Objective To purify,renature and explore the antiviral effects of recombinant cyanovirin-N(CV-N)on herpes simplex virus type 1(HSV-1).Methods The recombinant CV-N was purified with Ni Sepharose column and renatured by dilution method.The antiviral activities of CV-N were carried out in Vero cells by observing cytopathic effect(CPE)and by using MTT colorimetric assay for vital cell rate.Results SDS-PAGE showed that the purified protein was in the position of 11KDa with only one clear band.The renatured CV-N had little cytotoxic effect on Vero cells,it could not directly inactivate HSV-1 infectivity.CV-N not only interfered in adsorption of HSV-1 to Vero cells but also inhibited HSV-1 biosynthesis in the cells,which were more effective than the positive control Acyclovir.Conclusion CV-N exhibited pronounced antiviral activities agaist HSV-1,further development of CV-N might yield novel candidates of antiviral drugs.

In this study,a standard strain of HSV-1(strain SM44)was used to investigate the antiviral activity of the recombinant Cyanovirin-N(CV-N)against Herpes simplex virus type 1(HSV-1)in vitro and in vivo.Cytopathic effect(CPE)and MTT assays were used to evaluate the effect of CV-N on HSV-1 in Vero cells.The number of copies of HSV-DNA was detected by real-time fluorescence quantitative PCR(FQ-PCR).The results showed that CV-N had a low cytotoxicity on Vero cells with a CC50 of 359.03±0.56 μg/mL,and that it could not directly inactivate HSV-1 infectivity.CV-N not only reduced the CPE of HSV-1 when added before or after viral infection,with a 50% inhibitory concentration(IC50)with 2.26 and 30.16μg/mL respectively,but it also decreased the copies of HSV-1 DNA in infected host cells.The encephalitis model for HSV-1 infection was conducted in Kunming mice,and treated with three dosages of CV-N(0.5,5 & 10 mg/kg)which was administered intraperitoneally at 2h,3d,5d,7d post infection.The duration for the appearance of symptoms of encephalitis and the survival days were recorded and brain tissue samples were obtained for pathological examination(HEstaining).Compared with the untreated control group,in the 5mg/kg CV-N and 10mg/kg CV-N treated groups,the mice suffered light symptoms and the number of survival days were more than 9d and 14d respectively.HE staining also showed that in 5mg/kg CV-N and 10mg/kg CV-N treated groups,the brain cells did not show visible changes,except for a slight inflammation.Our results demonstrated that CV-N has pronounced antiviral activity against HSV-1 both in vitro and in vivo,and it would be a promising new candidate for anti-HSV therapeutics.

International academic conference is important for universities to participate in international cooperation and exchange. The several large-scale international conferences on military medicine held by the Third Medical Military University opened up international exchange in basic medical science, clinical medicine, military medicine, etc. We have adopted a diversity of means, such as taking initiative to hold international conference, facilitating conferences that conform to the current scientific trend and making use of all possible resources to gain the opportunities for holding the international conferences. As a result, we succeeded in absorbing the latest information in science and technology, building up academic reputation, promoting international cooperation, accelerating the training of personnels, creating a good atmosphere for international exchanges, et al.

Objective To explore the role of the DACT2 gene in the occurrence and development of renal cell carcinoma(RCC).Methods The samples of RCC tissues and corresponding tumor-adjacent tissues after radical operation and normal kidney tissues were collected.The methylation specific PCR (MSP) and real time fluorescence reverse transcriptase-PCR (RT-PCR) methods were adopted to detect the methylation status and mRNA expression of DACT2.The streptavidin-peroxidase (SP) method labeled by immunohistochemistry peroxidase was used to examine the expression of β-catenin protein.Then the relationship between DACT2 gene methylation status and mRNA expression with the clinicopathologic characteristics was analyzed.The relationship between DACT2 gene methylation with mRNA and β-catenin expression was analysed,as well.Results The DACT2 mRNA relative expression level in RCC tissues was 0.427±0.025,which was significantly lower than (0.801±0.047) in tumor-adjacent tissues and (0.872±0.022) in normal tissue,the positive rate of DACT2 gene methylation in RCC tissues was 45.76%,which was significantly higher than 6.78% in tumor-adjacent tissues and 5.08% in normal tissues,the difference was statistically significant (P<0.05),while the difference between tumor-adjacent tissues and normal tissues had no statistical significance (P>0.05).The DACT2 gene mRNA expression level in RCC tissues and promoter area methylation occurrence rate had no obvious correlation with the clinical data such as patients age,gender,tumor size,clinical stage and Fuhrman grade (P>0.05).The DACT2 gene mRNA relative level in the methylation group was lower than that in the non-methylation group,the difference was statistically significant (P<0.05).The expression rate of β-catenin protein in cytoplasma in RCC tissues was higher than that in the tumor-adjacent tissues and normal tissues,the difference was statistically significant (P<0.05),moreover,DACT2 gen methylation had a positive correlation with β-catenin protein expression (r=0.324,P=0.012).Conclusion The decrease of DACT2 gene promoter area methylation and mRNA relative expression level may participate in the RCC occurrence,but has no relationship with RCC clinical progression.Methylation occurred in DACT2 gene promoter area may be one of reasons causing mRNA relative expression decrase.DACT2 gene methylation occurrence in RCC tissue might be related to the high expression of β-catenin.

Objective To study the radiofrequency ablation (RA) and vagal denervafion (VD) in surgical treatment of long-standing atrial fibrillation (AF) associated with rheumatic heart disease (RHD).Methods Retrospective analysis the cardiac rhythm by 24-hour Holter monitoring during 5-year follow-up after total Maze procedure accompanied rheumatic mitral valve replacement.Between June 2006 and December 2007,a total of 173 consecutive patients with long-standing AF-associated RHD underwent mitral valve replacement and ablation maze procedure,92 cases had RA alone and 81 had RA + VD.Results Although Kaplan-Meier curve shows that the freedom from AF at 5 years follow-up time were similar(P =0.718),the percentage of antiarrhythmic drug therapy was significant higher in the RA group during early postoperative period(4th month,54.1％ vs.34.7％,P=0.017;5th month,39.2％ vs.21.3％,P=0.018;6th month,23.0％ vs.10.7％,P =0.044),and the percentage of those free by AF was significant lower(6th month,82.2％ vs.93.8％,P =0.023;1st year,76.1％ vs.89.9％,P=0.019).Conclusion Total maze procedure with bipolar radiofrequency ablation is effective to treat longstanding AF associated with rheumatic valve disease.Vagal denervation helped to maintain stable sinus rhythm and lower antiarrhythmic drug therapy at the early stage,but there was no additional benefit after the 1 st year of follow-up,it may be caused from the reactivation of vagal plexus electrical activity.

Objective To investigate the changes of intracellular GSH content in HepG2 cells infected with dengue virus(DV)or stably expressing E or NS3 protein.Methods HepG2 cells were cocultured with DV or inactivitated DV,l h later the viral supernatant was removed.The infected HepG2 cells were collected 10,20,30,40,60 min,and 2,6,12,24,48 h after the beginning coculture and intracellular GSH content was detected by spectrophotometry.Intracellular GSH content was also detected in HepG2 cells stably expressing E protein/NS3(pReceiver-E/HepG2,pReceiver-NS3/HepG2)and those containing empty plasmid(pReceiver/HepG2).Results GSH content showed a decreasing tendency after DV-2 infection.The lowest values were seen 30 min and 2,24 h after infection,which were of significant difference in comparison with those in inactivated DV infected HepG2 cells as well as at other time points.GSH levels in pReceiver-E/HepG2,pReceiver-NS3/HepG2 were significantly lower than those in pReceiver/HepG2.Conclusion DV-2 infection might lead to the GSH depletion in HepG2 cells,and GSH lost from HepG2 cells might undergo a three-step process:virus adsorption/penetration,protein synthesis and budding.E or NS3 protein stably expressed in HepG2 cells can also decrease the GSH levels.

Objective To obtain purified myosin-Vc (Myo5c) protein and prepare its polyclonal antibodies of mouse and rabbit. Methods The fragment encoding Myo5c specific protein (297 amino acids) was amplified with RT-PCR from human gastric mucosa. The product and the prokaryotic expression plasmid pQE-31 containing 6?His were used to construct pQE-31/Myo5c. The recombinant was transformed into E. coli JM109 and induced to express with IPTG. The objective product was purified. BALB/c mice and rabbits were immunized with the purified Myo5c protein and the antiserum was obtained. Titer and specificity of the polyclonal antibodies were determined by ELISA and Western blotting. Results pQE-31/Myo5c was successfully constructed. The fusion protein of Myo5c with molecular weight 42 000 was obtained and purified. The recombined protein showed immunogenicity. Mouse or rabbit antiserum with high level of specific antibodies for Myo5c was obtained. Indirect immunostaining and Western blotting analysis demonstrated that the antibodies could recognize native Myo5c protein. Conclusion Our results suggest that the prepared antibodies might be useful in studying the function of Myo5c in intracellular trafficking of endocytic vesicle, such as viruses.

Objective To evaluate the effect of perioperative administration of Ambroxol combined with Ipratropium on elderly lung cancer patients undergoing thoracoscopic surgery.Methods 82 lung cancer patients aged ≥70 years who were scheduled for thoracoscopic lung resection were randomly assigned into 2 groups:the observation group(n=42) and the control group(n=40).Patients in the observation group were treated with 90 mg Ambroxol(mucosolvan) by intravenous drip and Ipratropium(atrovent) by atomizing inhalation,while patients in the control group were treated with an equal volume of 0.9％ sodium chloride solution.Pulmonary function and changes of arterial blood gases at admission,before surgery and 5 days after surgery,incidences of postoperative atelectasis and pulmonary infection,the rate of return to the intensive care unit(ICU),and the length of postoperative hospital stay were compared between the 2 groups.Results Compared with the control group,the percent predicted forced expiratory volume in 1 second (FEV1％),the ratio of forced expiratory volume in 1 second to forced vital capacity(FEV1/FVC％),the percent predicted maximum ventilation volume per minute (MVV％),and the arterial oxygen pressure (PaO2) were increased in the observation group before surgery [(87.0±8.2)％ vs.(80.6±7.6) ％,(90.4±6.4)％ vs.(81.4±7.0)％,(87.1±5.6)％ vs.(74.6±6.9) ％,(86.6±6.2) mmHg vs.(81.7±6.9)mmHg,t=2.477,2.588,2.937,3.405,respectively,allP＜0.05].TheFEV1％,FEV1/FVC％,MVV％ and PaO2 were higher in the observation group than in the control group 5 days after treatment [(76.4±9.2) ％ vs.(67.3±10.2) ％,(74.7±9.1) ％ vs.(63.0±11.2) ％,(69.5±9.2)％ vs.(60.1±9.2) ％,(79.5±11.5) mmHg vs.(70.1±11.8) mmHg,t 2.583,2.987,2.778,2.666,respectively,all P＜0.05].The incidences of postoperative atelectasis,pulmonary infection and the length of postoperative hospital stay were lower or less in the observation group than in the controlgroup[7.1％ vs.25.0％,11.90％ vs.32.50％,(8.5±1.8) days vs.(12.1±2.6) days,x2=4.897,5.072,2.351,respectively,all P＜0.05].No significant difference in the rate of return to ICU was found between the two groups(P＞0.05).Conclusions The combination of perioperative Ambroxol and Ipratropium can effectively improve lung function by improving ventilation and gas exchange function,reduce postoperative pulmonary complications and shorten the length of postoperative hospital stay in lung cancer patients aged 70 years and over.

Background and purpose:MicroRNA (miRNA, miR) plays an important regulatory role in cancer. miR-222 is reported to be up-regulated in various tumors, but its role in renal cell carcinoma (RCC) remains unclear. In this study, we detected the expression of miR-222 in both RCC and adjacent tissue samples. The aim of this study was to investigate the role of miR-222 in RCC. Methods:The expression levels of miR-222 in RCC tissue samples were quantified by quantitative real-time polymerase chain reaction (qRT-PCR). DDIT4 and LC3-Ⅱ protein expressions were determined by Western blot. Dual luciferase assay was performed to verify the target of miR-222. EGFP-LC3 microscopy assay was performed to assess autophagy. Results:Results from qRT-PCR showed that the expression of miR-222 was up-regulated in RCC tissues. Knockdown of miR-222 with speciifc antagomiR decreased the cell viability of 786-O cells, whereas overexpression of miR-222 increased the cell viability (P<0.01). The levels of DDIT4 were up-regulated in 786-O cells transfected with miR-222 antagomiR, whereas overexpression of miR-222 induced the down-regulation of DDIT4 expression. Data from dual luciferase assay indicated that miR-222 directly targeted the expression of DDIT4. Consistently, the expression of DDIT4 in RCC tissues was down-regulated compared with adjacent tissues. Knockdown of miR-222 in 786-O cells induced a signiifcant increase of autophagosome formation and LC3 lipidation.These results supported that miR-222 could inhibit autophagy in RCC cells, which may affect the clinical characteristcs of RCC. Conclusion: miR-222 is up-regulated in RCC and can inhibit the autophagy of RCC cells through down-regulating the expression of DDIT4.

Objective: To investigate the effects of hinokitiol on the proliferative inhibition and apoptosis induction in human clear cell renal cancer 786-O cells. Methods:CCK-8 assays were performed to analyze the effects of hinokitiol on the proliferation of 786-O cells. The apoptosis rate was determined by flow cytometry. EGFP-LC3 microscopy assays were performed to assess the autoph-agy flux. Cleaved Caspase-3, LC3, and P62 were detected by Western blot. Results: Hinokitiol could inhibit the proliferation of the 786-O cells and could induce cell apoptosis via Caspase pathway. Hinokitiol induced the autophagy of 786-O cells, increased LC3 ex-pression, and downregulated P62 expression. Conclusion: Hinokitiol can inhibit the proliferation of 786-O cells and can induce cell apoptosis via autophagy induction.