基于Tet-On 3G的IFITM3诱导表达MDCK细胞系的建立及功能分析

利用Tet-On 3G系统构建了含人IFITM3基因的真核表达质粒pTRE3G-IFITM3,并将其与调控质粒pLVX-Tet3G共转染犬肾细胞(MDCK),转染后48 h用G418和嘌呤霉素进行筛选,用多西环素(Dox)对获得的细胞系进行诱导表达鉴定,并进行Dox敏感性分析、IFITM3~+细胞百分数及定位分析.用含萤光素酶(Luciferase)报告基因的禽流感病毒H5/H7蛋白或VSV G蛋白包裹的假型慢病毒颗粒进行感染实验,检测萤光素酶活性.结果表明,筛选获得了携带人IFITM3的MDCK诱导表达细胞系,且IFITM3表达量与Dox剂量和诱导时间相关;确定Dox工作浓度为2.5μg/mL,诱导12 h时IFITM3~+细胞数达75%以上,且IFITM3在晚期内体/溶酶体存在分布;假型病毒感染及萤光素酶活性分析表明,IFITM3可显著抑制禽流感病毒H5,H7和VSV G蛋白介导的病毒进入,为深入探究其具体的抑制机制奠定了基础.     

Design,Synthesis and Biological Evaluation of Pentacyclic Triterpene Dimers as HCV Entry Inhibitors

A series of triterpene dimers bearing different scaffold were designed and synthesized via CuAAC reaction. Their anti‐HCV entry activities were evaluated by HCVpp and VSVpp entry assays. It was found that echinocystic acid (EA) and its dimer were still necessary for maintaining anti‐HCV entry activity, and replacement of EA by other triterpenes might significantly decrease its anti‐viral activities. Using a linker bearing a piperazine group, compound 14 dramatically increased its potency with IC₅₀ at 2.87 nmol/L. In addition, the undesired hemolytic effect of all these compounds was removed.     

Downstream processing of Vero cell-derived human influenza A virus (H1N1) grown in serum-free medium

A downstream processing was examined for Vero cell-derived human influenza virus (H1N1) grown in serum free medium. Vero cell banks were established in serum free medium and characterized according to regulatory requirements. Serum free Vero cells were grown on Cytodex 3 microcarriers in 5L bioreactor and infected with influenza A virus (A/New Caledonia/99/55). The harvests were processed with the sequence of inactivation, clarification, anion exchange chromatography (DEAE FF), Cellufine Sulfate Chromatography (CSC) and size exclusion chromatography (Sepharose 6FF). Host cell DNA (hcDNA) was mainly removed with DEAE FF column and CSC by 40 and 223 fold, respectively. Most of Vero cell proteins were eliminated in CSC and Sepharose 6FF unit operation by about 13 fold. The overall scheme resulted in high recovery of hemagglutinin (HA) activity and the substantial removal of total protein, host protein and DNA. The total protein content and DNA content per 15 μg HA protein in final product was 89 μg and 33 pg, respectively, which complied with regulatory requirements for single strain influenza vaccines. SDS-PAGE analysis and Western blotting confirmed the purity of the final product. In conclusion, the suggested downstream process is suitable for the purification of microcarrier-based cell-derived influenza vaccine.     

Reduced surface area chromatography for flow-through purification of viruses and virus like particles

A method for flow-through purification of viruses and virus like nano-particles using a combination of binding and size-exclusion chromatography was developed. This technique relies on minimizing the external surface area per unit volume available for virus binding by increasing the mean diameter of the beads used in the column. At the same time the impurity binding capacity of the column is maximized by utilizing beads with multiple functionalities of the optimum size. Purification of different types of viruses and virus-like-particles could be achieved using this technique. Flow-through purification of influenza virus using this technique yielded virus recoveries greater than 70-80% coupled with impurity removal greater than 80%. Finally an approach to optimize and facilitate process development using this technology is presented. Since the impurity binding occurs via a non-specific mechanism and virus recovery is achieved through reduced surface area, the technique is not limited to specific types of viruses and offers the potential as a universal purification tool.     

Parallel differential mobility analysis for electrostatic characterization and manipulation of nanoparticles and viruses

The electrophoretic mobility of charged, airborne nanoparticles (NPs) or macromolecules and their specific complexes opens new avenues for their analysis and handling. The newly developed parallel differential mobility analyzer in combination with an electrostatic particle sampler enables not only the characterization of bio-NPs, but even their sampling while preserving their bioactivity (e.g., the enzyme activity of galactosidase). Precondition for the applicability of this technique is a well-defined charging status of the NPs in question. This charge conditioning can be achieved by means of a radioactive source, Po-210, even if the yield in terms of charged particles is low for sub-20-nm particles and the aging of the source influences the size spectra measured. Nevertheless, this technique enables size-defined sampling and enrichment, combined with real-time measurement of the size of both NPs and viruses. Furthermore, it allows determination of the number of attached biospecific antibodies, thereby providing information about the surface coverage of viruses by antibodies.     

Cellular automata approach for the dynamics of HIV infection under antiretroviral therapies: The role of the virus diffusion

We study a cellular automata model to test the timing of antiretroviral therapy strategies for the dynamics of infection with human immunodeficiency virus (HIV). We focus on the role of virus diffusion when its population is included in previous cellular automata model that describes the dynamics of the lymphocytes cells population during infection. This inclusion allows us to consider the spread of infection by the virus-cell interaction, beyond that which occurs by cell–cell contagion. The results show an acceleration of the infectious process in the absence of treatment, but show better efficiency in reducing the risk of the onset of AIDS when combined antiretroviral therapies are used even with drugs of low effectiveness. Comparison of results with clinical data supports the conclusions of this study.     

Delamination in sandwich panels due to local water slamming loads

We study delamination in a sandwich panel due to transient finite plane strain elastic deformations caused by local water slamming loads and use the boundary element method to analyze motion of water and the finite element method to determine deformations of the panel. The cohesive zone model is used to study delamination initiation and propagation. The fluid is assumed to be incompressible and inviscid, and undergo irrotational motion. A layer-wise third order shear and normal deformable plate/shell theory is employed to simulate deformations of the panel by considering all geometric nonlinearities (i.e., all non-linear terms in strain–displacement relations) and taking the panel material to be St. Venant–Kirchhoff (i.e., the second Piola–Kirchhoff stress tensor is a linear function of the Green–St. Venant strain tensor). The Rayleigh damping is introduced to account for structural damping that reduces oscillations in the pressure acting on the panel/water interface. Results have been computed for water entry of (i) straight and circular sandwich panels made of Hookean materials with and without consideration of delamination failure, and (ii) flat sandwich panels made of the St. Venant–Kirchhoff materials. The face sheets and the core of sandwich panels are made, respectively, of fiber reinforced composites and soft materials. It is found that for the same entry speed (i) the peak pressure for a curved panel is less than that for a straight panel, (ii) the consideration of geometric nonlinearities significantly increases the peak hydrodynamic pressure, (iii) delamination occurs in mode-II, and (iv) the delamination reduces the hydroelastic pressure acting on the panel surface and hence alters deformations of the panel.     

火星科学实验室气动特性数值分析

针对火星着陆探测器进入-下降-着陆过程的高超声速进入阶段,求解三维流体动力学Navier-Stokes 方程与化学反应动力学模型,分析火星科学实验室进入火星大气时的化学非平衡效应、探测器周围的流场结构和气动特性在化学非平衡效应影响下的变化. 结果表明,CO₂ 在激波后大量分解,消耗大量能量;在化学非平衡效应影响下,探测器头部激波脱体距离大幅减小,尾迹旋涡运动减弱;化学非平衡效应影响下探测器升力系数变化不大,阻力系数高于完全气体,升阻比略低,配平攻角小于完全气体.     

Oblique water entry of a cone by a fully three-dimensional nonlinear method

The hydrodynamic problem of a cone entering the water surface obliquely has been analyzed by the three-dimensional (3-D) incompressible velocity potential theory with the fully nonlinear boundary conditions on the moving free surface and body surface boundary. The time stepping method is used in the stretched coordinate system defined as the ratio of the physical system to the distance that the cone has travelled into water. The boundary element method is used to solve the potential at each time step. Both triangular element mesh and quadrilateral element mesh have been used. Discretisation of the body surface and the free surface is applied regularly during the simulation to account for their change and deformation, and data from the old mesh is transferred into the new one through interpolation. Both the dynamic and kinematic free surface boundary conditions are satisfied through the Eulerian form. In particular the free surface elevation and potential variation are traced at a given azimuth of the cylindrical coordinate system, in the direction parallel to the body surface or perpendicular to the free surface to avoid multi-valued function. Detailed convergence study with respect to time step and element size has been undertaken and high accuracy has been achieved. Results for the cone in vertical entry are compared with those obtained from the 2-D axisymmetric method and good agreement is found. Simulations are made for cones of various deadrise angles and different oblique entries and detailed results are provided.     

Evolution of three-dimensional cavitation following water entry of an inclined cylinder

The water entry of an inclined cylinder is firstly studied experimentally for low Froude number. The cylinder is 50 mm in diameter and 200 mm in length, with a moderate length to diameter ratio. As it is submerged below the water surface, the cavity is fully three-dimensional. Due to the rotation of the cylinder caused by the initial inclined impact, the cavity evolution is quite complicated and a new phenomenon is revealed. The cylinder moves along a curved trajectory in water, which greatly affects the evolution of the cavities. The cavity breaks up into two sub-cavities, and finally collapses because of hydrostatic pressure.