Objective To establish a rapid and accurate analysis method for food-derived ACE inhibitory peptides activity in vitro.Methods Reaction time of ACE and substrate was by measuring the hippuric acid liberated in the ACE reaction mixture at regular intervals;An optimal RP-HPLC method to measure food-derived ACE inhibitory peptides activity in vitro was set up.The hippuric acid from ACE reaction mixture(sea cucumber peptides were regarded as ACE inhibitor) was estimated by Zorbax SB-C_(18) analytical column with acetonitrile and ultrapure water as mobile phase.Results The reaction time of ACE with substrate was determined at sixty minutes;The elution was carried out with the ratio of acetonitrile to ultrapure water was 1:1(0.1%TFA) at a flow rate of 0.4 mL?min~(-1).The ahsorbance of the eluent was monitored at 228 nm,and column temperature was 25℃.The relationship between hippuric acid concentration and peak area exhibited a good linearity in the concentration ranges of 0～200?g?mL~(-1) and 200～800?g?mL~(-1).The RP-HPLC method was further validated by captopril,the oyster hydrolysate and the anchovy hydrolysate.Conclusion The method has been proved to be convenient,accurate and suitable for the analysis of foodderived ACE inhibitory peptides activity in vitro.

Objective To detect infection of Angiostrongylus cantonensis and examine effection of treatment to prepare monoclonal antibodies(McAbs). Methods Six-week-old BALB/c mice were imrnunized by the intraperitoneal injection of e/s antigens of Angiostrongylus cantonensis. Fusion of splecn cells from immunized mice with prepared SP2/0-Ag14 myeloma cells was performed in RPMI 1640. Fused cells were suspended in RPMI 1640 containing 1% HAT and 20% fetal calf serum and dispensed into 96-well cell culture plates. The supernatants of clones were screened by ELISA with sera of patients of angiostrongyliasis.Distribution of cohere antigen of 12D5 and 21B7 monoclonal antibodies was analyzed with immunohistochemistry. Two McAbs ( 12D5 and 21B7) were applied to detect the circulating antigen (CAg) in the sera of rats infected with A. cantonensis and angiostrongyliasis patients respectively by double antibody sandwich ELISA.Results 12D5 McAb was identified as IgG1 and 21 B7 McAb was IgM. Western blot result showed two McAbs could used to identified 55 × 103 protein of adult worms of A. cantonensis. Cohere antigen of 12D5 and 21B7 monoclonal antibodies were distributed on intestine surface of A. cantonensis. The detection rates of CAg in the sera of infected rats 100% (48/48), the detection rates of CAg in the sera of angiostrongyliasis patients was 100% (32/32). No cross-reaction to sera of patients with other infection of parasites, such as clonochiasis, fasiolopsiasis, ancylostomiasis, trichinosis, anisakiasis as well as schsitosomiasis, and health srea did not reacted with 12D5 and 21B7 McAbs,and detaction rate of antibody of angiostrongyliasis patients only reached 75% (24/32) with antigen of A. cantonensis. Conclusion Cohere antigen of 12D5 and 21B7monoclonal antibodies were antigens of enteric epithelium. Sandwich ELISA with 12D5 and 21B7 McAbs showed high specificity act as detecting CAg of A. cantonensis in sera of infection animal and patients. It is apparent that Sandwich ELISA with 12D5 and 21 B7 is not only rapid and simple without requirement of special instrument, but also rather sensitive and specific for the detection of current infection with A. cantonensis.

To identify the gene differentially expressed in female Anopheles anthropophagus and to analyze its gene sequence, this gene amplified by PCR was identified by real-time PCR and its cDNA was then amplified with rapid amplification of cDNA ends (RACE) technology. It was found that the expression ratio of the female differentially expressed gene in female and male mosquitoes was 267.49 according to the formula F=2~(-⊿⊿CT).The size of mRNA of the gene was 364 bp, and the amino acid sequence deduced from the open reading frame (ORF) was found to be similar to the sequence of tectin protein of Culex quinquefasciatus and proteins of other species. The mRNA sequence of this gene was submitted to NCBI with a accession number of FJ907236. This gene may provide a foundation for further studies on the biological functions of mosquitoes.

Objective To identify the species of Biomphalaria snails collected in Shenzhen reservoir,based on the mitochondrial 16S rDNA sequences.Methods The 16S rDNA fragments were amplified by PCR from the genome DNA of Biomphalaria snails,and inserted in plasmid pMD-18T for sequencing.The sequence of 16S rDNA fragment and its phylogenetic relationships with those of other species of Biomphalaria snails were analyzed with BLAST and MEGA4 software.Results The amplified 16S rDNA fragment of the Biomphalaria snails was about 466 bp in length.As aligned with the corresponding sequences of the related Biomphalaria species,the identity of nucleotides was 99％ with 1 isolate of Biomphaltria straminea (B.straminea),98％ with 3 isolates of B.kuhniana,95％ with 1 isolate of B.intermedia,and 94％ with 1 isolate of B.edisoni.Based on the 16S rDNA sequence,the results of phylogenetic analysis with neighbor-joining (NJ) and unweighted pair-group method with arithmetic means (UPGMA) indicated that the snails had close genetic relationships with the B.straminea isolate (Genbank accession NO.AY030213.1) Conclusion The Biomphalaria snails collected in Shenzhen reservoir could be classified as B.straminea based on the characteristics of 16S rDNA sequence.

Objective To investigate the clinical features of bilateral medial medullary infarction (BMMI)in elderly patients.Methods Clinical and imaging data of 8 elderly BMMI patients with different morphology on diffusion-weighted magnetic resonance imaging (DWI-MR) were retrospectively analyzed.All patients were diagnosed by MRI,while 4 patients received CTA and vascular ultrasound testing,and the other 4 patients received vascular ultrasound testing.Results All 8 cases(100.0％) had acute-onset BMMI.Patients showed varying degrees of acroparalysis(7/8,87.5 ％),dizziness (5/8,62.5 ％),dysarthria(6/8,80.0 ％),dysphagia(3/8,37.5 ％),deep or superficial sensory dysfunction(5/8,62.5 ％),consciousness disorders (2/8,25.0 ％),dyspnea (2/8,25.0 ％),and tinnitus(1/8,12.5 ％).Lesions in most patients were located in the upper part of medulla oblongata(7/8,87.5 ％).In the transverse direction of DWI,the lesions as the inverted V shape were seen in 3 cases (37.5％),the V shape(12.5％)in 1 case,the Y shape(37.5％)in 3 cases,and the heart shape(12.5％) in 1 case.All 8 patients were complicated with posterior cerebral artery stenosis or occlusion,of whom patients with heart-or Y-shaped lesions showed progressive exacerbation.After treatment,4 cases (50.0 ％) recovered,3 cases (37.5 ％) improved,and 1 case (12.5 ％) unrecovered before discharge from the hospital.Conclusions Most elderly BMMI patients have concurrent posterior circulation artery stenosis,and patients with heart-or Y-shaped lesions on MR-DWI show rapid progression and have a poor prognosis.Cranial examination with MR-DWI is helpful for early clinical diagnosis of BMMI,prediction of disease progression and effective prevention of complications.

Objective: To analyze the therapeutic effect on HBeAg-negative chronic hepatitis B patients treated with Peg-IFNα-2a combined with NAs to obtain the influencing factors for predicting HBsAg clearance. Methods: A retrospective study was conducted to investigate the effect of pegylated interferon alpha-2a combined with nucleoside analogues (lamivudine/adefovir dipivoxil) on HBeAg-negative chronic hepatitis B. The treatment course was 96 weeks. Patients were followed up 120 weeks after the treatment. HBsAg clearance at 120 weeks was taken as the objective of the study. Logistic regression and receiver operating characteristic curve analysis screened the related factors affecting HBsAg clearance. χ ² test was used to compare count data. Results: 111 patients were treated with pegylated interferon alpha-2a combined with nucleoside analogues, and 107 patients completed the scheduled course of treatment and follow-up. HBsAg clearance rate at120 week was 29.0% (31/107). The influencing factors for analysis were: (1) gender had no effect on HBsAg clearance rate; age and baseline levels of HBV DNA and alanine aminotransferase had no significant effect on HBsAg clearance; low baseline level of HBsAg (< 3.023 lgIU/ml) was beneficial to HBsAg clearance. The area under the working characteristic curve of the subjects was 0.746, the positive predictive value was 44.4%, and the negative predictive value was 86.8%. (2) HBsAg quantification or decline in 24 weeks and 48 weeks of treatment had a good predictive effect on HBsAg clearance, and the 48 weeks predicted value was higher than 24 weeks. When the HBsAg quantification was≤2.070 lgIU/ml at 48 weeks, the area under the receiver operating characteristic curve was 0.931, the positive predictive value was 52.8%, and the negative predictive value was 94.4%. When HBsAg decreased from baseline to≥0.991 lgIU/ml, the area under the receiver operating characteristic curve was 0.888, the positive predictive value was 50.8%, and the negative predictive value was 97.9%. (3) The analysis of HBsAg subgroup levels at 48 weeks suggested that the "interval analysis" can forecast HBsAg clearance more exactly than "nodal analysis" .The final HBsAg clearance rate of 100 IU/ml < HBsAg≤1 000 IU/ml, 10 IU/ml < HBsAg≤100 IU/ml and HBsAg≤10 IU/ml groups reached 6.7%, 31.8% and 67.7%, respectively. (4) The ALT abnormal group in the course of treatment obtained a higher HBsAg clearance rate (48.0%, 12/25). Conclusion 96-weeks long-term treatment with pegylated interferon-alpha -alpha-2a combined with nucleoside analogues for HBeAg-negative chronic hepatitis B has a good predictive value for HBsAg clearance at baseline and during treatment. The "interval level" of HBsAg at 48-weeks is more accurate in predicting HBsAg clearance, suggesting that HBeAg-negative chronic hepatitis B patients with low HBsAg levels at 48-weeks are the advantageous populations with HBsAg clearance. These patients are worthy of prolonged treatment to pursue "clinical cure".

Objective: Exploring the molecular characteristic of global and Shenzhen district H5N6 and H7N9 influenza viruses HA untranslated regions(UTRs). Methods: Mega7.0 and DNAStar 7.1.0 were used to construct phylogenetic tree and nucleotide analysis. Results: From 2014 to 2015, 3 strains of H5N6 influenza virus from Shenzhen were compared with the other H5NX influenza viruses, the nucleotide homology of HA-3’UTR was 77.4%-100%, which did not have obvious mutated sites. The nucleotide homology of H5N6-HA-5’UTR was 91.7%-100%, and the sites of 24 and 31 sites were mutated. From 2013 to 2014, 11 strains of H7N9 influenza virus from Shenzhen were compared with the other H7NX influenza viruses, the nucleotide homology of H7N9-HA-5’UTR was 76.8%-100%, which had multi-mutated sites on 2-6, 9, 10, 12 and 15-17 positions. Conclusions HA-UTR from human-infected H5N6 and H7N9 influenza viruses isolated in Shenzhen district has unique molecular characteristics, its conserved region has relatively high homology and the segment-specific region has genetic polymorphism.