Local RNA target structure influences siRNA efficacy: a systematic global analysis

The efficiency with which small interfering RNAs (siRNAs) down-regulate specific gene expression in living cells is variable and a number of sequence-governed, biochemical parameters of the siRNA duplex have been proposed for the design of an efficient siRNA. Some of these parameters have been clearly identified to influence the assembly of the RNA-induced silencing complex (RISC), or to favour the sequence preferences of the RISC endonuclease. For other parameters, it is difficult to ascertain whether the influence is a determinant of the siRNA per se, or a determinant of the target RNA, especially its local structural characteristics. In order to gain an insight into the effects of local target structure on the biological activity of siRNA, we have used large sets of siRNAs directed against local targets of the mRNAs of ICAM-1 and survivin. Target structures were classified as accessible or inaccessible using an original, iterative computational approach and by experimental RNase H mapping. The effectiveness of siRNA was characterized by measuring the IC50 values in cell culture and the maximal extent of target suppression. Mean IC50 values were tenfold lower for accessible local target sites, with respect to inaccessible ones. Mean maximal target suppression was improved. These data illustrate that local target structure does, indeed, influence the activity of siRNA. We suggest that local target screening can significantly improve the hit rate in the design of biologically active siRNAs.     

生物多样性野外调查地理信息管理、路线精细设计和精确导航方法

野外调查是生物多样性研究和保护的基础工作。在野外调查中, 自然地理环境十分复杂, 对相关地理信息的掌握程度、调查路线的设计质量和导航的准确性, 直接影响调查工作的成效, 甚至调查人员的人身安全。使用谷歌地球软件及相关数据转换工具, 能获取海量遥感影像和地形数据, 能将各种地理信息转换、编辑、叠加到三维地球模型上形成地理信息库, 进而精细设计野外考察路线、调查点和调查样方, 并以kml或kmz文件格式保存和分发设计结果。使用Android智能终端设备安装OruxMaps软件, 可以将多种网络地图下载制作成离线地图, 在卫星图上精确显示当前位置、记录走过的轨迹、导入kml或kmz文件进行导航。综合使用这些工具, 可以实现良好的地理信息管理、精细的生物多样性野外调查路线设计和高精度现场导航实施, 有效提高野外调查工作的成效和安全性。     

增强现实技术于洪水风险沟通中的应用

气候变迁,特别是洪水问题给地处三角洲的城市带来了与日俱增的风险。作为应对手段,提出一种整合性的洪水风险管理方法,该方法确保了洪水风险沟通在洪水风险管理战略中的重要地位。洪水的可视化作为洪水风险沟通的一种工具,在改变人们对洪水风险的观念方面有着强大的作用。它可以通过互动的方式,使当地的利益相关者了解洪水的剩余风险和未来风险。其中增强现实技术作为一种可视化手段,它的快速发展与应用为风景园林设计、规划与教育等领域提供了崭新的交互方式。首先,探讨增强现实技术（AR）在洪水风险沟通中的应用现状,并重点关注增强现实技术的2 个关键应用领域：对现场决策的支持和针对高层次设计的可视化与对景观干预的评估。随后,展示一种基于增强现实技术所开发的应用,该款创新性的应用为洪水风险沟通提供了一种轻量化的可视化手段。     

G‐LoSA: An efficient computational tool for local structure‐centric biological studies and drug design

Molecular recognition by protein mostly occurs in a local region on the protein surface. Thus, an efficient computational method for accurate characterization of protein local structural conservation is necessary to better understand biology and drug design. We present a novel local structure alignment tool, G‐LoSA. G‐LoSA aligns protein local structures in a sequence order independent way and provides a GA‐score, a chemical feature‐based and size‐independent structure similarity score. Our benchmark validation shows the robust performance of G‐LoSA to the local structures of diverse sizes and characteristics, demonstrating its universal applicability to local structure‐centric comparative biology studies. In particular, G‐LoSA is highly effective in detecting conserved local regions on the entire surface of a given protein. In addition, the applications of G‐LoSA to identifying template ligands and predicting ligand and protein binding sites illustrate its strong potential for computer‐aided drug design. We hope that G‐LoSA can be a useful computational method for exploring interesting biological problems through large‐scale comparison of protein local structures and facilitating drug discovery research and development. G‐LoSA is freely available to academic users at http://im.compbio.ku.edu/GLoSA/ .     

A new efficient mixture screening design for optimization of media

Screening ingredients for the optimization of media is an important first step to reduce the many potential ingredients down to the vital few components. In this study, we propose a new method of screening for mixture experiments called the centroid screening design. Comparison of the proposed design with Plackett‐Burman, fractional factorial, simplex lattice design, and modified mixture design shows that the centroid screening design is the most efficient of all the designs in terms of the small number of experimental runs needed and for detecting high‐order interaction among ingredients. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Ultraconserved element (UCE) probe set design: Base genome and initial design parameters critical for optimization

Targeted capture and enrichment approaches have proven effective for phylogenetic study. Ultraconserved elements (UCEs) in particular have exhibited great utility for phylogenomic analyses, with the software package phyluce being among the most utilized pipelines for UCE phylogenomics, including probe design. Despite the success of UCEs, it is becoming increasing apparent that diverse lineages require probe sets tailored to focal taxa in order to improve locus recovery. However, factors affecting probe design and methods for optimizing probe sets to focal taxa remain underexplored. Here, we use newly available beetle (Coleoptera) genomic resources to investigate factors affecting UCE probe set design using phyluce . In particular, we explore the effects of stringency during initial design steps, as well as base genome choice on resulting probe sets and locus recovery. We found that both base genome choice and initial bait design stringency parameters greatly alter the number of resultant probes included in final probe sets and strongly affect the number of loci detected and recovered during in silico testing of these probe sets. In addition, we identify attributes of base genomes that correlated with high performance in probe design. Ultimately, we provide a recommended workflow for using Phyluce to design an optimized UCE probe set that will work across a targeted lineage, and use our findings to develop a new, open‐source UCE probe set for beetles of the suborder Adephaga.     

Structural analysis, identification, and design of calcium-binding sites in proteins

Assigning proteins with functions based on the 3-D structure requires high-speed techniques to make a systematic survey of protein structures. Calcium regulates many biological systems by binding numerous proteins in different biological environments. Despite the great diversity in the composition of ligand residues and bond angles and lengths of calcium-binding sites, our structural analysis of 11 calcium-binding sites in different classes of proteins has shown that common local structural parameters can be used to identify and design calcium-binding proteins. Natural calcium-binding sites in both EF-hand proteins and non-EF-hand proteins can be described with the smallest deviation from the geometry of an ideal pentagonal bipyramid. Further, two different magnesium-binding sites in parvalbumin and calbindin(D9K) can also be identified using an octahedral geometry. Using the established method, we have designed de novo calcium-binding sites into the scaffold of non-calcium-binding proteins CD2 and Rop. Our results suggest that it is possible to identify calcium- and magnesium-binding sites in proteins and design de novo metal-binding sites.     

Farming system design for innovative crop-livestock integration in Europe

The development of integrated crop–livestock systems (ICLS) is a major challenge for the ecological modernisation of agriculture but appears difficult to implement at a large scale. A participatory method for ICLS design has been developed and implemented in 15 case studies across Europe, representing a range of production systems, challenges, constraints and resources for innovation. Local stakeholders, primarily farmers, but also cooperatives, environmental-association representatives and natural-resource managers, were involved in the identification of challenges and existing initiatives of crop-livestock integration; in the design of new options at field, farm and territory levels; and then in qualitative multicriteria assessment of these options. A conceptual framework based on a conceptual model (crops, grasslands, animals) was developed to act as a boundary object in the design step and invite innovative thinking in ‘metabolic’ and ‘ecosystemic’ approaches. A diversity of crops and grasslands interacting with animals appeared central for designing sustainable farming systems at the territory level, providing and benefitting from ecosystem services. Within this diversity, we define three types of integrated systems according to their degrees of spatial and temporal coordination: complementarity, local synergy, territorial synergy. Moreover, the options for cooperation and collective organisation between farmers and other stakeholders in territories to organise and manage this diversity of land use revealed opportunities for smart social innovation. The qualitative multicriteria assessment identified farmer workload as the main issue of concern while demonstrating expected benefits of ICLS simultaneously for economic, agronomic, environmental and social criteria. This study concludes that participatory design of ICLS based on a generic multi-level and multi-domain framework and a methodology to deal with a local context can identify new systems to be tested. Further assessment and redesign work will be performed in later stages of the European FP7 CANTOGETHER project.     

Dead‐end elimination with perturbations (DEEPer): A provable protein design algorithm with continuous sidechain and backbone flexibility

Computational protein and drug design generally require accurate modeling of protein conformations. This modeling typically starts with an experimentally determined protein structure and considers possible conformational changes due to mutations or new ligands. The DEE/A* algorithm provably finds the global minimum‐energy conformation (GMEC) of a protein assuming that the backbone does not move and the sidechains take on conformations from a set of discrete, experimentally observed conformations called rotamers. DEE/A* can efficiently find the overall GMEC for exponentially many mutant sequences. Previous improvements to DEE/A* include modeling ensembles of sidechain conformations and either continuous sidechain or backbone flexibility. We present a new algorithm, DEEPer (D ead‐E nd E limination with Per turbations), that combines these advantages and can also handle much more extensive backbone flexibility and backbone ensembles. DEEPer provably finds the GMEC or, if desired by the user, all conformations and sequences within a specified energy window of the GMEC. It includes the new abilities to handle arbitrarily large backbone perturbations and to generate ensembles of backbone conformations. It also incorporates the shear, an experimentally observed local backbone motion never before used in design. Additionally, we derive a new method to accelerate DEE/A*‐based calculations, indirect pruning, that is particularly useful for DEEPer. In 67 benchmark tests on 64 proteins, DEEPer consistently identified lower‐energy conformations than previous methods did, indicating more accurate modeling. Additional tests demonstrated its ability to incorporate larger, experimentally observed backbone conformational changes and to model realistic conformational ensembles. These capabilities provide significant advantages for modeling protein mutations and protein–ligand interactions. Proteins 2013. © 2012 Wiley Periodicals, Inc.     

The design of a parallax-correcting anthropometer for replication in nonspecialized machine shops

A fixed-rod, slider with a window, a double-scribed line for viewing a measuring tape affixed to the rod, and a sturdy footpiece are design features of an anthropometer for measuring projected lengths. The new instrument is designed to replace contemporary models which may be inaccessible to investigators with limited budgets or currency restrictions. None of the design features are beyond the ingenuity of local machinists to modify, find alternate materials, and use different machine procedures. The same principles of construction can be applied in making sliding calipers with straight and recurved branches. While there is some loss in portability of the fixed-length anthropometer, the improved stability and superior visual scale more than compensate for the disadvantages and make the new anthropometer a candidate for the instrument of choice in obtaining projected length measurements.     

Technical improvements in the computational target search for antisense oligonucleotides

A number of theoretical and experimental approaches to design biologically active antisense oligonucleotides (AS-ON) have proven their usefulness. This includes systematic computational strategies that are based on the understanding of antisense mechanisms. Here, we investigate in detail the relationship between computational parameters of the local target search for the theoretical design of AS-ON and the hit rate, that is, the biologic efficacy of AS-ON in cell culture. The computational design of AS-ON studied in this work is based on an established algorithm to predict structurally favorable local target sites along a given target RNA against which AS-ON are directed. Briefly, a sequence segment of a certain length (window) is used to predict a group of lowest-energy RNA secondary structures. Subsequently, this window is shifted along the target sequence by a certain step width. To date, those technical parameters of the systematic structural target analysis have been chosen arbitrarily. Here, we investigate their role for the successful design of AS-ON and suggest an optimized computer-based protocol for the selection of favorable local target sequences and, hence, an improved design of active AS-ON. Further, this study provides systematic insights into the structure- function relationship of AS-ON.     

The design heatmap: A simple visualization of -optimality design problems

Optimal experimental designs are often formal and specific, and not intuitively plausible to practical experimenters. However, even in theory, there often are many different possible design points providing identical or nearly identical information compared to the design points of a strictly optimal design. In practical applications, this can be used to find designs that are a compromise between mathematical optimality and practical requirements, including preferences of experimenters. For this purpose, we propose a derivative-based two-dimensional graphical representation of the design space that, given any optimal design is already known, will show which areas of the design space are relevant for good designs and how these areas relate to each other. While existing equivalence theorems already allow such an illustration in regard to the relevance of design points only, our approach also shows whether different design points contribute the same kind of information, and thus allows tweaking of designs for practical applications, especially in regard to the splitting and combining of design points. We demonstrate the approach on a toxicological trial where a -optimal design for a dose–response experiment modeled by a four-parameter log-logistic function was requested. As these designs require a prior estimate of the relevant parameters, which is difficult to obtain in a practical situation, we also discuss an adaption of our representations to the criterion of Bayesian -optimality. While we focus on -optimality, the approach is in principle applicable to different optimality criteria as well. However, much of the computational and graphical simplicity will be lost.     

基于乡土生态学知识理念设计濒危动物种群现状快速评估问卷——以极度濒危动物中华穿山甲为例

科学合理地制订濒危动物保护行动计划需要进行种群现状评估。问卷调查是基于受访者的乡土生态学知识的了解,用于广泛收集濒危动物种群现状评估数据的一种方法,具有经济、实用、快速的特点。本文基于濒危动物种群现状快速评估的需要,以极度濒危动物中华穿山甲Manis pentadactyla为例开发调查问卷。问卷由基本信息、引导语、受访者意愿、受访者动物辨别能力、受访者信息、补充问题和种群评估问题组成,其中设计了17个与种群评估有关的问题,包含地理分布、种群数量、栖息地分布、动物受胁因素、栖息地受胁因素、保护教育和社会经济状况。此问卷及问卷设计的思路与原则,能为其他濒危动物调查问卷的开发提供参考。     

Designing for emotion (among other things)

Using computational approaches to emotion in design appears problematic for a range of technical, cultural and aesthetic reasons. After introducing some of the reasons as to why I am sceptical of such approaches, I describe a prototype we built that tried to address some of these problems, using sensor-based inferencing to comment upon domestic ‘well-being’ in ways that encouraged users to take authority over the emotional judgements offered by the system. Unfortunately, over two iterations we concluded that the prototype we built was a failure. I discuss the possible reasons for this and conclude that many of the problems we found are relevant more generally for designs based on computational approaches to emotion. As an alternative, I advocate a broader view of interaction design in which open-ended designs serve as resources for individual appropriation, and suggest that emotional experiences become one of several outcomes of engaging with them.     

Design and Analysis of Field Experiments Incorporating Local and Remote Effects of Treatments

Design and analysis of field experiments in the presence of local and remote treatment effects are considered. The design criteria are optimality and balance for estimating local and remote effects. A nonlinear model, where remote effects are assumed to be proportional to the local effects, has been discussed.     

De novo design and Rosetta-based assessment of high-affinity antibody variable regions (Fv) against the SARS-CoV-2 spike receptor binding domain (RBD)

The continued emergence of new SARS-CoV-2 variants has accentuated the growing need for fast and reliable methods for the design of potentially neutralizing antibodies (Abs) to counter immune evasion by the virus. Here, we report on the de novo computational design of high-affinity Ab variable regions (Fv) through the recombination of VDJ genes targeting the most solvent-exposed hACE2-binding residues of the SARS-CoV-2 spike receptor binding domain (RBD) protein using the software tool OptMAVEn-2.0. Subsequently, we carried out computational affinity maturation of the designed variable regions through amino acid substitutions for improved binding with the target epitope. Immunogenicity of designs was restricted by preferring designs that match sequences from a 9-mer library of “human Abs” based on a human string content score. We generated 106 different antibody designs and reported in detail on the top five that trade-off the greatest computational binding affinity for the RBD with human string content scores. We further describe computational evaluation of the top five designs produced by OptMAVEn-2.0 using a Rosetta-based approach. We used Rosetta SnugDock for local docking of the designs to evaluate their potential to bind the spike RBD and performed “forward folding” with DeepAb to assess their potential to fold into the designed structures. Ultimately, our results identified one designed Ab variable region, P1.D1, as a particularly promising candidate for experimental testing. This effort puts forth a computational workflow for the de novo design and evaluation of Abs that can quickly be adapted to target spike epitopes of emerging SARS-CoV-2 variants or other antigenic targets.     

A new algorithm for RNA secondary structure design

The function of many RNAs depends crucially on their structure. Therefore, the design of RNA molecules with specific structural properties has many potential applications, e.g. in the context of investigating the function of biological RNAs, of creating new ribozymes, or of designing artificial RNA nanostructures. Here, we present a new algorithm for solving the following RNA secondary structure design problem: given a secondary structure, find an RNA sequence (if any) that is predicted to fold to that structure. Unlike the (pseudoknot-free) secondary structure prediction problem, this problem appears to be hard computationally. Our new algorithm, "RNA Secondary Structure Designer (RNA-SSD)", is based on stochastic local search, a prominent general approach for solving hard combinatorial problems. A thorough empirical evaluation on computationally predicted structures of biological sequences and artificially generated RNA structures as well as on empirically modelled structures from the biological literature shows that RNA-SSD substantially out-performs the best known algorithm for this problem, RNAinverse from the Vienna RNA Package. In particular, the new algorithm is able to solve structures, consistently, for which RNAinverse is unable to find solutions. The RNA-SSD software is publically available under the name of RNA Designer at the RNASoft website (www.rnasoft.ca).     

Experimental design for gene expression microarrays

We examine experimental design issues arising with gene expression microarray technology. Microarray experiments have multiple sources of variation, and experimental plans should ensure that effects of interest are not confounded with ancillary effects. A commonly used design is shown to violate this principle and to be generally inefficient. We explore the connection between microarray designs and classical block design and use a family of ANOVA models as a guide to choosing a design. We combine principles of good design and A-optimality to give a general set of recommendations for design with microarrays. These recommendations are illustrated in detail for one kind of experimental objective, where we also give the results of a computer search for good designs.     

Structural and dynamical properties of manganese catalase and the synthetic protein DF1 and their implication for reactivity from classical molecular dynamics calculations

There is a pressing need for accurate force fields to assist in metalloprotein analysis and design. Recent work on the design of mimics of dimetal proteins highlights the requirements for activity. DF1 is a de novo designed protein, which mimics the overall fold and active site geometry of a series of diiron and dimanganese proteins. Specifically, the dimanganese form of DF1 is a mimic of the natural enzyme manganese catalase, which catalyzes the dismutation reaction of hydrogen peroxide into water and oxygen. During catalytic turnover, the active site has to accommodate both the reduced and the oxidized state of the dimanganese core. The biomimetic protein DF1 is only stable in the reduced form and thus not active. Furthermore, the synthetic protein features an additional bridging glutamate sidechain, which occupies the substrate binding site. The goal of this study is to develop classical force fields appropriate for design of such important dimanganese proteins. To this aim, we use a nonbonded model to represent the metal-ligand interactions, which implicitly takes into account charge transfer and local polarization effects between the metal and its ligands. To calibrate this approach, we compare and contrast geometric and dynamical properties of manganese catalase and DF1. Having demonstrated a good correspondence with experimental structural data, we examine the effect of mutating the bridging glutamate to aspartate (M1) and serine (M2). Classical MD based on the refined forcefield shows that these point mutations affect not only the immediate coordination sphere of the manganese ions, but also the relative position of the helices, improving the similarity to Mn-catalase, especially in case of M2. On the basis of these findings, classical molecular dynamics calculations with the active site parameterization scheme introduced herein seem to be a promising addition to the protein design toolbox.