Objective To study the influence of the modification of the special neutralizing epitopes of the HIV-1 envelope (Env) on its assembly of functional pseudovirus and neutralizing activity. Methods Site-directed mutations were performed using cycling mutagenesis and selection of mutants with Dpn I . With this method, the 2G12 and 2F5 neutralizing epitopes were integrated into Env of subtype BC which was without the two epitopes, then the capability of forming pseudovirus and the neutralizing activity against 2G12 and 2F5 were compared with pre-modified Env. Results The special Env neutralizing epitopes of five HIV pseudovirus (BC02, BC03, BC04, BC05 and BC12) were modified. Among the five pseudovirus, BC04 and BC12 pseudovirus can't be formed after the 2G12 epitope was modified, whilst the BC02, BC03 and BC05 pseudovirus can be formed after the 2G12 and 2F5 epitopes were added, and there was no variation of the pseudovirus titer; On the aspect of neutralizing activity, BG03 pseudovirus against 2G12 and 2F5 was enhanced, BC02 and BC05 pseudovirus against 2F5 was enhanced while which against 2G12 was not changed. Conclusion The modification of 2G12 epitope influences the forming of pseudovirus and the addition of neutralizing epitopes can enhance the neutralizing activity of pseudovirus, which offers new approach for the optimization of HIV immunogen.

Objective To compare real-time fluorescence quantitative PCR with general PCR in detecting bovine and porcine derived materials in hydrolysate samples.Methods DNA were extracted from hydrolysate samples which prepared by different steps by real-time fluorescence quantitative PCR and general PCR.Results DNA of bovine and porcine could be detected by real-time fluorescence quantitative PCR and general PCR in samples prepared in the processes before enzymolysis solution, but not detected in samples from supermatant to the fourth ultrafiltrate.Conclusion Both real-time fluorescence quantitative PCR and general PCR can be applied to detect the fragments in hydrolysate samples.And real-time fluorescence quantitative PCR has the advantage such as rapid,convenient, non-environment-polluted, good repeatability, which improves the quality and efficiency.

Objective To investigate the dynamic expressions of epidermal growth factor receptor (EGFR) in mice with liver fibrosis and the effect of bone morphogenetic protein-7 (BMP-7) intervention on the expression of EGFR,and to explore a new therapy target for fibrosis.Methods A total of 30 healthy male ICR mice were randomly divided into three groups:6 mice in control group,18 mice in hepatic fibrosis group and 6 mice in BMP-7 intervention group.The model of mice with liver fibrosis was established by subcutaneous injection of carbon tetrachloride (CCl4) for 12 weeks.After administration of CCl4 for 8 weeks,human recombinant BMP-7 was given into mice in intervention group by intraperitoneal injection for 4 weeks.Hematoxylin-Eosin and Masson staining of liver tissues were employed to observe the pathological changes,and the semi-quantitative analysis of liver fibrosis was performed.Blood withdrawn from inferior vena cava was detected for levels of alanine aminotransferase (ALT),aspartate aminotransferase (AST) and albumin (Alb).The expressions of transforming growth factor-β1 (TGF-β1)mRNA and TGF-β1,EGFR,phosphorylation EGFR (pEGFR) protein in each group were detected using quantitative real time polymerase chain reaction and Western blot.Measurement date was compared using analysis of variance and Pearson correlation analysis.Results The model of mice with liver fibrosis was successfully established.In model group,the serum levels of ALT and AST increased,while the level of Alb decreased gradually.All these biochemical index improved after intervention of BMP-7 (ALT:[153.9±18.1] U/L vs [191.3±24.5] U/L;AST:[177.8±19.2] U/L vs [206.6±25.0] U/L;Alb:[25.4±0.9] g/L vs [22.2±1.2] g/L; all P＜0.05).With the progress of fibrosis,TGF-β1,EGFR and pEGFR protein expressions increased gradually in model group and reached a peak at week 12,which was significantly different compared to the control group (all P＜0.05).In BMP-7 intervention group,the expressions of the three proteins decreased significantly compared to model group (TGF-β1:0.256 ± 0.006 vs 0.287±0.014,EGFR:1.061±0.017 vs 1.094±0.014,pEGFR:0.855±0.053 vs 1.007±0.063;all P＜0.05).Additionally,linear correlation analysis showed that expressions of both EGFR and pEGFR proteins were positively correlated with TGF-β1 protein (rs =0.895 and 0.859,respectively; both P＜0.05).Conclusions BMP-7 can suppress the pathogenesis of mouse liver fibrosis.The mechanism may rely on the regulation of EGFR and TGF-β1 expressions.

Objective To pan and characterize anti-HIV-1 Fab by the phage antibody library technology. Methods Total RNA were extracted from lymphocytes which were isolated from peripheral blood collected from asymptomatic HIV-1 infected donors with high titer antibody against HIV-1. The genes of heavy chains Fd fragment and light chains of antibody were amplified by RT-PCR. The phagmids pComb3X cloned Fd and light chain genes were transformed into E. coli XL1-Blue by electroporation to construct phage Fab library. By three runs of "absorption-elution-neutralization-enrichment", the clones were induced by IPTG and characterized by ELISA. The positive clones were sequenced and analyzed the sequences. Subsequently, Fab antibodies of these positive clones were induced to expressed and purified, then the recombinant virus neutralization assay was performed. Results A phage Fab library was constructed with 8×106 members, and 11 positive clones were obtained by detecting IPTG-induced-expressing Fabs with ELISA. By analysis of the sequences, 10 light chain genes and 8 Fd genes were ensured to be obtained. Compared with the genes of anti-HIV-1 antibodies in HIV sequence database, the gene sequences we obtained were highly homologous to some patent genes of anti-HIV-1 gp120 antibodies in HIV sequence database( light chains with 60%-90% identity, Fd with 71%-85% identity); The CDRs of these positive clones were determined by comparing the positive clone genes with antibodies' genes in V base database, furthermore, CDRH3 of these positive clones has the length of 12-22 aa. Strand shift had little effect to improving affinity of our Fab clones. Fab antibodies were induced to express at the concentration of ＞ 10 mg/L. Three Fab antibodies neutralize HIV-1 virus to some extent. Conclusion The studies will provide the basis on further study on the anti-HIV-1 Fabs obtained successfully.

Objective To study the expressions of osteopontin (OPN),caspase-3 and mt-P53 proteins, and their relationship in gliomas. Methods Seventy gliomas specimens of patients (glioma group) were selected, and 10 samples of non-glioma brain tissue were used as control group. The SP method was used to detect the positive rates of protein expressions of OPN, caspase-3 and mt-P53 between two groups. The relationship between protein expressions of OPN, caspase-3 and mt-P53 in gliomas and grade of gliomas were detected by Western blot assay. Spearman rank correlation was compared between the positive expression of OPN, caspase-3 and rate mt-P53. Results The positive expression rates of OPN and mt-P53 were significantly higher in glioma group (64.29%and 60%) than those of control group (no positive expression), but the positive expression rate of caspase-3 was significantly lower than that of control group (47.14%vs. 90%, P<0.05). There were no significant differences in OPN, caspase-3 and mt-P53 expressions between different gender, age, tumor size and tumor position (P>0.05). The higher the WHO classification, the higher the positive expression rates of OPN and mt-P53 (P<0.001), and the lower the positive expression rate of caspase-3 (P<0.001). With the increased level of glioma grade, OPN and mt-P53 protein levels were increased, but caspase-3 protein expression level was decreased. There was a negatively correlation between OPN and the positive expression of caspase-3, but there was a positive correlation between OPN and the expression of mt-P53 (rs=-0.720 and 0.722, P<0.05). There was a negative correlation between caspase-3 and mt-P53 expressions (rs=-0.556, P<0.05). Conclusion The higher the WHO classification, the higher the positive expression rates of OPN and mt-P53, while the lower the positive expression rate of caspase-3. The study reveals that OPN, caspase-3 and mt-P53 expressions are associated with the occurrence and the progress of gliomas. The combined detection of them can contribute to the judgment of biological behavior of gliomas.

Objective To analyze the genetic characteristics of echovirus 6 ( E-6) strains isolated from patients with acute meningitis/encephalitis syndrome ( AMES) in 2014 and sewage samples in 2013—2014 in Shandong province and to investigate their correlations.Methods Enterovirus strains were isolated from cerebrospinal fluid, stool and throat swab samples collected from 940 cases of AMES and 96 sewage samples used for environmental surveillance.The positive isolates were identified by molecular typing meth-od.Homologous and phylogenetic analyses based on the VP1 sequences of E-6 isolates were performed.Re-sults Altogether 47 E-6 strains were isolated from patients with AMES in 2014, accounting for 29.56%of all isolated enteroviruses ( EVs) strains.No E-6 strains were isolated from AMES cases in 2013.Data of the environmental surveillance showed that E-6 virus strains had been frequently detected in sewage samples since the summer of 2013 to the end of 2014.In total, 40 E-6 virus strains were isolated (7.87% of total isolated EVs strains) in 2013 and 139 E-6 virus strains (26.18%) in 2014.Phylogenetic analysis indicated that the E-6 isolates recruited in this study belonged to clusters A and C with high intracluster sequence iden-tities between AMES and environmental isolates.The nucleotide identities were 98.3%-100% among cluster A E-6 virus strains isolated from AMES cases in 2014 and 96.6%-100% among cluster A E-6 virus environ-mental isolates during the surveillance year 2013—2014.The cluster A E-6 virus strains shared 97.1%-100% nucleotide identities between the AMES and environmental isolates.For cluster C E-6 virus strains, the nucleotide identities were 100%, 98.7%-100% and 99.1%-100%, respectively.Conclusion The epidemic of viral encephalitis in Shandong province in 2014 was associated the transmission of two lineages of E-6 virus.Environmental surveillance revealed the potential epidemic of E-6 virus even before the epidemic of viral encephalitis in Shandong province, indicating the possibility of using environmental surveillance for early warning of related diseases.

To analyze the genetic characteristics of a polio-I highly variant vaccine recombinant virus in Shandong Province (China) in 2011 and to identify isolates from healthy contacts, two stool specimens from one patient with acute flaccid paralysis (AFP) and 40 stool specimens from his contacts were collected for virus isolation. The complete genome of poliovirus and VP1 coding region of the non-polio enterovirus were sequenced. Homologous comparison and phylogenetic analyses based on VP1 sequences were undertaken among coxsackievirus (CV) B1, CV-B3 isolates, and those in GenBank. One poliovirus (P1/11186), CV-A4 and CV-A8 were isolated from the AFP patient; one CV-A2, Echovirus 3 (E-3), E-12 and E-14, ten CV-B1, and five CV-B3 strains were isolated from his contacts. These results led us to believe that there may be a human enterovirus epidemic in this area, and that surveillance must be enhanced. P1/11186 was a type-1 vaccine-related poliovirus; it combined with type-2 and type-3 polioviruses in 2A and 3A regions, respectively. There were 25 nucleotide mutations with 9 amino-acid alterations in the entire genome. There were 8 nucleotide mutations with 5 amino-acid alterations in the VP1 region compared with the corresponding Sabin strains. Homology analyses suggested that the ten CV-B1 isolates had 97.0%-100% nucleotide and 98.9%-100% amino-acid identities with each other, as well as 92.6%-100% nucleotide and 99.2%-100% amino-acid identities among the five CV-B3 isolates. Phylogenetic analyses on the complete sequences of VP1 among CV-B1 and CV-B3 isolates showed that Shandong strains, together with strains from other provinces in China, had a close relationship and belonged to the same group.
Base Sequence ; Capsid Proteins ; genetics ; immunology ; Child, Preschool ; China ; Humans ; Male ; Molecular Sequence Data ; Phylogeny ; Poliomyelitis ; etiology ; prevention & control ; virology ; Poliovirus ; classification ; genetics ; immunology ; isolation & purification ; Poliovirus Vaccines ; adverse effects ; genetics ; immunology

Objective To analyze the correlation among self efficacy, coping mode and negative mentality in youth with primary liver cancer.Methods Totally 203 young patients with primary liver cancer were investigated by adopting a cross-sectional survey method, and applying hospital anxiety and depression scale (HADS), general Self efficacy scale (GSES) and medical coping modes questionnaire (MCMQ).Results The positive rate of anxiety was 57. 14% in youth patients with primary liver cancer, and the positive rate of depression was 67.98%;the path analysis showed that self efficacy had a direct negative effect on depression and anxiety( Sβ=-0. 452,-0. 386;P<0. 05; self-efficacy had an indirect negative effect on depression and anxiety ( Sβ=-0.134,-0.120,P<0.05) . The facing coping mode had a direct negative effect on depression and anxiety (Sβ=-0.234,-0.244;P<0.05); the yielding coping dimension had a direct positive effect on anxiety and depression(Sβ=0.222,0.176;P<0.05).Conclusions The psychological health of young patients with primary liver cancer are in low status. Self-efficacy, facing coping and yielding coping are the important factors which influence patients′mental health.

Objective:To illustrate the effectiveness of Teachermate platform combined with problem-based learning (PBL) in gynecology and obstetrics teaching.Methods:Students from different classes were randomly divided into experimental group and control group, and the general data of the two groups were compared. The two groups respectively adopted Teachermate platform combined with PBL and traditional lecture teaching methods. The class attendance rate and the scores of relevant chapters in the final examination were analyzed through statistical methods for assessing the enthusiasm of students and teaching quality in class in terms of the same content and the same class hours. A questionnaire survey was conducted to know students' evaluation of this teaching mode, evaluating its teaching effect and satisfaction comprehensively.Results:There was no difference in general information between the two groups. The class attendance rate of the experimental group (274/279) was significantly higher than that of the control group (61/90) ( P=0.00), while the scores of the relevant chapters in the final exam were also significantly higher ( P=0.00). Questionnaire survey results among 93 students in the experimental group were taken back 93 valid questionnaires (the effective rate, 100%), showing that most of the students were very satisfied with this teaching mode (90/93) and hoped to apply it to more subjects teaching (89/93). Conclusion:The application of Teachermate platform combined with PBL in gynecology and obstetrics class teaching contributes to improve students' participating enthusiasm and can effectively improve the quality and efficiency of teaching. Most students highly recognize the teaching method, which is worth applying and popularizing.

Objective:To illustrate the effectiveness of Micro-Teachermate platform combined with problem-based learning (PBL) in gynecology and obstetrics teaching.Methods:Students from different classes were randomly divided into experimental group and control group, and the general data of the two groups were compared. The two groups respectively adopted Micro-Teachermate platform combined with PBL and traditional lecture teaching methods. The class attendance rate and the scores of relevant chapters in the final examination were analyzed through statistical methods for assessing the enthusiasm of students and teaching quality in class in terms of the same content and the same class hours. A questionnaire survey was conducted to know students' evaluation of this teaching mode, evaluating its teaching effect and satisfaction comprehensively. SPSS 17.0 was used for t test and chi-square test. Results:There was no difference in general information between the two groups. The class attendance rate of the experimental group (274/279) was significantly higher than that of the control group (61/90), while the scores of the relevant chapters in the final exam were also significantly higher ( P<0.01). Questionnaire survey results among 93 students in the experimental group were taken back 93 valid questionnaires (the effective rate, 100%), showing that most of the students were very satisfied with this teaching mode (90/93) and hoped to apply it to more subjects teaching (89/93). Conclusion:The application of Micro-Teachermate platform combined with PBL in gynecology and obstetrics class teaching contributes to improve students' participating enthusiasm and can effectively improve the quality and efficiency of teaching. Most students highly recognize the teaching method, which is worth applying and popularizing.