Screening for presenilin inhibitors using the free-living nematode, Caenorhabditis elegans

Caenorhabditis elegans contains 3 homologs of presenilin genes that are associated with Alzheimer s disease. Loss-of-function mutations in C. elegans genes cause a defect in egg laying. In humans, loss of presenilin-1 (PS1) function reduces amyloid-beta peptide processing from the amyloid protein precursor. Worms were screened for compounds that block egg laying, phenocopying presenilin loss of function. To accommodate even relatively high throughput screening, a semi-automated method to quantify egg laying was devised by measuring the chitinase released into the culture medium. Chitinase is released by hatching eggs, but little is shed into the medium from the body cavity of a hermaphrodite with an egg laying deficient (egl) phenotype. Assay validation involved measuring chitinase release from wild-type C. elegans (N2 strain), sel-12 presenilin loss-of-function mutants, and 2 strains of C. elegans with mutations in the egl-36 K(+) channel gene. Failure to find specific presenilin inhibitors in this collection likely reflects the small number of compounds tested, rather than a flaw in screening strategy. Absent defined biochemical pathways for presenilin, this screening method, which takes advantage of the genetic system available in C. elegans and its historical use for anthelminthic screening, permits an entry into mechanism-based discovery of drugs for Alzheimer's disease.     

There's no place like WormBase: an indispensable resource for Caenorhabditis elegans researchers

The nematode Caenorhabditis elegans is used extensively by scientists to study a wide variety of biological processes and is one of the most thoroughly characterized animals. Over the years, the community of C. elegans researchers has generated a wealth of information on the genetics, development, behaviour, and cellular and molecular biology of the worm. This body of data has grown even larger with the recent application of high throughput screening methodology to study gene function, expression and interactions. WormBase (http://www.wormbase.org) is the primary online source of biological data on C. elegans and related nematodes. Equipped with an assortment of powerful search tools, WormBase allows users to quickly extract a variety of information, including data on individual genes, DNA sequence, cell lineage and literature citations. As the database is well maintained and the functionalities constantly modified in response to evolving researcher needs, WormBase has become a vital component of the laboratories studying the worm and a model for other biological databases.     

Automated high-content live animal drug screening using C. elegans expressing the aggregation prone serpin α1-antitrypsin Z

The development of preclinical models amenable to live animal bioactive compound screening is an attractive approach to discovering effective pharmacological therapies for disorders caused by misfolded and aggregation-prone proteins. In general, however, live animal drug screening is labor and resource intensive, and has been hampered by the lack of robust assay designs and high throughput work-flows. Based on their small size, tissue transparency and ease of cultivation, the use of C. elegans should obviate many of the technical impediments associated with live animal drug screening. Moreover, their genetic tractability and accomplished record for providing insights into the molecular and cellular basis of human disease, should make C. elegans an ideal model system for in vivo drug discovery campaigns. The goal of this study was to determine whether C. elegans could be adapted to high-throughput and high-content drug screening strategies analogous to those developed for cell-based systems. Using transgenic animals expressing fluorescently-tagged proteins, we first developed a high-quality, high-throughput work-flow utilizing an automated fluorescence microscopy platform with integrated image acquisition and data analysis modules to qualitatively assess different biological processes including, growth, tissue development, cell viability and autophagy. We next adapted this technology to conduct a small molecule screen and identified compounds that altered the intracellular accumulation of the human aggregation prone mutant that causes liver disease in α1-antitrypsin deficiency. This study provides powerful validation for advancement in preclinical drug discovery campaigns by screening live C. elegans modeling α1-antitrypsin deficiency and other complex disease phenotypes on high-content imaging platforms.     

Caenorhabditis elegans functional genomics: omic resonance.

The nematode Caenorhabditis elegans is widely used as a model organism for studying many fundamental aspects of development and cell biology, including processes underlying human disease. The genome of C. elegans encodes over 19,000 protein-coding genes and hundreds of non-coding RNAs. The availability of whole genome sequence has facilitated the development of high throughput techniques for elucidating the function of individual genes and gene products. Furthermore, attempts can now be made to integrate these substantial functional genomics data collections and to understand at a global level how the flow of genomic information that is at the core of the central dogma leads to the development of a multicellular organism.     

基因芯片技术及应用研究进展

采用高速打印或光刻合成技术可在硅片、玻璃或尼龙膜上制造ＤＮＡ微阵列。样品ＤＮＡ／ＲＮＡ通过ＰＣＲ扩增、体外转录等技术掺入荧光标记分子,与微阵列杂交后通过荧光扫描仪器扫描及计算机分析即可获得样品中大量基因序列及表达的信息。该技术可应用于高通量基因表达平行分析、大规模基因发现及序列分析、基因多态性分析和基因组研究等 。     

Isolation of the interacting molecules with GEX-3 by a novel functional screening

To screen for important molecules that interact with a gene of interest in Caenorhabditis elegans (C. elegans), we established a novel functional screening system using the yeast two-hybrid system with the RNA interference technique. Our screening system makes it possible to identify the molecular machinery involved in the function of a gene of interest starting with the cDNA of this gene. As a model case, we examined the molecular machinery involved in the function of GEX-3, an essential factor of tissue morphogenesis. We identified many interacting molecules by yeast two-hybrid screening and could detect some functional interactions using this novel functional screening system.     

随机突变文库构建与筛选研究进展

定向进化是一个循环过程,在构建多样化基因序列和筛选功能基因变体之间交替进行.该技术目前已被广泛应用于DNA序列、基因功能和蛋白质结构的优化和分析.定向进化包括随机基因文库的生成、基因在合适宿主中的表达和突变文库的筛选.构建基因文库的关键是库容量和突变多样性,而筛选变体的关键是高灵敏度和高通量.文中讨论了定向进化技术的最...     

Evaluation of invertebrate infection models for pathogenic corynebacteria

For several pathogenic bacteria, model systems for host-pathogen interactions were developed, which provide the possibility of quick and cost-effective high throughput screening of mutant bacteria for genes involved in pathogenesis. A number of different model systems, including amoeba, nematodes, insects, and fish, have been introduced, and it was observed that different bacteria respond in different ways to putative surrogate hosts, and distinct model systems might be more or less suitable for a certain pathogen. The aim of this study was to develop a suitable invertebrate model for the human and animal pathogens Corynebacterium diphtheriae, Corynebacterium pseudotuberculosis, and Corynebacterium ulcerans. The results obtained in this study indicate that Acanthamoeba polyphaga is not optimal as surrogate host, while both Caenorhabtitis elegans and Galleria larvae seem to offer tractable models for rapid assessment of virulence between strains. Caenorhabtitis elegans gives more differentiated results and might be the best model system for pathogenic corynebacteria, given the tractability of bacteria and the range of mutant nematodes available to investigate the host response in combination with bacterial virulence. Nevertheless, Galleria will also be useful in respect to innate immune responses to pathogens because insects offer a more complex cell-based innate immune system compared with the simple innate immune system of C.?elegans.     

Genome-wide RNAi as a route to gene function in Drosophila.

With the sequencing of the human genome and the genomes of most major model organisms completed, the systematic characterisation of gene functions remains a key challenge. During the past few years, RNA interference (RNAi) has become a powerful tool to silence the expression of genes and analyse their loss-of-function phenotype when mutant alleles are not available. Genome-wide RNAi screens against all predicted genes have been successfully used to dissect a variety of biological processes in Caenorhabditis elegans. Recently, a genome-wide library of double-stranded RNAs, that target every gene in the Drosophila genome and that is suitable for high throughput cell-based assays, was published. In this paper, recent advances will be summarised. Screening strategies and applications as a route to comprehensively characterising gene function will be discussed.     

Biocatalytic conversion of unnatural substrates by recombinant almond R-HNL isoenzyme 5

Hydroxynitrile lyases (HNLs, EC 4.1.2.10, EC 4.1.2.11, EC 4.1.2.37, EC 4.1.2.39) enantioselectively catalyse the reversible addition of HCN to ketones or aldehydes, thereby forming chiral cyanohydrins, which is of special interest for industrial bio-conversions. We cloned the gene for the HNL isoenzyme 5 (PaHNL5) of the almond tree (Prunus amygdalus) and overexpressed it in the methylotrophic yeast Pichia pastoris. This opened new ways for the synthesis of (R)-cyanohydrins. The characterisation of PaHNL5 revealed high activity for the natural substrate and high enantioselectivity. For further improvement of enzyme properties such as higher activity for the conversion of unnatural substrates, a high throughput cultivation and screening system has been created, which allows the employment of P. pastoris as production host for high throughput cultivation and screening of thousands of enzyme variants. The synthesis and cleavage of 2-chlorobenzaldehyde cyanohydrin were used for the demonstration of enzyme activity of recombinant PaHNL5 with a non-natural substrate and for the development of a high throughput screening procedure.     

Genome-wide RNAi screening in Caenorhabditis elegans

In Caenorhabditis elegans, introduction of double-stranded RNA (dsRNA) results in the specific inactivation of an endogenous gene with corresponding sequence; this technique is known as RNA interference (RNAi). It has previously been shown that RNAi can be performed by direct microinjection of dsRNA into adult hermaphrodite worms, by soaking worms in a solution of dsRNA, or by feeding worms Escherichia coli expressing target-gene dsRNA. We have developed a simple optimized protocol exploiting this third mode of dsRNA introduction, RNAi by feeding, which allows rapid and effective analysis of gene function in C. elegans. Furthermore, we have constructed a library of bacterial strains corresponding to roughly 86% of the estimated 19,000 predicted genes in C. elegans, and we have used it to perform genome-wide analyses of gene function. This library is publicly available, reusable resource allowing for rapid large-scale RNAi experiments. We have used this library to perform genome-wide analyses of gene function in C. elegans. Here, we describe the protocols used for bacterial library construction and for high-throughput screening in C. elegans using RNAi by feeding.     

Conservation of microsatellites among tropical trees (Leguminosae)

Although microsatellites or simple sequence repeats (SSRs) have become a popular tool in genetic mapping and gene flow studies, their utility is limited due to paucity of information about DNA sequences in plants. We tested the utility of microsatellite markers characterized for the tropical tree Pithecellobium elegans as a genetic tool for related species. The results indicate that SSR loci are conserved among closely related species, and SSR primers developed for P. elegans could be successfully used as a genetic tool in several species of the tribe Ingeae. This study indicates that there is high potential for the transfer of SSR markers among closely related taxa, circumventing laborious cloning and screening procedures involved in characterizing SSR loci for many species.     

Adapting chromophore-assisted laser inactivation for high throughput functional proteomics.

Recent advances in genomics and proteomics have generated a change in emphasis from hypothesis-based to discovery-based investigations. Genomic and proteomic studies based on differential expression microarrays or comparative proteomics often provide many potential candidates for functionally important roles in normal and diseased cells. High throughput technologies to address protein and gene function in situ are still necessary to exploit these emerging advances in gene and protein discovery in order to validate these identified targets. The pharmaceutical industry is particularly interested in target validation, and has identified it as the critical early step in drug discovery. An especially powerful approach to target validation is a direct protein knockdown strategy called chromophore-assisted laser inactivation (CALI) which is a means of testing the role of specific proteins in particular cellular processes. Recent developments in CALI allow for its high throughput application to address many proteins in tandem. Thus, CALI may have applications for high throughput hypothesis testing, target validation or proteome-wide screening.     

Minireview: Challenges of High Throughput Screening Against Cell Surface Receptors

Abstract

Excessive or inappropriate activation of cell surface receptors can mediate the development of disease. Receptors, therefore, are a focus for drug discovery activities. Empirical screening is important in the search for novel compounds acting at receptors. Technical developments and the application of molecular biology have facilitated access to receptors of interest and have provided efficient screening methods capable of very high throughput. Reliability in high throughput screening requires the use of appropriate methodology, good screen design and effective validation and quality control processes. Validation should aim to establish that the basic experimental design is sound. In developing software to handle high throughput screening data, a fundamental requirement is to provide performance monitoring and error trapping facilities. Additional requirements are automatic data capture from instruments, on-line data reduction and analysis and transfer of results to central databases. As data volumes increase through effective high throughput screening, conventional interrogation methods become less appropriate and are being augmented by newer computing techniques referred to as knowledge mapping or database mining. Targeting cell surface receptors has been very successful as an approach to drug discovery. If the challenges of high throughput empirical screening are addressed effectively, cell surface receptors will provide new opportunities for improved therapy in the coming years.     

高通量药物筛选技术的研究现状及光学编码技术在其中的应用

高通量药物筛选是创新药物研究的重要内容,本文综合介绍了高通量药物筛选的研究现状及发展趋势,及目前遇到的一些问题,如随着样品的体积达到微升量级时,目前所采用的孔板筛选遇到了一些难以克服的障碍,使化合物和药物靶点的相互作用难以进行等。针对这些问题,本文介绍了一种新的以自编码光谱识别微球为基础的新型高通量药物筛选技术在高通量药物筛选中的应用,同时简要介绍了我们目前所做的一些工作。     

组胺H3受体激活剂高通量筛选细胞模型的研究

摘 要 目的 建立基于报告基因的组胺H3受体（H3R）激活剂的高通量筛选模型,用此模型对收集到的中草药化合物组分进行筛选,以发现新的组胺H3R激活剂。 方法 将H3R基因质粒（H3R/pCDNA3.1-hygro）与报告基因质粒（3XCRE-LUC）按3：1的比例共转染入HEK293细胞,建立了稳定的H3R配体的报告基因筛选细胞株。激活剂与细胞表面H3R结合后,激活相应的信号通路,调节Forskolin刺激后的报告基因的表达,通过测定荧光素酶报告基因表达水平的变化,评估激活剂影响H3受体的生物活性。 结果 通过对筛选条件,如激活剂孵育时间、Forskolin终浓度、化合物溶剂的选择、溶剂DMSO终浓度等的优化,建立了可靠的筛选方法,并对多种中草药萃取物进行了筛选,找到了两种对H3R有活性的中药组分。结论 建立的细胞模型可以有效的应用于以组胺H3受体为靶点的高通量药物筛选。     

Identification of a mouse orthologue of the CED-6 gene of Caenorhabditis elegans

The rapid engulfment of apoptotic cells is a specialized innate immune response used by organisms to remove apoptotic cells. In mammals, several receptors that recognize apoptotic cells have been identified. Previous analysis of the engulfment gene ced-6 in Caenorhabditis elegans (C. elegans) has suggested that CED-6 is an adapter protein that participates in signal transduction pathway that mediates the specific recognition and engulfment of apoptotic cells. Here, we describe our isolation and partial characterization of a mouse cDNA, which is like an orthologue of C. elegans CED-6. PCR screening of mouse cDNA pool with primers designed from the C. elegans CED-6 cDNA sequence resulted in about 300 bp PCR product which was partially sequenced and then screened to a mouse full-length cDNA library. Thus in this study we report the identification of a novel C. elegans CED-6-like orthologue in mouse, which has probable apoptotic like function.