Effect of Cu-Rich Phase Precipitation on the Microstructure and Mechanical Properties of CoCrNiCux Medium-Entropy Alloys Prepared via Laser Directed Energy Deposition

CoCrNiCux (x=0.16,0.33,0.75,and 1) without macro-segregation medium-entropy alloys (MEAs) was prepared using laser directed energy deposition (LDED).The microstructure and mechanical properties of CoCrNiCux alloys with increas-ing Cu content were investigated.The results indicate that a single matrix phase changes into a dual-phase structure and the tensile fracture behaviors convert from brittle to plastic pattern with increasing Cu content in CoCrNiCux alloys.In addi-tion,the tensile strength of CoCrNiCux alloys increased from 148 to 820 MPa,and the ductility increased from 1 to 11％with increasing Cu content.The nano-precipitated particles had a mean size of approximately 20 nm in the Cu-rich phase area,and a large number of neatly arranged misfit dislocations were observed at the interface between the two phases due to Cu-rich phase precipitation in the CoCrNiCu alloy.These misfit dislocations hinder the movement of dislocations during tensile deformation,as observed through transmission electron microscopy.This allows the CoCrNiCu alloy to reach the largest tensile strength and plasticity,and a new strengthening mechanism was achieved for the CoCrNiCu alloy.Moreover,twins were observed in the matrix phase after tensile fracture.Simultaneously,the dual-phase structure with different elastic moduli coordinated with each other during the deformation process,significantly improving the plasticity and strength of the CoCrNiCu alloy.     

面向二进制程序的导向性模糊测试方法

为了解决当前模糊测试技术中变异存在一定的盲目性以及变异生成的样本大多经过相同的高频路径的问题，提出并实现了一种基于轻量级程序分析技术的二进制程序模糊测试方法。首先对目标二进制程序进行静态分析来筛选在模糊测试过程中阻碍样本文件深入程序内部的比较指令；随后对目标文件进行插桩来获取比较指令中操作数的具体值，并根据该具体值为比较指令建立实时的比较进度信息，通过比较进度衡量样本的重要程度；然后基于模糊测试过程中实时的路径覆盖信息为经过稀有路径的样本增加其被挑选进行变异的概率；最后根据比较进度信息并结合启发式策略有针对性地对样本文件进行变异，通过变异引导提高模糊测试中生成能够绕过程序规约检查的有效样本的效率。实验结果表明，所提方法发现crash及发现新路径的能力均优于模糊测试工具AFL-Dyninst。     

直投式纳豆菌发酵剂制备工艺优化

为探索出一种工艺简便、成本低且活菌数高的直投式纳豆菌（Bacillus natto）发酵剂的制备方法，该研究以大米粉为基质替代传统的种子液培养基培养纳豆菌后直接将其烘干制成直投式纳豆菌发酵剂，通过单因素及正交试验优化直投式纳豆菌发酵剂的制备工艺。结果表明，用含1.5%蛋白胨的大米粉作为培养基，在接种量4.0%、加水量40%、培养温度43 ℃条件下培养纳豆菌28 h，菌体生长良好，将培养物烘干后制成的直投式纳豆菌发酵剂中的活菌数含量达4.71×109 CFU/g。此技术操作简便，可推广至工业生产中，简化生产过程。     

Engineered pH-inducible intein Mtu ΔI-CM variants with markedly reduced premature cleavage activity

Inteins have been widely exploited for the purification of tagless proteins. Among them, pH-inducible C-terminal-cleavage inteins enable the preparation of proteins and peptides with an authentic N-terminus. However, a severe premature cleavage around neutral pH has limited the application of these inteins, especially when used in recombinant hosts such as Escherichia coli. By targeting the microenvironment of the two key histidine residues H429 and H439, we engineered Mtu ΔI-CM intein to markedly reduce its premature cleavage. Kinetic analyses suggested that although the variants retained the pH dependence, they indeed cleaved slower, especially at pH 7.6. These variants resulted in higher yields for two model polypeptides than the original Mtu ΔI-CM intein, when used in conjunction with a cleavable self-assembling tag. This work suggests that more controllable pH-inducible inteins can be obtained by manipulating the residues in the self-cleavage sites and provide better performance for tag-based protein preparation strategies.     

Comprehensive mutagenesis on yeast cytosine deaminase yields improvements in 5-fluorocytosine toxicity in HT1080 cells

Improved prodrug-activating enzymes have the potential to increase the therapeutic efficacy of gene-directed enzyme prodrug therapy (GDEPT). Yeast cytosine deaminase (yCD) is commonly used to convert the prodrug 5-fluorocytosine (5-FC) to the chemotherapeutic 5-fluorouracil for GDEPT. Mutagenesis studies on yCD aimed at improving its application in GDEPT have been limited to subsets of residues or have sought to improve a single property of the enzyme. We performed comprehensive site-saturation mutagenesis (CSM) on yCD designed to create all 2,983 possible unique protein mutants with a single amino acid substitution. We identified active variants through Escherichia coli genetic complementation and screened these mutants, and combinations thereof, for increased ability to sensitize E. coli and HT1080 fibrosarcoma cells to 5-FC. Several mutants identified in this study showed increased sensitization ability for both E. coli and HT1080 cells indicating that CSM is an effective directed evolution tool for identifying unexpectedly beneficial mutations.     

Maximizing Convergence Speed for Second Order Consensus in Leaderless Multi-Agent Systems

The paper deals with the consensus problem in a leaderless network of agents that have to reach a common velocity while forming a uniformly spaced string. Moreover, the final common velocity (reference velocity) is determined by the agents in a distributed and leaderless way. Then, the consensus protocol parameters are optimized for networks characterized by a communication topology described by a class of directed graphs having a directed spanning tree, in order to maximize the convergence rate and avoid oscillations. The advantages of the optimized consensus protocol are enlightened by some simulation results and comparison with a protocol proposed in the related literature. The presented protocol can be applied to coordinate agents such as mobile robots, automated guided vehicles (AGVs) and autonomous vehicles that have to move with the same velocity and a common inter-space gap.      

Alpha‐Synuclein Binds to the Inner Membrane of Mitochondria in an α‐Helical Conformation

The human alpha‐Synuclein (αS) protein is of significant interest because of its association with Parkinson's disease and related neurodegenerative disorders. The intrinsically disordered protein (140 amino acids) is characterized by the absence of a well‐defined structure in solution. It displays remarkable conformational flexibility upon macromolecular interactions, and can associate with mitochondrial membranes. Site‐directed spin‐labeling in combination with electron paramagnetic resonance spectroscopy enabled us to study the local binding properties of αS on artificial membranes (mimicking the inner and outer mitochondrial membranes), and to evaluate the importance of cardiolipin in this interaction. With pulsed, twofrequency, double‐electron electron paramagnetic resonance (DEER) approaches, we examined, to the best of our knowledge for the first time, the conformation of αS bound to isolated mitochondria.     

Protein Engineering of the Antitumor Enzyme PpADI for Improved Thermal Resistance

Arginine deiminase (ADI, EC 3.5.3.6) is a potential antitumor drug for the treatment of arginine‐auxotrophic tumors such as hepatocellular carcinomas (HCCs) and melanomas. Studies in human lymphatic leukemia cell lines have confirmed the anti‐angiogenic activity of ADI. Activity and thermal resistance limit the efficacy of ADI in treatment of auxotrophic tumors. Previously, we reengineered ADI from Pseudomonas plecoglossicida (PpADI) for improved activity under physiological conditions (37 °C, PBS buffer, pH 7.4) by two rounds of directed evolution and combination of beneficial substitutions through site‐directed mutagenesis. The best variant, PpADI M6 (K5T/D38H/D44E/A128T/E296K/H404R), showed a 64.7‐fold improvement in k_cat value and a 37.6 % decreased S_0.5 value under physiological conditions. However, M6 lost rapidly its activity (half‐life of ～2 days at 37 °C). Here we report the re‐engineering of PpADI M6 for improved thermal resistance by directed evolution in order to increase its half‐life under physiological conditions. Directed evolution and recombination of the two most beneficial positions yielded variant PpADI M9 (K5T/D38H/D44E/A128T/V140L/E296K/F325L/H404R), for which the T_m value increased from 47 (M6) to 54 °C (M9); this corresponds to an increased half‐life from ～2 days (M6) to ～3.5 days (M9) under physiological conditions. Structure analysis of the homology model of M9 showed that the beneficial substitutions V140L and F325L likely promote the formation of tetrameric PpADI, which has greater thermal resistance than dimeric PpADI.     

基于FPGA的设计继承方法

FPGA的正确性不单单由代码来确定,还需要FPGA开发工具完成整个设计流程后形成正确的电路才能保证设计的正确性.为提高FPGA在实际工程中的开发效率,引入后端工具指导布线法、布局布线工具设计保持法这两种设计方法,通过设计工具使关键信号或者未更改部分的电路布局布线信息继续保留,因此大大减少因部分模块更替对以通过充分测试部分的设计的影响.在工程实践过程中,后端工具指导布线法仅对布线约束控制在40条以下的工程效果较好,但流程较简单;布局布线工具设计保持法流程较复杂,但保持的布线数量可达成百上千条.两种方法各有优劣的地方,设计人员应根据不同的工程需求,选择合适的方法来实现设计继承,以提高调试效率.