High-throughput crystallization-to-structure pipeline at RIKEN SPring-8 Center

A high-throughput crystallization-to-structure pipeline for structural genomics was recently developed at the Advanced Protein Crystallography Research Group of the RIKEN SPring-8 Center in Japan. The structure determination pipeline includes three newly developed technologies for automating X-ray protein crystallography: the automated crystallization and observation robot system "TERA", the SPring-8 Precise Automatic Cryosample Exchanger "SPACE" for automated data collection, and the Package of Expert Researcher's Operation Network "PERON" for automated crystallographic computation from phasing to model checking. During the 5 years following April, 2002, this pipeline was used by seven researchers to determine 138 independent crystal structures (resulting from 437 purified proteins, 234 cryoloop-mountable crystals, and 175 diffraction data sets). The protocols used in the high-throughput pipeline are described in this paper.     

Nonlinear transient computation as a potential "kernel trick" in cortical processing

Evidence has been found for the presence of chaotic dynamics at all levels of the mammalian brain. This has led to some searching questions about the potential role that nonlinear dynamics may have in neural information processing. We propose that chaos equips the brain with the equivalent of a kernel trick for solving hard nonlinear problems. The approach presented, which is described as nonlinear transient computation, uses the dynamics of a well known chaotic attractor. The paper provides experimental results to show that this approach can be used to solve some challenging pattern recognition tasks. The paper also offers evidence to suggest that the efficacy of nonlinear transient computation for nonlinear pattern classification is dependent only on the generic properties of chaotic attractors and is not sensitive to the particular dynamics of specific sub-regions of chaotic phase space. If, as this work suggests, nonlinear transient computation is independent of the particulars of any given chaotic attractor, then it could be offered as a possible explanation of how the chaotic dynamics that have been observed in brain structures contribute to neural information processing tasks.     

Outline of a novel architecture for cortical computation

In this paper a novel architecture for cortical computation has been proposed. This architecture is composed of computing paths consisting of neurons and synapses. These paths have been decomposed into lateral, longitudinal and vertical components. Cortical computation has then been decomposed into lateral computation (LaC), longitudinal computation (LoC) and vertical computation (VeC). It has been shown that various loop structures in the cortical circuit play important roles in cortical computation as well as in memory storage and retrieval, keeping in conformity with the molecular basis of short and long term memory. A new learning scheme for the brain has also been proposed and how it is implemented within the proposed architecture has been explained. A few mathematical results about the architecture have been proposed, some of which are without proof.     

A high-throughput fluorescent polarization assay for nuclear receptor binding utilizing crude receptor extract.

A homogenous high-throughput assay has been developed to measure the binding between nuclear receptors and test compounds. This assay applies a fluorescence polarization (FP) detection method using human glucocorticoid receptor (GR) as a model system. Crude receptor extract, which requires no additional purification, is used in the assay. The binding conditions (i.e., DMSO tolerance, temperature, stability, and variability) have been investigated and validated. At the optimized conditions, a signal-to-background ratio of 2:1 and a Z'-factor of 0.7 was achieved in a 384-well format. Several known strong and weak GR ligands have been evaluated in this system. Possible interference of fluorescent compounds and methods to identify false positives are also discussed. This FP-based assay system can potentially be used for many soluble nuclear receptors in high-throughput binding assays.     

From genes to proteins: high-throughput expression and purification of the human proteome

The development of high-throughput methods for gene discovery has paved the way for the design of new strategies for genome-scale protein analysis. Lawrence Livermore National Laboratory and Onyx Pharmaceuticals, Inc., have produced an automatable system for the expression and purification of large numbers of proteins encoded by cDNA clones from the IMAGE (Integrated Molecular Analysis of Genomes and Their Expression) collection. This high-throughput protein expression system has been developed for the analysis of the human proteome, the protein equivalent of the human genome, comprising the translated products of all expressed genes. Functional and structural analysis of novel genes identified by EST (Expressed Sequence Tag) sequencing and the Human Genome Project will be greatly advanced by the application of this high-throughput expression system for protein production. A prototype was designed to demonstrate the feasibility of our approach. Using a PCR-based strategy, 72 unique IMAGE cDNA clones have been used to create an array of recombinant baculoviruses in a 96-well microtiter plate format. Forty-two percent of these cDNAs successfully produced soluble, recombinant protein. All of the steps in this process, from PCR to protein production, were performed in 96-well microtiter plates, and are thus amenable to automation. Each recombinant protein was engineered to incorporate an epitope tag at the amino terminal end to allow for immunoaffinity purification. Proteins expressed from this system are currently being analyzed for functional and biochemical properties.     

A wheat germ cell-free system is a novel way to screen protein folding and function

For high-throughput protein structural analysis, it is indispensable to develop a reliable protein overexpression system. Although many protein overexpression systems, such as that involving Escherichia coli cells, have been developed, the number of overexpressed proteins showing the same biological activities as those of the native proteins is limited. A novel wheat germ cell-free protein synthesis system was developed recently, and most of the proteins functioning in solution were synthesized as soluble forms. This suggests the applicability of this protein synthesis method to determination of the solution structures of functional proteins. To examine this possibility, we have synthesized two (15)N-labeled proteins and obtained (1)H-(15)N HSQC spectra for them. The structural analysis of these proteins has already progressed with an E. coli overexpression system, and (1)H-(15)N HSQC spectra for biologically active proteins have already been obtained. Comparing the spectra, we have shown that proteins synthesized with a wheat germ cell-free system have the proper protein folding and enough biological activity. This is the first experimental evidence of the applicability of the wheat germ cell-free protein synthesis system to high-throughput protein structural analysis.     

A high-throughput search for electronic materials-thin-film dielectrics.

Parallel synthesis together with high-throughput screening was used to identify candidate materials for integrated circuit applications that demand a superior high permittivity dielectric thin film. Specifically, we developed a "continuous-composition spread" (CCS) technique to synthesize much of a pseudoternary oxide system in a single deposition and used this in conjunction with a high-throughput measurement protocol, thereby allowing each chemical system to be deposited and evaluated in about 24 h. This approach led to the identification of compositions in the Zr-Sn-Ti-O system with promising properties. The same technique was used to determine the optimum compositions as a function of processing parameters. Films with the composition Zr(.2)Sn(.2)Ti(.6)O(2) were then prepared using a conventional synthetic technique (on-axis sputtering) and were verified to have excellent properties. Thus, the CCS technique has demonstrated utility in rapidly identifying and developing a useful new material.     

Discovery of novel regulators of aldehyde dehydrogenase isoenzymes

Over the past three years we have been involved in high-throughput screening in an effort to discover novel small molecular modulators of aldehyde dehydrogenase (ALDH) activity. In particular, we have been interested in both the activation and inhibition of the three commonly studied isoenzymes, ALDH1A1, ALDH2 and ALDH3A1, as their distinct, yet overlapping substrate specificities, present a particularly difficult challenge for inhibitor discovery and design. Activation of ALDH2 has been shown to benefit cardiovascular outcome following periods of ischemia and renewed interest in specific inhibition of ALDH2 has application for alcohol aversion therapy, and more recently, in cocaine addiction. In contrast, inhibition of either ALDH1A1 or ALDH3A1 has application in cancer treatments where the isoenzymes are commonly over-expressed and serve as markers for cancer stem cells. We are taking two distinct approaches for these screens: in vitro enzyme activity screens using chemical libraries and virtual computational screens using the structures of the target enzymes as filters for identifying potential inhibitors, followed by in vitro testing of their ability to inhibit their intended targets. We have identified selective inhibitors of each of these three isoenzymes with inhibition constants in the high nanomolar to low micromolar range from these screening procedures. Together, these inhibitors provide proof for concept that selective inhibition of these broad specificity general detoxication enzymes through small molecule discovery and design is possible.     

A designed glycopeptide array for characterization of sugar-binding proteins toward a glycopeptide chip technology

For the realization of a practical high-throughput protein detection and analysis system, a novel peptide array has been constructed using a designed glycopeptide model library with an α-helical secondary structure. This study will contribute the increment of the diversity of such an array system and the application to focused proteomics and ligand screening by effective detection of sugar-binding proteins. Fluorescent glycopeptides with an α-helix, a β-strand, or a loop structure were designed initially to select a suitable scaffold for the detection of a model protein. After selection of the α-helical structure as the best scaffold, a small model library with various saccharides was constructed to have charge and hydrophobicity variations in the peptide sequences. When various sugar-binding proteins were added to the peptide library array, the fluorescent peptides showed different responses in fluorescence intensities depending on their sequences as well as saccharides. The patterns of these responses could be regarded as “protein fingerprints” (PFPs), which are able to establish the identities of the target proteins. The resulting PFPs reflected the recognition properties of the proteins. Furthermore, statistical data analysis from obtained PFPs was performed using a cluster analysis. The PFPs of sugar-binding proteins were clustered successfully depending on their families and binding properties. These studies demonstrate that arrays with glycopeptide libraries based on designed structures can be promising tools to detect and analyze the target proteins. Designed peptides with functional groups such as sugars will play roles as the capturing agents of high-throughput protein nano/micro arrays for focused proteomics and ligand screening studies.     

高通量技术在萜类合成酶筛选中的应用

萜类化合物种类繁多,生物活性多样,在食品、药品与化妆品等行业中具有广泛的应用。萜类化合物多来源于植物,然而随着合成生物学的快速发展,相较于传统的天然植物提取与化学合成方法,利用工程微生物进行萜类化合物异源合成的方法显得更为经济与环保。萜类合成酶的催化活性及合成产物的结构特性是萜类化合物异源合成的关键。通过蛋白定向进化与理性设计可以有针对性地优化萜类合成酶的催化性能及产物专一性,但该方案需要一个特异的筛选方法来实现蛋白突变体库的高通量筛选。近年来,一系列高通量筛选方法的建立使得萜类合成酶的筛选变得更加灵敏与高效。本文对近期建立的萜类合成酶高通量筛选方法进行了综述,简要概述了各种筛选方法的基本原理与优缺点,并对高通量筛选技术在萜类合成酶改造中的应用做出了展望。     

High-throughput Three-dimensional Gel Electrophoresis for Versatile Utilities： A Stacked Slice-gel System for Separation and Reactions （4SR）

Introduction The completion of the Human Genome Project has triggered large-scale screening of genomes (1) and proteomes (2) in aims to find out candidate genes related to diseases (3), perform expression analyses at the mRNA level (4) or at the protein level (5), discover new drugs (6), and analyze molecular in- teractions (7). For such purposes, technologies han- dling a tiny amount of samples should be developed, of which the importance has already been described as the ambient analyte th…     

膜片钳技术的新进展及其在药物高通量筛选中的应用

作为先进的细胞电生理技术,膜片钳一直被奉为研究离子通道的“金标准”。应用膜片钳技术可以证实细胞膜上离子通道的存在并能对其电生理特性、分子结构、药物作用机制等进行深入的研究。基因组学、蛋白质组学研究表明,以离子通道为靶标的药物研究在未来具有很大的发展空间。为了突破由于筛选技术所造成的针对离子通道为靶标的药物研发的瓶颈,近年来,对膜片钳技术进行了改进以适合药物高通量筛选的需求,由此产生了一些新的技术。本文就最近几年膜片钳技术的新进展及其在药物高通量筛选中的应用进行了综述。     

Single-cell phenomics in budding yeast

The demand for phenomics, a high-dimensional and high-throughput phenotyping method, has been increasing in many fields of biology. The budding yeast Saccharomyces cerevisiae, a unicellular model organism, provides an invaluable system for dissecting complex cellular processes using high-resolution phenotyping. Moreover, the addition of spatial and temporal attributes to subcellular structures based on microscopic images has rendered this cell phenotyping system more reliable and amenable to analysis. A well-designed experiment followed by appropriate multivariate analysis can yield a wealth of biological knowledge. Here we review recent advances in cell imaging and illustrate their broad applicability to eukaryotic cells by showing how these techniques have advanced our understanding of budding yeast.     

Development of a scintiplate assay for recombinant human alpha(2B)-adrenergic receptor

A high-throughput solid-phase platform for ligand-binding assays using microtiter plates (Scintiplates) has been developed using the scintillation proximity assay principle. The system has been developed using human alpha(2B)-adrenergic receptor (alpha(2B)-AR) expressed from Semliki Forest virus vectors in CHO cells. Alpha(2B)-AR bind natural (adrenaline and noradrenaline) and synthetic ligands with different affinities to mediate a variety of physiological and pharmacological responses. Antagonist radioligands were used for the binding experiments, and the values obtained for the binding constants with the Scintiplate system are in good agreement with those obtained by the traditional filter-binding assay system. The Scintiplate assay offers the advantages of a high-throughput format over the filter-binding assay and is amenable for screening many compounds rapidly for generation of leads.     

Eukaryotic membrane protein overproduction in Lactococcus lactis

Eukaryotic membrane proteins play many vital roles in the cell and are important drug targets. Approximately 25% of all genes identified in the genome are known to encode membrane proteins, but the vast majority have no assigned function. Although the generation of structures of soluble proteins has entered the high-throughput stage, for eukaryotic membrane proteins only a dozen high-resolution structures have been obtained so far. One major bottleneck for the functional and structural characterisation of membrane proteins is the overproduction of biologically active material. Recent advances in the development of the Lactococcus lactis expression system have opened the way for the high-throughput functional expression of eukaryotic membrane proteins.     

A topological framework for the computation of the HOMFLY polynomial and its application to proteins

Polymers can be modeled as open polygonal paths and their closure generates knots. Knotted proteins detection is currently achieved via high-throughput methods based on a common framework insensitive to the handedness of knots. Here we propose a topological framework for the computation of the HOMFLY polynomial, an handedness-sensitive invariant. Our approach couples a multi-component reduction scheme with the polynomial computation. After validation on tabulated knots and links the framework was applied to the entire Protein Data Bank along with a set of selected topological checks that allowed to discard artificially entangled structures. This led to an up-to-date table of knotted proteins that also includes two newly detected right-handed trefoil knots in recently deposited protein structures. The application range of our framework is not limited to proteins and it can be extended to the topological analysis of biological and synthetic polymers and more generally to arbitrary polygonal paths.     

High-throughput construction of expression system using yeast Pichia pastoris, and its application to membrane proteins

The well-established method for high-throughput construction of an expression system of the yeast Saccharomyces cerevisiae uses homologous recombination between an expression plasmid and a target gene (with homologous regions of the plasmid on both ends added by PCR). This method has been widely used for membrane proteins using plasmids containing GFP, and has been successfully used to investigate the cellular localization and solubilization conditions of the proteins. Although the methanol-utilizing yeast Pichia pastoris is known as an excellent expression host, a method for high-throughput construction of an expression system like that in S. cerevisiae has not been reported. In this study, we have attempted to construct expression systems via homologous recombination in P. pastoris. The insertion of genes into a plasmid could be easily checked by colony-PCR. Expression systems for seven membrane proteins of medaka fish (Oryzias latipes) and yeast (S. cerevisiae) were constructed, and the expression of proteins was analyzed by fluorescence spectra, fluorescence microscopy, and SDS-PAGE (in-gel fluorescence detection).     

Peptide arrays with designed secondary structures for protein characterization using fluorescent fingerprint patterns

To realize a practical high-throughput protein-detection system, novel peptide arrays have been constructed using designed peptide libraries with loop, alpha-helix, or beta-strand structures. Here, we describe the overview of the reported designed peptide arrays with loop and alpha-helix structures and the new results of those with beta-strand structures. Initially, several model peptides known to interact with model structured proteins were selected to establish the present strategy for high-throughput detection of proteins. The fluorescent probes and suitable scaffolds of peptides were examined for the effective detection of proteins. The detection methods were established in solution and in an immobilized manner using the model systems. In the case of alpha-helix peptide, the response of a peptide with fluorescent resonance energy transfer between two probes at both termini was several times higher than that of a peptide with a single probe. In the cases of peptides with other structures, however, proteins were effectively detectable even by the fluorescent change of one probe. Furthermore, structurally focused libraries consisting of a total of ca. 250 different peptides based on the model peptides with secondary and/or tertiary structures were constructed with systematic replacement of residues. Using these libraries, various proteins were characterized effectively to give their own fluorescent "protein fingerprint" patterns. The resulting protein fingerprints correlated with the recognition properties of the proteins. These studies demonstrate that arrays with peptide libraries based on designed structures can be promising tools for detecting the target proteins. Designed synthetic peptides play roles as the capturing agents to be developed for practical protein chips.     

Efficient Bayesian inference for mechanistic modelling with high-throughput data

Bayesian methods are routinely used to combine experimental data with detailed mathematical models to obtain insights into physical phenomena. However, the computational cost of Bayesian computation with detailed models has been a notorious problem. Moreover, while high-throughput data presents opportunities to calibrate sophisticated models, comparing large amounts of data with model simulations quickly becomes computationally prohibitive. Inspired by the method of Stochastic Gradient Descent, we propose a minibatch approach to approximate Bayesian computation. Through a case study of a high-throughput imaging scratch assay experiment, we show that reliable inference can be performed at a fraction of the computational cost of a traditional Bayesian inference scheme. By applying a detailed mathematical model of single cell motility, proliferation and death to a data set of 118 gene knockdowns, we characterise functional subgroups of gene knockdowns, each displaying its own typical combination of local cell density-dependent and -independent motility and proliferation patterns. By comparing these patterns to experimental measurements of cell counts and wound closure, we find that density-dependent interactions play a crucial role in the process of wound healing.