Ebola virus, the cause of severe and fatal hemorrahagic fever in humans, belongs to filovirus family. This study was designed to establish a cell-based screening and evaluation system in the pharmacological study of antivirus compounds. Three reporter systems were established with recombinant pseudoviral luciferase of HIV core (pNL4-3.Luc.R(-)E(-)) packed with filovirus glycoprotein (EBOV-Zaire GP/HIV-luc, EBOV-Sudan GP/HIV-luc and Marburg GP/HIV-luc), which are required for virus entry of cells. The level of filovirus entry was determined by the expression of luciferase reporter gene in the infected cells. For screening of filovirus entry inhibitors, the vesicular stomatitis G packed pseudovirions (VSVG/HIV-luc) was used to determine the compound specificity. The results of known filovirus entry inhibitors demonstrated successful establishment of the new model systems, which would be useful in high throughput screening of anti-filovirus drugs in the future.
Antiviral Agents ; pharmacology ; Drug Evaluation, Preclinical ; methods ; Ebolavirus ; drug effects ; physiology ; Genes, Reporter ; Glycoproteins ; genetics ; Hemorrhagic Fever, Ebola ; Humans ; Luciferases ; Viral Proteins ; genetics ; Virus Internalization ; drug effects

In traditional Chinese medicine,medical practice is combined with qi,which forms special qi-theory.Body fluid which includes blood and Jin-ye is the fluid structure of human being.Nutrient qi and defensive qi depend on these two kinds of body fluid.Defensive qi not only maintains physiological functions of Jin-ye,but also participates pathomechanism such as Jin-ye consumption,retention,etc.The identity of defensive qi and Jin-ye plays an important role in theory and clinic.

20 mg/L again.Children without treatment regulared exam for phe concentration level in blood and test for physical and mental capability development.Results Forty-eight children with PKU were diagnosed in 355 615 newborns who were collected from June 1999 to April 2005.The incidence rate of PKU was 1/7408,carriers with PKU gene was 1/48.There was no significant difference in physical and mental condition compared with that of normal children.Conclusions The treatment results for children with PKU is significant.It has good social and economic value.It is one of the important measures for reducing infant defect and improving population quality.

A L-histidine producing mutant was derived from Corynebacterium glutamicum HZ4221(TRA R DCP R AMT R histidase - )by means of mutagenesis with N-methy-N′-nitro- N-nitrosoguanidine(NTG).Contrast to original strain,the amount of histidine accumulation reached to a level of 5.31g/L in a medium containing 80g/Lglucose and 30g/L ammonium sulfate after cultured for 72 hours; the transketolase activity reduced to a degree of 15.7%.The utilization of the carbon sources,genetic stability,effect of metal ions were also been investigated in this paper.

Objective To quantitatively assess the left ventricular systolic dyssynchrony in patients with varied degrees of chronic congestive heart failure after old myocardial infarction(OMI) by real-time three-dimensional echocardiography(RT-3DE) and investigate the clinical value of the systolic dyssynchrony index(SDI). Methods Forty patients with congestive heart failure after OMI (infarction group) were divided into the severe dysfunction group (LVEF ≤35 %) and the mild dysfunction group (35 % ＜ LVEF＜50%) ,and 30 normal subjects served as the control. RT-3DE was performed on all subjects to obtain the 17-segmental time-volumetric curves and global systolic function. SDI changes in above groups and the correlation between SDI and LVEF were analyzed. Results The SDI of the infarction group was significantly higher than that of the normal control group ( P ＜0. 01 ). The SDI of the severe dysfunction group was significantly higher than that of the mild group (P＜0.01). SDI and LVEF were negatively correlated ( r = -0.84, P ＜0. 01 ). The dyssynchrony rate in the infarction group was 85 %,in the severe dysfunction group was 100%, in the mild group was 75%. Conclusions Left ventricular systolic dyssynchrony is prevalent in patients with OMI, and it is negatively correlated with the LVEF. SDI is a sensitive indicator in assessing left ventricular systolic dyssynchrony. RT-3DE has a unique advantage in the evaluation of the left ventricular systolic dyssynchrony,especially in the patients with myocardial infarction.

Objective To investigate the effect of hyperuricemia(HUA) on inflammation and endothelin-1 (ET-1) production and treatment of Benzbnomanone in hypertensive patients.Methods 90 initial hypertensive patients were enrolled from the inpatient division and clinic of our hospital,60 patients of them were identified HUA,and 30 patients were normal in uric acid as control.All these hypertensive patients with HUA were treated with basic anti-hypertensive drugs,of them 30 patients were additionally treated with Benzbromarone table 50mg for 8 weeks.The levels of inflammation indices and ET-1 were compared between these hypertensive patients with HUA and hypertensive patients with normal serum uric acid,also hypertensive patients with HUA treated with or without Benzbromarone for 8 weeks.Results Compared with the hypertensive patients with normal serum uric acid,levels of ET-1,interleukin-1 (IL-1) and high-sensitive C reactive protein (hsCRP) were higher in the hypertensive patients with HUA.Also,the levels of these indices were positively correlated with the level of serum uric acid [(86.6 ± 4.8) pg/ml vs (82.4 ±6.9)pg/ml; (47.6 ±6.2)mg/L vs (19.1 ±4.1) mg/L; (3.4 ±0.8)mg/L vs (2.9 ± 1.1)mg/L,r =0.81,0.74,0.83,all P ＜ 0.05].Benzbromarone could effectively decrease the levels of ET-1,IL-1and hsCRP in the hypertensive patients with HUA [(49.8 ± 5.0) pg/ml vs (87.5 ± 5.9) pg/ml ; (17.6 ±8.8) mg/L vs (48.2 ± 7.0) mg/L; (1.7 ± 0.7) mg/L vs (3.5 ± 0.9) mg/L,all P ＜ 0.05].Conclusions HUA could increase the levels of inflammation and ET-1,while Benzbromarone effectivelv decreased these changes.Decreasing the level of serum uric acid would retard the process of atherosclerosis in the hypertensive patients with HUA.

[ ABSTRACT] AIM:To investigate the effect of maxadilan, which specifically activates pituitary adenylate cycla-se-activating polypeptide type I receptor (PAC1 receptor), on the proliferation, apoptosis and differentiation potential of human adipose-derived stem cells ( ASCs) .METHODS:ASCs from human adipose tissue were isolated by enzymatic di-gestion and cultured.ASCs were confirmed by the analysis of the markers for cell phenotypes by flow cytometry ( FCM) and adipogenic/osteogenic induction.The effect of maxadilan on ASCs viability was analyzed by CCK-8 assay and FCM.ASCs were irradiated by ultraviolet C ( UVC) at 254 nm and the absorbance of apoptotic ASCs induced by various doses of UVC was measured by CCK-8 assay.ASCs were exposed to 702 J/m2 UVC for 24 h to induce apoptosis.The effect of maxadilan on ASC apoptosis was analyzed by FCM and the determination of caspase 3 and caspase 9 levels.RESULTS:Adipose-de-rived stem cells were confirmed by the detection of the positive expression of cell phenotypes including CD29, CD44, CD59 and CD105 by FCM.The data of CCK-8 assay revealed that ASCs treated with maxadilan (80 nmol/L) had the strongest ability of proliferation.The data of FCM also demonstrated that the addition of 80 nmol/L maxadilan to ASCs in experimen-tal group markedly improved the proliferation capacity of the cells compared with control group (P<0.05).The apoptosis of ASCs exposed to 702 J/m2UVC was dramatically inhibited by the treatment with maxadilan (80 nmol/L).Such process involved the caspase signaling pathway including caspase 3 and caspase 9.There was statistical significance (P<0.05) between experiment group ( ASCs irradiated by UVC and supplemented with maxadilan) and control group ( ASCs only irra-diated by UVC) .Meanwhile, adipogenic and osteogenic differentiation potentials were both positive in experiment group and control group.CONCLUSION:Maxadilan promotes proliferation and inhibits apoptosis of the ASCs.The differentia-tion potential of ASCs toward adipogenic and osteogenic lineages wouldn’ t be altered by maxadilan.Maxadilan would bene-fit to growth and expansion of ASCs in vitro.

[ABSTRACT]AIM:ToinvestigatethepromotingroleofTranswellcontactco-culturesysteminthegrowthand differentiation of single-dissociated induced pluripotent stem cells (iPSCs).METHODS:Bovine corneal endothelial cells (CECs) at passage 1~2 (P1~2) were seeded on the underside of Transwell inserts placed into culture plates and were cultured in 37 ℃and 5%CO2 for 8 h.Accutase digestion and 40μm filter process disaggregated colony-aggregated iPSCs into single-dissociated iPSCs , and the cells were seeded on the inside of Transwell inserts with CECs in medium of mTeSR 1 for 3 d and then in low-glucose DMEM supplemented with 10% FBS for 2 weeks.The characteristics and differentiation markers were evaluated by real-time fluorescence quantitative polymerase chain reaction ( qPCR ) , immunofluorescence staining, live&dead cell staining and alkaline phosphatase (ALP) staining.The group of iPSCs cultured in conventional medium was used as control group 1.The group of single-dissociated iPSCs co-cultured with CECs was set as experimental group, while single-dissociated iPSCs without co-culture were as control group 2.RESULTS: The bovine CECs showed typical hexagonal cobblestone shape .iPSCs showed colony-like growth , while became single-dissociated cells after Tran-swell contact co-culture with bovine CECs for 3 d.The single-dissociated iPSCs positively expressed the undifferentiated markers, Nanog and Oct4.The mRNA expression levels of Nanog , Oct4 and Sox2 between experimental group and control group 1 were both positive and had no statistical significance difference (P>0.05).The dead cells in experimental group decreased significantly, and there was statistically significant difference compared to control group 2 (P<0.01).After 14 d of induced differentiation co-culture , the single-dissociated iPSCs showed rather uniform polygonal morphology , increased dimension and no obvious colony existence .Negative ALP staining, positive immunofluorescence staining for ZO-1, AQP1 and CD31, and negative for CD34 and CD133 were also observed.The results of qPCR showed that the mRNA expression of Oct4, Nanog and Sox2 significantly decreased , and had statistically significant difference compared with control group 1 (P<0.01).CONCLUSION: When co-cultured with bovine CECs, iPSCs morphologically changed to endothelial-like cells and expressed some markers of CECs .Transwell contact co-culture system not only enhances the growth of single-dis-sociated iPSCs , but also promotes their differentiation .

Objective To investigate the transfection efficacy and expression of Ang-1 gene and proangiogenesis in vitro and vivo by ultrasound-mediated microbubble destruction.Methods 293T cells were divided into three groups:group A was given hAng-1 plasmid and microbubbles plus ultrasonic irradiation,group B was given hAng-1 plasmid and ultrasound,group C was given hAng-1 plasmid only (without ultrasound).Forty-eight hours after transfection,the transient expression rate was observed under fluorescence microscopy and flow cytometry.RT-PCR and Western blot analysis were taken to evaluate the mRNA and protein expression of Ang-1 respectively.Twenty-seven rabbit models of ligated left circumflex branch coronary artery were divided into 3 groups randomly as follow:group Ⅰ (accepted intravenous injection of SonoVue microbubble and Ang-1 plus ultrasonic irradiation),group Ⅱ (accepted intravenous injection of Ang-1 with ultrasound),group Ⅲ (control group).Myocardial contrast echocardiography (MCE) was executed on all animals before and after the treatment.Two weeks after gene delivery,RT-PCR and Western blot analysis were taken to evaluate mRNA and protein expression of Ang-1 respectively.Microvessel density (MVD) counting of infracted myocardium,observed by Factor Ⅷ immunochemical staining,was performed to value the proangiogenesis effect of Ang-1 delivered by ultrasound mediated cavitation of microbubble.Results Green fluorescence was observed in group A and B by fluorescence microscopy,which was negative in group C.The transfection expression rate was significantly improved in group A ( P ＜ 0.01).In vivo,Microbubbles could be observed in former ischemic myocardium in MCEexamination and the Ang-1 mRNA and protein could be detected in group Ⅰ.On the other hand,the contrast agent was defected obviously and none of the animals showed Ang-1 mRNA and protein expression in other two groups.The MVD counting showed significant improvement in group Ⅰ whereas other two groups didn't.ConclusionsMicrobubble-enhanced ultrasound exposure can improve the Ang-1 gene transfection expression rate observably both in vitro and in vivo.This strategy for delivering has great proangiogenesis effect in vivo.

Objective To certificate the effects on transfection ratio and cells viability of ultrasound (US) acoustic intensity, radiation duration, microbubbles concentration and DNA concentration in delivery human angiopioetin-1 gene (hAng-1) into 293T cells by SonoVue microbubbles and decide the optimal transfection parameters. Methods Mix 293T cells and SonoVue microbubbles linked with eGFP-C_3-hAng-1 in a different way, detect the gene transfection ratio and cells viability under the various US intensity, radiation duration, microbubbles and DNA concentrations. Results The gene expression would be increased if enhanced the intenstiy of US,radiation time,microbubbles and DNA concentrations,and the cells viability would be kept more than 90% ( P <0. 01). Whereas,if the US intensity increased over 1. 5 W/cm~2 ,the duration over 30 s and microbubbles and DNA concentrations over 20% and 15 mg/L respectively,the gene expression would not increase significantly ( P > 0. 05),whereas coupled with obviously decreased cells viability( P <0. 01). Conclusions The optimal conditions of deliver hAng-1 gene into 293T cells by SonoVue microbubbles was mixing cells and microbubbles in a cell wall-sticky way,US intensity was 1. 5 W/cm~2, duration 30 s,20% microbubbles and 15 mg/L DNA concentration.