiaH and rCiaR were prepared. The resistance to penicillin and cefotaxime of S.pneumoniae strains was examined after CiaH and CiaR were extracellularly and intracellularly blocked by the IgGs.

RESULTThe homogeneity of nucleotide and amino acid sequences of the cloned ciaH and ciaR genes with the reported sequences was 99.9-100% and 100%, respectively. The recombinant bacteria E.coli BL21DE3pET42a-ciaH and E.coli BL21DE3pET42a-ciaR were able to express the target recombinant proteins rCiaH and rCiaR with efficiency. The outputs of rCiaH and rCiaR were 33% and 45% of the total bacterial proteins, respectively. The double immunodiffusion titers of rCiaH antiserum,rCiaR antiserum,rCiaH-IgG and rCiaR-IgG were 1:4,1:4,1:1 and 1:1, respectively. After CiaH was extracellularly or intracellularly blocked by CiaH-IgG, and CiaR was intracellularly blocked by CiaR-IgG, the penicillin-sensitive or cefotaxime-sensitive strains developed resistance to the two antibiotics; but the blocks did not change that of penicillin-resisting or cefotaxime-resisting strains.

CONCLUSIONThe prokaryotic expression systems of S. pneumoniae ciaH/ciaR genes have been successfully constructed in this study. Both the CiaH and CiaR may be involved in penicillin and cefotaxime resistance of the bacterium.

Animals ; Bacterial Proteins ; genetics ; metabolism ; Cefotaxime ; pharmacology ; Escherichia coli ; genetics ; metabolism ; Gene Expression Regulation, Bacterial ; Genetic Vectors ; Penicillin Resistance ; genetics ; Protein Kinases ; genetics ; metabolism ; Rabbits ; Recombinant Fusion Proteins ; genetics ; metabolism ; Signal Transduction ; Streptococcus pneumoniae ; drug effects ; enzymology ; genetics ; beta-Lactam Resistance ; genetics