Trypsiligase-Catalyzed Labeling of Proteins on Living Cells

Fluorescent fusion proteins are powerful tools for studying biological processes in living cells, but universal application is limited due to the voluminous size of those tags, which might have an impact on the folding, localization or even the biological function of the target protein. The designed biocatalyst trypsiligase enables site-directed linkage of small-sized fluorescence dyes on the N terminus of integral target proteins located in the outer membrane of living cells through a stable native peptide bond. The function of the approach was tested by using the examples of covalent derivatization of the transmembrane proteins CD147 as well as the EGF receptor, both presented on human HeLa cells. Specific trypsiligase recognition of the site of linkage was mediated by the dipeptide sequence Arg-His added to the proteins’ native N termini, pointing outside the cell membrane. The labeling procedure takes only about 5 minutes, as demonstrated for couplings of the fluorescence dye tetramethyl rhodamine and the affinity label biotin as well.     

Fabrication and joining mechanism of Nano-Cu/Si3N4 ceramic substrates

A novel method for fabricating a nano-Cu/Si₃N₄ ceramic substrate is proposed. The nano-Cu/Si₃N₄ ceramic substrate is first fabricated using spark plasma sintering (SPS) with the addition of nanoscale multilayer films (Ti/TiN/Ti/TiN/Ti) as transition layers. The microstructures of the nano-Cu metal layer and the interface between Cu and Si₃N₄ are investigated. The results show that a higher SPS temperature increases the grain size of the nano-Cu metal layer and affects the hardness. The microstructure of the transition layer evolves significantly after SPS. Ti in the transition layer can react with Si₃N₄ and with nano-Cu to form interfacial reaction layers of TiN and Ti–Cu, respectively; these ensure stronger bonding between nano-Cu and Si₃N₄. Higher SPS temperatures improve the diffusion ability of Ti and Cu, inducing the formation of Ti₃Cu₃O compounds in the nano-Cu metal layer and Ti₂Cu in the transition layer. This study provides an important strategy for designing and constructing a new type of ceramic substrate.     

A stiffness parameter and truncation error criterion for adaptive path following in structural mechanics

We explore a truncation error criterion to steer adaptive step length refinement and coarsening in incremental-iterative path following procedures, applied to problems in large-deformation structural mechanics. Elaborating on ideas proposed by Bergan and collaborators in the 1970s, we first describe an easily computable scalar stiffness parameter whose sign and rate of change provide reliable information on the local behavior and complexity of the equilibrium path. We then derive a simple scaling law that adaptively adjusts the length of the next step based on the rate of change of the stiffness parameter at previous points on the path. We show that this scaling is equivalent to keeping a local truncation error constant in each step. We demonstrate with numerical examples that our adaptive method follows a path with a significantly reduced number of points compared to an analysis with uniform step length of the same fidelity level. A comparison with Abaqus illustrates that the truncation error criterion effectively concentrates points around the smallest-scale features of the path, which is generally not possible with automatic incrementation solely based on local convergence properties.     

粉土界面恒刚度循环剪切试验研究

竖向循环荷载作用下桩土界面的作用机理是研究桩土摩擦疲劳的关键。针对循环荷载作用下桩-粉土界面的剪切性能,使用改进的剪切试验装置在恒刚度条件下进行循环剪切试验,研究循环次数、累积位移和法向刚度对其摩擦疲劳性能、循环后单调剪切性能的影响。试验结果表明,粉土在循环剪切过程中,法向应力和剪应力在初始10个循环内随循环数增加快速衰减,随着循环进行,逐渐趋于稳定;单次循环内在剪切位移方向变化时,土体呈现表现出剪缩-剪胀-剪缩交替现象,总体变形呈现剪缩的趋势;循环荷载作用下,粉土界面的法向应力和剪应力随法向刚度增大衰减速率增大,达到稳定的累积循环位移越小;粉土循环后的单调剪切、法向应力恢复的单调剪切的剪应力比小于首次单调剪切试验值,且法向应力恢复的循环后剪切试验的剪胀程度较小,表明循环剪切过程中界面处粉土颗粒棱角破碎,颗粒变得光滑。在对试验数据分析的基础上,提出了与累积位移、法向刚度和初始应力相关的无量纲累积位移,建立了法向应力和界面摩擦角随累积位移的衰减方程。     

Design and Characterization of a New pVII Combinatorial Phage Display Peptide Library for Protease Substrate Mining Using Factor VII Activating Protease (FSAP) as Model

We describe a novel, easy and efficient combinatorial phage display peptide substrate-mining method to map the substrate specificity of proteases. The peptide library is displayed on the pVII capsid of the M13 bacteriophage, which renders pIII necessary for infectivity and efficient retrieval, in an unmodified state. As capture module, the 3XFLAG was chosen due to its very high binding efficiency to anti-FLAG mAbs and its independency of any post-translational modification. This library was tested with Factor-VII activating protease (WT-FSAP) and its single-nucleotide polymorphism variant Marburg-I (MI)-FSAP. The WT-FSAP results confirmed the previously reported Arg/Lys centered FSAP cleavage site consensus as dominant, as well as reinforcing MI-FSAP as a loss-of-function mutant. Surprisingly, rare substrate clones devoid of basic amino acids were also identified. Indeed one of these peptides was cleaved as free peptide, thus suggesting a broader range of WT-FSAP substrates than previously anticipated.     

二氧化锡溅射工艺研究

SnO2 是最早使用也是目前使用最广泛的一种气敏材料 ,使用该材料设计制作的气敏传感器具有许多优点。在简要介绍溅射镀膜的成膜过程和特点的基础上 ,着重介绍了SnO2 膜的制备流程 ,分析了功率和温度变化对成膜质量的影响     

底部两层框架-抗震墙砖房抗震能力的分析方法与设计控制

提出了底部两层框架 -抗震墙砖房框剪层和砖混层的极限受剪承载力、侧移刚度、极限剪力系数计算公式 ,并基于设置不同数量抗震墙的底部两层框架 -抗震墙砖房的实例计算和弹塑性时程分析 ,给出了结构砖混过渡层与相邻框剪层的极限剪力系数比和侧移刚度比的合理取值及薄弱楼层位置判别和破坏状态评定的方法和原则。     

A continuum-based modeling of MEMS devices for estimating their resonant frequencies

A continuum-based modeling of coupled electrostatics-structure interactions is presented for the frequency computations of MEMS devices. The present general formulation of electrostatics accounting for free space is validated first by specializing it to one-dimensional uniform motion of conducting surfaces and comparing the resulting electrostatics to conventional lumped models. The general coupled electrostatics-structure interactions are then applied for the prediction of resonant frequencies of MEMS devices due to bias-voltage changes and temperature variations. Comparisons of predicted resonant frequencies obtained by the present coupled electrostatics-structure interaction models with experimental results available in the literature demonstrate that the proposed continuum-based interaction modeling yields high-confidence predictions of resonant frequencies of MEMS devices.     

A direct stiffness analysis of a composite beam with partial interaction

The use of the conventional semi-analytical stiffness method in finite element analysis, in which interpolation polynomials are used to develop the stiffness relationships, leads to problems of curvature locking when beam-type elements are developed for composite members with partial interaction between the materials of which it is comprised. The curvature locking phenomenon that occurs for composite steel–concrete members is quite well reported, and the general approach to minimizing the undesirable ramifications of curvature locking has been to use higher-order polynomials with increasing numbers of internal nodes. This paper presents an alternate formulation based on a direct stiffness approach rather than starting from pre-defined interpolation polynomials, and which does not possess the undesirable locking characteristics. The formulation is based on a more general approach for a bi-material composite flexural member, whose constituent materials are joined by elastic shear connection so as to provide partial interaction. The stiffness relationships are derived, and these are applied to a simply supported and a continuous steel–concrete composite beam to demonstrate the efficacy of the method, and in particular its ability to model accurately both very flexible and very stiff shear connection that causes difficulties when implemented in competitive semi-analytical algorithms. Copyright © 2004 John Wiley & Sons, Ltd.