β-lactam antibiotic binding sites in Streptococcus pneumoniae StkP kinase extracellular region / 中华微生物学和免疫学杂志

Objective:To analyze the binding ability of motifs in the serine/threonine kinase StkP extracellular region (EC-StkP) of Streptococcus pneumoniae to β-lactam antibiotics. Methods:Three motifs (SXXK) in the EC-StkP were mutated into AXXA, respectively or simultaneously. Four mutant plasmids (EC- stkp-AXXA1, EC- stkp-AXXA2, EC- stkp-AXXA3 and EC- stkp-AXXA4) were transfected into recipient cells for cloning and expression. SDS-PAGE combined with gel image analysis was used to detect the expression of the recombinant mutant proteins (EC-rStkP-AXXA1, EC-rStkP-AXXA2, EC-rStkP-AXXA3 and EC-rStkP-AXXA4). The expressed mutated proteins were extracted and purified by Ni-NTA affinity chromatography. The binding abilities of the mutant proteins to penicillin (PCN) and cefotaxime (CTX) were detected by isothermal titration calorimetry (ITC 200) and surface plasmon resonance (Biacore t200). Results:PCN and CTX could not bind to the expressed proteins with mutations in the first or the third motif (EC-rStkP-AXXA1, EC-rStkP-AXXA3, EC-rStkP-AXXA4). EC-rStkP-AXXA2 could weakly bind to CTX, but not to PCN.Conclusions:All three motifs in the EC-StkP of Streptococcus pneumoniae could bind to β-lactam antibiotics with the first and the third motifs being more important.

Analysis and identification of a drug resistance-associated two-component signaling system composed of Streptococcus pneumoniae StkP/CiaR proteins / 中华微生物学和免疫学杂志

Objective:To identify a two-component signaling system (TCSS) composed of β-lactam antibiotic resistance-associated intracellular CiaR and transmembrane serine/threonine kinase StkP of Streptococcus pneumoniae ( S. pneumoniae). Methods:The intracellular segment of stkP gene (IC- stkP) was amplified by PCR and the PCR product was sequenced after T-A cloning. A prokaryotic expression system for IC- stkP segment was established. SDS-PAGE was used to detect the expression of the target recombinant proteins (rIC-StkP and rCiaR) by the established prokaryotic expression system and a previously established prokaryotic expression system for ciaR gene. Ni-NTA affinity chromatography was used to purify the recombinant proteins. The rIC-StkP-captured target proteins of S. pneumoniae were identified by LC-MS/MS after Co-IP. The ability of rCiaR to bind to rIC-StkP was detected by surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). Results:The established prokaryotic expression system for IC- stkP segment could effectively express rIC-StkP. Both rIC-StkP and rCiaR after purification showed a single protein band on SDS-PAGE. CiaR could be specifically co-precipitated with rIC-StkP. Three extracted cleaved peptides were found in CiaR molecule with exactly matched sequences. SPR and ITC showed that rCiaR could strongly bind to rIC-StkP with high affinity and the KD values were 1.526×10 -9 mol/L and 1.980×10 -6 mol/L, respectively. Conclusions:S. pneumoniae CiaR could act as the downstream response regulatory protein of StkP kinase to compose StkP/CiaR TCSS.

Predominant antigenic epitopes on Hap adhesin of nontypeable Haemophilus influenzae and their immunogenicity / 中华微生物学和免疫学杂志

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.

Streptococcus pneumoniae StkP kinase: binding ability with β-lactam antibiotics and correlation with drug resistance / 中华微生物学和免疫学杂志

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.

Advance in research of characteristics and action mechanism of microRNAs from prokaryotes / 中国人兽共患病学报

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.

Roles of Streptococcus pneumoniae CiaR in regulating the expression of pbps genes and csRNAs and its correlation with drug resistance / 中华微生物学和免疫学杂志

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.

Drug resistance and protoporphyrin ferrochelatase of / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To investigate the drug resistance, β-lactamase-encoding genes and protoporphyrin ferrochelatase-encoding genes of, and to explore its structure and pathogenic function.</p><p><b>METHODS</b>The strain was isolated by plate streaking method and identified by automatic bacteria detection system and 16S RNA gene PCR. Microdilution method was applied for drug susceptibility test. β-lactamases, extended spectrum β-lactamases (ESBL) and carbapenemases were detected using nitrocefin-disk, Kirby-Bauer disk, and Hodge test, respectively. Five β-lactamase-encoding genes and protoporphyrin ferrochelatase-encoding gene of the isolate were amplified by PCR for sequencing. Bioinformatic softwares were used to analyze the structure and function of the product of protoporphyrin ferrochelatase-encoding gene.</p><p><b>RESULTS</b>A strain belonging towas isolated. This isolate was sensitive to cefepime, ciprofloxacin, ofloxacin and tigecycline, but resistant to five penicillins, four cephalosporins and two carbapenems antibiotics. The isolate produced β-lactamases but did not produce ESBL and carbapenemases. The isolate had five distinct β-lactamase-encoding genes and protoporphyrin ferrochelatase-encoding gene. The product of protoporphyrin ferrochelatase-encoding gene contained two functional domains of protoporphyrin ferrochelatase belonging to type Ⅱ chelatase superfamily that presented the most closely genetic relationship with the protoporphyrin ferrochelatase of.</p><p><b>CONCLUSIONS</b>The isolate ofhas a higher resistance to β-lactam antibiotics and its β-lactamase-encoding genes are different with the common bacterial β-lactamase-encoding genes. Protoporphyrin ferrochelatase may act as an important virulence factor of.</p>

The positive role of insecurity / 国际中医中药杂志

There are many discussions and researches on the positive role of security and negative role of insecurity. However, from the view of biology and evolution, insecurity should be exist when people were born. It makes human to think, then produce relevant behaviors. Just these thinking and behaviors produce culture, maintain psychological individual balance and realize personal value.

The practice methods of TCM psychotherapy in community cospital / 国际中医中药杂志

To investigate how to promote the psychotherapy work of TCM in community hospitals. After a period of time developing the group psychotherapy of TCM and the community health education, Psychotherapy of TCM in community were promoted. In the community hospitals the psychotherapy work of TCM was carried out smoothly, and has been widely recognized and praised.

Analysis of application effect of evidence-based nursing in family planning surgery / 中国实用护理杂志

Objective To investigate the application effect of evidence-based nursing(EBN) in family planning surgery,evaluate the clinical nursing value and improve the quality of clinical nursing.Methods 240 patients who received family planning surgery were randomly divided into the control and the observation groups (120 patients in each group).The conventional nursing model was used in the control group and evidence-based nursing was implemented in the observation group.Self-rating Anxiety Scale(SAS),patients satisfaction(PA) and adverse reaction rate(ARR) were adopted to assess the two groups after different nursing.Results The score of SAS and ARR were significantly lower in the observation group than those in the control group.The patients satisfaction was significantly higher in the observation group than the control group.Conclusions Application of evidence-based nursing plays a significant role in treatment,nursing and life quality improvement of patients with family planning surgery,which is worthy of wide application.

Effect of heat preservation infusion on incidence of infection for patients after artificial abortion / 中国实用护理杂志

Objective To observe the control effect of heat preservation infusion on incidence of infection after artificial abortion.Methods 168 cases of patients after artificial abortion were randomly divided into the heat preservation infusion group and normal infusion group with 84 cases in each group.The normal infusion group received routine nursing,and was intravenously infused fluid with non heat preservation method,the heat preservation infusion group was intravenously infused constant temperature liquid heated to 37℃.The incidence of infection was observed in two groups of patients after artificial abortion.Results The infection rate of the heat preservation group was significantly less than the normal infusion group.Conclusions Heat preservation infusion can effectively reduce the infection rate after artificial abortion.

Effect of the constant temperature infusion on postoperative body temperature and blood coagulation of patients undergoing induced labor / 现代临床护理

Objective To explore the constant temperature infusion on postoperative body temperature and blood coagulation of patients undergoing induced labor.Methods Eighty patients undergoing induced labor were divided into the observation group and the control group randomly:In the former the infusion was done using fluids constantly kept at the temperature of 36℃and in the latter,the infused fluid was kept at room temperature.The two groups were compared in terms of changes of body temperatures,loss of energy,postoperative blood loss and blood coagulation.Results The temperatures of the controls were declined to different extents after infusion and the temperatures of the observation group showed no significant change,but the difference between the two groups was significant(P0.05),but the platelet(PLT)and fibrinogen(FIB) content were significantly decreased(P<0.05)and the blood loss of the observation group was significantly less than those in the control group(P<0.05).Conclusion The constant temperature infusion may maintain the temperature and blood coagulation of the patients, reduce blood loss and prevent adverse reactions induced by induced labor.

Analysis of psychological tests on newly employed staff in a Chinese comprehensive hospital / 国际中医中药杂志

Objective This study targeted on 298 newly employed staff in Guang'anmen Hospital,China Academy of Chinese Medical Sciences,who had finished the Minnesota Multiphasic Personality Inventory (MMPI).A retrospective study has been approached to investigate the mental states of the new employers in the Chinese comprehensive hospital.Methods SPSS statistics software 11.5 was used to do descriptive statistical analysis.Results Scores of all factors show no difference compared to the various norm groups studied.Most of the new employers represent calm and gentle personalities.They are confident,optimistic,independent,gentle,kind,decent,rational and objective,have self-control and self-discipline,barely have no doubt about their physical health,trust others; they are mature,able to maintain good interpersonal relationships and to adapt external pressure; they do not harm themselves psychologically; they are calm,stable and pragmatic.Conclusion Subjects are somehow over reporting or exaggerating the prevalence of being nice,which could be subconscious or intentional.However,this does not have impacts on reliability and validity.MMPI is a relatively comprehensive psychometric scale for employment.

Clinical Observation on 103 Cases of Traumatic Patellar Dislocation Treated with Integrative Traditional Chinese and Western Medicine / 国际中医中药杂志

Objective To observe clinical effect of traumatic patellar dislocation treated with integrative traditional Chinese and western.Methods 103 patients with traumatic patellar dislocation were randomly divided into a treatment group and a control group.The treatment group was given the therapy of traditional Chinese medicine combined with western medicine,while the control group was treated only with western medicine.After 3 months'treatment,observed it's the clinical effect.Results Efficacy of the treatment group was obviously higher than the control group,with a significant difference (P＜0.05).Conclusion The integrative traditional Chinese and western medicine exerts a better effect than the sole therapy of western medicine.