Objective:Analyzing the relationship between negative life events and depressive symptoms in university freshmen,and the mediating effects of neuroticism and the moderating role of exercise frequency.Meth-ods:A sampling of 8 079 university freshmen,and the Patient Health Questionnaire was used to assess depressive symptoms,the Eysenck Personality Inventory-Neuroticism subscale to assess neuroticism,the self-administered questionnaire to assess the number of negative life events that the participants had experienced and the exercise fre-quency.Model 4 in the Process plug-in was used to test the mediating effect of neuroticism,and Model 7 to test the moderating role of exercise frequency.Results:The numbers of negative life events were positively correlated with the depressive symptoms scores(r=0.16,P＜0.01),and were positively correlated with the neuroticism scores(r=0.26,P＜0.01).The neuroticism scores were positively correlated with the depressive symptoms scores(r=0.52,P＜0.01).Neuroticism score partially mediated between negative life events and depressive symptoms score,with a mediating effect of 78.4％,and exercise frequency score moderated between negative life events and neuroti-cism scores(β=-0.05,P=0.032).Conclusion:Negative life events are associated with depressive symptoms,neuroticism plays a mediating role,and exercise frequency could moderate negative life events and neuroticism.

Objective:To investigate the mechanism of cyclic AMP receptor protein (CRP) in regulating the siderophore enterobactin-related gene entC of carbapenem-resistant Klebsiella pneumoniae (CRKP). Methods:A mutant strain with crp gene deletion strain (Δ crp) and a complementary strain (c-Δ crp) were constructed using CRKP-27 as the wild-type strain. The influence of CRP on the secretion of siderophore by CRKP was analyzed by chrome azurol sulfonate (CAS) quantitative assay. RT-qPCR and lacZ reporter gene fusion assay were used to detect the regulatory effect of CRP on entC gene expression and its promoter. Electric mobility shift assay (EMSA) was performed to detect the binding of CRP to the entC promoter region and the binding sequence was analyzed by DNase Ⅰ footprinting assay. Results:The Δ crp and c-Δ crp strains were successfully constructed. Compared with the wild-type and c-Δ crp strains, the Δ crp strain could secrete more siderophore under both normal and iron-deficient conditions, but the difference was statistically significant only under normal condition ( P<0.05). The relative expression of entC gene at mRNA level was significantly lower in the Δ crp strain than that in the wild-type and c-Δ crp strains under both normal and iron-deficient conditions (both P<0.05). The promoter of entC gene in the Δ crp strain was less active than that in the wild-type and c-Δ crp strains under both normal and iron-deficient conditions (both P<0.05). EMSA showed that with the increase of CRP protein, the distance of entC probe from the positive pole was shortened and blocked. DNase Ⅰ footprinting assay further identified the specific binding site of the entC promoter region to CRP as 5′-AAGGTGATAAATGCGTCTCATTTTCAA-3′. Conclusions:The CRP protein in CRKP could specifically bind to the entC promoter region and directly promote its expression at transcriptional level.

Objective:To study the effect of the siderophore virulence gene entB on the virulence of carbapenem-resistant Klebsiella pneumonia (CRKP). Methods:CRKP-27 was selected as the experimental strain from 30 CRKP strains collected from the First Affiliated Hospital of Kunming Medical University. The knockdown strain (Δ entB) and complementing strain (C-Δ entB) were constructed by the clustered regularly interspaced short palindromic repeat-Cas9 technology, and verified by polymerase chain reaction (PCR). In order to initially understand the effect of entB on CRKP colony morphology and virulence phenotype, the colony morphology of CRKP-27, Δ entB, and C-Δ entB strains were observed and string test were tested. Draw the growth curve of the strains and determine the effect of entB on the growth of the CRKP strains. The siderophores production ability of the strains were detected quantitatively using chrome azurol S (CAS) detection solution. Mice model of inflammation was established to observe the survival rate of mice and intuitively understand the effect of entB on CRKP virulence. Results:The PCR results showed that the Δ entB strain and C-Δ entB stranin were constructed successfully. The entB has no significant effect on the colony morphology, capsule and virulence phenotype of CRKP. The growth rate of Δ entB was significantly faster than that of CRKP-27( P=0.008) and C-Δ entB ( P=0.001), which showed that entB weakened the growth ability of CRKP. Compared with CRKP-27( P=0.001) and C-Δ entB( P=0.001), the siderophore production of Δ entB was significantly decreased by 11.739 3% and 11.964 2%, indicating that entB gene increased the capacity of CRKP to produce siderophpres. In animal experiments, compared with CRKP-27( P=0.023) and C-Δ entB( P=0.024), the survival rate of mice in the Δ entB group was significantly increased, indicating that the entB increased the virulence of the CRKP. Conclusion:The siderophore virulence gene entB significantly weakened the growth ability of the strain, but clearly enhanced the siderophore production capacity and virulence of CRKP.

Objective To investigate the transmission of blaNDM-1 gene in carbapenem-resistant Citrobacter freundii. Methods A total of 18 strains of NDM-1-producing C. freundii were collected from the First Affiliated Hospital of Kunming Medical University during the period from June 2012 to October 2014. The isolates were identified and subjected to antimicrobial susceptibility testing with VITEK 2 System. Conjugation experiments, pulsed-field gel electrophoresis (PFGE) and Southern blot hybridization were performed to determine the transferability of plasmids. Results The antibiotic susceptibility results showed that all the NDM-1-producing C. freundii isolates were resistant to penicillins, cephalosporins and carbapenems. All isolates exhibited different level resistance to other antibiotics. Conjugation experiments revealed that the plasmids harboring blaNDM-1 in 13 strains were transformed into E. coli 600, and exhibited carbapenem resistance. PFGE and Southern blot hybridization found that blaNDM-1 was located on a 33.3 kb plasmid in 16 isolates and on 33.3-54.7 kb plasmid in 2 isolates. Conclusions Our findings suggest that plasmids contribute to the horizontal dissemination of blaNDM-1 gene in carbapenemresistant C. freundii.

Objective To detection the carrying situation of Clostridium difficile toxin gene A/B in stool specimens of the pa-tients with ulcerative colitis (UC) and persons undergoing physical examination and to research the relationship between Clostridium difficile toxin gene carrying and UC.Methods The stool specimens were collected from 53 cases of UC(32 cases of active stage and 21 cases of resting stage) and 45 persons undergoing physical examination.Total DNA was extracted from stool specimens.The Clostridium difficile toxin gene cdtA and cdtB were detected by real-time PCR ,then the PCR products were amplified and performed the agarose gel electrophoresis and gene sequencing for conducting the amplification products verification.Results 7 cases of cdtA and 4 cases of cdtB were checked out in the UC group ,in which 5 cases of cdtA and 2 cases of cdtB were in the UC active stage group ,2 cases of cdtA and 2 cases of cdtB were in the UC resting stage group.In the healthy control group ,5 cases of cdtA and 2 cases of cdtB were checked out.The detection rate of cdtA and cdtB had no statistically significant difference between the UC group and healthy control group(P>0.05).There was no statistically significant difference between UC active group and inactive group (P>0.05).Conclusion There is no appearant correlation between the carrying situation of cdtA and cdtB in stool with UC onset and UC stage.

Objective To construct a blaNDM-1gene deletion mutant of Enterobacter cloacae and to analyze its biological characteristics. Methods The blaNDM-1gene deletion mutant was constructed by using Red homologous recombination technology and verified by PCR and RT-qPCR. Antimicrobial susceptibility profiles,growth curves and in vitro competition abilities of the original strain and the blaNDM-1gene deletion mutant were analyzed. Results PCR,DNA sequencing and RT-qPCR showed that the blaNDM-1gene dele-tion mutant was successfully constructed. Antimicrobial susceptibility test showed that the original strain was resistant to imipenem,meropenem and ertapenem, while the blaNDM-1gene deletion mutant was sensitive to all. The original strain and the blaNDM-1gene deletion mutant had similar growth curves in Luria-Bertani liq-uid medium. In vitro competition experiment revealed that the competitive index of them was 0.69. Conclu-sion Red homologous recombination technology can be used to knockout the blaNDM-1gene of Enterobacter cloacae,which is associated with antimicrobial resistance and competitiveness.

Objective To investigate the distribution of integrons and ISCR1 elements in NDM-l-producing Citrobacterfreundii isolates,and analyze the genotypes of these strains to understand their homology.Methods A total of 18 strains of NDM-1-producing Citrobacterfreundii were collected from the First Affiliated Hospital of Kunming Medical University during the period from June 2012 and October 2014.The isolates were identified and subjected to antimicrobial susceptibility testing with VITEK 2 System.Class Ⅰ,Ⅱ,and 1Ⅲ integrons and ISCR1 elements were detected by PCR.Clonal relatedness was assessed by pulsed field gel electrophoresis (PFGE).Results Most (77.8％,14/18) strains were positive for class Ⅰ integron conserved region,27.8％ (5/18) isolates were positive for ISCR1 conserved region.No class Ⅱ or Ⅲ integron was detected.Most (72.2％,13/18) isolates were positive for class Ⅰ integron variable region.None of the strains harbored class Ⅱ integron or ISCR1 variable region.Integron variable regions included gene cassette encoding resistance to aminoglycosides (aadA1,aadA5,aac(6')-Ib-cr) and trimethoprim-sulfamethoxazole (dfrA,dfrA15,dfrA17).PFGE revealed 17 clusters among 18 NDM-l-producing Citrobacter freundii isolates.Conclusions The clonal dissemination of NDM-l-producing Citrobacterfreundii isolates is not significant.Class I integron is prevalent in NDM-l-producing Citrobacter freundii.The presence of ISCR1 is relatively rare.The two mobile elements are not related to the spread of NDM-1 gene in this hospital.

Objective To investigate the histopathologic characteristics of bladder tumor and provide theoretical basis for the reasonable selection of treatment modality.Methods This retrospective study collected the pathological data of 4 200 bladder tumor from May 2001 to October 2014.There were 3 443 male and 757 female, and the average diameter of these tumors was (1.8 ± 0.6) cm (ranged 0.2 to 6.5 cm).Among all cases, 3 214 (76.5％) cases were solitary tumor while 986 (23.5％) were multiple tumors.The histologic subtype, pathological grade and stage, the existence of vascular and lymphovascular invasion, tumor in situ, abnormal variants and rare subtypes were recorded and analyzed.Results 162 cases (3.9％)were benign tumors and 4 038 cases (96.1％)were malignant tumors including 4 008 cases of urothelial cancer (UC), 18 cases of primary adenocarcinoma and 12 cases of primary bladder squamous carcinoma.Furthermore, 2 460 (61.4％)cases were high grade UC while 1 548(38.6％)cases were low grade.320 cases were found intravascular tumor embolus or lymphovascular tumor thrombus and 391 (9.3％)cases were found metaplasia of squamous epithelium.Moreover, there were 230 cases of squamous differentiation, 120 cases of glandular differentiation, 110 cases of both squamous and glandular differentiation, and 39 cases (0.9％)of other rare subtypes or variations.On pathological stage, 112 (2.8 ％) cases were carcinoma in situ, 548 (13.7％)cases were Ta, 2 599(65.1％)cases were T1, 480(12％)cases were T2, 92 cases(2.3％)were T3 and 23 cases(0.6％)were T4 stage, with the rest cases being unable to be accurate staging.Multiple Logistic regression analysis revealed that lymphovascular invasion was related to tumor grade , pathological stage and abnormal differentiation (P ＜ 0.02).Moreover, UC with squamous and glandular differentiation were related with tumor recurrence and progression (P =0.02).Conclusions Most bladder tumors were high grade and low stage urothelial cancer with various forms of differentiation.Squamous and glandular differentiation were most common variation which should be avoided to diagnosed as hybrid carcinoma.Lymphovascular tumor thrombus and abnormal differentiation were correlated with tumor stage and grade.

Objective To verify the clinical applicability of the published standard intervals (WS/T402-2012) for WBC , RBC ,PLT and Hb based on the health examination results of Han and Uygur populations in Urumqi .Methods This was a retro-spective study .The results of WBC ,RBC ,PLT and Hb from health examination individuals of Han and Uygur populations from August 2013 to January 2015 were collected ,9 307 health subjects age range from 20 to 79 years of the two nationalities were cho-sen using health examination information system .The percents of health subjects failed falling in the published standard interval were calculated to verify the judgment criterion ,fail falling rate < 10 .00% was regard as qualified .Results The test of normality revealed that the Han and Uygur′s results of all verified items were skewed distributions .The 2 .5% and 97 .5% percentiles of the results of two nationalities :WBC Han(3 .6 ~ 9 .6) × 109 /L ,Uygur(3 .8 ~ 9 .9) × 109 /L ;RBC Han Male (4 .3 ~ 5 .9) × 1012 /L and Female (3 .9 ~ 5 .2) × 1012 /L ,Uygur Male (4 .4 ~ 5 .8) × 1012 /L and Female (3 .9 ~ 5 .2) × 1012 /L ;PLT Han(130 ~ 351) × 109 /L , Uygur(145 ~ 370) × 109 /L) ,and Hb Han :Male (133 ~ 178)g/L and Female (111 ~ 153)g/L ,Uygur :Male (133 ~ 174) g/L and Fe-male (110 ~ 152)g/L .The significant differences of each testing item were found in the different sex from the same nationality(P<0 .01) .The percents of health subjects failed falling in the published standard interval were < 10 .00% in both Han and Uygur na-tionality .Conclusion The published standard reference intervals for WBC ,RBC ,PLT and Hb are applicable to our laboratory for the detection of Han and Uygur populations .

Objective To investigate the clinical and pathological features of urothelial carcinoma with clear cell variant.Methods The pathological and clinical data of 7 cases pathological diagnosed urothelial carcinoma with clear cell variant between March 2005 and May 2014 were retrospectively reviewed.There were 6 males and 1 female,aged 46-75 years (mean,61 years).Clinical manifestations included gross hematuria in 5 cases,hematuria and backache in another 2 cases.The mean tumor size was 3.5 cm (ranged 2.0-6.0 cm).One case was multiple tumor and 6 cases were single tumor.Five cases were positive in urine cytology.All the 7 cases accepted surgical treatment,including radical nephroureterectomy in 2 cases,transurethral resection of bladder tumor plus pharmorubicin regular intravesicalinstillationin 1 case,and radical cystectomy in 4 patients.Results Pathological findings revealed that all the tumors were high-grade urothelial carcinoma with clear cell variant in different proportion.Among them,clear cell tumor was predominant in 1 case and focal in other 6 cases.Meanwhile,tumorsaccompanied by glandular differentiation were found in 2 cases,squamous differentiation was found in 1 case,and micropapillary variant was found in 1 case.Vascular tumor embolus was found in 4 cases.Pathological stage was pT2a (n =1),pT2b (n =3),and pT3a (n =3).Immunohistochemicalstaining revealed cytokeratin 7 (+),cytokeratin 20 (+),epithelial membrane antigen (+)and prostate specific antigen (-).Six cases were followed up.The bladder preservation case was followed up for 8 months without recurrence.In 3 radical cystectomy cases,1 died of cancer 25 months after surgery and another 2 case were followed up for 10 and 12 months after surgery without recurrence and metastasis.In 2 nephroureterectomy cases,1 died of tumor metastasis 18 months after surgery and the other case was followed up for 6 months without recurrence or metastasis.Conclusions Urothelialcarcinoma with clear cell variant is a malignancy often with advanced stage and poor prognosis.Radical surgery is recommended for the treatment.