Objective To express Japanese encephalitis virus (JEV) E protein in Escherichia coli. Methods The gene coding for JEV E protein was amplified by using RT-PCR, and cloned into plasmid pET-28a. The constructed plasmid was transformed into E.coli BL21 (DE3). Expression of E protein by the transformants was induced by IPTG and analyzed by SDS-PAGE and Western blot. Results Five nucleotide changes leading to amino substitutions were identify in the E protein gene of our laboratory strain of JEV when compared to a published gene sequence of JEV E protein. The yield of expressed JEV E protein with relative molecular mass approximately 52?10 3 was 35% of total bacterial proteins. Conclusions JEV E protein was expressed successfully in E.coli, which should be useful for the production of diagnostic reagents and the analysis of gene structure/function of the E protein.

Objective To isolate and identify the putative Japanese encephalitis virus (JEV) re-ceptors from C6/36 and Vero cells. Methods Molecules binding with JEV were isolated from C6/36 and Vero cells by co-immunoprecipitation (Co-IP) approach, identified by mass spectrometry, and detected by Western blot. The location of putative JEV receptor on cells membrane and the binding with JEV were ob-served by laser scanning confocal microscopy (LCM). Results Several molecules binding with JEV were isolated from C6/36 and Vero cells by Co-IP, and only one molecule was identified as heat shock cognate 70 (HSC70) by mass spectrometry. Antibody against HSC'70 was able to detect a 74 ×103 protein isolated by Co-IP from C6/36 and Vero cells membrane in Western blot assays. It was observed by LSCM that when JEV attached on the surface of C6/36 cells, JEV and HSCT0 protein were co-localization. Conclusion 74 x 103 molecular identified as HSC70 protein from C6/36 cells may be JEV receptor.

Objective To design and testify a novel strategy for acquiring mimetic epitope mapping by screening for a phage random peptide library using polyclonal anti keratin autoantibodies (AK auto Ab). Methods AK auto Ab were isolated and purified from pooled human sera by keratin affinity column in which keratin had been linked with CNBr Sepharose 4B,then biotinylated by the biotin ester. A 15 mer phage random peptide library was biopanned for 3 cycles and positive clones were identified by ELISA,competition assay and DNA sequencing. ResultsBy sequence comparison 23 positive clones were selected randomly and three epitopes were confirmed. Among the three epitopes SLSPMPTTNRR was the dominant epitope. The phages carrying positive clones reacted with AK auto Ab specifically and keratin could prevent interaction between AK auto Ab and positive phages. Conclusion The designed strategy is successfully applied in acquiring epitopes of polyclonal autoantibodies to keratin, which could provide a new approach for the discovery of epitope mapping which binds to natural autoantibodies.

Aim To study the mimicry peptide of JEV E protein by screening a phage 15-mer peptide library with anti-JEV E protein mAb 2H4. Methods After three rounds biopanning, the enriched positive phage clones were identified by ELISA. 10 positive phage clones were sequenced and compared homologously with JEV E protein. Results The short peptide displayed on screened positive phage could bind specifically to mAb 2H4, and the binding could be inhibited by natural JEV Ag. Amino acid sequences of the 10 positive phage clones were consensus, that is, RQDPQWPYANSTIAR. By homology analysis, two higher homologous sequences STXAR and WXXAXST were found in different regions of JEV E protein. The peptide displayed on positive phage could react specifically with the mouse antiserum against natural JEV Ag . Conclusion This peptide displayed on positive phage may mimic partial antigenicity of JEV E protein.

Objective To investigate the efficacy of pranoprofen drops on dry eye of patients with Sj(o)gren's syndrome (SS).Methods This is a prospective study.Sixty-eight inpatients with dry eye in our hospital were randomly divided into the experimental and control groups.Right eyes were taken for the trial,with 34 cases in each group.The experimental group was given pranoprofen eye drops combined with polyethylene glycol eye drops.Eyes of the control group were given polyethylene glycol drops only.Corneal fluorescein staining (FL),tear film breakup time (BUT) and Schirmer test (SIT) were tested before treatment and 1,2,4 weeks after treatment by the same care giver.The levels of IL-6 and TNF-α in tears were detected by ELISA.Analysis of variance of repeated data and t test were used for statistical analysis.Results The difference of FL,BUT,SIT and content IL-6 and TNF-α in tears in the experimental group patients before treatment and 1,2,4 weeks after treatment were signifcant (F=4.65,7.53,6.43,9.96,10.87; P＜0.05),which were statistically significantly different between the experimental group and the control group patients (F=3.27,5.85,4.36,8.36,7.23; P＜0.05).One week after treatment and before treatment,the difference of BUT and SIT of the two groups was not statistically significant (P＞0.05),those of the 2 weeks after treatment were statistically significantly different [BUT of the experimental group was (11.1±2.5) s,BUT of the control group was (9.7±1.9) s,t=2.594 8,P＜0.05; the SIT of the experimental group was (7.3±1.7) mm,the SIT of the control group was (5.9±1.7) mm,t=3.571 8,P＜0.05].BUT of the two groups at 4 weeks after treatment was statistically significantly different [BUT of the experimental group was (14.4±2.8) s,BUT of the control group was (11.4±2.6) s,t=4.469 4,P＜0.05; the SIT of the experimental group was (9.9±2.1) mm,the SIT of the control group was (8.7±1.9) mm,t=2.568 0,P＜0.05].The difference of FL and IL-6 and TNF-α in tears pretreatment between the two groups was not statistically significant (P＞0.05).At week 1,2,4 after treatment,the differences between the two groups were statistically significant (tFL=4.173 9,3.190 7,4.072 6; tIL-6=2.131 5,2.316 4,5.310 1; tTNF-α=2.216 4,4.871 9,8.175 0; P＜0.05).No significant discomfort and side effects were observed in the two groups.Conclusion Pranoprofen drops can significantly improve symptoms of dry eye in patients with pSS,in particular,the repair of the cornea,may be related to the inhibition of the expression of ocular inflammatory cytokines IL-6 and TNF-α,and thus reduce the ocular surface inflammatory reaction.

Objective:To investigate the predictive value of 18F-fluorodeoxyglucose (FDG) PET/CT imaging for the epidermal growth factor receptor (EGFR) mutations in patients with lung adenocarcinoma. Methods:From January 2013 to December 2017, a total of 146 patients (83 males, 63 females, age: (60.2±10.3) years) who were confirmed as lung adenocarcinoma by pathology and were examined by 18F-FDG PET/CT imaging and EGFR mutation testing in Shanxi Cancer Hospital were retrospectively analyzed. The differences of clinical characteristics (age, gender, smoking, tumor diameter, loymph node metastasis, distant metastasis, stage, thyroid transcripition factor-1 (TTF-1), NapsinA, cyiokeratin (CK)-7, Ki-67) and PET/CT parameters (maximun standardized uptake value (SUV max) of the primary tumor (pSUV max), SUV max of lymph node (nSUV max) and SUV max of distant metastasis (mSUV max)) between patients of EGFR mutation and EGFR wild type were analyzed using independent-sample t test, χ2 test and Fisher exact test. The predictors for EGFR mutation were analyzed by logistic regression analysis. The predictive value of pSUV max and pSUV max combined with gender, smoking and tumor diameter was determined by receiver operating characteristic (ROC) curve analysis. Results:There were 46.58%(68/146) patients with EGFR mutations and 53.42%(78/146) patients with wild type. Gender, smoking, lymph node metastasis, tumor diameter, pSUV max, nSUV max, TTF-1, NapsinA and Ki-67 were significantly different between patients with EGFR mutations and those with wild type ( t values: from -3.023 to -2.032, χ2 values: 4.725-33.749, all P<0.05). Female (odds ratio ( OR)=3.236, 95% CI: 1.213-8.779; P=0.029), non-smoker ( OR=4.947, 95% CI: 1.796-13.621; P=0.019), tumor diameter<3.5 cm ( OR=2.750, 95% CI: 1.109-6.818; P=0.001) and pSUV max<9.1( OR=2.960, 95% CI: 1.227-7.141; P=0.016) were predictors of EGFR mutations in lung adenocarcinoma. The area under the curve (AUC) of pSUV max was 0.640 with the specificity of 43.6%(34/78)and the sensitivity of 27.9%(19/68), while the AUC of the four independent factors was 0.83 with the specificity of 71.8%(56/78) and the sensitivity of 83.8%(19/68). Conclusions:pSUV max is associated with mutant EGFR status. Moreover, the combination of pSUV max, gender, smoking and tumor diameter can enhance the predictive value on EGFR mutation status in patients with lung adenocarcinoma.

OBJECTIVETo analyze the value of the diagnostic criteria for bladder outlet obstruction in benign prostatic hyperplasia (BPH).

METHODSA total of 358 patients with BPH were divided into 3 grades according to fibrous urethrocystoscopy information on the severity of obstructions, which were classified as Grade 1 (slight), Grade 2 (moderate), and Grade 3 (severe). By Schäfer's graph they were divided into 7 grades, represented by 0 to VI. We analyzed the volume of prostate, maximum flow rate (Qmax), residual urine volume, International Prostatic Symptom Score (IPSS) and detrusor instability. Statistical analysis ANOVA (analysis of variance) was made, spearman correlation evaluated and the coefficient of determination measured.

RESULTSOf all the patients, 27 were classified as Grade 1, 236 as Grade 2 and 95 as Grade 3. Eighty-four patients had detrusor instability. The volumes of the prostate ranged from 16 ml to 145 ml, averaging (47.04 +/- 15.61) ml. The mean maximum flow rate was (10.02 +/- 2.12) ml/min and the mean residual urine volume was (84.06 +/- 36.50) ml. With the increase of the severity of obstruction, the volume of the prostate increased (F = 4.216, P < 0.05), IPSS rose (F = 8.408, P < 0.001), the maximum flow rate decreased (F = 22.43, P < 0.001), the residual urine volume rose (F = 163.232, P < 0.001), the incidence of detrusor instability increased (F = 23.637, P < 0.001) and Schäfer's grades were elevated (F = 202.897, P < 0.001). The volume of the prostate, the maximum flow rate (Qmax), residual urine volume, IPSS detrusor instability and Schäfer's grades were all correlated significantly with the severity of the obstruction. The correlation index and coefficient of determination were r = 0.29, R2 = 0.08; r = 0.35, R2 = 0.12; r = -0.69, R2 = 0.47; r = 0.60, R2 = 0.36; r = 0.33, R2 = 0.11; r = 0.72, R2 = 0.52; respectively. The correlation between the urethrocystoscopy information and Schäfer's graph on the severity of the obstruction were the best criteria of all.

CONCLUSIONThe severity of the obstruction at urethrocystoscopy correlates well with that at urodynamic investigation. Such criteria could improve the sensitivity and specificity of the diagnosis of bladder outlet obstruction.

Aged ; Aged, 80 and over ; Humans ; Male ; Middle Aged ; Prostatic Hyperplasia ; complications ; diagnosis ; Retrospective Studies ; Urinary Bladder Neck Obstruction ; diagnosis ; etiology ; Urodynamics

OBJECTIVE To study the protective effect of phenolic acid components from Salvia deserta Schang on the oxidative stress injury of human renal tubular epithelial cells HK -2 induced by high glucose and high fat . METHODS HK-2 cells were divided into control group ，model group ，canagliflozin group （positive control group ，15 μmol/L），purified product of phenolic acids from S. deserta Schang group （10.8 μg/mL），4 monomers group （salvianic acid ，protocatechuic aldehyde ，caffeic acid，rosmarinic acid ，50 μmol/L）. In addition to the control group ，cell injury model of high glucose and high fat was established in other groups （500 μmol/L palmitic acid+ 30 mmol/L glucose for 48 h）and cultured for 48 h. The cell apoptotic rate ，the contents of malondialdehyde （MDA）and glutathione （GSH），and the activity of superoxide dismutase （SOD）were detected in each group ； the expression levels of nuclear erythroid 2-related factor 2（Nrf2），Kelch-like ECH -associated protein 1（Keap1）protein，heme oxygenase-1（HO-1）and NADH ：quinone acceptor oxidoreductase 1（NQO1）were determined in above 5 groups（except for salvianic acid ，protocatechuic aldehyde ，caffeic acid ）. RESULTS Compared with control group ，the apoptotic rate of HK -2 cells in model group was increased significantly （P＜0.01）；the content of MDA was increased significantly （P＜0.01），while the content of GSH and the activity of SOD were decreased significantly ；protein expressions of Nrf 2，NQO1 and HO -1 2018D01C169） were significantly down -regulated（P＜0.01），while the protein expression of Keap 1 was up -regulated significantly （P＜0.01）. Compared with model group ， the apoptotic rate and the content of MDA were decreased significantly in administration groups（P＜0.01）；the content of GSH in administration groups and the activity of SOD in purified product of phenolic acids group，protocatechuic aldehyde group and rosmarinic acid group were increased significantly （P＜0.01）. The protein expressions of Nrf2，HO-1 and NQO 1 in purified product of phenolic acids group as well as the protein expression of Nrf 2 in rosmarinic acid group were up -regulated significantly （P＜0.01），while the protein expression of Keap 1 was down -regulated significantly in purified product of phenolic acids group and rosmarinic acid group （P＜0.01）. CONCLUSIONS The phenolic acids components from S. deserta Schang can relieve oxidative stress injury of renal tubular epithelial cells induced by high glucose and high fat ，the mechanism of which may be associated with activating Keap 1/Nrf2 signaling pathway and inhibiting oxidative stress response .

Objective : To investigate the expression characteristics of mu opioid receptor ( MOR) in rat colon smooth muscle cells by cultured rat primary colonic smooth muscle cells . Methods : colonic smooth muscle cells were isolated , cultured and identified ; immunofluorescence double labeling method was used to observe the distribution characteristics of MOR , Endomorphin⁃2 (EM2) , and calmodulin (CaM) in colonic smooth muscle cells ; Western Blot method was used to detect the expression of MOR and EM2 in smooth muscle cells of the colon . After the intervention measures Acetylcholine (ACh) ( 1 × 10 - 3 mol/L) and EM2 (2 μmol/L) were applied , the changes of CaM protein expression were observed ; The calciumion imaging method was used to detect the changes of calciumi on concentration in smooth muscle cells. Results: The colonic smooth muscle cells were cultured and identified . The positive cells labeled with α ⁃smooth muscle actin ( α ⁃SMA) accounted for more than 95% of the total number of cells . Immunofluorescence double labeling showed that there were MOR and EM2 distributions in colonic smooth muscle cells , and all MOR and EM2 positive cells coexisted with α ⁃SMA . Western Blot results showed that there were MOR and EM2 expressions in colonic smooth muscle cells . CaM in the ACh group significantly increased at 10 minutes (P < 0. 05) , CaM in the EM2 group significantly decreased ( P < 0. 05) ; The calciumion imaging results showed that alone applied ACh , the calciumion concentration in smooth muscle cells significantly increased ( P <0. 05) ; Alone applied EM2 , the calciumion concentration in colonic smooth muscle cells was down⁃regulated (P <0. 05) ; Applied ACh and EM2 sequentially , EM2 significantly reduced the increase of the calciumion concentration in smooth muscle cells induced by ACh (P < 0. 05) . Conclusion MOR and EM2 are expressed in colonic smooth muscle cells , and EM2 may inhibit the expression of CaM and reduce the concentration of calcium ions through MOR .