Affinity maturation of a computationally designed binding protein affords a functional but disordered polypeptide

Computational methods have been recently applied to the design of protein–protein interfaces. Using this approach, a 61 amino acid long protein called Spider Roll was engineered to recognize the kinase domain of the human p21-activated kinase 1 (PAK1) with good specificity but modest affinity (K_D = 100 μM). Here we show that this artificial protein can be optimized by yeast surface display and fluorescence-activated cell sorting. After three rounds of mutagenesis and screening, a diverse set of tighter binding variants was obtained. A representative binder, MSR7, has a >10²-fold higher affinity for PAK1 when displayed on yeast and a 6 to 11-fold advantage when produced free in solution. In contrast to the starting Spider Roll protein, however, MSR7 unexpectedly exhibits characteristics typical of partially disordered proteins, including lower α-helical content, non-cooperative thermal denaturation, and NMR data showing peak broadening and poor signal dispersion. Although conformational disorder is increasingly recognized as an important property of proteins involved in cellular signaling and regulation, it is poorly modeled by current computational methods. Explicit consideration of structural flexibility may improve future protein designs and provide deeper insight into molecular events at protein–protein interfaces.     

Structure-based non-canonical amino acid design to covalently crosslink an antibody–antigen complex

Engineering antibodies to utilize non-canonical amino acids (NCAA) should greatly expand the utility of an already important biological reagent. In particular, introducing crosslinking reagents into antibody complementarity determining regions (CDRs) should provide a means to covalently crosslink residues at the antibody–antigen interface. Unfortunately, finding the optimum position for crosslinking two proteins is often a matter of iterative guessing, even when the interface is known in atomic detail. Computer-aided antibody design can potentially greatly restrict the number of variants that must be explored in order to identify successful crosslinking sites. We have therefore used Rosetta to guide the introduction of an oxidizable crosslinking NCAA, l-3,4-dihydroxyphenylalanine (l-DOPA), into the CDRs of the anti-protective antigen scFv antibody M18, and have measured crosslinking to its cognate antigen, domain 4 of the anthrax protective antigen. Computed crosslinking distance, solvent accessibility, and interface energetics were three factors considered that could impact the efficiency of l-DOPA-mediated crosslinking. In the end, 10 variants were synthesized, and crosslinking efficiencies were generally 10% or higher, with the best variant crosslinking to 52% of the available antigen. The results suggest that computational analysis can be used in a pipeline for engineering crosslinking antibodies. The rules learned from l-DOPA crosslinking of antibodies may also be generalizable to the formation of other crosslinked interfaces and complexes.     

Space Use as an Indicator of Enclosure Appropriateness in African Wild Dogs (Lycaon pictus)

A clear understanding of space use is required to more fully understand biological requirements of nonhuman animals in zoos, aid the design of exhibits, and maximize the animals' welfare. This study used electivity indexes to assess space use of two packs of African wild dogs (Lycaon pictus) and the appropriateness of two naturalistic, outdoor enclosures at the San Diego Zoo and Bronx Zoo. The results identified enclosure features that were both underutilized and overutilized. They suggest that replacing underutilized areas with features similar to areas that were overutilized may provide more preferred opportunities for the animals. Assessing space use of animals in human care may serve as an indicator of enclosure appropriateness and could have welfare implications. By looking at the possible reasons for area preferences, animal managers can get an idea of where improvements could be made. Designing future exhibits accordingly thus can provide possible welfare benefits for the animals concerned.     

DRUGSURV: a resource for repositioning of approved and experimental drugs in oncology based on patient survival information

The use of existing drugs for new therapeutic applications, commonly referred to as drug repositioning, is a way for fast and cost-efficient drug discovery. Drug repositioning in oncology is commonly initiated by in vitro experimental evidence that a drug exhibits anticancer cytotoxicity. Any independent verification that the observed effects in vitro may be valid in a clinical setting, and that the drug could potentially affect patient survival in vivo is of paramount importance. Despite considerable recent efforts in computational drug repositioning, none of the studies have considered patient survival information in modelling the potential of existing/new drugs in the management of cancer. Therefore, we have developed DRUGSURV; this is the first computational tool to estimate the potential effects of a drug using patient survival information derived from clinical cancer expression data sets. DRUGSURV provides statistical evidence that a drug can affect survival outcome in particular clinical conditions to justify further investigation of the drug anticancer potential and to guide clinical trial design. DRUGSURV covers both approved drugs (∼1700) as well as experimental drugs (∼5000) and is freely available at http://www.bioprofiling.de/drugsurv.     

Optimal surveillance strategy for invasive species management when surveys stop after detection

Invasive species are a cause for concern in natural and economic systems and require both monitoring and management. There is a trade‐off between the amount of resources spent on surveying for the species and conducting early management of occupied sites, and the resources that are ultimately spent in delayed management at sites where the species was present but undetected. Previous work addressed this optimal resource allocation problem assuming that surveys continue despite detection until the initially planned survey effort is consumed. However, a more realistic scenario is often that surveys stop after detection (i.e., follow a “removal” sampling design) and then management begins. Such an approach will indicate a different optimal survey design and can be expected to be more efficient. We analyze this case and compare the expected efficiency of invasive species management programs under both survey methods. We also evaluate the impact of mis‐specifying the type of sampling approach during the program design phase. We derive analytical expressions that optimize resource allocation between monitoring and management in surveillance programs when surveys stop after detection. We do this under a scenario of unconstrained resources and scenarios where survey budget is constrained. The efficiency of surveillance programs is greater if a “removal survey” design is used, with larger gains obtained when savings from early detection are high, occupancy is high, and survey costs are not much lower than early management costs at a site. Designing a surveillance program disregarding that surveys stop after detection can result in an efficiency loss. Our results help guide the design of future surveillance programs for invasive species. Addressing program design within a decision‐theoretic framework can lead to a better use of available resources. We show how species prevalence, its detectability, and the benefits derived from early detection can be considered.     

Viral clearance by flow‐through mode ion exchange columns and membrane adsorbers

Anion exchange (AEX) is a common downstream purification operation for biotechnology products manufactured in cell culture such as therapeutic monoclonal antibodies (mAbs) and Fc‐fusion proteins. We present a head‐to‐head comparison of the viral clearance efficiency of AEX adsorbers and column chromatography using the same process fluids and comparable run conditions. We also present overall trends from the CDER viral clearance database. In our comparison of multiple brands of resins and adsorbers, clearance of three model viruses (PPV, X‐MuLV, and PR772) was largely comparable, with some exceptions which may reflect run conditions that had not been optimized on a resin/membrane specific basis. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:124–131, 2014     

重大工程建设中生态安全格局构建基本原则和方法

随着人类活动的日益加强,生态安全成为全球关注的话题.生态安全的状态受到人类干扰活动的影响,而区域生态安全演变也会影响人类活动的功效.20世纪90年代以来,我国先后开展了长江三峡大坝、青藏铁路、西气东输、西电东送、南水北调等重大工程项目的建设.这些重大工程建设对区域生态安全的影响,以及区域生态系统演变对工程安全运行的影响成为我国政府关注的重要课题.如何在强度干扰下开展生态恢复与区域生态安全格局的构建,不仅关系到区域生态环境的保护,也关系到各项重大工程的顺利运行.本文系统分析了工程建设的类型、特征及其对区域生态环境的影响,在此基础上,提出了重大工程建设中生态恢复与安全格局构建的基本原则和方法.认为在工程建设中构建生态安全格局需要从6个方面着手：区域生态环境现状分析与评价、工程建设生态干扰与风险评价、生态安全格局预案构建、区域生态环境效应情景分析、区域生态恢复与安全格局再优化和生态系统管理方案的建立.讨论了我国重大工程建设中生态安全格局构建面临的突出问题.     

肇庆市景观生态规划研究

肇庆市生态资源丰富,自然保护现状良好,生态环境建设潜力大,但整个生态缺乏合理规划,与经济发展联系不大,对经济发展贡献率低,与旅游规划脱节,致使各地旅游形象不突出,特色不鲜明,景观整体性和连续性遭受破坏,景观、景区间协调不足,过渡不自然,景观视线范围内荒山和采矿点依稀可见,没有充分发挥生态在旅游中的作用。针对以上问题和矛盾,文章对肇庆市景观生态进行功能分区和规划设计,为肇庆市城市规划及景观生态建设提供一定的依据。     

蜜环菌诱导子对丹参冠瘿组织积累丹参酮的影响

以 6 7_V液体培养基为基本培养基 ,利用计算机软件“均匀设计、回归分析及优化系统” ,研究了蜜环菌(ArmillariamelleaKarst)诱导子浓度、诱导子的加入时间与处理的收获时间对丹参 (SalviamiltiorrhizaBunge)冠瘿组织积累丹参酮的影响。结果表明 ,蜜环菌诱导子浓度为 119mL/L、第 0天加入诱导子、第 2 9天收获培养物与培养液时可获得最高的丹参酮生产量 (147mg/L ,P <0 .0 5 ) ,蜜环菌诱导子浓度为 113mL/L、第 0天加入诱导子、第 2 6天收获培养液时可获得最高的丹参酮生产量 (6 2mg/L ,P <0 .0 5 ) ,蜜环菌诱导子浓度为 87mL/L、第 0天加入诱导子、第2 8天收获培养物时可获得最高的丹参酮生产量 (94mg/L ,P <0 .0 5 )。结果证实 ,计算机软件“均匀设计、回归分析及优化系统”对于观察值受多因素影响的研究非常有效和方便。     

AB5 toxins: structures and inhibitor design

High-resolution crystal structures of AB₅ toxins in their native form or in complex with a variety of ligands have led to the structure-based design and discovery of inhibitors targeting different areas of the toxins. The most significant progress is the development of highly potent multivalent ligands that block binding of the toxins to their receptors.