The expression cDNA library of A. tabescens was constructed by SMART technique, which useλTriplEx2 as a vector. The titer and the percentage of the constructed library were about 1.0 × 106pfu/mland 98.3% respectively, and the titer and the capacity of the amplified library were about 3.1 × 108pfu/mland 4.2 × 1010. The library was used to provide expressed sequence tags (ESTs). 147 Expressed SequenceTaqs (ESTs) were gained from 176 clones, which were selected randomly and sequenced at the 5'end. Thesequences were submitted to the EMBL database. Blasting the sequences in the GenBank, 43 of them werefound that they have significant similarity with data in GenBank. EST AJ620046 was has significantsimilarity with the arabinosidase of Bacteroides thetaiotaomicron. Using SMART-RACE a full-length cDNA ofAJ620046 was successfully obtained. In order to initially characterize the biochemical properties ofAJ620046, the ORF of AJ620046 named AF was cloned and expressed in Pichia Pastoris yeast.Recombinant pHIL-S1-AF constructed by inserting AF into pHIL-S1 was transformed into Pichia PastorisGS115. Preliminary experiments indicated that AJ620046 was expressed as a 32 kDa protein in recombinantyeast.

Objective To study the changes of Toll like receptor 4,7 (TLR4,TLR7) protein in cervical squamous cell carcinoma ( CSCC) tissues and the relationship between expression and clinical pathological characteristics of the patients. Methods A total of 67 pa-tients with CSCC carcinoma( CSCC group) ,30 patients with cervical intraepithelial neoplasia( CIN group) and 30 patients with normal cervi-cal tissues(normal group) were detected by immunohistochemical method. The expression of TLR4,TLR7 protein and its clinical pathological characteristics of patients with CSCC in the samples were analyzed. Results TLR4,TLR7 protein positive rate of CSCC group were respec-tively 80. 60% and 76. 12%,which were significantly higher than those of CIN group (46. 67%,40. 00%) and the normal group(13. 33%, 10. 00%),the difference was statistically significant (P<0. 05). TLR4,TLR7 protein positive expression rate in CIN group was significantly higher than that in the normal group (P<0. 05). The positive expression of TLR4 protein and CSCC in patients with FIGO stage,pathological grade,depth of invasion had a significant relationship(P<0. 05),pathological grade,the positive expression of TLR7 protein and CSCC in pa-tients with invasive depth had a significant relationship(P<0. 05). Conclusion There is increased expression of TLR4,TLR7 protein in cervical tissues of CSCC patients,and a certain relationship between the factors and clinical staging,pathological grading.

Objective To evaluate the clinical significance of quantitative sensory testing (QST) in screening diabetic peripheral neuropathy of the early stage. Methods One hundred patients with type 2 diabetes mellitus were examined by nerve conduction velocity (NCV) and QST examination. With the NCV positive as the gold criterion for screening diabetic peripheral neuropathy of the early stage, the sensitivity and specificity of QST was further analyzed for diagnosis of the early stage diabetic peripheral neuropathy. Results Among the 100 patients with type 2 diabetes mellitus,there were 41 cases positive and 59 cases negative in NCV examination. On the other hand,there were 74 cases positive,and 26 cases negative in QST. The sensitivity and specificity of QST for the diagnosis of early stage diabetic peripheral neuropathy was 97.56% (40/41) and 42.37% (25/59). Conclusions In the screening of early stage diabetic peripheral neuropathy,QST shows higher detection sensitivity,but lower specificity than NCV examination. Therefore, QST may be an examination for the supplement of the routine electromyography.

Objective:To explore the influence of total alkaloids of Corydalis Ochotensis(TAOCO) on the behavior and pathomorphology of brain tissue of the rats with Alzheimer's disease(AD) induced by β-amyloid protein 25-35(Aβ25-35),and to clarify its therapeutic effects on the AD rats.Methods:The Wistar rats were divided into mormal control group(treated with 0.5 mL·100 g-1 distilled water) (n=9),model group(treated with 0.5 mL·100 g-1 distilled water)(n=9),positive drug group(treated with 1.75 mg·kg-1 donepezil hydrochloride)(n=9),and low,middle and high doses (treated with 2.0,4.0 and 8.0 mg·kg-1) of TAOCO groups(n=8,n=9,n=9).The rat AD models were made by injecting Aβ25-35 into hippocampus.On the 14th day after operation,the rats were administered for 7 d.Morris water maze test was used to detect the spatial learning and memory ability of the rats;dark avoidance task was used to observe the passive avoidance ability of the rats;the pathomorphology of the cerebral cortex and hippocampus of the rats were detected.Results:The Morris water maze test results showed that compared with model group,the latency to platform of the rats in low dose of TAOCO group was decreased on the 4th and 5th days(P<0.05);the latency and swimming distance to reach the platform of the rats in middle dose of TAOCO group were decreased on the 5th day(P<0.05),and the starting angle of the rats was reduced on the 3th day(P<0.05);the latencies to reach the platform of the rats in high dose of TAOCO group were decreased on the 2nd and 5th days(P<0.05),and the starting angle of the rats was decreased on the 6th day(P<0.01).Compared with model group,on the 7th day,the time of staying on platform,distance of staying on platform,time of staying on platform/total time,distance of staying on platform/total distance of the rats in different doses of TAOCO groups were all increased significantly(P<0.05) within 1.5 min.Compared with model group,the times of crossing platform and time of staying in effective area of the rats in low and high doses of TAOCO groups were significantly increased(P<0.05).The dark avoidance task results showed that compared with model group,the error latencies and the error times of the rats in different doses of TAOCO groups had no significant differences on the 2nd day(P>0.05).Compared with model group,there was no obvious improvement of the cerebral cortex and hippocampus injury of the rats in low and middle doses of TAOCO groups.In high dose of TAOCO group,the cerebral cortex and hippocampus injury of the rats were significantly improved.Conclusion:TAOCO can improve the learning and memory function of the AD rats and reduce the pathological injury of brain tissue of AD rats.

Objective To compare the detection performance of the modified serum test (TRUST) method and the traditional method in syphilis screening.Methods A series of TRUST high titer syphilis serum was diluted,and the positive rate of each method was calculated by using the improved method and the traditional method.Comparison of two detection methods of C50,C5 ～ C95 interval,as well as the accuracy of the density curve,and the consistency of the two methods were compared,and diagnostic performance were compared.Results The improved method of C50 was less than the traditional method of C50,and the improved method of C5 ～ C95 range was narrower than the traditional method,compared with the traditional method.The improved method of the non precision density curve was steeper than the traditional method,and the two confidence interval of the consistency degree of the 95％ methods was 73.4％ to 95.8％.The diagnostic sensitivity (SEN),diagnostic specificity (SPE),positive predictive value (PPV),negative predictive value (NPV) and diagnostic efficiency(DF) of the improved method were 64.29 ％,99.1％,85.71％,97.05 ％ and 96.39 ％,respectively.The SEN,SPE,PPV,NPV and DF of the traditional methods were 3.75 ％,98.49 ％,75 ％,96.18 ％ and 95 ％,respectively.The improved method was superior to the traditional methods in the two aspects of SEN and PPV (x2 =8.25,10.03,all P＜0.05),with the statistically significant difference.The improved method was slightly higher than the traditional method in SPE,NPV and DF (x2 =2.39,3.45,4.03,all P＞0.05),with the no statistically significant difference.Conclusion The precision,diagnostic sensitivity and diagnostic specificity of the improved method was higher than that of the traditional method,and it can be applied to the detection of large batch samples with the aid of the full automatic enzyme immunoassay instrument.The improved method can be used to replace the traditional method for syphilis screening.

Objective To study the proliferation inhibition effect by silencing PLCε gene expression with RNA interference in BIU-87 cells. Methods The specific short hairpin RNA recombinant plasmids were constructed by gene clone technology.The expression level of PLCε protein and mRNA were detected by Western blot and RT-PCR respectively after transfected recombinant plasmids into BIU-87 cells.The influence on proliferation was check by MTT.The changes of proliferating cell nuclear antigen(PCNA)were analyzed by immunocytochemical method,and the distribution of cell cycle was analyzed using flow cytometry. Results After transfected with the specific recombinant plasmids,PCNA expression was decreased 33.08%,and the analysis of cell cycle indicated that cells of G0/G1 phase were increased comparision with(40.75±2.30)%and(40.00±1.76)0A,and its G2/M phase cells(8.16±0.51)%were decreased strikingly compared with group control(31.20±1.76)%and group NP(35.94±1.58)%.Cells were blocked at G0/G1 phase,the cell proliferation was inhibited obviously. Conclusion PLCε may play an important role in proliferation of bladder cancer cells,which could be a potential target of biological treatment on bladder cancer in the future.

The experiment was conducted by directed evolution strategy (error-prone PCR) to improve the activity of aflatoxin detoxifzyme with the high-throughput horse radish peroxidas and recessive brilliant green (HRP-RBG) screening system. We built up a mutant library to the order of 10(4). Two rounds of EP-PCR and HRP-RBG screening were used to obtain three optimum mutant strains A1773, A1476 and A2863. We found that mutant A1773 had upper temperature tolerance of 70 degrees C and that its enzyme activity was 6.5 times higher than that of the parent strain. Mutant strains A1476 worked well at pH 4.0 and its enzyme activity was 21 times higher than that of the parent strain. Mutant A2863 worked well at pH 4.0 and pH 7.5, and its enzyme activity was 12.6 times higher than that of the parent strain. With DNA sequencing we found that mutant A1773 revealed two amino acid substitutions, Glu127Lys and Gln613Arg. Mutant A1476 revealed four amino acid substitutions: Ser46Pro, Lys221Gln, Ile307Leu and Asn471lle. Mutant A2863 revealed four amino acid substitutions: Gly73Ser, Ile307Leu, Va1596Ala and Gln613Arg. The results provided a useful illustration for the deep understanding of the relationship between the function and structure of aflatoxin detoxifzyme.
Aflatoxin B1 ; antagonists & inhibitors ; chemistry ; Amino Acid Substitution ; Directed Molecular Evolution ; Enzyme Activation ; Enzyme Stability ; Multienzyme Complexes ; genetics ; metabolism ; Mutant Proteins ; genetics ; metabolism ; Point Mutation ; Polymerase Chain Reaction ; methods ; Protein Engineering

Objective: To investigate the role of des-gamma-carboxy prothrombin (DCP) in assessment of liver function and prognosis of patients with liver cirrhosis. Methods: From January 2013 to August 2016, a total of 137 patients with liver cirrhosis in Shanghai Changzheng Hospital were enrolled. The serum DCP level was measured, the clinical data was collected and the complication and survival situation was followed up. The 137 patients were divided into DCP negative group (DCP≤40 mAU/mL, 118 cases) and DCP positive group (DCP>40 mAU/mL, 19 cases). Forty-five patients with compensated liver cirrhosis were divided into high-level DCP group (DCP>16.5 mAU/mL, 32 cases) and low-level DCP group (DCP≤16.5 mAU/mL, 13 cases). Chi square test was used to analyze the difference in the positive rate of DCP in patients with different Child-Pugh classification. Spearman correlation test was performed to analyze the correlation between DCP and model for end-stage liver disease (MELD) scores. Kaplan-Meier survival curve was used to analyze the correlation between DCP and liver disease related mortality. Results: Compared to that of DCP negative group, albumin level of patients in DCP positive group decreased (35 g/L, 20 to 57 g/L vs. 29 g/L, 17 to 42 g/L), however, total bilirubin (TBil), prothrombin time (PT), and international normalized ratio all increased (12.9 mg/L, 1.80 to 83.0 mg/L vs.22.2 mg/L, 6.4 to 169.0 mg/L; 15.5 s, 11.7 to 35.7 s vs.17.5 s, 13.9 to 33.4 s; 1.24, 0.96 to 3.72 vs.1.44, 1.09 to 3.22), and the differences were statistically significant (Z=-2.785, -2.891, -2.945 and -2.879, all P<0.01). The DCP positive (DCP>40 mAU/mL) rates of Child-Pugh A, B and C patients were 1.8% (1/55), 21.2% (11/52) and 23.3% (7/30), respectively, and the difference was statistically significant (χ²=11.246, P=0.003). The DCP levels of patients with Child-Pugh class B and C cirrhosis were significantly correlated with MELD scores (r=0.259, P=0.021). There were 16 and three patients with ascites and spontaneous bacterial peritonitis (SBP) of DCP positive group, and the incidence was higher than that of DCP negative group (55.1%, 65/118 and 1.7%, 2/118), and the differences were statistically significant (χ²=5.744 and 97.636, both P<0.05). Patients in high-level DCP group had a higher proportion of clinical decompensation than low-level DCP group (53.1% (17/32) and two cases), the difference was statistically significant (χ²=5.397, P=0.024). The overall survival rate of DCP positive group (nine survival cases) were lower than that of DCP negative group (87.9%, 87/99), and the difference was statistically significant (χ²=5.442, P=0.020). Conclusions Serum DCP level is closely related to liver function, ascites and SBP in patients with liver cirrhosis. Furthermore, it is associated with occurrence of decompensation in compensated patients and liver-related mortality in patients with liver cirrhosis. DCP may be a useful serum marker for evaluation of disease severity and prognosis in patients with liver cirrhosis in clinical practice.

Objective: To compare the 10 years risk for ischemic cardiovascular disease among Han, Uygur, Kazak nationality residents of Xinjiang Uygur Autonomous Region. Methods: From October 2007 to October 2010,14 618 adult (aged ≥35 years) Han (n=5 757),Uygur (n=4 767) and Kazak (n=4 094) residents were selected to join this study through the four-stage stratified cluster sampling method from 7 cities and regions of Xinjiang Uygur Autonomous Region. The 10 years risk for ischemic cardiovascular disease was calculated according to the 10 years ischemic cardiovascular disease risk assessment form modified with Chinese characteristics and compared among the residents of 3 nationalities. Results: (1) There were significant differences in age, body mass index, systolic blood pressure, diastolic blood pressure,fasting blood glucose,triglycerides,total cholesterol,low-density lipoprotein,high-density lipoprotein cholesterol, smoking history, and drinking history among Han, Uygur, Kazak nationality population (all P< 0.001). (2) There were significant differences in 10 years risk for ischemic cardiovascular disease between different gender and age group including 35-39, 40-44, 45-49, 50-54, 55-59, and ≥60 years old between Han, Uygur, Kazak nationality population (all P<0.001). (3) There were significant differences in rates of 10%-20% and>20% of 10 years risk for ischemic cardiovascular disease between different gender in Han, Uygur, Kazak nationality population (P values were 0.013 and <0.001, respectively). There were no significant differences in rates of <5% and 5%-9% of 10 years risk for ischemic cardiovascular disease between different gender in Han,Uygur,Kazak nationality population (all P>0.05).(4) There were significant differences in detection rates of diabetes,hypertension,smoking,hypertriglyceridemia,and obesity in male and female Han,Uygur,Kazak nationality population with 10 years risk for ischemic cardiovascular disease ≥10% (P<0.01 or 0.05). Meanwhile,there was significant difference in detection rates of hypercholesteremia in male Han, Uygur, Kazak nationality adults(P<0.001). There were no significant differences in detection rates of elevated low density lipoprotein cholesterol and reduced high density lipoprotein cholesterol in male and female Han,Uygur,Kazak nationality adults (all P>0.05). Conclusion There are gender and age differences in the 10 years risk for ischemic cardiovascular disease in ≥35 years old Han,Uygur,Kazak nationality adults from Xinjiang Uygur Autonomous Region.