Objective:To analyze genetic diversity of Fluconazole(FLZ)-resistant and -susceptible Candida albicans(C. albicans) strains isolated from patients with Sjgren's Syndrome (SS). Methods:30 C.albicans strains were isolated from the patients with SS and identified using standard criteria. Microdilution method was performed to determine the minimum inhibitory concentrations (MICs) of C.albicans to FLZ. A composite genotype was generated for each strain through random amplification of polymorphic DNA (RAPD) using three different primers, RSD10, RSD11 and RSD12. Results:The DNA fingerprinting profiles indicated genetic diversity amongst both the FLZ-resistant as well as -susceptible isolates, and no specific features emerged distinguishing the drug-resistant and -susceptible groups. Conclusion:These observations cast doubt on the theory of a clonal origin of FLZ-resistant C. albicans isolates. The emergence of FLZ resistance in SS patients may be associated with continuous exposure to FLZ.

microRNAs is a group of small non-coding RNAs that play a negative regulation role in expression of target genes at post-transcriptional level by inhibition or degradation of target mRNAs after combination of the seed sequence (SS) in microRNAs with the SS-binding sequences usually located at 5'ends of target mRNAs.microRNAs was firstly found in Caenorhabditis elegans.Subsequently,many different microRNAs in eukaryocytes were revealed.In eukaryocytes,microRNA precursors are transcribed at first and then become functional microRNAs with 21-23 nt in size after splice.Most of eukaryocytic microRNAs combime with the sequences at 3'end of target mRNAs that cause the translation inhibition or degradation of the mRNAs.In the recent years,many different prokaryocytes,such as bacteria,have been confirmed to possess microRNAs.However,the microRNAs in prokaryotes such as bacteria are 50-400 nt in size and have the biological activity without splice.Moreover,the characteristics,action sites and mechanisms of the prokaryotic microRNAs have some certain diversity compared to the eukaryotic microRNAs.Our review briefly introduce the major regulation mechanisms of gene expression as well as the general characteristics of microRNAs and their regulation mechanisms of gene expression in prokaryocytes and eukaryocytes,which will provide a basis for further and profound study on the gene expression regulation and pathogenic mechanisms of prokaryotic microbial pathogens.

Objective To investigate the correlation between Streptococcus pneumoniae (S.pneumoniae) StkP kinase and drug resistance and to analyze the binding ability of StkP extracellular region (EC-StkP) to β-lactam antibiotics.Methods A stkP gene knockout (ΔstkP) mutant was constructed from S.pneumoniae strain ATCC6306 by insertional inactivation method.E-test was performed to detect the minimum inhibitory concentrations (MIC) of penicillin (PCN) and cefotaxime (CTX) against ΔstkP mutant and its wild-type strain.Bioinformatic softwares were used to predict the EC-StkP of S.pneumonia strain ATCC6306,to generate the three-dimensional structure model of EC-StkP and to analyze the correlation between the structure and functions of EC-StkP.PCR was performed to amplify the extracellular segment of stkP (EC-stkP) gene and the product of it was sequenced after T-A cloning.A prokaryotic expression system of EC-stkP gene was constructed.SDS-PAGE in combination with a gel image analysis system was used to detect the expression of the recombinant EC-StkP (EC-rStkP).The expressed EC-rStkP was extracted by Ni-NTA affinity chromatography.The binding abilities of EC-rStkP to PCN and CTX were detected by isothermal titration calorimetry (VT-ITC) and surface plasmon resonance (Biacore).Results S.pneumonia strain ATCC6306 was sensitive to PCN (MIC=0.06 μg/ml) and CTX (MIC=0.12 μg/ml),but its ΔstkP mutant was resistant to the two antibiotics (PCN MIC=16 μg/ml,CTX MIC=32 μg/ml).The 295 aa segment was predicted as the extracellular region at C-end of StkP of S.pneumoniae strain ATCC6306,containing four penicillin-binding proteins and Ser/Thr kinase-associated (PASTA) domains.The cloned EC-stkP segment and the EC-stkP segment in GenBank shared 99.6% similarity in nucleotide sequence and 100% in amino acid sequence.The constructed prokaryotic expression system for EC-stkP gene expressed EC-rStkP in soluble form.Both PCN and CTX could bind to EC-rStkP and CTX was better than PCN in term of binding ability.Conclusion The stkP gene of S.pneumonia is closely related to drug resistance and the encoded protein,Ser/Thr kinase StkP,can recognize and bind to β-lactam antibiotics.

OBJECTIVE To investigate the value of acute phase protein in the diagnosis and therapy for nosocomial infection monitoring. METHODS The levels of serum C-reactive protein(CRP),serum amyloid A(SAA),?_1-acid glycoprotein(AAG),and ?_1-antitrypsin(AAT) were measured in 71 gynecological patients with nosocomial infection during chemotherapy,Thirty normal controls and 33 chemotherapy patient controls were detected by nephelometry and compared with white blood cell counts. RESULTS The nosocomial infection rate of chemotherapy patients was 16.7%.Main pathogens were Gram-negative bacteria(60.6%).Fungi infection rate was 19.7%.The most frequent hospital infection sites were respiratory tract,gastrointestinal tract and wound.Compared with controls,the levels of serum CRP,SAA,AAG and AAT were significantly higher in chemotherapy patients with bacteria infection(P

Objective To research and develop a model of the representative active component's content by NIR spectroscopy,so as to realize the on-line quality control of extracting process for multiple herbal medicine system in product scale.Methods The on-line NIR detection of extracting process was used to obtain the NIR spectrum,HPLC detection of the extracts was carried out to determine the content of danshensu,and PLS method was used to establish the relationship between the information of NIR and HPLC.Results The optimum NIR wavelength range was 9 715-7 082 cm-1,R=0.959 4,RMSEC=0.049 4,the average relative error was 7.2%.Conclusion NIR Technique could be used in the on-line quality control of Compound Danshen's extracting process.

Objective To provide an experimental evidence for the clinical applications of glyceryl trinatrate(GTN)and isosorbide dinitrate(ISDN)against Coxsackievirus B(CVB)-related myocarditis.Methods Coxsackievirus B3 was propagated in HeLa cells.Virus yields were determinded by 50% tissue culture infective dosage(TCID50).BALB/c mice were attacked with 5000 TCID50 of CVB3,meanwhile,the mice were administrated with GTN and ISDN.All mice were killed at the 14th day.The myocardial tissues were harvested for histologic evaluation.Results The infection plaques in the myocardial tissues obtained from CVB3-infected BALB/c mice treated with GTN were siginificantly reduced(0.89?0.18 in GTN group and 1.25?0.22 in ISDN group)compared with that of the untreated CVB3-infected mice(P

AIM:To explore whether histamine can regulate the expression of early growth response factor-1 (Egr-1) in the cerebral cortex astrocytes.METHODS:Normal wild-type (WT) mice, histidine decarboxylase knockout ( HDC-KO) mice and histamine treated HDC-KO mice were sacrificed for extracting the total RNA of the cerebral cortex. Primary cultured rat cortical astrocytes were treated with histamine at concentrations of 10 -8 , 10 -7 , 10 -6 , 10 -5 or 10 -4 mol/L for 15, 30, 60, 120 or 240 min.H1 or H2 receptor antagonists were pretreated for 15 min before histamine treat-ment.After histamine treatment, the cell total RNA or protein was extracted.The expression of Egr-1 at mRNA and protein levels was determined by real-time PCR and Western blot.RESULTS:The mRNA level of Egr-1 in cerebral cortex of HDC-KO mice was significantly lower than that in WT mice, while exogenous histamine induced the mRNA expression of Egr-1 in HDC-KO mice.In cultured astrocytes, histamine induced the mRNA expression of Egr-1.The maximum increase in the mRNA level of Egr-1 was produced by histamine at concentration of 10 -5 mol/L.In addition, histamine-induced Egr-1 mRNA accumulation peaked at 30 min after commencing stimulation, while histamine significantly increased Egr-1 protein expression at 60 min.Furthermore, histamine-induced Egr-1 expression was inhibited by H1 receptor antagonist but not H2 receptor antagonist.CONCLUSION:Histamine up-regulates the Egr-1 expression in cerebral cortex and cultured astrocytes, which may attribute to H1 receptor activation.

Objective To investigate the distribution and sequence conservation of Hap adhensin encoding gene (hap) in clinical isolates of nontypeable Haemophilus influenzae (NTHi), to screen out and identify the predominant T-and B-cell (T-B) combined antigenic epitopes on Hap protein and to analyze their immunogenicity.Methods Sequence conservation of hap genes in NTHi strains and T-B combined antigenic epitopes were predicted using bioinformatic softwares.PCR was used to amplify the 156 bp segment at 5′-end and the 855 bp segment at 3′-end of hap gene (hap-5′-156 and hap-3′-855) and the amplified products were sequenced.Phage display systems of seven T-B combined antigenic epitopes located on the 55 aa segment at N-terminal and the 285 aa segment at C-terminal of Hap protein (Hap-N52 and Hap-C285) were constructed.Western blot assay and ELISA were performed to detect the antigenicity and immunoreactivity of different T-B combined epitopes displayed by recombinant phage PⅢ protein (rPⅢ).Results Hap protein encoded by the hap gene in NTHi was located on membrane surface.Sequences of the 156 bp segment at 5′-end and the 855 bp segment at 3′-end of hap genes extracted from different NTHi strains were relatively conservative, but many mutations were found in sequences at the middle regions of these hap genes.All of the 56 NTHi strains carried hap-5′-156 and hap-3′-855 segments and shared 92.3％-100％ identities in nucleotide and amino acid sequences of these segements.Hap-N5-24 in the Hap-N52 segment as well as Hap-C4-27, Hap-C28-47, Hap-C114-129, Hap-C150-173, Hap-C200-227 and Hap-C241-267 in the Hap-C285 segment was predicted as the T-B combined antigenic epitope with a higher score and less mutations.Results of Western blot assay and ELISA confirmed that the rPⅢ-displayed Hap-C4-27 and Hap-C150-173 epitopes presented clear hybridization bands with NTHi antisera, and 96.9％ (63/65) and 92.3％ (60/65) of serum samples from children with NTHi infection were positive for antibodies against Hap-C4-27 and Hap-C150-173 epitopes, respectively.Conclusion The gene of hap is widely distributed in clinical isolates of NTHi.Moreover, sequences of the 156 pb segment at 5′-end and the 855 bp segment at 3′-end of hap gene are conservative.Hap-C4-27 and Hap-C150-173 are the predominant T-B combined antigenic epitopes on Hap protein, suggesting that they can be used as epitope candidates for developing multiple antigenic peptide vaccines against NTHi.

Objective To construct a ciaR gene-knockout (ΔciaR) mutant of Streptococcus pneu-moniae ( S. pneumoniae) and to investigate the effects of CiaR in CiaH/CiaR, a streptococcal two-component signal-transducing system, on the expression of genes encoding penicillin-binding proteins ( pbps genes) and cia-dependent small RNAs (csRNAs). Methods Electrophoretic mobility shift assay (ESMA) was per-formed to detect the recombinant CiaR (rCiaR)-binding pbps genes. A suicide plasmid pEVP3ciaR for ciaR gene knockout was constructed and then aΔciaR mutant was obtained through homologous recombination and insertion inactivation of the suicide plasmid, and screening with chloromycin. The mutant was identified using PCR and sequencing analysis. E-test was used to detect the minimal inhibitory concentrations ( MIC) of penicillin ( PCN) and cefotaxime ( CTX) against S. pneumoniae strains. Changes and differences in the expression of pbps genes and csRNAs in theΔciaR mutant and its wild-type strain before and after treatment with 1/4 MIC of PCN or CTX were detected using real-time quantitative RT-PCR. Results The rCiaR could bind to the promoter regions in pbp1a, pbp1b and pbp2b genes of S. pneumoniae. The ciaR gene in ΔciaR mutant was inactivated by insertion according to the results of PCR and sequencing analysis. After treatment with 1/4 MIC of PCN or CTX, the expression of pbps genes at mRNA level ( pbps-mRNAs) in theΔciaR mu-tant was significantly increased (P<0. 05), but the levels of csRNAs were significantly decreased (P<0. 05);whereas a significantly decreased pbps-mRNAs (P<0. 05) and increased csRNAs (P<0. 05) were observed in its wild-type strain. The result of E-test showed that the MICs of PCN and CTX against ΔciaR mutant were increased by 250-fold as compared with those against its wild-type strain. Conclusion The CiaR can enhance the drug resistance of S. pneumoniae to PCN and CTX through down-regulating the expres-sion of PBP1a, PBP1b and PBP2b and up-regulating the expression of csRNAs to inhibit the expression of PBPs.

Objective To investigate the effect of prevention and treatment of urinary retention for patients after abdominal surgery using electroacupuncture unit model 6805.Methods 180 cases of ab-dominal surgery patients were randomly divided into three groups, the treatment group(64 patients)were treated with electroacupuneture at the points of sanyinjiao, zhongji, pangguangyu, the control group 1(57patients)Was treated with moxibustion and foment were used in the control group 2(259 patients).The incidence rate of urinary retention Was compared between the three groups. Results The rate of spontaneously emiction was 56.25%in 30 minutes and 35.94%in 40 minutes in the treatment group, the results in the control group 1 and 2 were 22.81% and 15.79%,23.73% and 13.56% respectively. There were significant differences between three groups. Conclusions Electroacupuncture Can effectively prevent and treat urinary retention after abdominal operation.