WorldWideScience

Sample records for chemical genomics screens

  1. Genome-wide genetic screening with chemically mutagenized haploid embryonic stem cells

    OpenAIRE

    Forment, Josep V.; Herzog, Mareike; Coates, Julia; Konopka, Tomasz; Gapp, Bianca V.; Nijman, Sebastian M.; Adams, David J; Keane, Thomas M.; Jackson, Stephen P.

    2016-01-01

    This is the author accepted manuscript. In model organisms, classical genetic screening via random mutagenesis provides key insights into the molecular bases of genetic interactions, helping to define synthetic lethality, synthetic viability and drug-resistance mechanisms. The limited genetic tractability of diploid mammalian cells, however, precludes this approach. Here, we demonstrate the feasibility of classical genetic screening in mammalian systems by using haploid cells, chemical mut...

  2. Screening of genomic libraries.

    Science.gov (United States)

    Novelli, Valdenice M; Cristofani-Yaly, Mariângela; Bastianel, Marinês; Palmieri, Dario A; Machado, Marcos A

    2013-01-01

    Microsatellites, or simple sequence repeats (SSRs), have proven to be an important molecular marker in plant genetics and breeding research. The main strategies to obtain these markers can be through genomic DNA and from expressed sequence tags (ESTs) from mRNA/cDNA libraries. Genetic studies using microsatellite markers have increased rapidly because they can be highly polymorphic, codominant markers and they show heterozygous conserved sequences. Here, we describe a methodology to obtain microsatellite using the enrichment library of DNA genomic sequences. This method is highly efficient to development microsatellite markers especially in plants that do not have available ESTs or genome databases. This methodology has been used to enrich SSR marker libraries in Citrus spp., an important tool to genotype germplasm, to select zygotic hybrids, and to saturate genetic maps in breeding programs.

  3. A Global Genomic Screening Strategy Reveals Genetic and Chemical Activators ofPeroxisome Proliferator-Activated Receptor alpha (PPARalpha)

    Science.gov (United States)

    A comprehensive survey of chemical, diet and genetic perturbations that activate PPARalpha in the mouse liver has not been carried out but would be useful to identify the factors that may contribute to PPARalpha-dependent liver tumors. A gene signature dependent on PPARalpha ac...

  4. Chemical compatibility screening test results

    Energy Technology Data Exchange (ETDEWEB)

    Nigrey, P.J.; Dickens, T.G.

    1997-12-01

    A program for evaluating packaging components that may be used in transporting mixed-waste forms has been developed and the first phase has been completed. This effort involved the screening of ten plastic materials in four simulant mixed-waste types. These plastics were butadiene-acrylonitrile copolymer rubber, cross-linked polyethylene (XLPE), epichlorohydrin rubber, ethylene-propylene rubber (EPDM), fluorocarbon (Viton or Kel-F), polytetrafluoroethylene, high-density polyethylene (HDPE), isobutylene-isoprene copolymer rubber (butyl), polypropylene, and styrene-butadiene rubber (SBR). The selected simulant mixed wastes were (1) an aqueous alkaline mixture of sodium nitrate and sodium nitrite; (2) a chlorinated hydrocarbon mixture; (3) a simulant liquid scintillation fluid; and (4) a mixture of ketones. The testing protocol involved exposing the respective materials to 286,000 rads of gamma radiation followed by 14-day exposures to the waste types at 60{degrees}C. The seal materials were tested using vapor transport rate (VTR) measurements while the liner materials were tested using specific gravity as a metric. For these tests, a screening criterion of 0.9 g/hr/m{sup 2} for VTR and a specific gravity change of 10% was used. Based on this work, it was concluded that while all seal materials passed exposure to the aqueous simulant mixed waste, EPDM and SBR had the lowest VTRs. In the chlorinated hydrocarbon simulant mixed waste, only Viton passed the screening tests. In both the simulant scintillation fluid mixed waste and the ketone mixture simulant mixed waste, none of the seal materials met the screening criteria. For specific gravity testing of liner materials, the data showed that while all materials with the exception of polypropylene passed the screening criteria, Kel-F, HDPE, and XLPE offered the greatest resistance to the combination of radiation and chemicals.

  5. Microelectroporation device for genomic screening

    Energy Technology Data Exchange (ETDEWEB)

    Perroud, Thomas D.; Renzi, Ronald F.; Negrete, Oscar; Claudnic, Mark R.

    2014-09-09

    We have developed an microelectroporation device that combines microarrays of oligonucleotides, microfluidic channels, and electroporation for cell transfection and high-throughput screening applications (e.g. RNA interference screens). Microarrays allow the deposition of thousands of different oligonucleotides in microscopic spots. Microfluidic channels and microwells enable efficient loading of cells into the device and prevent cross-contamination between different oligonucleotides spots. Electroporation allows optimal transfection of nucleic acids into cells (especially hard-to-transfect cells such as primary cells) by minimizing cell death while maximizing transfection efficiency. This invention has the advantage of a higher throughput and lower cost, while preventing cross-contamination compared to conventional screening technologies. Moreover, this device does not require bulky robotic liquid handling equipment and is inherently safer given that it is a closed system.

  6. Overview of chemical genomics and proteomics.

    Science.gov (United States)

    Zanders, Edward D

    2012-01-01

    Chemical genetics, genomics, and proteomics have been in existence as distinct offshoots of chemical biology for about 20 years. This review provides a brief definition of each, followed by some examples of how each technology is being used to advance basic research and drug discovery.

  7. 75 FR 70248 - Endocrine Disruptor Screening Program; Second List of Chemicals for Tier 1 Screening

    Science.gov (United States)

    2010-11-17

    .... List of Subjects Environmental protection, Chemicals, Drinking water, Endocrine disruptors, Pesticides... AGENCY Endocrine Disruptor Screening Program; Second List of Chemicals for Tier 1 Screening AGENCY... chemicals and substances for which EPA intends to issue test orders under the Endocrine Disruptor...

  8. Genomic libraries: I. Construction and screening of fosmid genomic libraries.

    Science.gov (United States)

    Quail, Mike A; Matthews, Lucy; Sims, Sarah; Lloyd, Christine; Beasley, Helen; Baxter, Simon W

    2011-01-01

    Large insert genome libraries have been a core resource required to sequence genomes, analyze haplotypes, and aid gene discovery. While next generation sequencing technologies are revolutionizing the field of genomics, traditional genome libraries will still be required for accurate genome assembly. Their utility is also being extended to functional studies for understanding DNA regulatory elements. Here, we present a detailed method for constructing genomic fosmid libraries, testing for common contaminants, gridding the library to nylon membranes, then hybridizing the library membranes with a radiolabeled probe to identify corresponding genomic clones. While this chapter focuses on fosmid libraries, many of these steps can also be applied to bacterial artificial chromosome libraries.

  9. 75 FR 77869 - Endocrine Disruptor Screening Program; Second List of Chemicals for Tier 1 Screening; Extension...

    Science.gov (United States)

    2010-12-14

    ... water, Endocrine disruptors, Pesticides and pests. Dated: December 8, 2010. Stephen A. Owens, Assistant... AGENCY Endocrine Disruptor Screening Program; Second List of Chemicals for Tier 1 Screening; Extension of... Endocrine Disruptor Screening Program's (EDSP) second list of chemicals for Tier 1 screening. This...

  10. High-content screening of functional genomic libraries.

    Science.gov (United States)

    Rines, Daniel R; Tu, Buu; Miraglia, Loren; Welch, Genevieve L; Zhang, Jia; Hull, Mitchell V; Orth, Anthony P; Chanda, Sumit K

    2006-01-01

    Recent advances in functional genomics have enabled genome-wide genetic studies in mammalian cells. These include the establishment of high-throughput transfection and viral propagation methodologies, the production of large-scale cDNA and siRNA libraries, and the development of sensitive assay detection processes and instrumentation. The latter has been significantly facilitated by the implementation of automated microscopy and quantitative image analysis, collectively referred to as high-content screening (HCS), toward cell-based functional genomics application. This technology can be applied to whole genome analysis of discrete molecular and phenotypic events at the level of individual cells and promises to significantly expand the scope of functional genomic analyses in mammalian cells. This chapter provides a comprehensive guide for curating and preparing function genomics libraries and performing HCS at the level of the genome.

  11. Determination of sample size in genome-scale RNAi screens.

    Science.gov (United States)

    Zhang, Xiaohua Douglas; Heyse, Joseph F

    2009-04-01

    For genome-scale RNAi research, it is critical to investigate sample size required for the achievement of reasonably low false negative rate (FNR) and false positive rate. The analysis in this article reveals that current design of sample size contributes to the occurrence of low signal-to-noise ratio in genome-scale RNAi projects. The analysis suggests that (i) an arrangement of 16 wells per plate is acceptable and an arrangement of 20-24 wells per plate is preferable for a negative control to be used for hit selection in a primary screen without replicates; (ii) in a confirmatory screen or a primary screen with replicates, a sample size of 3 is not large enough, and there is a large reduction in FNRs when sample size increases from 3 to 4. To search a tradeoff between benefit and cost, any sample size between 4 and 11 is a reasonable choice. If the main focus is the selection of siRNAs with strong effects, a sample size of 4 or 5 is a good choice. If we want to have enough power to detect siRNAs with moderate effects, sample size needs to be 8, 9, 10 or 11. These discoveries about sample size bring insight to the design of a genome-scale RNAi screen experiment.

  12. Virtual screening: an in silico tool for interlacing the chemical universe with the proteome.

    Science.gov (United States)

    Westermaier, Yvonne; Barril, Xavier; Scapozza, Leonardo

    2015-01-01

    In silico screening both in the forward (traditional virtual screening) and reverse sense (inverse virtual screening (IVS)) are helpful techniques for interlacing the chemical universe of small molecules with the proteome. The former, which is using a protein structure and a large chemical database, is well-known by the scientific community. We have chosen here to provide an overview on the latter, focusing on validation and target prioritization strategies. By comparing it to complementary or alternative wet-lab approaches, we put IVS in the broader context of chemical genomics, target discovery and drug design. By giving examples from the literature and an own example on how to validate the approach, we provide guidance on the issues related to IVS. Copyright © 2014 Elsevier Inc. All rights reserved.

  13. RAPID SCREENING OF ENVIRONMENTAL CHEMICALS FOR ESTROGEN RECEPTOR BINDING CAPACITY

    Science.gov (United States)

    Over the last few years, an increased awareness of endocrine disrupting chemicals (EDCs) and their potential to affect wildlife and humans has produced a demand for practical screening methods to identify endocrine activity in a wide range of environmental and industrial chemical...

  14. Efficient exploration of chemical space by fragment-based screening.

    Science.gov (United States)

    Hall, Richard J; Mortenson, Paul N; Murray, Christopher W

    2014-01-01

    Screening methods seek to sample a vast chemical space in order to identify starting points for further chemical optimisation. Fragment based drug discovery exploits the superior sampling of chemical space that can be achieved when the molecular weight is restricted. Here we show that commercially available fragment space is still relatively poorly sampled and argue for highly sensitive screening methods to allow the detection of smaller fragments. We analyse the properties of our fragment library versus the properties of X-ray hits derived from the library. We particularly consider properties related to the degree of planarity of the fragments.

  15. Chemical Safety Alert: Identifying Chemical Reactivity Hazards Preliminary Screening Method

    Science.gov (United States)

    Introduces small-to-medium-sized facilities to a method developed by Center for Chemical Process Safety (CCPS), based on a series of twelve yes-or-no questions to help determine hazards in warehousing, repackaging, blending, mixing, and processing.

  16. Genome-wide screening and identification of antigens for rickettsial vaccine development

    Science.gov (United States)

    The capacity to identify immunogens for vaccine development by genome-wide screening has been markedly enhanced by the availability of complete microbial genome sequences coupled to rapid proteomic and bioinformatic analysis. Critical to this genome-wide screening is in vivo testing in the context o...

  17. A whole genome RNAi screen identifies replication stress response genes.

    Science.gov (United States)

    Kavanaugh, Gina; Ye, Fei; Mohni, Kareem N; Luzwick, Jessica W; Glick, Gloria; Cortez, David

    2015-11-01

    Proper DNA replication is critical to maintain genome stability. When the DNA replication machinery encounters obstacles to replication, replication forks stall and the replication stress response is activated. This response includes activation of cell cycle checkpoints, stabilization of the replication fork, and DNA damage repair and tolerance mechanisms. Defects in the replication stress response can result in alterations to the DNA sequence causing changes in protein function and expression, ultimately leading to disease states such as cancer. To identify additional genes that control the replication stress response, we performed a three-parameter, high content, whole genome siRNA screen measuring DNA replication before and after a challenge with replication stress as well as a marker of checkpoint kinase signalling. We identified over 200 replication stress response genes and subsequently analyzed how they influence cellular viability in response to replication stress. These data will serve as a useful resource for understanding the replication stress response.

  18. Adapting CRISPR/Cas9 for functional genomics screens.

    Science.gov (United States)

    Malina, Abba; Katigbak, Alexandra; Cencic, Regina; Maïga, Rayelle Itoua; Robert, Francis; Miura, Hisashi; Pelletier, Jerry

    2014-01-01

    The use of CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein) for targeted genome editing has been widely adopted and is considered a "game changing" technology. The ease and rapidity by which this approach can be used to modify endogenous loci in a wide spectrum of cell types and organisms makes it a powerful tool for customizable genetic modifications as well as for large-scale functional genomics. The development of retrovirus-based expression platforms to simultaneously deliver the Cas9 nuclease and single guide (sg) RNAs provides unique opportunities by which to ensure stable and reproducible expression of the editing tools and a broad cell targeting spectrum, while remaining compatible with in vivo genetic screens. Here, we describe methods and highlight considerations for designing and generating sgRNA libraries in all-in-one retroviral vectors for such applications.

  19. RAPD-based screening of genomic libraries for positional cloning.

    Science.gov (United States)

    Dioh, W; Tharreau, D; Lebrun, M H

    1997-12-15

    RAPD markers are frequently used for positional cloning. However, RAPD markers often contain repeated sequences which prevent genomic library screening by hybridisation. We have developed a simple RAPD analysis of genomic libraries based on the identification of cosmid pools and clones amplifying the RAPD marker of interest. Our method does not require the cloning or characterisation of the RAPD marker as it relies on the analysis of cosmid pools or clones using a simple RAPD protocol. We applied this strategy using four RAPD markers composed of single copy or repeated sequences linked to avirulence genes of the rice blast fungus Magnaporthe grisea . Cosmids containing these RAPD markers were easily and rapidly identified allowing the construction of physical contigs at these loci.

  20. Functional genomic screening approaches in mechanistic toxicology and potential future applications of CRISPR-Cas9.

    Science.gov (United States)

    Shen, Hua; McHale, Cliona M; Smith, Martyn T; Zhang, Luoping

    2015-01-01

    Characterizing variability in the extent and nature of responses to environmental exposures is a critical aspect of human health risk assessment. Chemical toxicants act by many different mechanisms, however, and the genes involved in adverse outcome pathways (AOPs) and AOP networks are not yet characterized. Functional genomic approaches can reveal both toxicity pathways and susceptibility genes, through knockdown or knockout of all non-essential genes in a cell of interest, and identification of genes associated with a toxicity phenotype following toxicant exposure. Screening approaches in yeast and human near-haploid leukemic KBM7 cells have identified roles for genes and pathways involved in response to many toxicants but are limited by partial homology among yeast and human genes and limited relevance to normal diploid cells. RNA interference (RNAi) suppresses mRNA expression level but is limited by off-target effects (OTEs) and incomplete knockdown. The recently developed gene editing approach called clustered regularly interspaced short palindrome repeats-associated nuclease (CRISPR)-Cas9, can precisely knock-out most regions of the genome at the DNA level with fewer OTEs than RNAi, in multiple human cell types, thus overcoming the limitations of the other approaches. It has been used to identify genes involved in the response to chemical and microbial toxicants in several human cell types and could readily be extended to the systematic screening of large numbers of environmental chemicals. CRISPR-Cas9 can also repress and activate gene expression, including that of non-coding RNA, with near-saturation, thus offering the potential to more fully characterize AOPs and AOP networks. Finally, CRISPR-Cas9 can generate complex animal models in which to conduct preclinical toxicity testing at the level of individual genotypes or haplotypes. Therefore, CRISPR-Cas9 is a powerful and flexible functional genomic screening approach that can be harnessed to provide

  1. Chemical and genomic evolution of enzyme-catalyzed reaction networks.

    Science.gov (United States)

    Kanehisa, Minoru

    2013-09-02

    There is a tendency that a unit of enzyme genes in an operon-like structure in the prokaryotic genome encodes enzymes that catalyze a series of consecutive reactions in a metabolic pathway. Our recent analysis shows that this and other genomic units correspond to chemical units reflecting chemical logic of organic reactions. From all known metabolic pathways in the KEGG database we identified chemical units, called reaction modules, as the conserved sequences of chemical structure transformation patterns of small molecules. The extracted patterns suggest co-evolution of genomic units and chemical units. While the core of the metabolic network may have evolved with mechanisms involving individual enzymes and reactions, its extension may have been driven by modular units of enzymes and reactions.

  2. Genome-Wide Screening of Genes Required for Glycosylphosphatidylinositol Biosynthesis.

    Directory of Open Access Journals (Sweden)

    Yao Rong

    Full Text Available Glycosylphosphatidylinositol (GPI is synthesized and transferred to proteins in the endoplasmic reticulum (ER. GPI-anchored proteins are then transported from the ER to the plasma membrane through the Golgi apparatus. To date, at least 17 steps have been identified to be required for the GPI biosynthetic pathway. Here, we aimed to establish a comprehensive screening method to identify genes involved in GPI biosynthesis using mammalian haploid screens. Human haploid cells were mutagenized by the integration of gene trap vectors into the genome. Mutagenized cells were then treated with a bacterial pore-forming toxin, aerolysin, which binds to GPI-anchored proteins for targeting to the cell membrane. Cells that showed low surface expression of CD59, a GPI-anchored protein, were further enriched for. Gene trap insertion sites in the non-selected population and in the enriched population were determined by deep sequencing. This screening enriched 23 gene regions among the 26 known GPI biosynthetic genes, which when mutated are expected to decrease the surface expression of GPI-anchored proteins. Our results indicate that the forward genetic approach using haploid cells is a useful and powerful technique to identify factors involved in phenotypes of interest.

  3. Application of high-throughput affinity-selection mass spectrometry for screening of chemical compound libraries in lead discovery.

    Science.gov (United States)

    Zehender, Hartmut; Mayr, Lorenz M

    2007-02-01

    High-throughput screening of chemical libraries for compounds that interfere with a particular molecular target is among the most powerful methodologies applied in lead discovery at present. In this review, the authors describe a label-free, homogeneous, affinity-selection-based technology developed at Novartis, termed SpeedScreen, which is compared with similar technologies used for high-throughput screening in the pharmaceutical and biotechnology industries. The focus at present of SpeedScreen is twofold: first, this technology is applied to orphan genomic targets and to those targets that are non-tractable by a functional assay; second, this technology is applied complementary to the well-established traditional methodologies for the screening of molecular targets. In summary, the authors discuss the value of affinity-selection-based high-throughput screening as a complementary technology to the common functional screening platforms and the benefits as well as the limitations of this new technology are outlined.

  4. Application of Plagiarism Screening Software in the Chemical Engineering Curriculum

    Science.gov (United States)

    Cooper, Matthew E.; Bullard, Lisa G.

    2014-01-01

    Plagiarism is an area of increasing concern for written ChE assignments, such as laboratory and design reports, due to ease of access to text and other materials via the internet. This study examines the application of plagiarism screening software to four courses in a university chemical engineering curriculum. The effectiveness of plagiarism…

  5. Application of Plagiarism Screening Software in the Chemical Engineering Curriculum

    Science.gov (United States)

    Cooper, Matthew E.; Bullard, Lisa G.

    2014-01-01

    Plagiarism is an area of increasing concern for written ChE assignments, such as laboratory and design reports, due to ease of access to text and other materials via the internet. This study examines the application of plagiarism screening software to four courses in a university chemical engineering curriculum. The effectiveness of plagiarism…

  6. Genome-wide Escherichia coli stress response and improved tolerance towards industrially relevant chemicals

    DEFF Research Database (Denmark)

    Rau, Martin Holm; Calero Valdayo, Patricia; Lennen, Rebecca

    2016-01-01

    of transcription changes within and between chemical groups, with functions such as energy metabolism, stress response, membrane modification, transporters and iron metabolism being affected. Regulon enrichment analysis identified key regulators likely mediating the transcriptional response, including CRP, Rpo...... approach was employed to understand the chemical stress response of Escherichia coli, including a genome-wide screen for mutants with increased fitness during chemical stress. Twelve chemicals with significant production potential were selected, consisting of organic solvent-like chemicals (butanol...... stress identified 294 enriched and 336 depleted mutants and experimental validation revealed up to 60 % increase in mutant growth rates. Mutants enriched in several conditions contained, among others, insertions in genes of the Mar-Sox-Rob regulon as well as transcription and translation related gene...

  7. ScreenBEAM: a novel meta-analysis algorithm for functional genomics screens via Bayesian hierarchical modeling | Office of Cancer Genomics

    Science.gov (United States)

    Functional genomics (FG) screens, using RNAi or CRISPR technology, have become a standard tool for systematic, genome-wide loss-of-function studies for therapeutic target discovery. As in many large-scale assays, however, off-target effects, variable reagents' potency and experimental noise must be accounted for appropriately control for false positives.

  8. Functional genomics down under: RNAi screening in the Victorian Centre for Functional Genomics.

    Science.gov (United States)

    Thomas, Daniel W; Gould, Cathryn M; Handoko, Yanny; Simpson, Kaylene J

    2014-05-01

    The Victorian Centre for Functional Genomics (VCFG) is an RNAi screening facility housed at the Peter MacCallum Cancer Centre in Melbourne, Australia. The Peter Mac is Australia's largest dedicated Cancer Research Institute, home to a team of over 520 scientists that focus on understanding the genetic risk of cancer, the molecular events regulating cancer growth and dissemination and improving detection through new diagnostic tools (www.petermac.org). Peter Mac is a well recognised technology leader and established the VCFG with a view to enabling researchers Australia and New Zealand-wide access to cutting edge functional genomics technology, infrastructure and expertise. This review documents the technology platforms operated within the VCFG and provides insight into the workflows and analysis pipelines currently in operation.

  9. Chemical microarray: a new tool for drug screening and discovery.

    Science.gov (United States)

    Ma, Haiching; Horiuchi, Kurumi Y

    2006-07-01

    HTS with microtiter plates has been the major tool used in the pharmaceutical industry to explore chemical diversity space and to identify active compounds and pharmacophores for specific biological targets. However, HTS faces a daunting challenge regarding the fast-growing numbers of drug targets arising from genomic and proteomic research, and large chemical libraries generated from high-throughput synthesis. There is an urgent need to find new ways to profile the activity of large numbers of chemicals against hundreds of biological targets in a fast, low-cost fashion. Chemical microarray can rise to this challenge because it has the capability of identifying and evaluating small molecules as potential therapeutic reagents. During the past few years, chemical microarray technology, with different surface chemistries and activation strategies, has generated many successes in the evaluation of chemical-protein interactions, enzyme activity inhibition, target identification, signal pathway elucidation and cell-based functional analysis. The success of chemical microarray technology will provide unprecedented possibilities and capabilities for parallel functional analysis of tremendous amounts of chemical compounds.

  10. From structure prediction to genomic screens for novel non-coding RNAs

    DEFF Research Database (Denmark)

    Gorodkin, Jan; Hofacker, Ivo L.

    2011-01-01

    . This and the increased amount of available genomes have made it possible to employ structure-based methods for genomic screens. The field has moved from folding prediction of single sequences to computational screens for ncRNAs in genomic sequence using the RNA structure as the main characteristic feature. Whereas early...... methods focused on energy-directed folding of single sequences, comparative analysis based on structure preserving changes of base pairs has been efficient in improving accuracy, and today this constitutes a key component in genomic screens. Here, we cover the basic principles of RNA folding and touch...

  11. Improved set of short-tandem-repeat polymorphisms for screening the human genome

    Energy Technology Data Exchange (ETDEWEB)

    Yuan, Bo; Vaske, D.; Weber, J.L. [Marshfield Medical Research Foundation, WI (United States)] [and others

    1997-02-01

    Short-tandem-repeat (microsatellite) DNA polymorphisms are widely used for screening the human and other genomes in initial linkage mapping. Since the average spacing between polymorphisms in genome screens is usually {ge}10 cM and since many thousands of human short-tandem-repeat polymorphisms (STRPs) are now available, optimal subsets of STRPs must be selected for screening. Two screening sets of STRPs for humans have been described in the literature, both of which are based primarily on dinucleotide-repeat polymorphisms. Here we describe our eighth and most recent human screening set, which is based almost entirely on trinucleotide-and tetranucleotide-repeat polymorphisms. 7 refs., 1 tab.

  12. A primer on using pooled shRNA libraries for functional genomic screens

    Institute of Scientific and Technical Information of China (English)

    Guang Hu; Ji Luo

    2012-01-01

    The discovery of RNA interference (RNAi) has revolutionized genetic analysis in mammalian cells.Loss-of-function RNAi screens enable rapid,functional annotation of the genome.Of the various RNAi approaches,pooled shRNA libraries have received considerable attention because of their versatility.A number of genome-wide shRNA libraries have been constructed against the human and mouse genomes,and these libraries can be readily applied to a variety of screens to interrogate the function of human and mouse genes in an unbiased fashion.We provide an introduction to the technical aspects of using pooled shRNA libraries for genetic screens.

  13. Functional genomic and high-content screening for target discovery and deconvolution

    Science.gov (United States)

    Heynen-Genel, Susanne; Pache, Lars; Chanda, Sumit K

    2014-01-01

    Introduction Functional genomic screens apply knowledge gained from the sequencing of the human genome toward rapid methods of identifying genes involved in cellular function based on a specific phenotype. This approach has been made possible through the use of advances in both molecular biology and automation. The utility of this approach has been further enhanced through the application of image-based high content screening, an automated microscopy and quantitative image analysis platform. These approaches can significantly enhance acquisition of novel targets for drug discovery. Areas covered Both the utility and potential issues associated with functional genomic screening approaches are discussed along with examples that illustrate both. The considerations for high content screening applied to functional genomics are also presented. Expert opinion Functional genomic and high content screening are extremely useful in the identification of new drug targets. However, the technical, experimental, and computational parameters have an enormous influence on the results. Thus, although new targets are identified, caution should be applied toward interpretation of screening data in isolation. Genomic screens should be viewed as an integral component of a target identification campaign that requires both the acquisition of orthogonal data, as well as a rigorous validation strategy. PMID:22860749

  14. Expression of heterologous sigma factors enables functional screening of metagenomic and heterologous genomic libraries.

    Science.gov (United States)

    Gaida, Stefan M; Sandoval, Nicholas R; Nicolaou, Sergios A; Chen, Yili; Venkataramanan, Keerthi P; Papoutsakis, Eleftherios T

    2015-05-06

    A key limitation in using heterologous genomic or metagenomic libraries in functional genomics and genome engineering is the low expression of heterologous genes in screening hosts, such as Escherichia coli. To overcome this limitation, here we generate E. coli strains capable of recognizing heterologous promoters by expressing heterologous sigma factors. Among seven sigma factors tested, RpoD from Lactobacillus plantarum (Lpl) appears to be able of initiating transcription from all sources of DNA. Using the promoter GFP-trap concept, we successfully screen several heterologous and metagenomic DNA libraries, thus enlarging the genomic space that can be functionally sampled in E. coli. For an application, we show that screening fosmid-based Lpl genomic libraries in an E. coli strain with a chromosomally integrated Lpl rpoD enables the identification of Lpl genetic determinants imparting strong ethanol tolerance in E. coli. Transcriptome analysis confirms increased expression of heterologous genes in the engineered strain.

  15. Versatility of chemically synthesized guide RNAs for CRISPR-Cas9 genome editing.

    Science.gov (United States)

    Kelley, Melissa L; Strezoska, Žaklina; He, Kaizhang; Vermeulen, Annaleen; Smith, Anja van Brabant

    2016-09-10

    The CRISPR-Cas9 system has become the most popular and efficient method for genome engineering in mammalian cells. The Streptococcus pyogenes Cas9 nuclease can function with two types of guide RNAs: the native dual crRNA and tracrRNA (crRNA:tracrRNA) or a chimeric single guide RNA (sgRNA). Although sgRNAs expressed from a DNA vector are predominant in the literature, guide RNAs can be rapidly generated by chemical synthesis and provide equivalent functionality in gene editing experiments. This review highlights the attributes and advantages of chemically synthesized guide RNAs including the incorporation of chemical modifications to enhance gene editing efficiencies in certain applications. The use of synthetic guide RNAs is also uniquely suited to genome-scale high throughput arrayed screening, particularly when using complex phenotypic assays for functional genomics studies. Finally, the use of synthetic guide RNAs along with DNA-free sources of Cas9 (mRNA or protein) allows for transient CRISPR-Cas9 presence in the cell, thereby resulting in a decreased probability of off-target events.

  16. Mining Chemical Activity Status from High-Throughput Screening Assays

    KAUST Repository

    Soufan, Othman

    2015-12-14

    High-throughput screening (HTS) experiments provide a valuable resource that reports biological activity of numerous chemical compounds relative to their molecular targets. Building computational models that accurately predict such activity status (active vs. inactive) in specific assays is a challenging task given the large volume of data and frequently small proportion of active compounds relative to the inactive ones. We developed a method, DRAMOTE, to predict activity status of chemical compounds in HTP activity assays. For a class of HTP assays, our method achieves considerably better results than the current state-of-the-art-solutions. We achieved this by modification of a minority oversampling technique. To demonstrate that DRAMOTE is performing better than the other methods, we performed a comprehensive comparison analysis with several other methods and evaluated them on data from 11 PubChem assays through 1,350 experiments that involved approximately 500,000 interactions between chemicals and their target proteins. As an example of potential use, we applied DRAMOTE to develop robust models for predicting FDA approved drugs that have high probability to interact with the thyroid stimulating hormone receptor (TSHR) in humans. Our findings are further partially and indirectly supported by 3D docking results and literature information. The results based on approximately 500,000 interactions suggest that DRAMOTE has performed the best and that it can be used for developing robust virtual screening models. The datasets and implementation of all solutions are available as a MATLAB toolbox online at www.cbrc.kaust.edu.sa/dramote and can be found on Figshare.

  17. Mining Chemical Activity Status from High-Throughput Screening Assays.

    Directory of Open Access Journals (Sweden)

    Othman Soufan

    Full Text Available High-throughput screening (HTS experiments provide a valuable resource that reports biological activity of numerous chemical compounds relative to their molecular targets. Building computational models that accurately predict such activity status (active vs. inactive in specific assays is a challenging task given the large volume of data and frequently small proportion of active compounds relative to the inactive ones. We developed a method, DRAMOTE, to predict activity status of chemical compounds in HTP activity assays. For a class of HTP assays, our method achieves considerably better results than the current state-of-the-art-solutions. We achieved this by modification of a minority oversampling technique. To demonstrate that DRAMOTE is performing better than the other methods, we performed a comprehensive comparison analysis with several other methods and evaluated them on data from 11 PubChem assays through 1,350 experiments that involved approximately 500,000 interactions between chemicals and their target proteins. As an example of potential use, we applied DRAMOTE to develop robust models for predicting FDA approved drugs that have high probability to interact with the thyroid stimulating hormone receptor (TSHR in humans. Our findings are further partially and indirectly supported by 3D docking results and literature information. The results based on approximately 500,000 interactions suggest that DRAMOTE has performed the best and that it can be used for developing robust virtual screening models. The datasets and implementation of all solutions are available as a MATLAB toolbox online at www.cbrc.kaust.edu.sa/dramote and can be found on Figshare.

  18. Mining Chemical Activity Status from High-Throughput Screening Assays.

    Science.gov (United States)

    Soufan, Othman; Ba-alawi, Wail; Afeef, Moataz; Essack, Magbubah; Rodionov, Valentin; Kalnis, Panos; Bajic, Vladimir B

    2015-01-01

    High-throughput screening (HTS) experiments provide a valuable resource that reports biological activity of numerous chemical compounds relative to their molecular targets. Building computational models that accurately predict such activity status (active vs. inactive) in specific assays is a challenging task given the large volume of data and frequently small proportion of active compounds relative to the inactive ones. We developed a method, DRAMOTE, to predict activity status of chemical compounds in HTP activity assays. For a class of HTP assays, our method achieves considerably better results than the current state-of-the-art-solutions. We achieved this by modification of a minority oversampling technique. To demonstrate that DRAMOTE is performing better than the other methods, we performed a comprehensive comparison analysis with several other methods and evaluated them on data from 11 PubChem assays through 1,350 experiments that involved approximately 500,000 interactions between chemicals and their target proteins. As an example of potential use, we applied DRAMOTE to develop robust models for predicting FDA approved drugs that have high probability to interact with the thyroid stimulating hormone receptor (TSHR) in humans. Our findings are further partially and indirectly supported by 3D docking results and literature information. The results based on approximately 500,000 interactions suggest that DRAMOTE has performed the best and that it can be used for developing robust virtual screening models. The datasets and implementation of all solutions are available as a MATLAB toolbox online at www.cbrc.kaust.edu.sa/dramote and can be found on Figshare.

  19. Visual genome-wide RNAi screening to identify human host factors required for Trypanosoma cruzi infection

    CSIR Research Space (South Africa)

    Genovesio, A

    2011-05-01

    Full Text Available cellular microarray-based RNAi screening over glass slides method was first described by Erfle and collaborators in 2004 [29] and was further developed for high-throughput scale in genome-wide screens investigating mitosis, cell cycle progression...

  20. Wildlife ecological screening levels for inhalation of volatile organic chemicals.

    Science.gov (United States)

    Gallegos, Patricia; Lutz, Jill; Markwiese, James; Ryti, Randall; Mirenda, Rich

    2007-06-01

    For most chemicals, evaluation of ecological risk typically does not address inhalation because ingestion dominates exposure. However, burrowing ecological receptors have an increased exposure potential from inhalation at sites contaminated with volatile chemicals in the subsurface. Evaluation of ecological risk from contaminants like volatile organic chemicals (VOCs) is constrained by a lack of relevant ecological screening levels (ESLs). To address this need, inhalation ESLs were developed for 16 VOCs: Acetone, benzene, carbon tetrachloride, chloroform, chloromethane, dichlorodifluoromethane, 1,1-dichloroethane, 1,2-dichloroethane, 1,1-dichloroethene, methylene chloride, tetrachloroethene, toluene, 1,1,1-trichloroethane, trichloroethene, trichlorofluoromethane, and total xylene. These ESLs are based on Botta's pocket gopher (Thomomys bottae) as a representative fossorial receptor. The ESLs are presented with an emphasis on the process for developing inhalation toxicity reference values to illustrate the selection of suitable toxicity data and effect levels from the literature. The resulting ESLs provide a quantitative method for evaluating ecological risk of VOCs through comparison to relevant exposure data such as direct burrow-air measurements. The toxicity reference value development and ESL calculation processes and assumptions detailed here are provided as bases from which risk assessors can use or refine to suit site-specific needs with respect to toxicity and exposure inputs.

  1. Improved genome-scale multi-target virtual screening via a novel collaborative filtering approach to cold-start problem

    Science.gov (United States)

    Lim, Hansaim; Gray, Paul; Xie, Lei; Poleksic, Aleksandar

    2016-12-01

    Conventional one-drug-one-gene approach has been of limited success in modern drug discovery. Polypharmacology, which focuses on searching for multi-targeted drugs to perturb disease-causing networks instead of designing selective ligands to target individual proteins, has emerged as a new drug discovery paradigm. Although many methods for single-target virtual screening have been developed to improve the efficiency of drug discovery, few of these algorithms are designed for polypharmacology. Here, we present a novel theoretical framework and a corresponding algorithm for genome-scale multi-target virtual screening based on the one-class collaborative filtering technique. Our method overcomes the sparseness of the protein-chemical interaction data by means of interaction matrix weighting and dual regularization from both chemicals and proteins. While the statistical foundation behind our method is general enough to encompass genome-wide drug off-target prediction, the program is specifically tailored to find protein targets for new chemicals with little to no available interaction data. We extensively evaluate our method using a number of the most widely accepted gene-specific and cross-gene family benchmarks and demonstrate that our method outperforms other state-of-the-art algorithms for predicting the interaction of new chemicals with multiple proteins. Thus, the proposed algorithm may provide a powerful tool for multi-target drug design.

  2. The TSCA interagency testing committee`s approaches to screening and scoring chemicals and chemical groups: 1977-1983

    Energy Technology Data Exchange (ETDEWEB)

    Walker, J.D. [Environmental Protection Agency, Washington, DC (United States)

    1990-12-31

    This paper describes the TSCA interagency testing committee`s (ITC) approaches to screening and scoring chemicals and chemical groups between 1977 and 1983. During this time the ITC conducted five scoring exercises to select chemicals and chemical groups for detailed review and to determine which of these chemicals and chemical groups should be added to the TSCA Section 4(e) Priority Testing List. 29 refs., 1 fig., 2 tabs.

  3. Chemoinformatics and chemical genomics: potential utility of in silico methods.

    Science.gov (United States)

    Valerio, Luis G; Choudhuri, Supratim

    2012-11-01

    Computational life sciences and informatics are inseparably intertwined and they lie at the heart of modern biology, predictive quantitative modeling and high-performance computing. Two of the applied biological disciplines that are poised to benefit from such progress are pharmacology and toxicology. This review will describe in silico chemoinformatics methods such as (quantitative) structure-activity relationship modeling and will overview how chemoinformatic technologies are considered in applied regulatory research. Given the post-genomics era and large-scale repositories of omics data that are available, this review will also address potential applications of in silico techniques in chemical genomics. Chemical genomics utilizes small molecules to explore the complex biological phenomena that may not be not amenable to straightforward genetic approach. The reader will gain the understanding that chemoinformatics stands at the interface of chemistry and biology with enabling systems for mapping, statistical modeling, pattern recognition, imaging and database tools. The great potential of these technologies to help address complex issues in the toxicological sciences is appreciated with the applied goal of the protection of public health.

  4. Broad target chemical screening approach used as tool for rapid assessment of groundwater quality

    NARCIS (Netherlands)

    ter Laak, T.L.; Puijker, L.M.; van Leerdam, J.A.; Raat, K.J.; Kolkman, A.; de Voogt, P.; van Wezel, A.P.

    2012-01-01

    The chemical water quality is often assessed by screening for a limited set of target chemicals. This ‘conventional’ target analysis approach inevitably misses chemicals present in the samples. In this study a ‘broad’ target screening approach for water quality assessment using high resolution and a

  5. What’s old is new again: yeast mutant screens in the era of pooled segregant analysis by genome sequencing

    Directory of Open Access Journals (Sweden)

    Chris Curtin

    2016-04-01

    Full Text Available While once de-rigueur for identification of genes involved in biological processes, screening of chemically induced mutant populations is an approach that has largely been superseded for model organisms such as Saccharomyces cerevisiae. Availability of single gene deletion/overexpression libraries and combinatorial synthetic genetic arrays provide yeast researchers more structured ways to probe genetic networks. Furthermore, in the age of inexpensive DNA sequencing, methodologies such as mapping of quantitative trait loci (QTL by pooled segregant analysis and genome-wide association enable the identification of multiple naturally occurring allelic variants that contribute to polygenic phenotypes of interest. This is, however, contingent on the capacity to screen large numbers of individuals and existence of sufficient natural phenotypic variation within the available population. The latter cannot be guaranteed and non-selectable, industrially relevant phenotypes, such as production of volatile aroma compounds, pose severe limitations on the use of modern genetic techniques due to expensive and time-consuming downstream analyses. An interesting approach to overcome these issues can be found in Den Abt et al.[1] (this issue of Microbial Cell, where a combination of repeated rounds of chemical mutagenesis and pooled segregant analysis by whole genome sequencing was applied to identify genes involved in ethyl acetate formation, demonstrating a new path for industrial yeast strain development and bringing classical mutant screens into the 21st century.

  6. Factors affecting reproducibility between genome-scale siRNA-based screens

    Science.gov (United States)

    Barrows, Nicholas J.; Le Sommer, Caroline; Garcia-Blanco, Mariano A.; Pearson, James L.

    2011-01-01

    RNA interference-based screening is a powerful new genomic technology which addresses gene function en masse. To evaluate factors influencing hit list composition and reproducibility, we performed two identically designed small interfering RNA (siRNA)-based, whole genome screens for host factors supporting yellow fever virus infection. These screens represent two separate experiments completed five months apart and allow the direct assessment of the reproducibility of a given siRNA technology when performed in the same environment. Candidate hit lists generated by sum rank, median absolute deviation, z-score, and strictly standardized mean difference were compared within and between whole genome screens. Application of these analysis methodologies within a single screening dataset using a fixed threshold equivalent to a p-value ≤ 0.001 resulted in hit lists ranging from 82 to 1,140 members and highlighted the tremendous impact analysis methodology has on hit list composition. Intra- and inter-screen reproducibility was significantly influenced by the analysis methodology and ranged from 32% to 99%. This study also highlighted the power of testing at least two independent siRNAs for each gene product in primary screens. To facilitate validation we conclude by suggesting methods to reduce false discovery at the primary screening stage. In this study we present the first comprehensive comparison of multiple analysis strategies, and demonstrate the impact of the analysis methodology on the composition of the “hit list”. Therefore, we propose that the entire dataset derived from functional genome-scale screens, especially if publicly funded, should be made available as is done with data derived from gene expression and genome-wide association studies. PMID:20625183

  7. Construction of the BAC Library of Small Abalone (Haliotis diversicolor) for Gene Screening and Genome Characterization.

    Science.gov (United States)

    Jiang, Likun; You, Weiwei; Zhang, Xiaojun; Xu, Jian; Jiang, Yanliang; Wang, Kai; Zhao, Zixia; Chen, Baohua; Zhao, Yunfeng; Mahboob, Shahid; Al-Ghanim, Khalid A; Ke, Caihuan; Xu, Peng

    2016-02-01

    The small abalone (Haliotis diversicolor) is one of the most important aquaculture species in East Asia. To facilitate gene cloning and characterization, genome analysis, and genetic breeding of it, we constructed a large-insert bacterial artificial chromosome (BAC) library, which is an important genetic tool for advanced genetics and genomics research. The small abalone BAC library includes 92,610 clones with an average insert size of 120 Kb, equivalent to approximately 7.6× of the small abalone genome. We set up three-dimensional pools and super pools of 18,432 BAC clones for target gene screening using PCR method. To assess the approach, we screened 12 target genes in these 18,432 BAC clones and identified 16 positive BAC clones. Eight positive BAC clones were then sequenced and assembled with the next generation sequencing platform. The assembled contigs representing these 8 BAC clones spanned 928 Kb of the small abalone genome, providing the first batch of genome sequences for genome evaluation and characterization. The average GC content of small abalone genome was estimated as 40.33%. A total of 21 protein-coding genes, including 7 target genes, were annotated into the 8 BACs, which proved the feasibility of PCR screening approach with three-dimensional pools in small abalone BAC library. One hundred fifty microsatellite loci were also identified from the sequences for marker development in the future. The BAC library and clone pools provided valuable resources and tools for genetic breeding and conservation of H. diversicolor.

  8. Flow method and apparatus for screening chemicals using micro x-ray fluorescence

    Energy Technology Data Exchange (ETDEWEB)

    Warner, Benjamin P. (Los Alamos, NM); Havrilla, George J. (Los Alamos, NM); Miller, Thomasin C. (Bartlesville, OK); Lewis, Cris (Los Alamos, NM); Mahan, Cynthia A. (Los Alamos, NM); Wells, Cyndi A. (Los Alamos, NM)

    2011-04-26

    Method and apparatus for screening chemicals using micro x-ray fluorescence. A method for screening a mixture of potential pharmaceutical chemicals for binding to at least one target binder involves flow separating a solution of chemicals and target binders into separated components, exposing them to an x-ray excitation beam, detecting x-ray fluorescence signals from the components, and determining from the signals whether or not a binding event between a chemical and target binder has occurred.

  9. Flow method and apparatus for screening chemicals using micro x-ray fluorescence

    Energy Technology Data Exchange (ETDEWEB)

    Warner, Benjamin P. (Los Alamos, NM); Havrilla, George J. (Los Alamos, NM); Miller, Thomasin C. (Bartlesville, OK); Lewis, Cris (Los Alamos, NM); Mahan, Cynthia A. (Los Alamos, NM); Wells, Cyndi A. (Los Alamos, NM)

    2009-04-14

    Method and apparatus for screening chemicals using micro x-ray fluorescence. A method for screening a mixture of potential pharmaceutical chemicals for binding to at least one target binder involves flow-separating a solution of chemicals and target binders into separated components, exposing them to an x-ray excitation beam, detecting x-ray fluorescence signals from the components, and determining from the signals whether or not a binding event between a chemical and target binder has occurred.

  10. Genetic screens and functional genomics using CRISPR/Cas9 technology.

    Science.gov (United States)

    Hartenian, Ella; Doench, John G

    2015-04-01

    Functional genomics attempts to understand the genome by perturbing the flow of information from DNA to RNA to protein, in order to learn how gene dysfunction leads to disease. CRISPR/Cas9 technology is the newest tool in the geneticist's toolbox, allowing researchers to edit DNA with unprecedented ease, speed and accuracy, and representing a novel means to perform genome-wide genetic screens to discover gene function. In this review, we first summarize the discovery and characterization of CRISPR/Cas9, and then compare it to other genome engineering technologies. We discuss its initial use in screening applications, with a focus on optimizing on-target activity and minimizing off-target effects. Finally, we comment on future challenges and opportunities afforded by this technology.

  11. Public attitudes towards genomic risk profiling as a component of routine population screening.

    Science.gov (United States)

    Nicholls, S G; Wilson, B J; Craigie, S M; Etchegary, H; Castle, D; Carroll, J C; Potter, B K; Lemyre, L; Little, J

    2013-10-01

    Including low penetrance genomic variants in population-based screening might enable personalization of screening intensity and follow up. The application of genomics in this way requires formal evaluation. Even if clinically beneficial, uptake would still depend on the attitudes of target populations. We developed a deliberative workshop on two hypothetical applications (in colorectal cancer and newborn screening) in which we applied stepped, neutrally-framed, information sets. Data were collected using nonparticipant observation, free-text comments by individual participants, and a structured survey. Qualitative data were transcribed and analyzed using thematic content analysis. Eight workshops were conducted with 170 individuals (120 colorectal cancer screening and 50 newborn screening for type 1 diabetes). The use of information sets promoted informed deliberation. In both contexts, attitudes appeared to be heavily informed by assessments of the likely validity of the test results and its personal and health care utility. Perceived benefits included the potential for early intervention, prevention, and closer monitoring while concerns related to costs, education needs regarding the probabilistic nature of risk, the potential for worry, and control of access to personal genomic information. Differences between the colorectal cancer and newborn screening groups appeared to reflect different assessments of potential personal utility, particularly regarding prevention.

  12. Inducing mutations in the mouse genome with the chemical mutagen ethylnitrosourea

    Directory of Open Access Journals (Sweden)

    S.M.G. Massironi

    2006-09-01

    Full Text Available When compared to other model organisms whose genome is sequenced, the number of mutations identified in the mouse appears extremely reduced and this situation seriously hampers our understanding of mammalian gene function(s. Another important consequence of this shortage is that a majority of human genetic diseases still await an animal model. To improve the situation, two strategies are currently used: the first makes use of embryonic stem cells, in which one can induce knockout mutations almost at will; the second consists of a genome-wide random chemical mutagenesis, followed by screening for mutant phenotypes and subsequent identification of the genetic alteration(s. Several projects are now in progress making use of one or the other of these strategies. Here, we report an original effort where we mutagenized BALB/c males, with the mutagen ethylnitrosourea. Offspring of these males were screened for dominant mutations and a three-generation breeding protocol was set to recover recessive mutations. Eleven mutations were identified (one dominant and ten recessives. Three of these mutations are new alleles (Otop1mlh, Foxn1sepe and probably rodador at loci where mutations have already been reported, while 4 are new and original alleles (carc, eqlb, frqz, and Sacc. This result indicates that the mouse genome, as expected, is far from being saturated with mutations. More mutations would certainly be discovered using more sophisticated phenotyping protocols. Seven of the 11 new mutant alleles induced in our experiment have been localized on the genetic map as a first step towards positional cloning.

  13. Development of chemical compositions for impervious screens in rocks

    Science.gov (United States)

    Kurlenya, MV; Serdyukov, SV; Shilova, TV; Patutin, AV

    2017-02-01

    The paper presents the method to create anti-seepage screens by hydraulic fracturing with three-component polyurethane mixture. The proposed working fluids and their pumping circuits allow creation of a fracture and an adjacent insulation layer. Gas permeability of porous medium is determined at limit consumption of reagents per insulating screen unit area.

  14. Chemical genetics and drug screening in Drosophila cancer models

    Institute of Scientific and Technical Information of China (English)

    Mara Gladstone; Tin Tin Su

    2011-01-01

    Drug candidates often fail in preclinical and clinical testing because of reasons of efficacy and/or safety.It would be time- and cost-efficient to have screening models that reduce the rate of such false positive candidates that appear promising at first but fail later.In this regard,it would be particularly useful to have a rapid and inexpensive whole animal model that can pre-select hits from high-throughput screens but before testing in costly rodent assays.Drosophila melanogaster has emerged as a potential whole animal model for drug screening.Of particular interest have been drugs that must act in the context of multi-cellularity such as those for neurological disorders and cancer.A recent review provides a comprehensive summary of drug screening in Drosophila,but with an emphasis on neurodegenerative disorders.Here,we review Drosophila screens in the literature aimed at cancer therapeutics.

  15. Use of the bioaccumulation factor to screen chemicals for bioaccumulation potential.

    Science.gov (United States)

    Costanza, Jed; Lynch, David G; Boethling, Robert S; Arnot, Jon A

    2012-10-01

    The fish bioconcentration factor (BCF), as calculated from controlled laboratory tests, is commonly used in chemical management programs to screen chemicals for bioaccumulation potential. The bioaccumulation factor (BAF), as calculated from field-caught fish, is more ecologically relevant because it accounts for dietary, respiratory, and dermal exposures. The BCFBAF™ program in the U.S. Environmental Protection Agency's Estimation Programs Interface Suite (EPI Suite™ Ver 4.10) screening-level tool includes the Arnot-Gobas quantitative structure-activity relationship model to estimate BAFs for organic chemicals in fish. Bioaccumulation factors can be greater than BCFs, suggesting that using the BAF rather than the BCF for screening bioaccumulation potential could have regulatory and resource implications for chemical assessment programs. To evaluate these potential implications, BCFBAF was used to calculate BAFs and BCFs for 6,034 U.S. high- and medium-production volume chemicals. The results indicate no change in the bioaccumulation rating for 86% of these chemicals, with 3% receiving lower and 11% receiving higher bioaccumulation ratings when using the BAF rather than the BCF. All chemicals that received higher bioaccumulation ratings had log K(OW ) values greater than 4.02, in which a chemical's BAF was more representative of field-based bioaccumulation than its BCF. Similar results were obtained for 374 new chemicals. Screening based on BAFs provides ecologically relevant results without a substantial increase in resources needed for assessments or the number of chemicals screened as being of concern for bioaccumulation potential.

  16. An integrated hybrid microfluidic device for oviposition-based chemical screening of adult Drosophila melanogaster.

    Science.gov (United States)

    Leung, Jacob C K; Hilliker, Arthur J; Rezai, Pouya

    2016-02-21

    Chemical screening using Drosophila melanogaster (the fruit fly) is vital in drug discovery, agricultural, and toxicological applications. Oviposition (egg laying) on chemically-doped agar plates is an important read-out metric used to quantitatively assess the biological fitness and behavioral responses of Drosophila. Current oviposition-based chemical screening studies are inaccurate, labor-intensive, time-consuming, and inflexible due to the manual chemical doping of agar. In this paper, we have developed a novel hybrid agar-polydimethylsiloxane (PDMS) microfluidic device for single- and multi-concentration chemical dosing and on-chip oviposition screening of free-flying adult stage Drosophila. To achieve this, we have devised a novel technique to integrate agar with PDMS channels using ice as a sacrificial layer. Subsequently, we have conducted single-chemical toxicity and multiple choice chemical preference assays on adult Drosophila melanogaster using zinc and acetic acid at various concentrations. Our device has enabled us to 1) demonstrate that Drosophila is capable of sensing the concentration of different chemicals on a PDMS-agar microfluidic device, which plays significant roles in determining oviposition site selection and 2) investigate whether oviposition preference differs between single- and multi-concentration chemical environments. This device may be used to study fundamental and applied biological questions in Drosophila and other egg laying insects. It can also be extended in design to develop sophisticated and dynamic chemical dosing and high-throughput screening platforms in the future that are not easily achievable with the existing oviposition screening techniques.

  17. Chemical Genomics and Emerging DNA Technologies in the Identification of Drug Mechanisms and Drug Targets

    DEFF Research Database (Denmark)

    Olsen, Louise Cathrine Braun; Færgeman, Nils J.

    2012-01-01

    critical roles in the genomic age of biological research and drug discovery. In the present review we discuss how simple biological model organisms can be used as screening platforms in combination with emerging genomic technologies to advance the identification of potential drugs and their molecular...

  18. A whole genome screen for HIV restriction factors

    Directory of Open Access Journals (Sweden)

    Liu Li

    2011-11-01

    Full Text Available Abstract Background Upon cellular entry retroviruses must avoid innate restriction factors produced by the host cell. For human immunodeficiency virus (HIV human restriction factors, APOBEC3 (apolipoprotein-B-mRNA-editing-enzyme, p21 and tetherin are well characterised. Results To identify intrinsic resistance factors to HIV-1 replication we screened 19,121 human genes and identified 114 factors with significant inhibition of infection. Those with a known function are involved in a broad spectrum of cellular processes including receptor signalling, vesicle trafficking, transcription, apoptosis, cross-nuclear membrane transport, meiosis, DNA damage repair, ubiquitination and RNA processing. We focused on the PAF1 complex which has been previously implicated in gene transcription, cell cycle control and mRNA surveillance. Knockdown of all members of the PAF1 family of proteins enhanced HIV-1 reverse transcription and integration of provirus. Over-expression of PAF1 in host cells renders them refractory to HIV-1. Simian Immunodeficiency Viruses and HIV-2 are also restricted in PAF1 expressing cells. PAF1 is expressed in primary monocytes, macrophages and T-lymphocytes and we demonstrate strong activity in MonoMac1, a monocyte cell line. Conclusions We propose that the PAF1c establishes an anti-viral state to prevent infection by incoming retroviruses. This previously unrecognised mechanism of restriction could have implications for invasion of cells by any pathogen.

  19. From structure prediction to genomic screens for novel non-coding RNAs.

    Directory of Open Access Journals (Sweden)

    Jan Gorodkin

    2011-08-01

    Full Text Available Non-coding RNAs (ncRNAs are receiving more and more attention not only as an abundant class of genes, but also as regulatory structural elements (some located in mRNAs. A key feature of RNA function is its structure. Computational methods were developed early for folding and prediction of RNA structure with the aim of assisting in functional analysis. With the discovery of more and more ncRNAs, it has become clear that a large fraction of these are highly structured. Interestingly, a large part of the structure is comprised of regular Watson-Crick and GU wobble base pairs. This and the increased amount of available genomes have made it possible to employ structure-based methods for genomic screens. The field has moved from folding prediction of single sequences to computational screens for ncRNAs in genomic sequence using the RNA structure as the main characteristic feature. Whereas early methods focused on energy-directed folding of single sequences, comparative analysis based on structure preserving changes of base pairs has been efficient in improving accuracy, and today this constitutes a key component in genomic screens. Here, we cover the basic principles of RNA folding and touch upon some of the concepts in current methods that have been applied in genomic screens for de novo RNA structures in searches for novel ncRNA genes and regulatory RNA structure on mRNAs. We discuss the strengths and weaknesses of the different strategies and how they can complement each other.

  20. University of Texas Southwestern Medical Center: U01 Natural Products Screening | Office of Cancer Genomics

    Science.gov (United States)

    The goal of this project was to enlarge the chemical space probed by Project 1 (High-Throughput siRNA Screening of a Non-Small Cell Lung Cancer Cell Line Panel) by screening an expanded natural products library (~40,000) in an effort to further define vulnerabilities and therapeutic targets in non-small cell lung cancer. This new library is derived from a diverse collection of marine bacteria (prepared by Dr. John MacMillan, University of Texas Southwestern).

  1. A novel method to screen genomic libraries that combines genomic immunization with the prime-boost strategy.

    Science.gov (United States)

    Yero, Daniel; Pajón, Rolando; Caballero, Evelin; González, Sonia; Cobas, Karem; Fariñas, Mildrey; Lopez, Yamilé; Acosta, Armando

    2007-08-01

    We employed a prime-boost regimen in combination with the expression library immunization protocol to improve the protective effectiveness of a genomic library used as immunogen. To demonstrate the feasibility of this novel strategy, we used as a prime a serogroup B Neisseria meningitidis random genomic library constructed in a eukaryotic expression vector. Mice immunized with different fractions of this library and boosted with a single dose of meningococcal outer membrane vesicles elicited higher bactericidal antibody titers compared with mice primed with the empty vector. After the boost, passive administration of sera from mice primed with two of these fractions significantly reduced the number of viable bacteria in the blood of infant rats challenged with live N. meningitidis. The method proposed could be applied to the identification of subimmunogenic antigens during vaccine candidate screening by employing expression library immunization.

  2. 76 FR 49473 - Petition to Maximize Practical Utility of List 1 Chemicals Screened Through EPA's Endocrine...

    Science.gov (United States)

    2011-08-10

    ... (11) Tier 1 screening assays is to determine the potential for a chemical to interact with estrogen, androgen and thyroid hormone systems. Based on the data from the tier 1 assays, should the determination be...

  3. A new age in functional genomics using CRISPR/Cas9 in arrayed library screening.

    Science.gov (United States)

    Agrotis, Alexander; Ketteler, Robin

    2015-01-01

    CRISPR technology has rapidly changed the face of biological research, such that precise genome editing has now become routine for many labs within several years of its initial development. What makes CRISPR/Cas9 so revolutionary is the ability to target a protein (Cas9) to an exact genomic locus, through designing a specific short complementary nucleotide sequence, that together with a common scaffold sequence, constitute the guide RNA bridging the protein and the DNA. Wild-type Cas9 cleaves both DNA strands at its target sequence, but this protein can also be modified to exert many other functions. For instance, by attaching an activation domain to catalytically inactive Cas9 and targeting a promoter region, it is possible to stimulate the expression of a specific endogenous gene. In principle, any genomic region can be targeted, and recent efforts have successfully generated pooled guide RNA libraries for coding and regulatory regions of human, mouse and Drosophila genomes with high coverage, thus facilitating functional phenotypic screening. In this review, we will highlight recent developments in the area of CRISPR-based functional genomics and discuss potential future directions, with a special focus on mammalian cell systems and arrayed library screening.

  4. A New Age in Functional Genomics Using CRISPR/Cas9 in Arrayed Library Screening

    Directory of Open Access Journals (Sweden)

    Alexander eAgrotis

    2015-09-01

    Full Text Available CRISPR technology has rapidly changed the face of biological research, such that precise genome editing has now become routine for many labs within several years of its initial development. What makes CRISPR/Cas9 so revolutionary is the ability to target a protein (Cas9 to an exact genomic locus, through designing a specific short complementary nucleotide sequence, that together with a common scaffold sequence, constitute the guide RNA bridging the protein and the DNA. Wild-type Cas9 cleaves both DNA strands at its target sequence, but this protein can also be modified to exert many other functions. For instance, by attaching an activation domain to catalytically inactive Cas9 and targeting a promoter region, it is possible to stimulate the expression of a specific endogenous gene. In principle, any genomic region can be targeted, and recent efforts have successfully generated pooled guide RNA libraries for coding and regulatory regions of human, mouse and Drosophila genomes with high coverage, thus facilitating functional phenotypic screening. In this review, we will highlight recent developments in the area of CRISPR-based functional genomics and discuss potential future directions, with a special focus on mammalian cell systems and arrayed library screening.

  5. Human genomic library screened with 17-base oligonucleotide probes yields a novel interferon gene.

    OpenAIRE

    Torczynski, R M; Fuke, M; Bollon, A P

    1984-01-01

    A method is presented that has permitted a human genomic library to be screened for low-copy genes using 17-base synthetic oligonucleotides as probes. Parallel screening with two different 17-base probes permitted the unambiguous identification of clones containing interferon-alpha (IFN-alpha) genes. The isolated human IFN-alpha genes were sequenced, and one appears to be IFN-alpha L; the other is one not previously described, which we have designated IFN-alpha WA. The IFN-alpha WA sequence d...

  6. Microfluidic screening and whole-genome sequencing identifies mutations associated with improved protein secretion by yeast

    DEFF Research Database (Denmark)

    Huang, Mingtao; Bai, Yunpeng; Sjostrom, Staffan L.

    2015-01-01

    interest in improving its protein secretion capacity. Due to the complexity of the secretory machinery in eukaryotic cells, it is difficult to apply rational engineering for construction of improved strains. Here we used high-throughput microfluidics for the screening of yeast libraries, generated by UV...... to construct efficient cell factories for protein secretion. The combined use of microfluidics screening and whole-genome sequencing to map the mutations associated with the improved phenotype can easily be adapted for other products and cell types to identify novel engineering targets, and this approach could...

  7. Causes of genome instability: the effect of low dose chemical exposures in modern society

    Science.gov (United States)

    Langie, Sabine A.S.; Koppen, Gudrun; Desaulniers, Daniel; Al-Mulla, Fahd; Al-Temaimi, Rabeah; Amedei, Amedeo; Azqueta, Amaya; Bisson, William H.; Brown, Dustin; Brunborg, Gunnar; Charles, Amelia K.; Chen, Tao; Colacci, Annamaria; Darroudi, Firouz; Forte, Stefano; Gonzalez, Laetitia; Hamid, Roslida A.; Knudsen, Lisbeth E.; Leyns, Luc; Lopez de Cerain Salsamendi, Adela; Memeo, Lorenzo; Mondello, Chiara; Mothersill, Carmel; Olsen, Ann-Karin; Pavanello, Sofia; Raju, Jayadev; Rojas, Emilio; Roy, Rabindra; Ryan, Elizabeth; Ostrosky-Wegman, Patricia; Salem, Hosni K.; Scovassi, Ivana; Singh, Neetu; Vaccari, Monica; Van Schooten, Frederik J.; Valverde, Mahara; Woodrick, Jordan; Zhang, Luoping; van Larebeke, Nik; Kirsch-Volders, Micheline; Collins, Andrew R.

    2015-01-01

    Genome instability is a prerequisite for the development of cancer. It occurs when genome maintenance systems fail to safeguard the genome’s integrity, whether as a consequence of inherited defects or induced via exposure to environmental agents (chemicals, biological agents and radiation). Thus, genome instability can be defined as an enhanced tendency for the genome to acquire mutations; ranging from changes to the nucleotide sequence to chromosomal gain, rearrangements or loss. This review raises the hypothesis that in addition to known human carcinogens, exposure to low dose of other chemicals present in our modern society could contribute to carcinogenesis by indirectly affecting genome stability. The selected chemicals with their mechanisms of action proposed to indirectly contribute to genome instability are: heavy metals (DNA repair, epigenetic modification, DNA damage signaling, telomere length), acrylamide (DNA repair, chromosome segregation), bisphenol A (epigenetic modification, DNA damage signaling, mitochondrial function, chromosome segregation), benomyl (chromosome segregation), quinones (epigenetic modification) and nano-sized particles (epigenetic pathways, mitochondrial function, chromosome segregation, telomere length). The purpose of this review is to describe the crucial aspects of genome instability, to outline the ways in which environmental chemicals can affect this cancer hallmark and to identify candidate chemicals for further study. The overall aim is to make scientists aware of the increasing need to unravel the underlying mechanisms via which chemicals at low doses can induce genome instability and thus promote carcinogenesis. PMID:26106144

  8. SCREENING CHEMICALS FOR ESTROGEN RECEPTOR BIOACTIVITY USING A COMPUTATIONAL MODEL

    Science.gov (United States)

    The U.S. Environmental Protection Agency (EPA) is considering the use high-throughput and computational methods for regulatory applications in the Endocrine Disruptor Screening Program (EDSP). To use these new tools for regulatory decision making, computational methods must be a...

  9. Human Genome-Wide RNAi Screen for Host Factors That Modulate Intracellular Salmonella Growth

    OpenAIRE

    Thornbrough, Joshua M.; Tom Hundley; Raphael Valdivia; Worley, Micah J.

    2012-01-01

    Salmonella enterica is a bacterial pathogen of humans that can proliferate within epithelial cells as well as professional phagocytes of the immune system. While much has been learned about the microbial genes that influence the infectious process through decades of intensive research, relatively little is known about the host factors that affect infection. We performed a genome-wide siRNA screen to identify host genes that Salmonella enterica serovar Typhimurium (S. typhimurium) utilizes to ...

  10. A genome-wide screen and linkage mapping for a large pedigree with episodic ataxia.

    Science.gov (United States)

    Cader, M Z; Steckley, J L; Dyment, D A; McLachlan, R S; Ebers, G C

    2005-07-12

    Episodic ataxias are ion channel disorders characterized by attacks of incoordination. The authors performed a genome-wide screen in a large pedigree segregating a novel episodic ataxia and found significant linkage on 1q42 with a multipoint lod score of 3.65. Haplotype analysis and fine mapping yielded a peak 2-point lod score of 4.14 and indicated a 4-cM region on 1q42 that is likely to harbor an episodic ataxia gene.

  11. Study on the Mitochondrial Genome of Sea Island Cotton (Gossypium barbadense) by BAC Library Screening

    Institute of Scientific and Technical Information of China (English)

    SU Ai-guo; LI Shuang-shuang; LIU Guo-zheng; LEI Bin-bin; KANG Ding-ming; LI Zhao-hu; MA Zhi-ying; HUA Jin-ping

    2014-01-01

    The plant mitochondrial genome displays complex features, particularly in terms of cytoplasmic male sterility (CMS). Therefore, research on the cotton mitochondrial genome may provide important information for analyzing genome evolution and exploring the molecular mechanism of CMS. In this paper, we present a preliminary study on the mitochondrial genome of sea island cotton (Gossypium barbadense) based on positive clones from the bacterial artiifcial chromosome (BAC) library. Thirty-ifve primers designed with the conserved sequences of functional genes and exons of mitochondria were used to screen positive clones in the genome library of the sea island cotton variety called Pima 90-53. Ten BAC clones were obtained and veriifed for further study. A contig was obtained based on six overlapping clones and subsequently laid out primarily on the mitochondrial genome. One BAC clone, clone 6 harbored with the inserter of approximate 115 kb mtDNA sequence, in which more than 10 primers fragments could be ampliifed, was sequenced and assembled using the Solexa strategy. Fifteen mitochondrial functional genes were revealed in clone 6 by gene annotation. The characteristics of the syntenic gene/exon of the sequences and RNA editing were preliminarily predicted.

  12. BYSTANDER EFFECTS GENOMIC INSTABILITY, ADAPTIVE RESPONSE AND CANCER RISK ASSESSMENT FOR RADIAION AND CHEMICAL EXPOSURES

    Science.gov (United States)

    BYSTANDER EFFECTS, GENOMIC INSTABILITY, ADAPTIVE RESPONSE AND CANCER RISK ASSESSMENT FOR RADIATION AND CHEMICAL EXPOSURESR. Julian PrestonEnvironmental Carcinogenesis Division, U.S. Environmental Protection Agency, Research Triangle Park, N.C. 27711, USAThere ...

  13. Largest Common Chemical Feature Subtree as a Virtual Screening Method

    DEFF Research Database (Denmark)

    Kristensen, Thomas Greve; Pedersen, Christian Storm; Thomsen, Rene

    We investigate the effectiveness of using a tree comparison based method to screen for drug candidates. Molecules are represented as trees in which ring systems are reduced to single nodes. These trees are compared to the tree of a selected known binder and the molecules are ranked according...... to the normalized size of their largest common subtree. The nodes of the molecular trees contains information about the atoms or ring systems they represent (e.g. charge and hydrogen donor/acceptor properties). In this way we can restrict which nodes are matched when calculating the size of the largest common...

  14. Statistically designed experiments to screen chemical mixtures for possible interactions

    NARCIS (Netherlands)

    Groten, J.P.; Tajima, O.; Feron, V.J.; Schoen, E.D.

    1998-01-01

    For the accurate analysis of possible interactive effects of chemicals in a defined mixture, statistical designs are necessary to develop clear and manageable experiments. For instance, factorial designs have been successfully used to detect two-factor interactions. Particularly useful for this purp

  15. Statistically designed experiments to screen chemical mixtures for possible interactions

    NARCIS (Netherlands)

    Groten, J.P.; Tajima, O.; Feron, V.J.; Schoen, E.D.

    1998-01-01

    For the accurate analysis of possible interactive effects of chemicals in a defined mixture, statistical designs are necessary to develop clear and manageable experiments. For instance, factorial designs have been successfully used to detect two-factor interactions. Particularly useful for this purp

  16. Systematic Identification of MCU Modulators by Orthogonal Interspecies Chemical Screening.

    Science.gov (United States)

    Arduino, Daniela M; Wettmarshausen, Jennifer; Vais, Horia; Navas-Navarro, Paloma; Cheng, Yiming; Leimpek, Anja; Ma, Zhongming; Delrio-Lorenzo, Alba; Giordano, Andrea; Garcia-Perez, Cecilia; Médard, Guillaume; Kuster, Bernhard; García-Sancho, Javier; Mokranjac, Dejana; Foskett, J Kevin; Alonso, M Teresa; Perocchi, Fabiana

    2017-08-17

    The mitochondrial calcium uniporter complex is essential for calcium (Ca(2+)) uptake into mitochondria of all mammalian tissues, where it regulates bioenergetics, cell death, and Ca(2+) signal transduction. Despite its involvement in several human diseases, we currently lack pharmacological agents for targeting uniporter activity. Here we introduce a high-throughput assay that selects for human MCU-specific small-molecule modulators in primary drug screens. Using isolated yeast mitochondria, reconstituted with human MCU, its essential regulator EMRE, and aequorin, and exploiting a D-lactate- and mannitol/sucrose-based bioenergetic shunt that greatly minimizes false-positive hits, we identify mitoxantrone out of more than 600 clinically approved drugs as a direct selective inhibitor of human MCU. We validate mitoxantrone in orthogonal mammalian cell-based assays, demonstrating that our screening approach is an effective and robust tool for MCU-specific drug discovery and, more generally, for the identification of compounds that target mitochondrial functions. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. Next-generation libraries for robust RNA interference-based genome-wide screens.

    Science.gov (United States)

    Kampmann, Martin; Horlbeck, Max A; Chen, Yuwen; Tsai, Jordan C; Bassik, Michael C; Gilbert, Luke A; Villalta, Jacqueline E; Kwon, S Chul; Chang, Hyeshik; Kim, V Narry; Weissman, Jonathan S

    2015-06-30

    Genetic screening based on loss-of-function phenotypes is a powerful discovery tool in biology. Although the recent development of clustered regularly interspaced short palindromic repeats (CRISPR)-based screening approaches in mammalian cell culture has enormous potential, RNA interference (RNAi)-based screening remains the method of choice in several biological contexts. We previously demonstrated that ultracomplex pooled short-hairpin RNA (shRNA) libraries can largely overcome the problem of RNAi off-target effects in genome-wide screens. Here, we systematically optimize several aspects of our shRNA library, including the promoter and microRNA context for shRNA expression, selection of guide strands, and features relevant for postscreen sample preparation for deep sequencing. We present next-generation high-complexity libraries targeting human and mouse protein-coding genes, which we grouped into 12 sublibraries based on biological function. A pilot screen suggests that our next-generation RNAi library performs comparably to current CRISPR interference (CRISPRi)-based approaches and can yield complementary results with high sensitivity and high specificity.

  18. Hierarchical dose-response modeling for high-throughput toxicity screening of environmental chemicals.

    Science.gov (United States)

    Wilson, Ander; Reif, David M; Reich, Brian J

    2014-03-01

    High-throughput screening (HTS) of environmental chemicals is used to identify chemicals with high potential for adverse human health and environmental effects from among the thousands of untested chemicals. Predicting physiologically relevant activity with HTS data requires estimating the response of a large number of chemicals across a battery of screening assays based on sparse dose-response data for each chemical-assay combination. Many standard dose-response methods are inadequate because they treat each curve separately and under-perform when there are as few as 6-10 observations per curve. We propose a semiparametric Bayesian model that borrows strength across chemicals and assays. Our method directly parametrizes the efficacy and potency of the chemicals as well as the probability of response. We use the ToxCast data from the U.S. Environmental Protection Agency (EPA) as motivation. We demonstrate that our hierarchical method provides more accurate estimates of the probability of response, efficacy, and potency than separate curve estimation in a simulation study. We use our semiparametric method to compare the efficacy of chemicals in the ToxCast data to well-characterized reference chemicals on estrogen receptor α (ERα) and peroxisome proliferator-activated receptor γ (PPARγ) assays, then estimate the probability that other chemicals are active at lower concentrations than the reference chemicals.

  19. Visual Genome-Wide RNAi Screening to Identify Human Host Factors Required for Trypanosoma cruzi Infection

    Science.gov (United States)

    de Macedo Dossin, Fernando; Choi, Seo Yeon; Kim, Nam Youl; Kim, Hi Chul; Jung, Sung Yong; Schenkman, Sergio; Almeida, Igor C.; Emans, Neil; Freitas-Junior, Lucio H.

    2011-01-01

    The protozoan parasite Trypanosoma cruzi is the etiologic agent of Chagas disease, a neglected tropical infection that affects millions of people in the Americas. Current chemotherapy relies on only two drugs that have limited efficacy and considerable side effects. Therefore, the development of new and more effective drugs is of paramount importance. Although some host cellular factors that play a role in T. cruzi infection have been uncovered, the molecular requirements for intracellular parasite growth and persistence are still not well understood. To further study these host-parasite interactions and identify human host factors required for T. cruzi infection, we performed a genome-wide RNAi screen using cellular microarrays of a printed siRNA library that spanned the whole human genome. The screening was reproduced 6 times and a customized algorithm was used to select as hits those genes whose silencing visually impaired parasite infection. The 162 strongest hits were subjected to a secondary screening and subsequently validated in two different cell lines. Among the fourteen hits confirmed, we recognized some cellular membrane proteins that might function as cell receptors for parasite entry and others that may be related to calcium release triggered by parasites during cell invasion. In addition, two of the hits are related to the TGF-beta signaling pathway, whose inhibition is already known to diminish levels of T. cruzi infection. This study represents a significant step toward unveiling the key molecular requirements for host cell invasion and revealing new potential targets for antiparasitic therapy. PMID:21625474

  20. Visual genome-wide RNAi screening to identify human host factors required for Trypanosoma cruzi infection.

    Directory of Open Access Journals (Sweden)

    Auguste Genovesio

    Full Text Available The protozoan parasite Trypanosoma cruzi is the etiologic agent of Chagas disease, a neglected tropical infection that affects millions of people in the Americas. Current chemotherapy relies on only two drugs that have limited efficacy and considerable side effects. Therefore, the development of new and more effective drugs is of paramount importance. Although some host cellular factors that play a role in T. cruzi infection have been uncovered, the molecular requirements for intracellular parasite growth and persistence are still not well understood. To further study these host-parasite interactions and identify human host factors required for T. cruzi infection, we performed a genome-wide RNAi screen using cellular microarrays of a printed siRNA library that spanned the whole human genome. The screening was reproduced 6 times and a customized algorithm was used to select as hits those genes whose silencing visually impaired parasite infection. The 162 strongest hits were subjected to a secondary screening and subsequently validated in two different cell lines. Among the fourteen hits confirmed, we recognized some cellular membrane proteins that might function as cell receptors for parasite entry and others that may be related to calcium release triggered by parasites during cell invasion. In addition, two of the hits are related to the TGF-beta signaling pathway, whose inhibition is already known to diminish levels of T. cruzi infection. This study represents a significant step toward unveiling the key molecular requirements for host cell invasion and revealing new potential targets for antiparasitic therapy.

  1. Total Chemical Synthesis,Assembly of Human Torque Teno Virus Genome

    Institute of Scientific and Technical Information of China (English)

    Zheng Hou; Gengfu Xiao

    2011-01-01

    Torque teno virus(TTV)is a nonenveloped virus containing a single-stranded,circular DNA genome of approximately 3.8kb.We completely synthesized the 3808 nucleotides of the TTV(SANBAN isolate)genome,which contains a hairpin structure and a GC-rich region.More than 100 overlapping oligonucleotides were chemically synthesized and assembled by polymerise chain assembly reaction(PCA),and the synthesis was completed with splicing by overlap extension(SOEing).This study establishes the methodological basis of the chemical synthesis of a viral genome for use as a live attenuated vaccine or gene therapy vector.

  2. Modifying culture conditions in chemical library screening identifies alternative inhibitors of mycobacteria.

    Science.gov (United States)

    Miller, Christopher H; Nisa, Shahista; Dempsey, Sandi; Jack, Cameron; O'Toole, Ronan

    2009-12-01

    In this study, application of a dual absorbance/fluorescence assay to a chemical library screen identified several previously unknown inhibitors of mycobacteria. In addition, growth conditions had a significant effect on the activity profile of the library. Some inhibitors such as Se-methylselenocysteine were detected only when screening was performed under nutrient-limited culture conditions as opposed to nutrient-rich culture conditions. We propose that multiple culture condition library screening is required for complete inhibitory profiling and for maximal antimycobacterial compound detection.

  3. Recovery of a soybean urease genomic clone by sequential library screening with two synthetic oligodeoxynucleotides.

    Science.gov (United States)

    Krueger, R W; Holland, M A; Chisholm, D; Polacco, J C

    1987-01-01

    We report the first isolation of a low-copy-number gene from a complex higher plant (soybean) genome by direct screening with synthetic oligodeoxynucleotide (oligo) probes. A synthetic, mixed, 21-nucleotide (nt) oligo (21-1) based on a seven amino acid (aa) sequence from soybean seed urease, was used to screen genomic libraries of soybean (Glycine max [L.] Merr.) in the lambda Charon 4 vector. Twenty homologous clones were recovered from a screen of 500,000 plaques. These were counterscreened with embryo-specific cDNA (15-2 cDNA) made by priming with a second, mixed 15-nt oligo (15-2), based on a Jack bean (Canavalia ensiformis) urease peptide [Takishima et al., J. Natl. Def. Med. Coll. 5 (1980) 19-23]. Five out of 20 clones were homologous to 15-2 cDNA and proved to be identical. Nucleotide sequence analysis of representative clone E15 confirmed that it contained urease sequences. Subclones of E15 homologous to the oligo probes contain a deduced amino acid sequence which matches 108 of 130 aa residues of an amino acid run in a recently published [Mamiya et al., Proc. Jap. Acad. 61B (1985) 359-398] complete protein sequence for Jack-bean seed urease. Using clone E15 as a probe of soybean embryonic mRNA revealed a homologous 3.8-kb species that is the size of the urease messenger. This species is absent from mRNA of embryos of a soybean seed urease-null mutant. However, both urease-positive and urease-null genomes contain the 11-kb DNA fragment bearing urease sequences.

  4. A genome-wide screen for Schizosaccharomyces pombe deletion mutants that affect telomere length

    Institute of Scientific and Technical Information of China (English)

    Ning-Ning Liu; Tian Xu Han; Li-Lin Du; Jin-Qiu Zhou

    2010-01-01

    @@ Dear Editor, Both the fission yeast Schizosaccharomyces pombe and the budding yeast Saccharomyces cerevisiae are popular model organisms, and studies using these models have provided many informative clues for solving fundamental biological questions [1], such as DNA replication,cell cycle regulation and gene transcription. Since the completion of genome sequencing of these fungi [2, 3],systematic genetic modification, e.g. gene deletion, has become possible, and genome-wide phenotypic screening for gene function has been widely carried out. For example, Askree et al. and Gatbonton et al. examined the telomere-length change in about 4 800 non-essential gene deletion mutants of S. cerevisiae, and found that about 250 genes are involved in telomere-length regulation.

  5. The screening of chemicals for juvenoid-related endocrine activity using the water flea Daphnia magna.

    Science.gov (United States)

    Wang, Helen Ying; Olmstead, Allen W; Li, Hong; Leblanc, Gerald A

    2005-09-10

    U.S. Environmental Protection Agency is charged with developing a screening and testing paradigm for detecting endocrine toxicity of chemicals that are subject to regulation under the Food Quality Protection and the Safe Drinking Water Acts. In this study, we developed and evaluated a screening assay that could be employed to detect juvenoid-related endocrine-modulating activity in an invertebrate species. Juvenoid activity, anti-juvenoid activity, and juvenoid potentiator activity of chemicals was assessed using the water flea Daphnia magna. Male sex determination is under the regulatory control of juvenoid hormone, presumably methyl farnesoate, and this endpoint was used to detect juvenoid modulating activity of chemicals. Eighteen chemicals were evaluated for juvenoid agonist activity. Positive responses were detected with the juvenoid hormones methyl farnesoate and juvenile hormone III along with the insect growth regulating insecticides pyriproxyfen, fenoxycarb, and methoprene. Weak juvenoid activity also was detected with the cyclodiene insecticide dieldrin. Assays performed repetitively with compounds that gave either strong positive, weak positive, or negative response were 100% consistent indicating that the assay is not prone to false positive or negative responses. Five candidate chemicals were evaluated for anti-juvenoid activity and none registered positive. Four chemicals (all trans-retinoic acid, methoprene, kinoprene, bisphenol A) also were evaluated for their ability to potentiate the activity of methyl farnesoate. All registered positive. Results demonstrate that an in vivo assay with a crustacean species customarily employed in toxicity testing can be used to effectively screen chemicals for juvenoid-modulating activity.

  6. A multistep genomic screen identifies new genes required for repair of DNA double-strand breaks in Saccharomyces cerevisiae.

    Science.gov (United States)

    McKinney, Jennifer Summers; Sethi, Sunaina; Tripp, Jennifer DeMars; Nguyen, Thuy N; Sanderson, Brian A; Westmoreland, James W; Resnick, Michael A; Lewis, L Kevin

    2013-04-15

    Efficient mechanisms for rejoining of DNA double-strand breaks (DSBs) are vital because misrepair of such lesions leads to mutation, aneuploidy and loss of cell viability. DSB repair is mediated by proteins acting in two major pathways, called homologous recombination and nonhomologous end-joining. Repair efficiency is also modulated by other processes such as sister chromatid cohesion, nucleosome remodeling and DNA damage checkpoints. The total number of genes influencing DSB repair efficiency is unknown. To identify new yeast genes affecting DSB repair, genes linked to gamma radiation resistance in previous genome-wide surveys were tested for their impact on repair of site-specific DSBs generated by in vivo expression of EcoRI endonuclease. Eight members of the RAD52 group of DNA repair genes (RAD50, RAD51, RAD52, RAD54, RAD55, RAD57, MRE11 and XRS2) and 73 additional genes were found to be required for efficient repair of EcoRI-induced DSBs in screens utilizing both MATa and MATα deletion strain libraries. Most mutants were also sensitive to the clastogenic chemicals MMS and bleomycin. Several of the non-RAD52 group genes have previously been linked to DNA repair and over half of the genes affect nuclear processes. Many proteins encoded by the protective genes have previously been shown to associate physically with each other and with known DNA repair proteins in high-throughput proteomics studies. A majority of the proteins (64%) share sequence similarity with human proteins, suggesting that they serve similar functions. We have used a genetic screening approach to detect new genes required for efficient repair of DSBs in Saccharomyces cerevisiae. The findings have spotlighted new genes that are critical for maintenance of genome integrity and are therefore of greatest concern for their potential impact when the corresponding gene orthologs and homologs are inactivated or polymorphic in human cells.

  7. A Genome-Wide RNAi Screen for Factors Involved in Neuronal Specification in Caenorhabditis elegans.

    Directory of Open Access Journals (Sweden)

    Richard J Poole

    2011-06-01

    Full Text Available One of the central goals of developmental neurobiology is to describe and understand the multi-tiered molecular events that control the progression of a fertilized egg to a terminally differentiated neuron. In the nematode Caenorhabditis elegans, the progression from egg to terminally differentiated neuron has been visually traced by lineage analysis. For example, the two gustatory neurons ASEL and ASER, a bilaterally symmetric neuron pair that is functionally lateralized, are generated from a fertilized egg through an invariant sequence of 11 cellular cleavages that occur stereotypically along specific cleavage planes. Molecular events that occur along this developmental pathway are only superficially understood. We take here an unbiased, genome-wide approach to identify genes that may act at any stage to ensure the correct differentiation of ASEL. Screening a genome-wide RNAi library that knocks-down 18,179 genes (94% of the genome, we identified 245 genes that affect the development of the ASEL neuron, such that the neuron is either not generated, its fate is converted to that of another cell, or cells from other lineage branches now adopt ASEL fate. We analyze in detail two factors that we identify from this screen: (1 the proneural gene hlh-14, which we find to be bilaterally expressed in the ASEL/R lineages despite their asymmetric lineage origins and which we find is required to generate neurons from several lineage branches including the ASE neurons, and (2 the COMPASS histone methyltransferase complex, which we find to be a critical embryonic inducer of ASEL/R asymmetry, acting upstream of the previously identified miRNA lsy-6. Our study represents the first comprehensive, genome-wide analysis of a single neuronal cell fate decision. The results of this analysis provide a starting point for future studies that will eventually lead to a more complete understanding of how individual neuronal cell types are generated from a single

  8. STRP Screening Sets for the human genome at 5 cM density

    Directory of Open Access Journals (Sweden)

    Marth Gabor

    2003-02-01

    Full Text Available Abstract Background Short tandem repeat polymorphisms (STRPs are powerful tools for gene mapping and other applications. A STRP genome scan of 10 cM is usually adequate for mapping single gene disorders. However mapping studies involving genetically complex disorders and especially association (linkage disequilibrium often require higher STRP density. Results We report the development of two separate 10 cM human STRP Screening Sets (Sets 12 and 52 which span all chromosomes. When combined, the two Sets contain a total of 782 STRPs, with average STRP spacing of 4.8 cM, average heterozygosity of 0.72, and total sex-average coverage of 3535 cM. The current Sets are comprised almost entirely of STRPs based on tri- and tetranucleotide repeats. We also report correction of primer sequences for many STRPs used in previous Screening Sets. Detailed information for the new Screening Sets is available from our web site: http://research.marshfieldclinic.org/genetics. Conclusion Our new human STRP Screening Sets will improve the quality and cost effectiveness of genotyping for gene mapping and other applications.

  9. Novel Data Mining Methods for Virtual Screening of Biological Active Chemical Compounds

    KAUST Repository

    Soufan, Othman M.

    2016-11-23

    Drug discovery is a process that takes many years and hundreds of millions of dollars to reveal a confident conclusion about a specific treatment. Part of this sophisticated process is based on preliminary investigations to suggest a set of chemical compounds as candidate drugs for the treatment. Computational resources have been playing a significant role in this part through a step known as virtual screening. From a data mining perspective, availability of rich data resources is key in training prediction models. Yet, the difficulties imposed by big expansion in data and its dimensionality are inevitable. In this thesis, I address the main challenges that come when data mining techniques are used for virtual screening. In order to achieve an efficient virtual screening using data mining, I start by addressing the problem of feature selection and provide analysis of best ways to describe a chemical compound for an enhanced screening performance. High-throughput screening (HTS) assays data used for virtual screening are characterized by a great class imbalance. To handle this problem of class imbalance, I suggest using a novel algorithm called DRAMOTE to narrow down promising candidate chemicals aimed at interaction with specific molecular targets before they are experimentally evaluated. Existing works are mostly proposed for small-scale virtual screening based on making use of few thousands of interactions. Thus, I propose enabling large-scale (or big) virtual screening through learning millions of interaction while exploiting any relevant dependency for a better accuracy. A novel solution called DRABAL that incorporates structure learning of a Bayesian Network as a step to model dependency between the HTS assays, is showed to achieve significant improvements over existing state-of-the-art approaches.

  10. A New Method for Rapid Screening of End-Point PCR Products: Application to Single Genome Amplified HIV and SIV Envelope Amplicons.

    Directory of Open Access Journals (Sweden)

    Laurent Houzet

    Full Text Available PCR is the most widely applied technique for large scale screening of bacterial clones, mouse genotypes, virus genomes etc. A drawback of large PCR screening is that amplicon analysis is usually performed using gel electrophoresis, a step that is very labor intensive, tedious and chemical waste generating. Single genome amplification (SGA is used to characterize the diversity and evolutionary dynamics of virus populations within infected hosts. SGA is based on the isolation of single template molecule using limiting dilution followed by nested PCR amplification and requires the analysis of hundreds of reactions per sample, making large scale SGA studies very challenging. Here we present a novel approach entitled Long Amplicon Melt Profiling (LAMP based on the analysis of the melting profile of the PCR reactions using SYBR Green and/or EvaGreen fluorescent dyes. The LAMP method represents an attractive alternative to gel electrophoresis and enables the quick discrimination of positive reactions. We validate LAMP for SIV and HIV env-SGA, in 96- and 384-well plate formats. Because the melt profiling allows the screening of several thousands of PCR reactions in a cost-effective, rapid and robust way, we believe it will greatly facilitate any large scale PCR screening.

  11. Screening of perfluorinated chemicals (PFCs) in various aquatic organisms

    Energy Technology Data Exchange (ETDEWEB)

    Fernandez-Sanjuan, Maria; Meyer, Johan; Damasio, Joana; Faria, Melissa; Barata, Carlos; Lacorte, Silvia [IDAEA-CSIC, Department of Environmental Chemistry, Barcelona (Spain)

    2010-10-15

    The aim of this study was to evaluate the occurrence of five perfluorinated chemicals (perfluorooctane sulfonic acid (PFOS), perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), perfluorohexane sulfonic acid (PFHxS), and perfluorobutane sulfonic acid) in aquatic organisms dwelling in either freshwater or marine ecosystems. Organisms selected were insect larvae, oysters, zebra mussels, sardines, and crabs, which are widespread in the environment and may represent potential bioindicators of exposure to PFCs. The study comprises the optimization of a solid-liquid extraction method and determination by high-performance liquid chromatography coupled to tandem mass spectrometry. Using spiked zebra mussels at 10 and 100 ng/g level, the method developed provided recoveries of 96% and 122%, and 82% to 116%, respectively, and a limit of detection between 0.07 and 0.22 ng/g ww. The method was highly sensitivity and robust to determine PFC compounds in a wide array of biological matrices, and no matrix interferents nor blank contamination was observed. Among organisms studied, none of the bivalves accumulated PFCs, and contrarily, insect larvae, followed by fish and crabs contained levels ranging from 0.23 to 144 ng/g ww of PFOS, from 0.14 to 4.3 ng/g ww of PFOA, and traces of PFNA and PFHxS. Assessment of the potential use of aquatic organisms for biomonitoring studies is further discussed. (orig.)

  12. Improved exposure estimation in soil screening and clean-up criteria for volatile organic chemicals.

    Science.gov (United States)

    DeVaull, George E

    2017-02-18

    Soil clean-up criteria define acceptable concentrations of organic chemical constituents for exposed humans. These criteria sum the estimated soil exposure over multiple pathways. Assumptions for ingestion, dermal contact, and dust exposure generally presume a chemical persists in surface soils at a constant concentration level for the entire exposure duration. For volatile chemicals this is an unrealistic assumption. A calculation method is presented for surficial soil criteria which include volatile depletion of chemical for these uptake pathways. The depletion estimates compare favorably with measured concentration profiles and with field measurements of soil concentration. Corresponding volatilization estimates compare favorably with measured data for a wide range of volatile and semi-volatile chemicals, including instances with and without the presence of a mixed-chemical residual phase. Selected examples show application of the revised factors in estimating screening levels for benzene in surficial soils. This article is protected by copyright. All rights reserved.

  13. Development of a consumer product ingredient database for chemical exposure screening and prioritization.

    Science.gov (United States)

    Goldsmith, M-R; Grulke, C M; Brooks, R D; Transue, T R; Tan, Y M; Frame, A; Egeghy, P P; Edwards, R; Chang, D T; Tornero-Velez, R; Isaacs, K; Wang, A; Johnson, J; Holm, K; Reich, M; Mitchell, J; Vallero, D A; Phillips, L; Phillips, M; Wambaugh, J F; Judson, R S; Buckley, T J; Dary, C C

    2014-03-01

    Consumer products are a primary source of chemical exposures, yet little structured information is available on the chemical ingredients of these products and the concentrations at which ingredients are present. To address this data gap, we created a database of chemicals in consumer products using product Material Safety Data Sheets (MSDSs) publicly provided by a large retailer. The resulting database represents 1797 unique chemicals mapped to 8921 consumer products and a hierarchy of 353 consumer product "use categories" within a total of 15 top-level categories. We examine the utility of this database and discuss ways in which it will support (i) exposure screening and prioritization, (ii) generic or framework formulations for several indoor/consumer product exposure modeling initiatives, (iii) candidate chemical selection for monitoring near field exposure from proximal sources, and (iv) as activity tracers or ubiquitous exposure sources using "chemical space" map analyses. Chemicals present at high concentrations and across multiple consumer products and use categories that hold high exposure potential are identified. Our database is publicly available to serve regulators, retailers, manufacturers, and the public for predictive screening of chemicals in new and existing consumer products on the basis of exposure and risk.

  14. A genome-wide RNAi screen to dissect centriole duplication and centrosome maturation in Drosophila.

    Directory of Open Access Journals (Sweden)

    Jeroen Dobbelaere

    2008-09-01

    Full Text Available Centrosomes comprise a pair of centrioles surrounded by an amorphous pericentriolar material (PCM. Here, we have performed a microscopy-based genome-wide RNA interference (RNAi screen in Drosophila cells to identify proteins required for centriole duplication and mitotic PCM recruitment. We analysed 92% of the Drosophila genome (13,059 genes and identified 32 genes involved in centrosome function. An extensive series of secondary screens classified these genes into four categories: (1 nine are required for centriole duplication, (2 11 are required for centrosome maturation, (3 nine are required for both functions, and (4 three genes regulate centrosome separation. These 32 hits include several new centrosomal components, some of which have human homologs. In addition, we find that the individual depletion of only two proteins, Polo and Centrosomin (Cnn can completely block centrosome maturation. Cnn is phosphorylated during mitosis in a Polo-dependent manner, suggesting that the Polo-dependent phosphorylation of Cnn initiates centrosome maturation in flies.

  15. Genome-wide functional screen identifies a compendium of genes affecting sensitivity to tamoxifen.

    Science.gov (United States)

    Mendes-Pereira, Ana M; Sims, David; Dexter, Tim; Fenwick, Kerry; Assiotis, Ioannis; Kozarewa, Iwanka; Mitsopoulos, Costas; Hakas, Jarle; Zvelebil, Marketa; Lord, Christopher J; Ashworth, Alan

    2012-02-21

    Therapies that target estrogen signaling have made a very considerable contribution to reducing mortality from breast cancer. However, resistance to tamoxifen remains a major clinical problem. Here we have used a genome-wide functional profiling approach to identify multiple genes that confer resistance or sensitivity to tamoxifen. Combining whole-genome shRNA screening with massively parallel sequencing, we have profiled the impact of more than 56,670 RNA interference reagents targeting 16,487 genes on the cellular response to tamoxifen. This screen, along with subsequent validation experiments, identifies a compendium of genes whose silencing causes tamoxifen resistance (including BAP1, CLPP, GPRC5D, NAE1, NF1, NIPBL, NSD1, RAD21, RARG, SMC3, and UBA3) and also a set of genes whose silencing causes sensitivity to this endocrine agent (C10orf72, C15orf55/NUT, EDF1, ING5, KRAS, NOC3L, PPP1R15B, RRAS2, TMPRSS2, and TPM4). Multiple individual genes, including NF1, a regulator of RAS signaling, also correlate with clinical outcome after tamoxifen treatment.

  16. Generation of Mouse Haploid Somatic Cells by Small Molecules for Genome-wide Genetic Screening

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    Zheng-Quan He

    2017-08-01

    Full Text Available The recent success of derivation of mammalian haploid embryonic stem cells (haESCs has provided a powerful tool for large-scale functional analysis of the mammalian genome. However, haESCs rapidly become diploidized after differentiation, posing challenges for genetic analysis. Here, we show that the spontaneous diploidization of haESCs happens in metaphase due to mitotic slippage. Diploidization can be suppressed by small-molecule-mediated inhibition of CDK1 and ROCK. Through ROCK inhibition, we can generate haploid somatic cells of all three germ layers from haESCs, including terminally differentiated neurons. Using piggyBac transposon-based insertional mutagenesis, we generated a haploid neural cell library harboring genome-wide mutations for genetic screening. As a proof of concept, we screened for Mn2+-mediated toxicity and identified the Park2 gene. Our findings expand the applications of mouse haploid cell technology to somatic cell types and may also shed light on the mechanisms of ploidy maintenance.

  17. Data-driven prioritization of chemicals for various water types using suspect screening LC-HRMS

    NARCIS (Netherlands)

    Sjerps, Rosa M.A.; Vughs, Dennis; van Leerdam, Jan A.; ter Laak, Thomas L.; van Wezel, Annemarie P.

    2016-01-01

    For the prioritization of more than 5200 anthropogenic chemicals authorized on the European market, we use a large scale liquid chromatography-high resolution mass spectrometry (LC-HRMS) suspect screening study. The prioritization is based on occurrence in 151 water samples including effluent, surfa

  18. PLASMA PROTEIN PROFILING AS A HIGH THROUGHPUT TOOL FOR CHEMICAL SCREENING USING A SMALL FISH MODEL

    Science.gov (United States)

    Hudson, R. Tod, Michael J. Hemmer, Kimberly A. Salinas, Sherry S. Wilkinson, James Watts, James T. Winstead, Peggy S. Harris, Amy Kirkpatrick and Calvin C. Walker. In press. Plasma Protein Profiling as a High Throughput Tool for Chemical Screening Using a Small Fish Model (Abstra...

  19. Use of whole genome expression analysis in the toxicity screening of nanoparticles

    Energy Technology Data Exchange (ETDEWEB)

    Fröhlich, Eleonore, E-mail: eleonore.froehlich@medunigraz.at [Center for Medical Research, Medical University of Graz, Stiftingtalstr. 24, 8010 Graz (Austria); Meindl, Claudia; Wagner, Karin [Center for Medical Research, Medical University of Graz, Stiftingtalstr. 24, 8010 Graz (Austria); Leitinger, Gerd [Center for Medical Research, Medical University of Graz, Stiftingtalstr. 24, 8010 Graz (Austria); Institute for Cell Biology, Histology and Embryology, Medical University of Graz, Harrachgasse 21, 8010 Graz (Austria); Roblegg, Eva [Institute of Pharmaceutical Sciences, Department of Pharmaceutical Technology, Karl-Franzens-University of Graz, Universitätsplatz 1, 8010 Graz (Austria)

    2014-10-15

    The use of nanoparticles (NPs) offers exciting new options in technical and medical applications provided they do not cause adverse cellular effects. Cellular effects of NPs depend on particle parameters and exposure conditions. In this study, whole genome expression arrays were employed to identify the influence of particle size, cytotoxicity, protein coating, and surface functionalization of polystyrene particles as model particles and for short carbon nanotubes (CNTs) as particles with potential interest in medical treatment. Another aim of the study was to find out whether screening by microarray would identify other or additional targets than commonly used cell-based assays for NP action. Whole genome expression analysis and assays for cell viability, interleukin secretion, oxidative stress, and apoptosis were employed. Similar to conventional assays, microarray data identified inflammation, oxidative stress, and apoptosis as affected by NP treatment. Application of lower particle doses and presence of protein decreased the total number of regulated genes but did not markedly influence the top regulated genes. Cellular effects of CNTs were small; only carboxyl-functionalized single-walled CNTs caused appreciable regulation of genes. It can be concluded that regulated functions correlated well with results in cell-based assays. Presence of protein mitigated cytotoxicity but did not cause a different pattern of regulated processes. - Highlights: • Regulated functions were screened using whole genome expression assays. • Polystyrene particles regulated more genes than short carbon nanotubes. • Protein coating of polystyrene particles did not change regulation pattern. • Functions regulated by microarray were confirmed by cell-based assay.

  20. Genome-wide screening for genetic loci associated with noise-induced hearing loss.

    Science.gov (United States)

    White, Cory H; Ohmen, Jeffrey D; Sheth, Sonal; Zebboudj, Amina F; McHugh, Richard K; Hoffman, Larry F; Lusis, Aldons J; Davis, Richard C; Friedman, Rick A

    2009-04-01

    Noise-induced hearing loss (NIHL) is one of the more common sources of environmentally induced hearing loss in adults. In a mouse model, Castaneous (CAST/Ei) is an inbred strain that is resistant to NIHL, while the C57BL/6J strain is susceptible. We have used the genome-tagged mice (GTM) library of congenic strains, carrying defined segments of the CAST/Ei genome introgressed onto the C57BL/6J background, to search for loci modifying the noise-induced damage seen in the C57BL/6J strain. NIHL was induced by exposing 6-8-week old mice to 108 dB SPL intensity noise. We tested the hearing of each mouse strain up to 23 days after noise exposure using auditory brainstem response (ABR). This study identifies a number of genetic loci that modify the initial response to damaging noise, as well as long-term recovery. The data suggest that multiple alleles within the CAST/Ei genome modify the pathogenesis of NIHL and that screening congenic libraries for loci that underlie traits of interest can be easily carried out in a high-throughput fashion.

  1. Integrating experimental and analytic approaches to improve data quality in genome-wide RNAi screens.

    Science.gov (United States)

    Zhang, Xiaohua Douglas; Espeseth, Amy S; Johnson, Eric N; Chin, Jayne; Gates, Adam; Mitnaul, Lyndon J; Marine, Shane D; Tian, Jenny; Stec, Eric M; Kunapuli, Priya; Holder, Dan J; Heyse, Joseph F; Strulovici, Berta; Ferrer, Marc

    2008-06-01

    RNA interference (RNAi) not only plays an important role in drug discovery but can also be developed directly into drugs. RNAi high-throughput screening (HTS) biotechnology allows us to conduct genome-wide RNAi research. A central challenge in genome-wide RNAi research is to integrate both experimental and computational approaches to obtain high quality RNAi HTS assays. Based on our daily practice in RNAi HTS experiments, we propose the implementation of 3 experimental and analytic processes to improve the quality of data from RNAi HTS biotechnology: (1) select effective biological controls; (2) adopt appropriate plate designs to display and/or adjust for systematic errors of measurement; and (3) use effective analytic metrics to assess data quality. The applications in 5 real RNAi HTS experiments demonstrate the effectiveness of integrating these processes to improve data quality. Due to the effectiveness in improving data quality in RNAi HTS experiments, the methods and guidelines contained in the 3 experimental and analytic processes are likely to have broad utility in genome-wide RNAi research.

  2. Genome-wide association screens for Achilles tendon and ACL tears and tendinopathy

    Science.gov (United States)

    Roos, Thomas R.; Roos, Andrew K.; Kleimeyer, John P.; Ahmed, Marwa A.; Goodlin, Gabrielle T.; Fredericson, Michael; Ioannidis, John P. A.; Avins, Andrew L.; Dragoo, Jason L.

    2017-01-01

    Achilles tendinopathy or rupture and anterior cruciate ligament (ACL) rupture are substantial injuries affecting athletes, associated with delayed recovery or inability to return to competition. To identify genetic markers that might be used to predict risk for these injuries, we performed genome-wide association screens for these injuries using data from the Genetic Epidemiology Research on Adult Health and Aging (GERA) cohort consisting of 102,979 individuals. We did not find any single nucleotide polymorphisms (SNPs) associated with either of these injuries with a p-value that was genome-wide significant (pAchilles tendon injury and ACL rupture, respectively. We then tested SNPs previously reported to be associated with either Achilles tendon injury or ACL rupture. None showed an association in our cohort with a false discovery rate of less than 5%. We obtained, however, moderate to weak evidence for replication in one case; specifically, rs4919510 in MIR608 had a p-value of 5.1x10-3 for association with Achilles tendon injury, corresponding to a 7% chance of false replication. Finally, we tested 2855 SNPs in 90 candidate genes for musculoskeletal injury, but did not find any that showed a significant association below a false discovery rate of 5%. We provide data containing summary statistics for the entire genome, which will be useful for future genetic studies on these injuries. PMID:28358823

  3. Screening the budding yeast genome reveals unique factors affecting K2 toxin susceptibility.

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    Elena Servienė

    Full Text Available BACKGROUND: Understanding how biotoxins kill cells is of prime importance in biomedicine and the food industry. The budding yeast (S. cerevisiae killers serve as a convenient model to study the activity of biotoxins consistently supplying with significant insights into the basic mechanisms of virus-host cell interactions and toxin entry into eukaryotic target cells. K1 and K2 toxins are active at the cell wall, leading to the disruption of the plasma membrane and subsequent cell death by ion leakage. K28 toxin is active in the cell nucleus, blocking DNA synthesis and cell cycle progression, thereby triggering apoptosis. Genome-wide screens in the budding yeast S. cerevisiae identified several hundred effectors of K1 and K28 toxins. Surprisingly, no such screen had been performed for K2 toxin, the most frequent killer toxin among industrial budding yeasts. PRINCIPAL FINDINGS: We conducted several concurrent genome-wide screens in S. cerevisiae and identified 332 novel K2 toxin effectors. The effectors involved in K2 resistance and hypersensitivity largely map in distinct cellular pathways, including cell wall and plasma membrane structure/biogenesis and mitochondrial function for K2 resistance, and cell wall stress signaling and ion/pH homeostasis for K2 hypersensitivity. 70% of K2 effectors are different from those involved in K1 or K28 susceptibility. SIGNIFICANCE: Our work demonstrates that despite the fact that K1 and K2 toxins share some aspects of their killing strategies, they largely rely on different sets of effectors. Since the vast majority of the host factors identified here is exclusively active towards K2, we conclude that cells have acquired a specific K2 toxin effectors set. Our work thus indicates that K1 and K2 have elaborated different biological pathways and provides a first step towards the detailed characterization of K2 mode of action.

  4. Genome-wide screen of Pseudomonas aeruginosa In Saccharomyces cerevisiae identifies new virulence factors

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    Rafat eZrieq

    2015-11-01

    Full Text Available Pseudomonas aeruginosa is a human opportunistic pathogen that causes mortality in cystic fibrosis and immunocompromised patients. While many virulence factors of this pathogen have already been identified, several remain to be discovered. In this respect we set an unprecedented genome-wide screen of a P. aeruginosa expression library based on a yeast growth phenotype. 51 candidates were selected in a three-round screening process. The robustness of the screen was validated by the selection of three well known secreted proteins including one demonstrated virulence factor, the protease LepA. Further in silico sorting of the 51 candidates highlighted three potential new Pseudomonas effector candidates (Pec. By testing the cytotoxicity of wild type P. aeruginosa vs pec mutants towards macrophages and the virulence in the Caenorhabditis elegans model, we demonstrated that the three selected Pecs are novel virulence factors of P. aeruginosa. Additional cellular localization experiments in the host revealed specific localization for Pec1 and Pec2 that could inform about their respective functions.

  5. Negative regulators of insulin signaling revealed in a genome-wide functional screen.

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    Shih-Min A Huang

    Full Text Available BACKGROUND: Type 2 diabetes develops due to a combination of insulin resistance and beta-cell failure and current therapeutics aim at both of these underlying causes. Several negative regulators of insulin signaling are known and are the subject of drug discovery efforts. We sought to identify novel contributors to insulin resistance and hence potentially novel targets for therapeutic intervention. METHODOLOGY: An arrayed cDNA library encoding 18,441 human transcripts was screened for inhibitors of insulin signaling and revealed known inhibitors and numerous potential novel regulators. The novel hits included proteins of various functional classes such as kinases, phosphatases, transcription factors, and GTPase associated proteins. A series of secondary assays confirmed the relevance of the primary screen hits to insulin signaling and provided further insight into their modes of action. CONCLUSION/SIGNIFICANCE: Among the novel hits was PALD (KIAA1274, paladin, a previously uncharacterized protein that when overexpressed led to inhibition of insulin's ability to down regulate a FOXO1A-driven reporter gene, reduced upstream insulin-stimulated AKT phosphorylation, and decreased insulin receptor (IR abundance. Conversely, knockdown of PALD gene expression resulted in increased IR abundance, enhanced insulin-stimulated AKT phosphorylation, and an improvement in insulin's ability to suppress FOXO1A-driven reporter gene activity. The present data demonstrate that the application of arrayed genome-wide screening technologies to insulin signaling is fruitful and is likely to reveal novel drug targets for insulin resistance and the metabolic syndrome.

  6. Chemical and Biophysical Modulation of Cas9 for Tunable Genome Engineering.

    Science.gov (United States)

    Nuñez, James K; Harrington, Lucas B; Doudna, Jennifer A

    2016-03-18

    The application of the CRISPR-Cas9 system for genome engineering has revolutionized the ability to interrogate genomes of mammalian cells. Programming the Cas9 endonuclease to induce DNA breaks at specified sites is achieved by simply modifying the sequence of its cognate guide RNA. Although Cas9-mediated genome editing has been shown to be highly specific, cleavage events at off-target sites have also been reported. Minimizing, and eventually abolishing, unwanted off-target cleavage remains a major goal of the CRISPR-Cas9 technology before its implementation for therapeutic use. Recent efforts have turned to chemical biology and biophysical approaches to engineer inducible genome editing systems for controlling Cas9 activity at the transcriptional and protein levels. Here, we review recent advancements to modulate Cas9-mediated genome editing by engineering split-Cas9 constructs, inteins, small molecules, protein-based dimerizing domains, and light-inducible systems.

  7. Identification of previously unrecognized antiestrogenic chemicals using a novel virtual screening approach.

    Science.gov (United States)

    Wang, Ching Y; Ai, Ni; Arora, Sonia; Erenrich, Eric; Nagarajan, Karthigeyan; Zauhar, Randy; Young, Douglas; Welsh, William J

    2006-12-01

    The physiological roles of estrogen in sexual differentiation and development, female and male reproductive processes, and bone health are complex and diverse. Numerous natural and synthetic chemical compounds, commonly known as endocrine disrupting chemicals (EDCs), have been shown to alter the physiological effects of estrogen in humans and wildlife. As such, these EDCs may cause unanticipated and even undesirable effects. Large-scale in vitro and in vivo screening of chemicals to assess their estrogenic activity would demand a prodigious investment of time, labor, and money and would require animal testing on an unprecedented scale. Approaches in silico are increasingly recognized as playing a vital role in screening and prioritizing chemicals to extend limited resources available for experimental testing. Here, we evaluated a multistep procedure that is suitable for in silico (virtual) screening of large chemical databases to identify compounds exhibiting estrogenic activity. This procedure incorporates Shape Signatures, a novel computational tool that rapidly compares molecules on the basis of similarity in shape, polarity, and other bio-relevant properties. Using 4-hydroxy tamoxifen (4-OH TAM) and diethylstilbestrol (DES) as input queries, we employed this scheme to search a sample database of approximately 200,000 commercially available organic chemicals for matches (hits). Of the eight compounds identified computationally as potentially (anti)estrogenic, biological evaluation confirmed two as heretofore unknown estrogen antagonists. Subsequent radioligand binding assays confirmed that two of these three compounds exhibit antiestrogenic activities comparable to 4-OH TAM. Molecular modeling studies of these ligands docked inside the binding pocket of estrogen receptor alpha (ERalpha) elucidated key ligand-receptor interactions that corroborate these experimental findings. The present study demonstrates the utility of our computational scheme for this and

  8. Chemical compatibility screening results of plastic packaging to mixed waste simulants

    Energy Technology Data Exchange (ETDEWEB)

    Nigrey, P.J.; Dickens, T.G.

    1995-12-01

    We have developed a chemical compatibility program for evaluating transportation packaging components for transporting mixed waste forms. We have performed the first phase of this experimental program to determine the effects of simulant mixed wastes on packaging materials. This effort involved the screening of 10 plastic materials in four liquid mixed waste simulants. The testing protocol involved exposing the respective materials to {approximately}3 kGy of gamma radiation followed by 14 day exposures to the waste simulants of 60 C. The seal materials or rubbers were tested using VTR (vapor transport rate) measurements while the liner materials were tested using specific gravity as a metric. For these tests, a screening criteria of {approximately}1 g/m{sup 2}/hr for VTR and a specific gravity change of 10% was used. It was concluded that while all seal materials passed exposure to the aqueous simulant mixed waste, EPDM and SBR had the lowest VTRs. In the chlorinated hydrocarbon simulant mixed waste, only VITON passed the screening tests. In both the simulant scintillation fluid mixed waste and the ketone mixture simulant mixed waste, none of the seal materials met the screening criteria. It is anticipated that those materials with the lowest VTRs will be evaluated in the comprehensive phase of the program. For specific gravity testing of liner materials the data showed that while all materials with the exception of polypropylene passed the screening criteria, Kel-F, HDPE, and XLPE were found to offer the greatest resistance to the combination of radiation and chemicals.

  9. Functional screening of metagenome and genome libraries for detection of novel flavonoid-modifying enzymes.

    Science.gov (United States)

    Rabausch, U; Juergensen, J; Ilmberger, N; Böhnke, S; Fischer, S; Schubach, B; Schulte, M; Streit, W R

    2013-08-01

    The functional detection of novel enzymes other than hydrolases from metagenomes is limited since only a very few reliable screening procedures are available that allow the rapid screening of large clone libraries. For the discovery of flavonoid-modifying enzymes in genome and metagenome clone libraries, we have developed a new screening system based on high-performance thin-layer chromatography (HPTLC). This metagenome extract thin-layer chromatography analysis (META) allows the rapid detection of glycosyltransferase (GT) and also other flavonoid-modifying activities. The developed screening method is highly sensitive, and an amount of 4 ng of modified flavonoid molecules can be detected. This novel technology was validated against a control library of 1,920 fosmid clones generated from a single Bacillus cereus isolate and then used to analyze more than 38,000 clones derived from two different metagenomic preparations. Thereby we identified two novel UDP glycosyltransferase (UGT) genes. The metagenome-derived gtfC gene encoded a 52-kDa protein, and the deduced amino acid sequence was weakly similar to sequences of putative UGTs from Fibrisoma and Dyadobacter. GtfC mediated the transfer of different hexose moieties and exhibited high activities on flavones, flavonols, flavanones, and stilbenes and also accepted isoflavones and chalcones. From the control library we identified a novel macroside glycosyltransferase (MGT) with a calculated molecular mass of 46 kDa. The deduced amino acid sequence was highly similar to sequences of MGTs from Bacillus thuringiensis. Recombinant MgtB transferred the sugar residue from UDP-glucose effectively to flavones, flavonols, isoflavones, and flavanones. Moreover, MgtB exhibited high activity on larger flavonoid molecules such as tiliroside.

  10. Molecular, chemical and biological screening of soil actinomycete isolates in seeking bioactive peptide metabolites

    Directory of Open Access Journals (Sweden)

    Javad Hamedi

    2015-10-01

    Full Text Available Background and Objective: Due to the evolution of multidrug-resistant strains, screening of natural resources, especially actinomycetes, for new therapeutic agents discovery has become the interests of researchers. In this study, molecular, chemical and biological screening of soil actinomycetes was carried out in order to search for peptide-producing actinomycetes.Materials and Methods: 60 actinomycetes were isolated from soils of Iran. The isolates were subjected to molecular screening for detection NRPS (non-ribosomal peptide synthetases gene. Phylogenic identification of NRPS containing isolates was performed. Chemical screening of the crude extracts was performed using chlorine o-dianisidine as peptide detector reagent and bioactivity of peptide producing strains was determined by antimicrobial bioassay. High pressure liquid chromatography- mass spectrometry (HPLC-MS with UV-visible spectroscopy was performed for detection of the metabolite diversity in selected strain.Results: Amplified NRPS adenylation gene (700 bp was detected among 30 strains. Phylogenic identification of these isolates showed presence of rare actinomycetes genera among the isolates and 10 out of 30 strains were subjected to chemical screening. Nocardia sp. UTMC 751 showed antimicrobial activity against bacterial and fungal test pathogens. HPLC-MSand UV-visible spectroscopy results from the crude extract showed that this strain has probably the ability to produce new metabolites.Conclusion: By application of a combined approach, including molecular, chemical and bioactivity analysis, a promising strain of Nocardia sp. UTMC 751 was obtained. This strain had significant activity against Staphylococcus aureus and Pseudomonas aeruginosa. Strain Nocardia sp. UTMC 751 produce five unknown and most probably new metabolites with molecular weights of 274.2, 390.3, 415.3, 598.4 and 772.5. This strain had showed 99% similarity to Nocardia ignorata DSM 44496 T.

  11. Development of Screening Tools for the Interpretation of Chemical Biomonitoring Data

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    Richard A. Becker

    2012-01-01

    Full Text Available Evaluation of a larger number of chemicals in commerce from the perspective of potential human health risk has become a focus of attention in North America and Europe. Screening-level chemical risk assessment evaluations consider both exposure and hazard. Exposures are increasingly being evaluated through biomonitoring studies in humans. Interpreting human biomonitoring results requires comparison to toxicity guidance values. However, conventional chemical-specific risk assessments result in identification of toxicity-based exposure guidance values such as tolerable daily intakes (TDIs as applied doses that cannot directly be used to evaluate exposure information provided by biomonitoring data in a health risk context. This paper describes a variety of approaches for development of screening-level exposure guidance values with translation from an external dose to a biomarker concentration framework for interpreting biomonitoring data in a risk context. Applications of tools and concepts including biomonitoring equivalents (BEs, the threshold of toxicologic concern (TTC, and generic toxicokinetic and physiologically based toxicokinetic models are described. These approaches employ varying levels of existing chemical-specific data, chemical class-specific assessments, and generic modeling tools in response to varying levels of available data in order to allow assessment and prioritization of chemical exposures for refined assessment in a risk management context.

  12. Mining Natural-Products Screening Data for Target-Class Chemical Motifs.

    Science.gov (United States)

    Coma, Isabel; Bandyopadhyay, Deepak; Diez, Emilio; Ruiz, Emilio Alvarez; de los Frailes, Maria Teresa; Colmenarejo, Gonzalo

    2014-06-01

    In this article, we describe two complementary data-mining approaches used to characterize the GlaxoSmithKline (GSK) natural-products set (NPS) based on information from the high-throughput screening (HTS) databases. Both methods rely on the aggregation and analysis of a large set of single-shot screening data for a number of biological assays, with the goal to reveal natural-product chemical motifs. One of them is an established method based on the data-driven clustering of compounds using a wide range of descriptors,(1)whereas the other method partitions and hierarchically clusters the data to identify chemical cores.(2,3)Both methods successfully find structural scaffolds that significantly hit different groups of discrete drug targets, compared with their relative frequency of demonstrating inhibitory activity in a large number of screens. We describe how these methods can be applied to unveil hidden information in large single-shot HTS data sets. Applied prospectively, this type of information could contribute to the design of new chemical templates for drug-target classes and guide synthetic efforts for lead optimization of tractable hits that are based on natural-product chemical motifs. Relevant findings for 7TM receptors (7TMRs), ion channels, class-7 transferases (protein kinases), hydrolases, and oxidoreductases will be discussed.

  13. Reevaluation of 1999 Health-Based Environmental Screening Levels (HBESLs) for Chemical Warfare Agents

    Energy Technology Data Exchange (ETDEWEB)

    Watson, Annetta Paule [ORNL; Dolislager, Fredrick G [ORNL

    2007-05-01

    This report evaluates whether new information and updated scientific models require that changes be made to previously published health-based environmental soil screening levels (HBESLs) and associated environmental fate/breakdown information for chemical warfare agents (USACHPPM 1999). Specifically, the present evaluation describes and compares changes that have been made since 1999 to U.S. Environmental Protection Agency (EPA) risk assessment models, EPA exposure assumptions, as well as to specific chemical warfare agent parameters (e.g., toxicity values). Comparison was made between screening value estimates recalculated with current assumptions and earlier health-based environmental screening levels presented in 1999. The chemical warfare agents evaluated include the G-series and VX nerve agents and the vesicants sulfur mustard (agent HD) and Lewisite (agent L). In addition, key degradation products of these agents were also evaluated. Study findings indicate that the combined effect of updates and/or changes to EPA risk models, EPA default exposure parameters, and certain chemical warfare agent toxicity criteria does not result in significant alteration to the USACHPPM (1999) health-based environmental screening level estimates for the G-series and VX nerve agents or the vesicant agents HD and L. Given that EPA's final position on separate Tier 1 screening levels for indoor and outdoor worker screening assessments has not yet been released as of May 2007, the study authors find that the 1999 screening level estimates (see Table ES.1) are still appropriate and protective for screening residential as well as nonresidential sites. As such, risk management decisions made on the basis of USACHPPM (1999) recommendations do not require reconsideration. While the 1999 HBESL values are appropriate for continued use as general screening criteria, the updated '2007' estimates (presented below) that follow the new EPA protocols currently under development

  14. A genome-wide siRNA screen to identify modulators of insulin sensitivity and gluconeogenesis.

    Directory of Open Access Journals (Sweden)

    Ruojing Yang

    Full Text Available BACKGROUND: Hepatic insulin resistance impairs insulin's ability to suppress hepatic glucose production (HGP and contributes to the development of type 2 diabetes (T2D. Although the interests to discover novel genes that modulate insulin sensitivity and HGP are high, it remains challenging to have a human cell based system to identify novel genes. METHODOLOGY/PRINCIPAL FINDINGS: To identify genes that modulate hepatic insulin signaling and HGP, we generated a human cell line stably expressing beta-lactamase under the control of the human glucose-6-phosphatase (G6PC promoter (AH-G6PC cells. Both beta-lactamase activity and endogenous G6PC mRNA were increased in AH-G6PC cells by a combination of dexamethasone and pCPT-cAMP, and reduced by insulin. A 4-gene High-Throughput-Genomics assay was developed to concomitantly measure G6PC and pyruvate-dehydrogenase-kinase-4 (PDK4 mRNA levels. Using this assay, we screened an siRNA library containing pooled siRNA targeting 6650 druggable genes and identified 614 hits that lowered G6PC expression without increasing PDK4 mRNA levels. Pathway analysis indicated that siRNA-mediated knockdown (KD of genes known to positively or negatively affect insulin signaling increased or decreased G6PC mRNA expression, respectively, thus validating our screening platform. A subset of 270 primary screen hits was selected and 149 hits were confirmed by target gene KD by pooled siRNA and 7 single siRNA for each gene to reduce G6PC expression in 4-gene HTG assay. Subsequently, pooled siRNA KD of 113 genes decreased PEPCK and/or PGC1alpha mRNA expression thereby demonstrating their role in regulating key gluconeogenic genes in addition to G6PC. Last, KD of 61 of the above 113 genes potentiated insulin-stimulated Akt phosphorylation, suggesting that they suppress gluconeogenic gene by enhancing insulin signaling. CONCLUSIONS/SIGNIFICANCE: These results support the proposition that the proteins encoded by the genes identified in

  15. Establishment of IL-7 Expression Reporter Human Cell Lines, and Their Feasibility for High-Throughput Screening of IL-7-Upregulating Chemicals

    Science.gov (United States)

    Sim, Chan Kyu; Kim, Inki

    2016-01-01

    Interleukin-7 (IL-7) is a cytokine essential for T cell homeostasis, and is clinically important. However, the regulatory mechanism of IL-7 gene expression is not well known, and a systematic approach to screen chemicals that regulate IL-7 expression has not yet been developed. In this study, we attempted to develop human reporter cell lines using CRISPR/Cas9-mediated genome editing technology. For this purpose, we designed donor DNA that contains an enhanced green fluorescent protein (eGFP) gene, drug selection cassette, and modified homologous arms which are considered to enhance the translation of the eGFP reporter transcript, and also a highly efficient single-guide RNA with a minimal off-target effect to target the IL-7 start codon region. By applying this system, we established IL-7 eGFP reporter cell lines that could report IL-7 gene transcription based on the eGFP protein signal. Furthermore, we utilized the cells to run a pilot screen campaign for IL-7-upregulating chemicals in a high-throughput format, and identified a chemical that can up-regulate IL-7 gene transcription. Collectively, these results suggest that our IL-7 reporter system can be utilized in large-scale chemical library screening to reveal novel IL-7 regulatory pathways and to identify potential drugs for development of new treatments in immunodeficiency disease. PMID:27589392

  16. Genome-wide screen for differential DNA methylation associated with neural cell differentiation in mouse.

    Directory of Open Access Journals (Sweden)

    Rene Cortese

    Full Text Available Cellular differentiation involves widespread epigenetic reprogramming, including modulation of DNA methylation patterns. Using Differential Methylation Hybridization (DMH in combination with a custom DMH array containing 51,243 features covering more than 16,000 murine genes, we carried out a genome-wide screen for cell- and tissue-specific differentially methylated regions (tDMRs in undifferentiated embryonic stem cells (ESCs, in in-vitro induced neural stem cells (NSCs and 8 differentiated embryonic and adult tissues. Unsupervised clustering of the generated data showed distinct cell- and tissue-specific DNA methylation profiles, revealing 202 significant tDMRs (p1.96 enrichment for genes involved in neural differentiation, including, for example, Jag1 and Tcf4. Our results provide robust evidence for the relevance of DNA methylation in early neural development and identify novel marker candidates for neural cell differentiation.

  17. Integrated, genome-wide screening for hypomethylated oncogenes in salivary gland adenoid cystic carcinoma

    Science.gov (United States)

    Shao, Chunbo; Sun, Wenyue; Tan, Marietta; Glazer, Chad A.; Bhan, Sheetal; Zhong, Xiaoli; Fakhry, Carole; Sharma, Rajni; Westra, William H.; Hoque, Mohammad O.; Moskaluk, Christopher A.; Sidransky, David; Califano, Joseph A.; Ha, Patrick K.

    2011-01-01

    Purpose Salivary gland adenoid cystic carcinoma (ACC) is a rare malignancy that is poorly understood. In order to look for relevant oncogene candidates under the control of promoter methylation, an integrated, genome-wide screen was performed. Experimental Design Global demethylation of normal salivary gland cell strains using 5-aza-2′-deoxycytidine (5-Aza dC) and Trichostatin A (TSA), followed by expression array analysis was performed. ACC-specific expression profiling was generated using expression microarray analysis of primary ACC and normal samples. Next, the two profiles were integrated to identify a subset of genes for further validation of promoter demethylation in ACC versus normal. Finally, promising candidates were further validated for mRNA, protein, and promoter methylation levels in larger ACC cohorts. Functional validation was then performed in cancer cell lines. Results We found 159 genes that were significantly re-expressed after 5-Aza dC/TSA treatment and overexpressed in ACC. After initial validation, eight candidates showed hypomethylation in ACC: AQP1, CECR1, C1QR1, CTAG2, P53AIP1, TDRD12, BEX1, and DYNLT3. Aquaporin 1 (AQP1) showed the most significant hypomethylation and was further validated. AQP1 hypomethylation in ACC was confirmed with two independent cohorts. Of note, there was significant overexpression of AQP1 in both mRNA and protein in the paraffin-embedded ACC cohort. Furthermore, AQP1 was up-regulated in 5-Aza dC/TSA treated SACC83. Lastly, AQP1 promoted cell proliferation and colony formation in SACC83. Conclusions Our integrated, genome-wide screening method proved to be an effective strategy for detecting novel oncogenes in ACC. AQP1 is a promising oncogene candidate for ACC and is transcriptionally regulated by promoter hypomethylation. PMID:21551254

  18. Genome-wide association study of coronary and aortic calcification in lung cancer screening CT

    Science.gov (United States)

    de Vos, Bob D.; van Setten, Jessica; de Jong, Pim A.; Mali, Willem P.; Oudkerk, Matthijs; Viergever, Max A.; Išgum, Ivana

    2016-03-01

    Arterial calcification has been related to cardiovascular disease (CVD) and osteoporosis. However, little is known about the role of genetics and exact pathways leading to arterial calcification and its relation to bone density changes indicating osteoporosis. In this study, we conducted a genome-wide association study of arterial calcification burden, followed by a look-up of known single nucleotide polymorphisms (SNPs) for coronary artery disease (CAD) and myocardial infarction (MI), and bone mineral density (BMD) to test for a shared genetic basis between the traits. The study included a subcohort of the Dutch-Belgian lung cancer screening trial comprised of 2,561 participants. Participants underwent baseline CT screening in one of two hospitals participating in the trial. Low-dose chest CT images were acquired without contrast enhancement and without ECG-synchronization. In these images coronary and aortic calcifications were identified automatically. Subsequently, the detected calcifications were quantified using coronary artery calcium Agatston and volume scores. Genotype data was available for these participants. A genome-wide association study was conducted on 10,220,814 SNPs using a linear regression model. To reduce multiple testing burden, known CAD/MI and BMD SNPs were specifically tested (45 SNPs from the CARDIoGRAMplusC4D consortium and 60 SNPS from the GEFOS consortium). No novel significant SNPs were found. Significant enrichment for CAD/MI SNPs was observed in testing Agatston and coronary artery calcium volume scores. Moreover, a significant enrichment of BMD SNPs was shown in aortic calcium volume scores. This may indicate genetic relation of BMD SNPs and arterial calcification burden.

  19. Human genome-wide RNAi screen for host factors that modulate intracellular Salmonella growth.

    Science.gov (United States)

    Thornbrough, Joshua M; Hundley, Tom; Valdivia, Raphael; Worley, Micah J

    2012-01-01

    Salmonella enterica is a bacterial pathogen of humans that can proliferate within epithelial cells as well as professional phagocytes of the immune system. While much has been learned about the microbial genes that influence the infectious process through decades of intensive research, relatively little is known about the host factors that affect infection. We performed a genome-wide siRNA screen to identify host genes that Salmonella enterica serovar Typhimurium (S. typhimurium) utilizes to facilitate growth within human epithelial cells. In this screen, with siRNAs targeting every predicted gene in the human genome, we identified 252 new human-host-susceptibility factors (HSFs) for S. typhimurium. We also identified 39 genes whose silencing results in increased intracellular growth of S. typhimurium. The HSFs identified are regulated most centrally by NFκB and associate with each other through an extremely dense network of interactions that center around a group of kinases. Most genes identified were not previously appreciated as playing roles in the intracellular lifecycle of S. enterica. Numerous HSFs identified with interesting characteristics that could play plausible roles in mediating intracellular microbial growth are discussed. Importantly, this study reveals significant overlap between the host network that supports S. typhimurium growth within human epithelial cells and the one that promotes the growth of Mycobacterium tuberculosis within human macrophages. In addition to providing much new information about the molecular mechanisms underlying S. enterica-host cell interplay, all 252 HSFs identified are candidates for new anti-microbial targets for controlling S. enterica infections, and some may provide broad-spectrum anti-microbial activity.

  20. A whole-genome RNA interference screen for human cell factors affecting myxoma virus replication.

    Science.gov (United States)

    Teferi, Wondimagegnehu M; Dodd, Kristopher; Maranchuk, Rob; Favis, Nicole; Evans, David H

    2013-04-01

    Myxoma virus (MYXV) provides an important model for investigating host-pathogen interactions. Recent studies have also highlighted how mutations in transformed human cells can expand the host range of this rabbit virus. Although virus growth depends upon interactions between virus and host proteins, the nature of these interactions is poorly understood. To address this matter, we performed small interfering RNA (siRNA) screens for genes affecting MYXV growth in human MDA-MB-231 cells. By using siRNAs targeting the whole human genome (21,585 genes), a subset of human phosphatases and kinases (986 genes), and also a custom siRNA library targeting selected statistically significant genes ("hits") and nonsignificant genes ("nonhits") of the whole human genome screens (88 genes), we identified 711 siRNA pools that promoted MYXV growth and 333 that were inhibitory. Another 32 siRNA pools (mostly targeting the proteasome) were toxic. The overall overlap in the results was about 25% for the hits and 75% for the nonhits. These pro- and antiviral genes can be clustered into pathways and related groups, including well-established inflammatory and mitogen-activated protein kinase pathways, as well as clusters relating to β-catenin and the Wnt signaling cascade, the cell cycle, and cellular metabolism. The validity of a subset of these hits was independently confirmed. For example, treating cells with siRNAs that might stabilize cells in G(1), or inhibit passage into S phase, stimulated MYXV growth, and these effects were reproduced by trapping cells at the G(1)/S boundary with an inhibitor of cyclin-dependent kinases 4/6. By using 2-deoxy-D-glucose and plasmids carrying the gene for phosphofructokinase, we also confirmed that infection is favored by aerobic glycolytic metabolism. These studies provide insights into how the growth state and structure of cells affect MYXV growth and how these factors might be manipulated to advantage in oncolytic virus therapy.

  1. An isothermal primer extension method for whole genome amplification of fresh and degraded DNA: applications in comparative genomic hybridization, genotyping and mutation screening.

    Science.gov (United States)

    Lee, Cheryl I P; Leong, Siew Hong; Png, Adrian E H; Choo, Keng Wah; Syn, Christopher; Lim, Dennis T H; Law, Hai Yang; Kon, Oi Lian

    2006-01-01

    We describe a protocol that uses a bioinformatically optimized primer in an isothermal whole genome amplification (WGA) reaction. Overnight incubation at 37 degrees C efficiently generates several hundred- to several thousand-fold increases in input DNA. The amplified product retains reasonably faithful quantitative representation of unamplified whole genomic DNA (gDNA). We provide protocols for applying this isothermal primer extension WGA protocol in three different techniques of genomic analysis: comparative genomic hybridization (CGH), genotyping at simple tandem repeat (STR) loci and screening for single base mutations in a common monogenic disorder, beta-thalassemia. gDNA extracted from formalin-fixed paraffin-embedded (FFPE) tissues can also be amplified with this protocol.

  2. Genome-wide uniparental disomy screen in human discarded morphologically abnormal embryos.

    Science.gov (United States)

    Xu, Jiawei; Zhang, Meixiang; Niu, Wenbin; Yao, Guidong; Sun, Bo; Bao, Xiao; Wang, Linlin; Du, Linqing; Sun, Yingpu

    2015-07-21

    Uniparental disomy (UPD) has been shown to be rare in human normal blastocysts, but its frequency in discarded morphologically abnormal embryos and its relevance to embryonic self-correction of aneuploid remains unknown. The aim of this study was to detect UPD in discarded morphologically abnormal embryos. Both discarded morphologically abnormal embryos, including zero-pronuclear zygotes (0PN), one-pronuclear zygotes (1PN), three-pronuclear zygotes (3PN) and 2PN embryos scored as low development potential were cultured into blastocysts then underwent trophectoderm biopsy. Genome-wide UPD screening of the trophectoderm of 241 discarded morphologically abnormal embryo sourced blastocysts showed that UPD occurred in nine embryos. Five embryos exhibited UPDs with euploid chromosomes, and four displayed UPDs with chromosomal aneuploid. The percentage of UPDs among the morphologically abnormal sourced blastocysts was 3.73%, which is significant higher than the percentage observed in normal blastocysts. The frequency of UPD in 3PN-sourced blastocysts was 7.69%, which is significantly higher than that in normal blastocysts. This study provides the first systematic genome-wide profile of UPD in discarded morphologically abnormal embryos. Our results indicated that UPD may be a common phenomenon in discarded morphologically abnormal embryos and may be relevant to human embryonic self-correction.

  3. A primary screen of the bovine genome for quantitative trait loci affecting carcass and growth traits.

    Science.gov (United States)

    Stone, R T; Keele, J W; Shackelford, S D; Kappes, S M; Koohmaraie, M

    1999-06-01

    A primary genomic screen for quantitative trait loci (QTL) affecting carcass and growth traits was performed by genotyping 238 microsatellite markers on 185 out of 300 total progeny from a Bos indicus x Bos taurus sire mated to Bos taurus cows. The following traits were analyzed for QTL effects: birth weight (BWT), weaning weight (WW), yearling weight (YW), hot carcass weight (HCW), dressing percentage (DP), fat thickness (FT), marbling score (MAR), longissimus muscle area (LMA), rib bone (RibB), rib fat (RibF), and rib muscle (RibM), and the predicted whole carcass traits, retail product yield (RPYD), fat trim yield (FATYD), bone yield (BOYD), retail product weight (RPWT), fat weight (FATWT), and bone weight (BOWT). Data were analyzed by generating an F-statistic profile computed at 1-cM intervals for each chromosome by the regression of phenotype on the conditional probability of receiving the Brahman allele from the sire. There was compelling evidence for a QTL allele of Brahman origin affecting an increase in RibB and a decrease in DP on chromosome 5 (BTA5). Putative QTL at or just below the threshold for genome-wide significance were as follows: an increase in RPYD and component traits on BTA2 and BTA13, an increase in LMA on BTA14, and an increase in BWT on BTA1. Results provided represent a portion of our efforts to identify and characterize QTL affecting carcass and growth traits.

  4. NMD Microarray Analysis for Rapid Genome-Wide Screen of Mutated Genes in Cancer

    Directory of Open Access Journals (Sweden)

    Maija Wolf

    2005-01-01

    Full Text Available Gene mutations play a critical role in cancer development and progression, and their identification offers possibilities for accurate diagnostics and therapeutic targeting. Finding genes undergoing mutations is challenging and slow, even in the post-genomic era. A new approach was recently developed by Noensie and Dietz to prioritize and focus the search, making use of nonsense-mediated mRNA decay (NMD inhibition and microarray analysis (NMD microarrays in the identification of transcripts containing nonsense mutations. We combined NMD microarrays with array-based CGH (comparative genomic hybridization in order to identify inactivation of tumor suppressor genes in cancer. Such a “mutatomics” screening of prostate cancer cell lines led to the identification of inactivating mutations in the EPHB2 gene. Up to 8% of metastatic uncultured prostate cancers also showed mutations of this gene whose loss of function may confer loss of tissue architecture. NMD microarray analysis could turn out to be a powerful research method to identify novel mutated genes in cancer cell lines, providing targets that could then be further investigated for their clinical relevance and therapeutic potential.

  5. Array-based comparative genomic hybridization for genome-wide screening of DNA copy number in bladder tumors.

    NARCIS (Netherlands)

    Veltman, J.A.; Fridlyand, J.; Pejavar, S.; Olshen, A.B.; Korkola, J.E.; Vries, S. de; Carroll, P.; Kuo, W.L.; Pinkel, D.; Albertson, D.; Cordon-Cardo, C.; Jain, A.N.; Waldman, F.M.

    2003-01-01

    Genome-wide copy number profiles were characterized in 41 primary bladder tumors using array-based comparative genomic hybridization (array CGH). In addition to previously identified alterations in large chromosomal regions, alterations were identified in many small genomic regions, some with high-l

  6. Genome-wide RNAi screen identifies broadly-acting host factors that inhibit arbovirus infection.

    Directory of Open Access Journals (Sweden)

    Ari Yasunaga

    2014-02-01

    Full Text Available Vector-borne viruses are an important class of emerging and re-emerging pathogens; thus, an improved understanding of the cellular factors that modulate infection in their respective vertebrate and insect hosts may aid control efforts. In particular, cell-intrinsic antiviral pathways restrict vector-borne viruses including the type I interferon response in vertebrates and the RNA interference (RNAi pathway in insects. However, it is likely that additional cell-intrinsic mechanisms exist to limit these viruses. Since insects rely on innate immune mechanisms to inhibit virus infections, we used Drosophila as a model insect to identify cellular factors that restrict West Nile virus (WNV, a flavivirus with a broad and expanding geographical host range. Our genome-wide RNAi screen identified 50 genes that inhibited WNV infection. Further screening revealed that 17 of these genes were antiviral against additional flaviviruses, and seven of these were antiviral against other vector-borne viruses, expanding our knowledge of invertebrate cell-intrinsic immunity. Investigation of two newly identified factors that restrict diverse viruses, dXPO1 and dRUVBL1, in the Tip60 complex, demonstrated they contributed to antiviral defense at the organismal level in adult flies, in mosquito cells, and in mammalian cells. These data suggest the existence of broadly acting and functionally conserved antiviral genes and pathways that restrict virus infections in evolutionarily divergent hosts.

  7. Nickel-resistance determinants in Acidiphilium sp. PM identified by genome-wide functional screening.

    Directory of Open Access Journals (Sweden)

    Patxi San Martin-Uriz

    Full Text Available Acidiphilium spp. are conspicuous dwellers of acidic, metal-rich environments. Indeed, they are among the most metal-resistant organisms; yet little is known about the mechanisms behind the metal tolerance in this genus. Acidiphilium sp. PM is an environmental isolate from Rio Tinto, an acidic, metal-laden river located in southwestern Spain. The characterization of its metal resistance revealed a remarkable ability to tolerate high Ni concentrations. Here we report the screening of a genomic library of Acidiphilium sp. PM to identify genes involved in Ni resistance. This approach revealed seven different genes conferring Ni resistance to E. coli, two of which form an operon encoding the ATP-dependent protease HslVU (ClpQY. This protease was found to enhance resistance to both Ni and Co in E. coli, a function not previously reported. Other Ni-resistance determinants include genes involved in lipopolysaccharide biosynthesis and the synthesis of branched amino acids. The diversity of molecular functions of the genes recovered in the screening suggests that Ni resistance in Acidiphilium sp. PM probably relies on different molecular mechanisms.

  8. Genome-wide RNAi screen for nuclear actin reveals a network of cofilin regulators.

    Science.gov (United States)

    Dopie, Joseph; Rajakylä, Eeva K; Joensuu, Merja S; Huet, Guillaume; Ferrantelli, Evelina; Xie, Tiao; Jäälinoja, Harri; Jokitalo, Eija; Vartiainen, Maria K

    2015-07-01

    Nuclear actin plays an important role in many processes that regulate gene expression. Cytoplasmic actin dynamics are tightly controlled by numerous actin-binding proteins, but regulation of nuclear actin has remained unclear. Here, we performed a genome-wide RNA interference (RNAi) screen in Drosophila cells to identify proteins that influence either nuclear polymerization or import of actin. We validate 19 factors as specific hits, and show that Chinmo (known as Bach2 in mammals), SNF4Aγ (Prkag1 in mammals) and Rab18 play a role in nuclear localization of actin in both fly and mammalian cells. We identify several new regulators of cofilin activity, and characterize modulators of both cofilin kinases and phosphatase. For example, Chinmo/Bach2, which regulates nuclear actin levels also in vivo, maintains active cofilin by repressing the expression of the kinase Cdi (Tesk in mammals). Finally, we show that Nup98 and lamin are candidates for regulating nuclear actin polymerization. Our screen therefore reveals new aspects of actin regulation and links nuclear actin to many cellular processes.

  9. Genome-wide screening for genes associated with valproic acid sensitivity in fission yeast.

    Directory of Open Access Journals (Sweden)

    Lili Zhang

    Full Text Available We have been studying the action mechanisms of valproic acid (VPA in fission yeast Schizosaccharomyces pombe by developing a genetic screen for mutants that show hypersensitivity to VPA. In the present study, we performed a genome-wide screen of 3004 haploid deletion strains and confirmed 148 deletion strains to be VPA sensitive. Of the 148 strains, 93 strains also showed sensitivity to another aliphatic acids HDAC inhibitor, sodium butyrate (SB, and 55 strains showed sensitivity to VPA but not to SB. Interestingly, we found that both VPA and SB treatment induced a marked increase in the transcription activity of Atf1 in wild-type cells. However, in clr6-1, a mutant allele the clr6(+ gene encoding class I HDAC, neither VPA- nor SB induced the activation of Atf1 transcription activity. We also found that VPA, but not SB, caused an increase in cytoplasmic Ca(2+ level. We further found that the cytoplasmic Ca(2+ increase was caused by Ca(2+ influx from extracellular medium via Cch1-Yam8 channel complex. Altogether, our present study indicates that VPA and SB play similar but distinct roles in multiple physiological processes in fission yeast.

  10. Genomic analysis of thermophilic Bacillus coagulans strains: efficient producers for platform bio-chemicals.

    Science.gov (United States)

    Su, Fei; Xu, Ping

    2014-01-29

    Microbial strains with high substrate efficiency and excellent environmental tolerance are urgently needed for the production of platform bio-chemicals. Bacillus coagulans has these merits; however, little genetic information is available about this species. Here, we determined the genome sequences of five B. coagulans strains, and used a comparative genomic approach to reconstruct the central carbon metabolism of this species to explain their fermentation features. A novel xylose isomerase in the xylose utilization pathway was identified in these strains. Based on a genome-wide positive selection scan, the selection pressure on amino acid metabolism may have played a significant role in the thermal adaptation. We also researched the immune systems of B. coagulans strains, which provide them with acquired resistance to phages and mobile genetic elements. Our genomic analysis provides comprehensive insights into the genetic characteristics of B. coagulans and paves the way for improving and extending the uses of this species.

  11. Genome-wide CRISPR screens reveal a Wnt-FZD5 signaling circuit as a druggable vulnerability of RNF43-mutant pancreatic tumors

    NARCIS (Netherlands)

    Steinhart, Zachary; Pavlovic, Zvezdan; Chandrashekhar, Megha; Hart, Traver; Wang, Xiaowei; Zhang, Xiaoyu; Robitaille, Mélanie; Brown, Kevin R; Jaksani, Sridevi; Overmeer, René; Boj, Sylvia F; Adams, Jarrett; Pan, James; Clevers, Hans; Sidhu, Sachdev; Moffat, Jason; Angers, Stéphane

    2016-01-01

    Forward genetic screens with CRISPR-Cas9 genome editing enable high-resolution detection of genetic vulnerabilities in cancer cells. We conducted genome-wide CRISPR-Cas9 screens in RNF43-mutant pancreatic ductal adenocarcinoma (PDAC) cells, which rely on Wnt signaling for proliferation. Through thes

  12. High throughput screening in duchenne muscular dystrophy: from drug discovery to functional genomics.

    Science.gov (United States)

    Gintjee, Thomas J J; Magh, Alvin S H; Bertoni, Carmen

    2014-11-14

    Centers for the screening of biologically active compounds and genomic libraries are becoming common in the academic setting and have enabled researchers devoted to developing strategies for the treatment of diseases or interested in studying a biological phenomenon to have unprecedented access to libraries that, until few years ago, were accessible only by pharmaceutical companies. As a result, new drugs and genetic targets have now been identified for the treatment of Duchenne muscular dystrophy (DMD), the most prominent of the neuromuscular disorders affecting children. Although the work is still at an early stage, the results obtained to date are encouraging and demonstrate the importance that these centers may have in advancing therapeutic strategies for DMD as well as other diseases. This review will provide a summary of the status and progress made toward the development of a cure for this disorder and implementing high-throughput screening (HTS) technologies as the main source of discovery. As more academic institutions are gaining access to HTS as a valuable discovery tool, the identification of new biologically active molecules is likely to grow larger. In addition, the presence in the academic setting of experts in different aspects of the disease will offer the opportunity to develop novel assays capable of identifying new targets to be pursued as potential therapeutic options. These assays will represent an excellent source to be used by pharmaceutical companies for the screening of larger libraries providing the opportunity to establish strong collaborations between the private and academic sectors and maximizing the chances of bringing into the clinic new drugs for the treatment of DMD.

  13. High Throughput Screening in Duchenne Muscular Dystrophy: From Drug Discovery to Functional Genomics

    Directory of Open Access Journals (Sweden)

    Thomas J.J. Gintjee

    2014-11-01

    Full Text Available Centers for the screening of biologically active compounds and genomic libraries are becoming common in the academic setting and have enabled researchers devoted to developing strategies for the treatment of diseases or interested in studying a biological phenomenon to have unprecedented access to libraries that, until few years ago, were accessible only by pharmaceutical companies. As a result, new drugs and genetic targets have now been identified for the treatment of Duchenne muscular dystrophy (DMD, the most prominent of the neuromuscular disorders affecting children. Although the work is still at an early stage, the results obtained to date are encouraging and demonstrate the importance that these centers may have in advancing therapeutic strategies for DMD as well as other diseases. This review will provide a summary of the status and progress made toward the development of a cure for this disorder and implementing high-throughput screening (HTS technologies as the main source of discovery. As more academic institutions are gaining access to HTS as a valuable discovery tool, the identification of new biologically active molecules is likely to grow larger. In addition, the presence in the academic setting of experts in different aspects of the disease will offer the opportunity to develop novel assays capable of identifying new targets to be pursued as potential therapeutic options. These assays will represent an excellent source to be used by pharmaceutical companies for the screening of larger libraries providing the opportunity to establish strong collaborations between the private and academic sectors and maximizing the chances of bringing into the clinic new drugs for the treatment of DMD.

  14. An siRNA-based functional genomics screen for the identification of regulators of ciliogenesis and ciliopathy genes

    NARCIS (Netherlands)

    Wheway, G.; Schmidts, M.; Mans, D.A.; Szymanska, K.; Nguyen, T.M.; Racher, H.; Phelps, I.G.; Toedt, G.; Kennedy, J.; Wunderlich, K.A.; Sorusch, N.; Abdelhamed, Z.A.; Natarajan, S.; Herridge, W.; Reeuwijk, J. van; Horn, N.; Boldt, K.; Parry, D.A.; Letteboer, S.J.F.; Roosing, S.; Adams, M.; Bell, S.M.; Bond, J.; Higgins, J.; Morrison, E.E.; Tomlinson, D.C.; Slaats, G.G.; Dam, T.J.P. van; Huang, L.; Kessler, K.; Giessl, A.; Logan, C.V.; Boyle, E.A.; Shendure, J.; Anazi, S.; Aldahmesh, M.; Hazzaa, S. Al; Hegele, R.A.; Ober, C.; Frosk, P.; Mhanni, A.A.; Chodirker, B.N.; Chudley, A.E.; Lamont, R.; Bernier, F.P.; Beaulieu, C.L.; Gordon, P.; Pon, R.T.; Donahue, C.; Barkovich, A.J.; Wolf, L.; Toomes, C.; Thiel, C.T.; Boycott, K.M.; McKibbin, M.; Inglehearn, C.F.; Stewart, F.; Omran, H.; Huynen, M.A.; Sergouniotis, P.I.; Alkuraya, F.S.; Parboosingh, J.S.; Innes, A.M.; Willoughby, C.E.; Giles, R.H.; Webster, A.R.; Ueffing, M.; Blacque, O.; Gleeson, J.G.; Wolfrum, U.; Beales, P.L.; Gibson, T.; Doherty, D.; Mitchison, H.M.; Roepman, R.; Johnson, C.A.

    2015-01-01

    Defects in primary cilium biogenesis underlie the ciliopathies, a growing group of genetic disorders. We describe a whole-genome siRNA-based reverse genetics screen for defects in biogenesis and/or maintenance of the primary cilium, obtaining a global resource. We identify 112 candidate ciliogenesis

  15. Exploiting Chemical Libraries, Structure, and Genomics in the Search for Kinase Inhibitors

    NARCIS (Netherlands)

    Gray, Nathanael S.; Wodicka, Lisa; Thunnissen, Andy-Mark W.H.; Norman, Thea C.; Kwon, Soojin; Espinoza, F. Hernan; Morgan, David O.; Barnes, Georjana; LeClerc, Sophie; Meijer, Laurent; Kim, Sung-Hou; Lockhart, David J.; Schultz, Peter G.

    1998-01-01

    Selective protein kinase inhibitors were developed on the basis of the unexpected binding mode of 2,6,9-trisubstituted purines to the adenosine triphosphate-binding site of the human cyclin-dependent kinase 2 (CDK2). By iterating chemical library synthesis and biological screening, potent inhibitors

  16. Patients' ratings of genetic conditions validate a taxonomy to simplify decisions about preconception carrier screening via genome sequencing.

    Science.gov (United States)

    Leo, Michael C; McMullen, Carmit; Wilfond, Benjamin S; Lynch, Frances L; Reiss, Jacob A; Gilmore, Marian J; Himes, Patricia; Kauffman, Tia L; Davis, James V; Jarvik, Gail P; Berg, Jonathan S; Harding, Cary; Kennedy, Kathleen A; Simpson, Dana Kostiner; Quigley, Denise I; Richards, C Sue; Rope, Alan F; Goddard, Katrina A B

    2016-03-01

    Advances in genome sequencing and gene discovery have created opportunities to efficiently assess more genetic conditions than ever before. Given the large number of conditions that can be screened, the implementation of expanded carrier screening using genome sequencing will require practical methods of simplifying decisions about the conditions for which patients want to be screened. One method to simplify decision making is to generate a taxonomy based on expert judgment. However, expert perceptions of condition attributes used to classify these conditions may differ from those used by patients. To understand whether expert and patient perceptions differ, we asked women who had received preconception genetic carrier screening in the last 3 years to fill out a survey to rate the attributes (predictability, controllability, visibility, and severity) of several autosomal recessive or X-linked genetic conditions. These conditions were classified into one of five taxonomy categories developed by subject experts (significantly shortened lifespan, serious medical problems, mild medical problems, unpredictable medical outcomes, and adult-onset conditions). A total of 193 women provided 739 usable ratings across 20 conditions. The mean ratings and correlations demonstrated that participants made distinctions across both attributes and categories. Aggregated mean attribute ratings across categories demonstrated logical consistency between the key features of each attribute and category, although participants perceived little difference between the mild and serious categories. This study provides empirical evidence for the validity of our proposed taxonomy, which will simplify patient decisions for results they would like to receive from preconception carrier screening via genome sequencing.

  17. Small molecule screening in zebrafish: an in vivo approach to identifying new chemical tools and drug leads

    Directory of Open Access Journals (Sweden)

    Patton E Elizabeth

    2010-06-01

    Full Text Available Abstract In the past two decades, zebrafish genetic screens have identified a wealth of mutations that have been essential to the understanding of development and disease biology. More recently, chemical screens in zebrafish have identified small molecules that can modulate specific developmental and behavioural processes. Zebrafish are a unique vertebrate system in which to study chemical genetic systems, identify drug leads, and explore new applications for known drugs. Here, we discuss some of the advantages of using zebrafish in chemical biology, and describe some important and creative examples of small molecule screening, drug discovery and target identification.

  18. Screening of herbal extracts influencing hematopoiesis and their chemical genetic effects in embryonic zebrafish

    Institute of Scientific and Technical Information of China (English)

    Rajaretinam Rajesh Kannan; Samuel Gnana Prakash Vincent

    2012-01-01

    Objective: To screen the herbal extracts influencing the hematopoietic stem cells (HSC) in zebrafish embryos and their chemical genetic effects. Methods: The herbals used in this study had been widely applicable in Siddha medicines in South India. Herbal extracts were treated in zebrafish embryos at 4 d post fertilization and the extracts inducing the HSC were enumerated in hemocytometer. The biocompatibility and the organogenesis of the screened extracts were assessed in the zebrafish embryos for their chemical genetic effects. The LC50 values were calculated with their parallel control. The blood cells were enumerated. Results: The level of RBC was found increased in the Bergera koenigii (B. koenigii) at 15 μg/mL (P<0.05), Mimosa pudica (M. pudica) at 20 μg/mL (P<0.05) and Solanum trilobatum (S. trilobatum) at 25 μg/mL (P<0.05) and decreased RBC level was found in Phyllanthus niruri (P. niruri) at 30 μg/mL (P<0.05). The WBC count was found increased in S. trilobatum at 20 μg/mL (P<0.05) and Annona muricata (Annona muricata) at 15 μg/mL (P<0.05) and the Vitis quadrangularis (V. quadrangularis) at 20 μg/mL (P<0.05) decreased the WBC level. There were no notable effects in heart beats and the chemical genetic effects were observed at higher concentration of the extract resulting in Pericardial bulging, trunk tail flexure with heart edema, fin fold deformities etc. Conclusions: This in vivo based screening of Hematopoiesis is an inexpensive assay to screen herbal compounds and found that S. trilobatum extract influenced embryonic HSC in zebrafish, which could be a therapeutic for blood related disorders.

  19. Screening values for Non-Carcinogenic Hanford Waste Tank Vapor Chemicals that Lack Established Occupational Exposure Limits

    Energy Technology Data Exchange (ETDEWEB)

    Poet, Torka S.; Mast, Terryl J.; Huckaby, James L.

    2006-02-06

    Over 1,500 different volatile chemicals have been reported in the headspaces of tanks used to store high-level radioactive waste at the U.S. Department of Energy's Hanford Site. Concern about potential exposure of tank farm workers to these chemicals has prompted efforts to evaluate their toxicity, identify chemicals that pose the greatest risk, and incorporate that information into the tank farms industrial hygiene worker protection program. Established occupation exposure limits for individual chemicals and petroleum hydrocarbon mixtures have been used elsewhere to evaluate about 900 of the chemicals. In this report headspace concentration screening values were established for the remaining 600 chemicals using available industrial hygiene and toxicological data. Screening values were intended to be more than an order of magnitude below concentrations that may cause adverse health effects in workers, assuming a 40-hour/week occupational exposure. Screening values were compared to the maximum reported headspace concentrations.

  20. Screening chemicals for the potential to be persistent organic pollutants: a case study of Arctic contaminants.

    Science.gov (United States)

    Brown, Trevor N; Wania, Frank

    2008-07-15

    A large and ever-increasing number of chemicals are used in commerce, and researchers and regulators have struggled to ascertain that these chemicals do not threaten human health or cause environmental or ecological damage. The presence of persistent organic pollutants (POPs) in remote environments such as the Arctic is of special concern and has international regulatory implications. Responding to the need for a way to identify chemicals of high concern, a methodology has been developed which compares experimentally measured properties, or values predicted from chemical structure alone, to a set of screening criteria. These criteria include partitioning properties that allow for accumulation in the physical Arctic environment and in the Arctic human food chain, and resistance to atmospheric oxidation. Atthe same time we quantify the extent of structural resemblance to a group of known Arctic contaminants. Comparison of the substances that are identified by a mechanistic description of the processes that lead to Arctic contamination with those substances that are structurally similar to known Arctic contaminants reveals the strengths and limitations of either approach. Within a data set of more than 100,000 distinct industrial chemicals, the methodology identifies 120 high production volume chemicals which are structurally similarto known Arctic contaminants and/or have partitioning properties that suggest they are potential Arctic contaminants.

  1. In silico screening of estrogen-like chemicals based on different nonlinear classification models.

    Science.gov (United States)

    Liu, Huanxiang; Papa, Ester; Walker, John D; Gramatica, Paola

    2007-07-01

    Increasing concern is being shown by the scientific community, government regulators, and the public about endocrine-disrupting chemicals that are adversely affecting human and wildlife health through a variety of mechanisms. There is a great need for an effective means of rapidly assessing endocrine-disrupting activity, especially estrogen-simulating activity, because of the large number of such chemicals in the environment. In this study, quantitative structure activity relationship (QSAR) models were developed to quickly and effectively identify possible estrogen-like chemicals based on 232 structurally-diverse chemicals (training set) by using several nonlinear classification methodologies (least-square support vector machine (LS-SVM), counter-propagation artificial neural network (CP-ANN), and k nearest neighbour (kNN)) based on molecular structural descriptors. The models were externally validated by 87 chemicals (prediction set) not included in the training set. All three methods can give satisfactory prediction results both for training and prediction sets, and the most accurate model was obtained by the LS-SVM approach through the comparison of performance. In addition, our model was also applied to about 58,000 discrete organic chemicals; about 76% were predicted not to bind to Estrogen Receptor. The obtained results indicate that the proposed QSAR models are robust, widely applicable and could provide a feasible and practical tool for the rapid screening of potential estrogens.

  2. From DNA Sequences to Chemical Structures – Methods for Mining Microbial Genomic and Metagenomic Data Sets for New Natural Products

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    Jurica Zucko

    2010-01-01

    Full Text Available Rapid mining of large genomic and metagenomic data sets for modular polyketide synthases, non-ribosomal peptide synthetases and hybrid polyketide synthase/non-ribosomal peptide synthetase biosynthetic gene clusters has been achieved using the generic computer program packages ClustScan and CompGen. These program packages perform the annotation with the hierarchical structuring into polypeptides, modules and domains, as well as storage and graphical presentations of the data. This aims to achieve the most accurate predictions of the activities and specificities of catalytically active domains that can be made with present knowledge, leading to a prediction of the most likely chemical structures produced by these enzymes. The program packages also allow generation of novel clusters by homologous recombination of the annotated genes in silico. ClustScan and CompGen were used to construct a custom database of known compounds (CSDB and of predicted entirely novel recombinant products (r-CSDB that can be used for in silico screening with computer aided drug design technology. The use of these programs has been exemplified by analysing genomic sequences from terrestrial prokaryotes and eukaryotic microorganisms, a marine metagenomic data set and a newly discovered example of a 'shared metabolic pathway' in marine-microbial endosymbiosis.

  3. Towards personalized agriculture: What chemical genomics can bring to plant biotechnology

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    Michael E Stokes

    2014-07-01

    Full Text Available In contrast to the dominant drug paradigm in which compounds were developed to fit all, new models focused around personalized medicine are appearing where treatments are customized for individual patients. The agricultural biotechnology industry should also think about these new personalized models. For example, most common herbicides are generic in action, which led to the development of genetically modified crops to add specificity. The ease and accessibility of modern genomic analysis should facilitate the discovery of chemicals that are more selective in their utility. Is it possible to develop species-selective herbicides and growth regulators? More generally put, is plant research at a stage where chemicals can be developed that streamline plant development and growth to various environments? We believe the advent of chemical genomics now opens up these and other opportunities to personalize agriculture. Furthermore, chemical genomics does not necessarily require genetically tractable plant models, which in principle should allow quick translation to practical applications. For this to happen, however, will require collaboration between the Ag-biotech industry and academic labs for early-stage research and development.

  4. Fluorescence-based assay as a new screening tool for toxic chemicals

    Science.gov (United States)

    Moczko, Ewa; Mirkes, Evgeny M.; Cáceres, César; Gorban, Alexander N.; Piletsky, Sergey

    2016-09-01

    Our study involves development of fluorescent cell-based diagnostic assay as a new approach in high-throughput screening method. This highly sensitive optical assay operates similarly to e-noses and e-tongues which combine semi-specific sensors and multivariate data analysis for monitoring biochemical processes. The optical assay consists of a mixture of environmental-sensitive fluorescent dyes and human skin cells that generate fluorescence spectra patterns distinctive for particular physico-chemical and physiological conditions. Using chemometric techniques the optical signal is processed providing qualitative information about analytical characteristics of the samples. This integrated approach has been successfully applied (with sensitivity of 93% and specificity of 97%) in assessing whether particular chemical agents are irritating or not for human skin. It has several advantages compared with traditional biochemical or biological assays and can impact the new way of high-throughput screening and understanding cell activity. It also can provide reliable and reproducible method for assessing a risk of exposing people to different harmful substances, identification active compounds in toxicity screening and safety assessment of drugs, cosmetic or their specific ingredients.

  5. A Genome-wide screen identifies frequently methylated genes in haematological and epithelial cancers

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    Maher Eamonn R

    2010-02-01

    Full Text Available Abstract Background Genetic as well as epigenetic alterations are a hallmark of both epithelial and haematological malignancies. High throughput screens are required to identify epigenetic markers that can be useful for diagnostic and prognostic purposes across malignancies. Results Here we report for the first time the use of the MIRA assay (methylated CpG island recovery assay in combination with genome-wide CpG island arrays to identify epigenetic molecular markers in childhood acute lymphoblastic leukemia (ALL on a genome-wide scale. We identified 30 genes demonstrating methylation frequencies of ≥25% in childhood ALL, nine genes showed significantly different methylation frequencies in B vs T-ALL. For majority of the genes expression could be restored in methylated leukemia lines after treatment with 5-azaDC. Forty-four percent of the genes represent targets of the polycomb complex. In chronic myeloid leukemia (CML two of the genes, (TFAP2A and EBF2, demonstrated increased methylation in blast crisis compared to chronic phase (P ATG16L2 was associated with poorer prognosis in terms of molecular response to Imatinib treatment. Lastly we demonstrated that ten of these genes were also frequently methylated in common epithelial cancers. Conclusion In summary we have identified a large number of genes showing frequent methylation in childhood ALL, methylation status of two of these genes is associated with advanced disease in CML and methylation status of another gene is associated with prognosis. In addition a subset of these genes may act as epigenetic markers across hematological malignancies as well as common epithelial cancers.

  6. Chemical library screening for WNK signalling inhibitors using fluorescence correlation spectroscopy.

    Science.gov (United States)

    Mori, Takayasu; Kikuchi, Eriko; Watanabe, Yuko; Fujii, Shinya; Ishigami-Yuasa, Mari; Kagechika, Hiroyuki; Sohara, Eisei; Rai, Tatemitsu; Sasaki, Sei; Uchida, Shinichi

    2013-11-01

    WNKs (with-no-lysine kinases) are the causative genes of a hereditary hypertensive disease, PHAII (pseudohypoaldosteronism type II), and form a signal cascade with OSR1 (oxidative stress-responsive 1)/SPAK (STE20/SPS1-related proline/alanine-rich protein kinase) and Slc12a (solute carrier family 12) transporters. We have shown that this signal cascade regulates blood pressure by controlling vascular tone as well as renal NaCl excretion. Therefore agents that inhibit this signal cascade could be a new class of antihypertensive drugs. Since the binding of WNK to OSR1/SPAK kinases was postulated to be important for signal transduction, we sought to discover inhibitors of WNK/SPAK binding by screening chemical compounds that disrupt the binding. For this purpose, we developed a high-throughput screening method using fluorescent correlation spectroscopy. As a result of screening 17000 compounds, we discovered two novel compounds that reproducibly disrupted the binding of WNK to SPAK. Both compounds mediated dose-dependent inhibition of hypotonicity-induced activation of WNK, namely the phosphorylation of SPAK and its downstream transporters NKCC1 (Na/K/Cl cotransporter 1) and NCC (NaCl cotransporter) in cultured cell lines. The two compounds could be the promising seeds of new types of antihypertensive drugs, and the method that we developed could be applied as a general screening method to identify compounds that disrupt the binding of two molecules.

  7. A genome-scale CRISPR-Cas9 screening method for protein stability reveals novel regulators of Cdc25A.

    Science.gov (United States)

    Wu, Yuanzhong; Zhou, Liwen; Wang, Xin; Lu, Jinping; Zhang, Ruhua; Liang, Xiaoting; Wang, Li; Deng, Wuguo; Zeng, Yi-Xin; Huang, Haojie; Kang, Tiebang

    2016-01-01

    The regulation of stability is particularly crucial for unstable proteins in cells. However, a convenient and unbiased method of identifying regulators of protein stability remains to be developed. Recently, a genome-scale CRISPR-Cas9 library has been established as a genetic tool to mediate loss-of-function screening. Here, we developed a protein stability regulators screening assay (Pro-SRSA) by combining the whole-genome CRISPR-Cas9 library with a dual-fluorescence-based protein stability reporter and high-throughput sequencing to screen for regulators of protein stability. Using Cdc25A as an example, Cul4B-DDB1(DCAF8) was identified as a new E3 ligase for Cdc25A. Moreover, the acetylation of Cdc25A at lysine 150, which was acetylated by p300/CBP and deacetylated by HDAC3, prevented the ubiquitin-mediated degradation of Cdc25A by the proteasome. This is the first study to report that acetylation, as a novel posttranslational modification, modulates Cdc25A stability, and we suggest that this unbiased CRISPR-Cas9 screening method at the genome scale may be widely used to globally identify regulators of protein stability.

  8. Evaluating the Impact of Uncertainties in Clearance and Exposure When Prioritizing Chemicals Screened in High-Throughput Assays

    Science.gov (United States)

    The toxicity-testing paradigm has evolved to include high-throughput (HT) methods for addressing the increasing need to screen hundreds to thousands of chemicals rapidly. Approaches that involve in vitro screening assays, in silico predictions of exposure concentrations, and phar...

  9. Chemical screen identifies FDA-approved drugs and target pathways that induce precocious pancreatic endocrine differentiation.

    Science.gov (United States)

    Rovira, Meritxell; Huang, Wei; Yusuff, Shamila; Shim, Joong Sup; Ferrante, Anthony A; Liu, Jun O; Parsons, Michael J

    2011-11-29

    Pancreatic β-cells are an essential source of insulin and their destruction because of autoimmunity causes type I diabetes. We conducted a chemical screen to identify compounds that would induce the differentiation of insulin-producing β-cells in vivo. To do this screen, we brought together the use of transgenic zebrafish as a model of β-cell differentiation, a unique multiwell plate that allows easy visualization of lateral views of swimming larval fish and a library of clinical drugs. We identified six hits that can induce precocious differentiation of secondary islets in larval zebrafish. Three of these six hits were known drugs with a considerable background of published data on mechanism of action. Using pharmacological approaches, we have identified and characterized two unique pathways in β-cell differentiation in the zebrafish, including down-regulation of GTP production and retinoic acid biosynthesis.

  10. A Multiplexed Single-Cell CRISPR Screening Platform Enables Systematic Dissection of the Unfolded Protein Response. | Office of Cancer Genomics

    Science.gov (United States)

    Functional genomics efforts face tradeoffs between number of perturbations examined and complexity of phenotypes measured. We bridge this gap with Perturb-seq, which combines droplet-based single-cell RNA-seq with a strategy for barcoding CRISPR-mediated perturbations, allowing many perturbations to be profiled in pooled format. We applied Perturb-seq to dissect the mammalian unfolded protein response (UPR) using single and combinatorial CRISPR perturbations. Two genome-scale CRISPR interference (CRISPRi) screens identified genes whose repression perturbs ER homeostasis.

  11. A genome-wide RNAi screen identifies regulators of cholesterol-modified hedgehog secretion in Drosophila.

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    Reid Aikin

    Full Text Available Hedgehog (Hh proteins are secreted molecules that function as organizers in animal development. In addition to being palmitoylated, Hh is the only metazoan protein known to possess a covalently-linked cholesterol moiety. The absence of either modification severely disrupts the organization of numerous tissues during development. It is currently not known how lipid-modified Hh is secreted and released from producing cells. We have performed a genome-wide RNAi screen in Drosophila melanogaster cells to identify regulators of Hh secretion. We found that cholesterol-modified Hh secretion is strongly dependent on coat protein complex I (COPI but not COPII vesicles, suggesting that cholesterol modification alters the movement of Hh through the early secretory pathway. We provide evidence that both proteolysis and cholesterol modification are necessary for the efficient trafficking of Hh through the ER and Golgi. Finally, we identified several putative regulators of protein secretion and demonstrate a role for some of these genes in Hh and Wingless (Wg morphogen secretion in vivo. These data open new perspectives for studying how morphogen secretion is regulated, as well as provide insight into regulation of lipid-modified protein secretion.

  12. Genome-wide RNAi screen identifies novel host proteins required for alphavirus entry.

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    Yaw Shin Ooi

    Full Text Available The enveloped alphaviruses include important and emerging human pathogens such as Chikungunya virus and Eastern equine encephalitis virus. Alphaviruses enter cells by clathrin-mediated endocytosis, and exit by budding from the plasma membrane. While there has been considerable progress in defining the structure and function of the viral proteins, relatively little is known about the host factors involved in alphavirus infection. We used a genome-wide siRNA screen to identify host factors that promote or inhibit alphavirus infection in human cells. Fuzzy homologue (FUZ, a protein with reported roles in planar cell polarity and cilia biogenesis, was required for the clathrin-dependent internalization of both alphaviruses and the classical endocytic ligand transferrin. The tetraspanin membrane protein TSPAN9 was critical for the efficient fusion of low pH-triggered virus with the endosome membrane. FUZ and TSPAN9 were broadly required for infection by the alphaviruses Sindbis virus, Semliki Forest virus, and Chikungunya virus, but were not required by the structurally-related flavivirus Dengue virus. Our results highlight the unanticipated functions of FUZ and TSPAN9 in distinct steps of alphavirus entry and suggest novel host proteins that may serve as targets for antiviral therapy.

  13. Drosophila genome-wide RNAi screen identifies multiple regulators of HIF-dependent transcription in hypoxia.

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    Andrés Dekanty

    2010-06-01

    Full Text Available Hypoxia-inducible factors (HIFs are a family of evolutionary conserved alpha-beta heterodimeric transcription factors that induce a wide range of genes in response to low oxygen tension. Molecular mechanisms that mediate oxygen-dependent HIF regulation operate at the level of the alpha subunit, controlling protein stability, subcellular localization, and transcriptional coactivator recruitment. We have conducted an unbiased genome-wide RNA interference (RNAi screen in Drosophila cells aimed to the identification of genes required for HIF activity. After 3 rounds of selection, 30 genes emerged as critical HIF regulators in hypoxia, most of which had not been previously associated with HIF biology. The list of genes includes components of chromatin remodeling complexes, transcription elongation factors, and translational regulators. One remarkable hit was the argonaute 1 (ago1 gene, a central element of the microRNA (miRNA translational silencing machinery. Further studies confirmed the physiological role of the miRNA machinery in HIF-dependent transcription. This study reveals the occurrence of novel mechanisms of HIF regulation, which might contribute to developing novel strategies for therapeutic intervention of HIF-related pathologies, including heart attack, cancer, and stroke.

  14. Drosophila genome-wide RNAi screen identifies multiple regulators of HIF-dependent transcription in hypoxia.

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    Andrés Dekanty

    2010-06-01

    Full Text Available Hypoxia-inducible factors (HIFs are a family of evolutionary conserved alpha-beta heterodimeric transcription factors that induce a wide range of genes in response to low oxygen tension. Molecular mechanisms that mediate oxygen-dependent HIF regulation operate at the level of the alpha subunit, controlling protein stability, subcellular localization, and transcriptional coactivator recruitment. We have conducted an unbiased genome-wide RNA interference (RNAi screen in Drosophila cells aimed to the identification of genes required for HIF activity. After 3 rounds of selection, 30 genes emerged as critical HIF regulators in hypoxia, most of which had not been previously associated with HIF biology. The list of genes includes components of chromatin remodeling complexes, transcription elongation factors, and translational regulators. One remarkable hit was the argonaute 1 (ago1 gene, a central element of the microRNA (miRNA translational silencing machinery. Further studies confirmed the physiological role of the miRNA machinery in HIF-dependent transcription. This study reveals the occurrence of novel mechanisms of HIF regulation, which might contribute to developing novel strategies for therapeutic intervention of HIF-related pathologies, including heart attack, cancer, and stroke.

  15. A genome-wide screen indicates correlation between differentiation and expression of metabolism related genes.

    Science.gov (United States)

    Roy, Priti; Kumar, Brijesh; Shende, Akhilesh; Singh, Anupama; Meena, Anil; Ghosal, Ritika; Ranganathan, Madhav; Bandyopadhyay, Amitabha

    2013-01-01

    Differentiated tissues may be considered as materials with distinct properties. The differentiation program of a given tissue ensures that it acquires material properties commensurate with its function. It may be hypothesized that some of these properties are acquired through production of tissue-specific metabolites synthesized by metabolic enzymes. To establish correlation between metabolism and organogenesis we have carried out a genome-wide expression study of metabolism related genes by RNA in-situ hybridization. 23% of the metabolism related genes studied are expressed in a tissue-restricted but not tissue-exclusive manner. We have conducted the screen on whole mount chicken (Gallus gallus) embryos from four distinct developmental stages to correlate dynamic changes in expression patterns of metabolic enzymes with spatio-temporally unique developmental events. Our data strongly suggests that unique combinations of metabolism related genes, and not specific metabolic pathways, are upregulated during differentiation. Further, expression of metabolism related genes in well established signaling centers that regulate different aspects of morphogenesis indicates developmental roles of some of the metabolism related genes. The database of tissue-restricted expression patterns of metabolism related genes, generated in this study, should serve as a resource for systematic identification of these genes with tissue-specific functions during development. Finally, comprehensive understanding of differentiation is not possible unless the downstream genes of a differentiation cascade are identified. We propose, metabolic enzymes constitute a significant portion of these downstream target genes. Thus our study should help elucidate different aspects of tissue differentiation.

  16. A genome-wide screen indicates correlation between differentiation and expression of metabolism related genes.

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    Priti Roy

    Full Text Available Differentiated tissues may be considered as materials with distinct properties. The differentiation program of a given tissue ensures that it acquires material properties commensurate with its function. It may be hypothesized that some of these properties are acquired through production of tissue-specific metabolites synthesized by metabolic enzymes. To establish correlation between metabolism and organogenesis we have carried out a genome-wide expression study of metabolism related genes by RNA in-situ hybridization. 23% of the metabolism related genes studied are expressed in a tissue-restricted but not tissue-exclusive manner. We have conducted the screen on whole mount chicken (Gallus gallus embryos from four distinct developmental stages to correlate dynamic changes in expression patterns of metabolic enzymes with spatio-temporally unique developmental events. Our data strongly suggests that unique combinations of metabolism related genes, and not specific metabolic pathways, are upregulated during differentiation. Further, expression of metabolism related genes in well established signaling centers that regulate different aspects of morphogenesis indicates developmental roles of some of the metabolism related genes. The database of tissue-restricted expression patterns of metabolism related genes, generated in this study, should serve as a resource for systematic identification of these genes with tissue-specific functions during development. Finally, comprehensive understanding of differentiation is not possible unless the downstream genes of a differentiation cascade are identified. We propose, metabolic enzymes constitute a significant portion of these downstream target genes. Thus our study should help elucidate different aspects of tissue differentiation.

  17. New Tools for Embryo Selection: Comprehensive Chromosome Screening by Array Comparative Genomic Hybridization

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    Lorena Rodrigo

    2014-01-01

    Full Text Available The objective of this study was to evaluate the usefulness of comprehensive chromosome screening (CCS using array comparative genomic hybridization (aCGH. The study included 1420 CCS cycles for recurrent miscarriage (n=203; repetitive implantation failure (n=188; severe male factor (n=116; previous trisomic pregnancy (n=33; and advanced maternal age (n=880. CCS was performed in cycles with fresh oocytes and embryos (n=774; mixed cycles with fresh and vitrified oocytes (n=320; mixed cycles with fresh and vitrified day-2 embryos (n=235; and mixed cycles with fresh and vitrified day-3 embryos (n=91. Day-3 embryo biopsy was performed and analyzed by aCGH followed by day-5 embryo transfer. Consistent implantation (range: 40.5–54.2% and pregnancy rates per transfer (range: 46.0–62.9% were obtained for all the indications and independently of the origin of the oocytes or embryos. However, a lower delivery rate per cycle was achieved in women aged over 40 years (18.1% due to the higher percentage of aneuploid embryos (85.3% and lower number of cycles with at least one euploid embryo available per transfer (40.3%. We concluded that aneuploidy is one of the major factors which affect embryo implantation.

  18. TDP-43 identified from a genome wide RNAi screen for SOD1 regulators.

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    Balajee R Somalinga

    Full Text Available Amyotrophic Lateral Sclerosis (ALS is a late-onset, progressive neurodegenerative disease affecting motor neurons in the brain stem and spinal cord leading to loss of voluntary muscular function and ultimately, death due to respiratory failure. A subset of ALS cases are familial and associated with mutations in superoxide dismutase 1 (SOD1 that destabilize the protein and predispose it to aggregation. In spite of the fact that sporadic and familial forms of ALS share many common patho-physiological features, the mechanistic relationship between SOD1-associated and sporadic forms of the disease if any, is not well understood. To better understand any molecular connections, a cell-based protein folding assay was employed to screen a whole genome RNAi library for genes that regulate levels of soluble SOD1. Statistically significant hits that modulate SOD1 levels, when analyzed by pathway analysis revealed a highly ranked network containing TAR DNA binging protein (TDP-43, a major component of aggregates characteristic of sporadic ALS. Biochemical experiments confirmed the action of TDP-43 on SOD1. These results highlight an unexpected relationship between TDP-43 and SOD1 which may have implications in disease pathogenesis.

  19. Genomic screening for Chlamydophila pneumoniae-specific antigens using serum samples from patients with primary infection.

    Science.gov (United States)

    Yasui, Yumiko; Yanatori, Izumi; Kawai, Yasuhiro; Miura, Koshiro; Suminami, Yoshinori; Hirota, Tomomitsu; Tamari, Mayumi; Ouchi, Kazunobu; Kishi, Fumio

    2012-04-01

    Chlamydophila pneumoniae, an obligate intracellular human pathogen, causes respiratory tract infections. The most common techniques used for the serological diagnosis of C. pneumoniae infections are microimmunofluorescence tests and commercial serological ELISA tests; these are based on the detection of antibodies against whole chlamydial elementary bodies and lipopolysaccharide/outer membrane protein, respectively. Identification of more specific and highly immunodominant antigens is essential for the development of new serodiagnostic assays. To identify novel specific antigens from C. pneumoniae, we screened 455 genes with unknown function in the genome of C. pneumoniae J138. Extracts of Saccharomyces cerevisiae cells expressing GFP-tagged C. pneumoniae proteins were subjected to Western blot analysis using serum samples from C. pneumoniae-infected patients as the primary antibodies. From this comprehensive analysis, 58 clones expressing C. pneumoniae open reading frames, including hypothetical proteins, were identified as antigens. These results have provided useful information for the development of new serological tools for the diagnosis for C. pneumoniae infections and for the development of vaccines in future.

  20. A systematic identification of multiple toxin-target interactions based on chemical, genomic and toxicological data.

    Science.gov (United States)

    Zhou, Wei; Huang, Chao; Li, Yan; Duan, Jinyou; Wang, Yonghua; Yang, Ling

    2013-02-01

    Although the assessment of toxicity of various agents, -omics (genomic, proteomic, metabolomic, etc.) data has been accumulated largely, the acquirement of toxicity information of variety of molecules through experimental methods still remains a difficult task. Presently, a systems toxicology approach that integrates massive diverse chemical, genomic and toxicological information was developed for prediction of the toxin targets and their related networks. The procedures are: (1) by use of two powerful statistical methods, i.e., support vector machine (SVM) and random forest (RF), a systemic model for prediction of multiple toxin-target interactions using the extracted chemical and genomic features has been developed with its reliability and robustness estimated. And the qualitative classification of targets according to the phenotypic diseases has been taken into account to further uncover the biological meaning of the targets, as well as to validate the robustness of the in silico models. (2) Based on the predicted toxin-target interactions, a genome-scale toxin-target-disease network exampled by cardiovascular disease is generated. (3) A topological analysis of the network is carried out to identify those targets that are most susceptible in human to topical agents including the most critical toxins, as well as to uncover both the toxin-specific mechanisms and pathways. The methodologies presented herein for systems toxicology will make drug development, toxin environmental risk assessment more efficient, acceptable and cost-effective.

  1. An effective method for controlling false discovery and false nondiscovery rates in genome-scale RNAi screens.

    Science.gov (United States)

    Zhang, Xiaohua Douglas

    2010-10-01

    In most genome-scale RNA interference (RNAi) screens, the ultimate goal is to select siRNAs with a large inhibition or activation effect. The selection of hits typically requires statistical control of 2 errors: false positives and false negatives. Traditional methods of controlling false positives and false negatives do not take into account the important feature in RNAi screens: many small-interfering RNAs (siRNAs) may have very small but real nonzero average effects on the measured response and thus cannot allow us to effectively control false positives and false negatives. To address for deficiencies in the application of traditional approaches in RNAi screening, the author proposes a new method for controlling false positives and false negatives in RNAi high-throughput screens. The false negatives are statistically controlled through a false-negative rate (FNR) or false nondiscovery rate (FNDR). FNR is the proportion of false negatives among all siRNAs examined, whereas FNDR is the proportion of false negatives among declared nonhits. The author also proposes new concepts, q*-value and p*-value, to control FNR and FNDR, respectively. The proposed method should have broad utility for hit selection in which one needs to control both false discovery and false nondiscovery rates in genome-scale RNAi screens in a robust manner.

  2. A spectroscopic screening of the chemical speciation of europium(III) in gastrointestinal tract. The intestine

    Energy Technology Data Exchange (ETDEWEB)

    Wilke, Claudia; Barkleit, Astrid [Helmholtz-Zentrum Dresden-Rossendorf e.V., Dresden (Germany). Div. Chemistry of the F-Elements

    2016-07-01

    To evaluate the health risks of lanthanides (Ln) and radiotoxic actinides (An), investigations into the chemical reactions of these metals in the human gastrointestinal tract are necessary. In order to identify the dominant binding partners (i.e. counter ions and/or ligands) of An/Ln in the gastrointestinal tract, a spectroscopic screening was performed by Time-Resolved Laser-induced Fluorescence Spectroscopy (TRLFS) using artificial digestive juices containing Eu(III), a representative of Ln(III) and An(III). In the intestine, Eu(III) show a strong complexation especially with organic substances of the pancreatic and bile juice like the protein mucin.

  3. Chemical Screening Method for the Rapid Identification of Microbial Sources of Marine Invertebrate-Associated Metabolites

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    Russell G. Kerr

    2011-03-01

    Full Text Available Marine invertebrates have proven to be a rich source of secondary metabolites. The growing recognition that marine microorganisms associated with invertebrate hosts are involved in the biosynthesis of secondary metabolites offers new alternatives for the discovery and development of marine natural products. However, the discovery of microorganisms producing secondary metabolites previously attributed to an invertebrate host poses a significant challenge. This study describes an efficient chemical screening method utilizing a 96-well plate-based bacterial cultivation strategy to identify and isolate microbial producers of marine invertebrate-associated metabolites.

  4. Discovery of antagonists of tick dopamine receptors via chemical library screening and comparative pharmacological analyses.

    Science.gov (United States)

    Ejendal, Karin F K; Meyer, Jason M; Brust, Tarsis F; Avramova, Larisa V; Hill, Catherine A; Watts, Val J

    2012-11-01

    Ticks transmit a wide variety of disease causing pathogens to humans and animals. Considering the global health impact of tick-borne diseases, there is a pressing need to develop new methods for vector control. We are exploring arthropod dopamine receptors as novel targets for insecticide/acaricide development because of their integral roles in neurobiology. Herein, we developed a screening assay for dopamine receptor antagonists to further characterize the pharmacological properties of the two D₁-like dopamine receptors (Isdop1 and Isdop2) identified in the Lyme disease vector, Ixodes scapularis, and develop a screening assay for receptor antagonists. A cell-based, cyclic AMP luciferase reporter assay platform was implemented to screen the LOPAC(1280) small molecule library for Isdop2 receptor antagonists, representing the first reported chemical library screen for any tick G protein-coupled receptor. Screening resulted in the identification of 85 "hit" compounds with antagonist activity at the Isdop2 receptor. Eight of these chemistries were selected for confirmation assays using a direct measurement of cAMP, and the effects on both Isdop1 and Isdop2 were studied for comparison. Each of these eight compounds showed antagonistic activity at both Isdop1 and Isdop2, although differences were observed regarding their relative potencies. Furthermore, comparison of the pharmacological properties of the tick dopamine receptors with that of the AaDOP2 receptor from the yellow fever mosquito and the human dopamine D₁ receptor (hD₁) revealed species-specific pharmacological profiles of these receptors. Compounds influencing dopaminergic functioning, such as the dopamine receptor antagonists discovered here, may provide lead chemistries for discovery of novel acaricides useful for vector control

  5. Chemical Biology Approaches to Genome Editing: Understanding, Controlling, and Delivering Programmable Nucleases.

    Science.gov (United States)

    Hu, Johnny H; Davis, Kevin M; Liu, David R

    2016-01-21

    Programmable DNA nucleases have provided scientists with the unprecedented ability to probe, regulate, and manipulate the human genome. Zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeat-Cas9 system (CRISPR-Cas9) represent a powerful array of tools that can bind to and cleave a specified DNA sequence. In their canonical forms, these nucleases induce double-strand breaks at a DNA locus of interest that can trigger cellular DNA repair processes that disrupt or replace genes. The fusion of these programmable nucleases with a variety of other protein domains has led to a rapidly growing suite of tools for activating, repressing, visualizing, and modifying loci of interest. Maximizing the usefulness and therapeutic relevance of these tools, however, requires precisely controlling their activity and specificity to minimize potentially toxic side effects arising from off-target activities. This need has motivated the application of chemical biology principles and methods to genome-editing proteins, including the engineering of variants of these proteins with improved or altered specificities, and the development of genetic, chemical, optical, and protein delivery methods that control the activity of these agents in cells. Advancing the capabilities, safety, effectiveness, and therapeutic relevance of genome-engineering proteins will continue to rely on chemical biology strategies that manipulate their activity, specificity, and localization. Copyright © 2016 Elsevier Ltd. All rights reserved.

  6. Identification of structure-activity relationships from screening a structurally compact DNA-encoded chemical library.

    Science.gov (United States)

    Franzini, Raphael M; Ekblad, Torun; Zhong, Nan; Wichert, Moreno; Decurtins, Willy; Nauer, Angela; Zimmermann, Mauro; Samain, Florent; Scheuermann, Jörg; Brown, Peter J; Hall, Jonathan; Gräslund, Susanne; Schüler, Herwig; Neri, Dario

    2015-03-23

    Methods for the rapid and inexpensive discovery of hit compounds are essential for pharmaceutical research and DNA-encoded chemical libraries represent promising tools for this purpose. We here report on the design and synthesis of DAL-100K, a DNA-encoded chemical library containing 103 200 structurally compact compounds. Affinity screening experiments and DNA-sequencing analysis provided ligands with nanomolar affinities to several proteins, including prostate-specific membrane antigen and tankyrase 1. Correlations of sequence counts with binding affinities and potencies of enzyme inhibition were observed and enabled the identification of structural features critical for activity. These results indicate that libraries of this type represent a useful source of small-molecule binders for target proteins of pharmaceutical interest and information on structural features important for binding. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. Quantitative Chemical-Genetic Interaction Map Connects Gene Alterations to Drug Responses | Office of Cancer Genomics

    Science.gov (United States)

    In a recent Cancer Discovery report, CTD2 researchers at the University of California in San Francisco developed a new quantitative chemical-genetic interaction mapping approach to evaluate drug sensitivity or resistance in isogenic cell lines. Performing a high-throughput screen with isogenic cell lines allowed the researchers to explore the impact of a panel of emerging and established drugs on cells overexpressing a single cancer-associated gene in isolation.

  8. Chemical characterization of chars developed from thermochemical treatment of Kentucky bluegrass seed screenings.

    Science.gov (United States)

    Griffith, Stephen M; Banowetz, Gary M; Gady, David

    2013-08-01

    Seed mill screenings would be a considerable biofeedstock source for bioenergy and char production. Char produced from the gasification of residues resulting from cleaning of grass seed and small grains could be recycled to a cropping system as a soil amendment if chemical characterization determined that the gasification process had not produced or concentrated deleterious chemical or physical factors that might harm the environment, crop growth or yield. Previous reports have shown that char derived from the pyrolysis of a variety of biomass feedstocks has potential to enhance soil quality by pH adjustment, mineral amendment, and improved soil porosity. The objective of this research was to characterize char produced from Kentucky bluegrass seed mill screenings (KBss) by a small-scale gasification unit, operated at temperatures between 600 and 650°C, with respect to polycyclic aromatic hydrocarbons, selected heavy metals, as well as other physical and chemical characteristics, and determine its suitability for agricultural application as a soil amendment. We utilized KBss as a model for seed and grain-cleaning residues with the understanding that chemical and physical characteristics of char produced by gasification or other cleaning residues may differ based on soil and environmental conditions under which the crops were produced. Our results support the hypothesis that KBss char could be applied in a cropping system without toxic environmental consequences and serve multiple purposes, such as; recycling critical plant macro- and micro-nutrients back to existing cropland, enhancing soil carbon sequestration, managing soil pH, and improving water holding capacity. Crop field trails need to be implemented to further test these hypotheses.

  9. Dermal permeation data and models for the prioritization and screening-level exposure assessment of organic chemicals

    Science.gov (United States)

    High throughput screening (HTS) models are being developed and applied to prioritize chemicals for more comprehensive exposure and risk assessment. Dermal pathways are possible exposure routes to humans for thousands of chemicals found in personal care products and the indoor env...

  10. Volatile organic chemical emissions from carpet cushions: Screening measurements. Final report

    Energy Technology Data Exchange (ETDEWEB)

    Hodgson, A.T.; Phan, T.A.

    1994-05-01

    The US Consumer Product Safety Commission (CPSC) has received complaints from consumers regarding the occurrence of adverse health effects following the installation of new carpeting (Schachter, 1990). Carpet systems are suspected of emitting chemicals which may be the cause of these complaints, as well as objectionable odors. Carpets themselves have been shown to emit a variety of volatile organic compounds (VOCs). The objective of this study was to screen the representative samples of carpet cushions for emissions of individual VOCS, total VOCs (TVOC), formaldehyde, and, for the two types of polyurethane cushions, isomers of toluene diisocyanate (TDI). The measurements of VOCS, TVOC and formaldehyde were made over six-hour periods using small-volume (4-L) dynamic chambers. Sensitive gas chromatography-mass spectrometry (GC-MS) techniques were used to identify many of the VOCs emitted by the cushion samples and to obtain quantitative estimates of the emission rates of selected compounds. Separate screening measurements were conducted for TDI. The data from the screening measurements were used by the CPSC`s Health Sciences Laboratory to help design and conduct week-long measurements of emission rates of selected compounds.

  11. Data-driven prioritization of chemicals for various water types using suspect screening LC-HRMS.

    Science.gov (United States)

    Sjerps, Rosa M A; Vughs, Dennis; van Leerdam, Jan A; ter Laak, Thomas L; van Wezel, Annemarie P

    2016-04-15

    For the prioritization of more than 5200 anthropogenic chemicals authorized on the European market, we use a large scale liquid chromatography-high resolution mass spectrometry (LC-HRMS) suspect screening study. The prioritization is based on occurrence in 151 water samples including effluent, surface water, ground water and drinking water. The suspect screening linked over 700 detected compounds with known accurate masses to one or multiple suspects. Using a prioritization threshold and removing false positives reduced this to 113 detected compounds linked to 174 suspects, 24 compounds reflect a confirmed structure by comparison with the pure reference standard. The prioritized compounds and suspects are relevant for detailed risk assessments after confirmation of their identity. Only one of the 174 prioritized compounds and suspects is mentioned in water quality regulations, and only 20% is mentioned on existing lists of potentially relevant chemicals. This shows the complementarity to commonly used target-based methods. The semi-quantitative total concentration, expressed as internal standard equivalents of detected compounds linked to suspects, in effluents is approximately 10 times higher than in surface waters, while ground waters and drinking waters show the lowest response. The average retention time, a measure for hydrophobicity, of the detected compounds per sample decreased from effluent to surface- and groundwater to drinking water, confirming the occurrence of more polar compounds in drinking water. The semi-quantitative total concentrations exceed the conservative and precautionary threshold of toxicological concern. Therefore, adverse effects of mixtures cannot be neglected without a more thorough risk assessment.

  12. A genome-wide deletion mutant screen identifies pathways affected by nickel sulfate in Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Dai Wei

    2009-11-01

    Full Text Available Abstract Background The understanding of the biological function, regulation, and cellular interactions of the yeast genome and proteome, along with the high conservation in gene function found between yeast genes and their human homologues, has allowed for Saccharomyces cerevisiae to be used as a model organism to deduce biological processes in human cells. Here, we have completed a systematic screen of the entire set of 4,733 haploid S. cerevisiae gene deletion strains (the entire set of nonessential genes for this organism to identify gene products that modulate cellular toxicity to nickel sulfate (NiSO4. Results We have identified 149 genes whose gene deletion causes sensitivity to NiSO4 and 119 genes whose gene deletion confers resistance. Pathways analysis with proteins whose absence renders cells sensitive and resistant to nickel identified a wide range of cellular processes engaged in the toxicity of S. cerevisiae to NiSO4. Functional categories overrepresented with proteins whose absence renders cells sensitive to NiSO4 include homeostasis of protons, cation transport, transport ATPases, endocytosis, siderophore-iron transport, homeostasis of metal ions, and the diphthamide biosynthesis pathway. Functional categories overrepresented with proteins whose absence renders cells resistant to nickel include functioning and transport of the vacuole and lysosome, protein targeting, sorting, and translocation, intra-Golgi transport, regulation of C-compound and carbohydrate metabolism, transcriptional repression, and chromosome segregation/division. Interactome analysis mapped seven nickel toxicity modulating and ten nickel-resistance networks. Additionally, we studied the degree of sensitivity or resistance of the 111 nickel-sensitive and 72 -resistant strains whose gene deletion product has a similar protein in human cells. Conclusion We have undertaken a whole genome approach in order to further understand the mechanism(s regulating the cell

  13. UV-visible spectroscopy method for screening the chemical stability of potential antioxidants for proton exchange membrane fuel cells

    Science.gov (United States)

    Banham, Dustin; Ye, Siyu; Knights, Shanna; Stewart, S. Michael; Wilson, Mahlon; Garzon, Fernando

    2015-05-01

    A novel method based on UV-visible spectroscopy is reported for screening the chemical stability of potential antioxidant additives for proton exchange membrane fuel cells, and the chemical stabilities of three CeOx samples of varying crystallite sizes (6, 13, or 25 nm) are examined. The chemical stabilities predicted by this new screening method are compared to in-situ membrane electrode assembly (MEA) accelerated stress testing, with the results confirming that this rapid and inexpensive method can be used to accurately predict performance impacts of antioxidants.

  14. Whole Genome Sequencing and a New Bioinformatics Platform Allow for Rapid Gene Identification in D. melanogaster EMS Screens

    Directory of Open Access Journals (Sweden)

    Jeannette Osterloh

    2012-12-01

    Full Text Available Forward genetic screens in Drosophila melanogaster using ethyl methanesulfonate (EMS mutagenesis are a powerful approach for identifying genes that modulate specific biological processes in an in vivo setting. The mapping of genes that contain randomly-induced point mutations has become more efficient in Drosophila thanks to the maturation and availability of many types of genetic tools. However, classic approaches to gene mapping are relatively slow and ultimately require extensive Sanger sequencing of candidate chromosomal loci. With the advent of new high-throughput sequencing techniques, it is increasingly efficient to directly re-sequence the whole genome of model organisms. This approach, in combination with traditional chromosomal mapping, has the potential to greatly simplify and accelerate mutation identification in mutants generated in EMS screens. Here we show that next-generation sequencing (NGS is an accurate and efficient tool for high-throughput sequencing and mutation discovery in Drosophila melanogaster. As a test case, mutant strains of Drosophila that exhibited long-term survival of severed peripheral axons were identified in a forward EMS mutagenesis. All mutants were recessive and fell into a single lethal complementation group, which suggested that a single gene was responsible for the protective axon degenerative phenotype. Whole genome sequencing of these genomes identified the underlying gene ect4. To improve the process of genome wide mutation identification, we developed Genomes Management Application (GEM.app, https://genomics.med.miami.edu, a graphical online user interface to a custom query framework. Using a custom GEM.app query, we were able to identify that each mutant carried a unique non-sense mutation in the gene ect4 (dSarm, which was recently shown by Osterloh et al. to be essential for the activation of axonal degeneration. Our results demonstrate the current advantages and limitations of NGS in Drosophila

  15. Chemical biology drug sensitivity screen identifies sunitinib as synergistic agent with disulfiram in prostate cancer cells.

    Directory of Open Access Journals (Sweden)

    Kirsi Ketola

    Full Text Available BACKGROUND: Current treatment options for castration- and treatment-resistant prostate cancer are limited and novel approaches are desperately needed. Our recent results from a systematic chemical biology sensitivity screen covering most known drugs and drug-like molecules indicated that aldehyde dehydrogenase inhibitor disulfiram is one of the most potent cancer-specific inhibitors of prostate cancer cell growth, including TMPRSS2-ERG fusion positive cancers. However, the results revealed that disulfiram alone does not block tumor growth in vivo nor induce apoptosis in vitro, indicating that combinatorial approaches may be required to enhance the anti-neoplastic effects. METHODS AND FINDINGS: In this study, we utilized a chemical biology drug sensitivity screen to explore disulfiram mechanistic details and to identify compounds potentiating the effect of disulfiram in TMPRSS2-ERG fusion positive prostate cancer cells. In total, 3357 compounds including current chemotherapeutic agents as well as drug-like small molecular compounds were screened alone and in combination with disulfiram. Interestingly, the results indicated that androgenic and antioxidative compounds antagonized disulfiram effect whereas inhibitors of receptor tyrosine kinase, proteasome, topoisomerase II, glucosylceramide synthase or cell cycle were among compounds sensitizing prostate cancer cells to disulfiram. The combination of disulfiram and an antiangiogenic agent sunitinib was studied in more detail, since both are already in clinical use in humans. Disulfiram-sunitinib combination induced apoptosis and reduced androgen receptor protein expression more than either of the compounds alone. Moreover, combinatorial exposure reduced metastatic characteristics such as cell migration and 3D cell invasion as well as induced epithelial differentiation shown as elevated E-cadherin expression. CONCLUSIONS: Taken together, our results propose novel combinatorial approaches to inhibit

  16. Chemical Biology Drug Sensitivity Screen Identifies Sunitinib as Synergistic Agent with Disulfiram in Prostate Cancer Cells

    Science.gov (United States)

    Ketola, Kirsi; Kallioniemi, Olli; Iljin, Kristiina

    2012-01-01

    Background Current treatment options for castration- and treatment-resistant prostate cancer are limited and novel approaches are desperately needed. Our recent results from a systematic chemical biology sensitivity screen covering most known drugs and drug-like molecules indicated that aldehyde dehydrogenase inhibitor disulfiram is one of the most potent cancer-specific inhibitors of prostate cancer cell growth, including TMPRSS2-ERG fusion positive cancers. However, the results revealed that disulfiram alone does not block tumor growth in vivo nor induce apoptosis in vitro, indicating that combinatorial approaches may be required to enhance the anti-neoplastic effects. Methods and Findings In this study, we utilized a chemical biology drug sensitivity screen to explore disulfiram mechanistic details and to identify compounds potentiating the effect of disulfiram in TMPRSS2-ERG fusion positive prostate cancer cells. In total, 3357 compounds including current chemotherapeutic agents as well as drug-like small molecular compounds were screened alone and in combination with disulfiram. Interestingly, the results indicated that androgenic and antioxidative compounds antagonized disulfiram effect whereas inhibitors of receptor tyrosine kinase, proteasome, topoisomerase II, glucosylceramide synthase or cell cycle were among compounds sensitizing prostate cancer cells to disulfiram. The combination of disulfiram and an antiangiogenic agent sunitinib was studied in more detail, since both are already in clinical use in humans. Disulfiram-sunitinib combination induced apoptosis and reduced androgen receptor protein expression more than either of the compounds alone. Moreover, combinatorial exposure reduced metastatic characteristics such as cell migration and 3D cell invasion as well as induced epithelial differentiation shown as elevated E-cadherin expression. Conclusions Taken together, our results propose novel combinatorial approaches to inhibit prostate cancer cell

  17. A genome-screen experiment to detect quantitative trait loci affecting resistance to facial eczema disease in sheep.

    Science.gov (United States)

    Phua, S H; Dodds, K G; Morris, C A; Henry, H M; Beattie, A E; Garmonsway, H G; Towers, N R; Crawford, A M

    2009-02-01

    Facial eczema (FE) is a secondary photosensitization disease arising from liver cirrhosis caused by the mycotoxin sporidesmin. The disease affects sheep, cattle, deer and goats, and costs the New Zealand sheep industry alone an estimated NZ$63M annually. A long-term sustainable solution to this century-old FE problem is to breed for disease-resistant animals by marker-assisted selection. As a step towards finding a diagnostic DNA test for FE sensitivity, we have conducted a genome-scan experiment to screen for quantitative trait loci (QTL) affecting this trait in Romney sheep. Four F(1) sires, obtained from reciprocal matings of FE resistant and susceptible selection-line animals, were used to generate four outcross families. The resulting half-sib progeny were artificially challenged with sporidesmin to phenotype their FE traits measured in terms of their serum levels of liver-specific enzymes, namely gamma-glutamyl transferase and glutamate dehydrogenase. In a primary screen using selective genotyping on extreme progeny of each family, a total of 244 DNA markers uniformly distributed over all 26 ovine autosomes (with an autosomal genome coverage of 79-91%) were tested for linkage to the FE traits. Data were analysed using Haley-Knott regression. The primary screen detected one significant and one suggestive QTL on chromosomes 3 and 8 respectively. Both the significant and suggestive QTL were followed up in a secondary screen where all progeny were genotyped and analysed; the QTL on chromosome 3 was significant in this analysis.

  18. The non-genomic effects of endocrine-disrupting chemicals on mammalian sperm.

    Science.gov (United States)

    Tavares, R S; Escada-Rebelo, S; Correia, M; Mota, P C; Ramalho-Santos, J

    2016-01-01

    Exposure to toxicants present in the environment, especially the so-called endocrine-disrupting chemicals (EDCs), has been associated with decreased sperm quality and increased anomalies in male reproductive organs over the past decades. Both human and animal populations are continuously exposed to ubiquitous synthetic and natural-occurring EDCs through diet, dermal contact and/or inhalation, therefore potentially compromising male reproductive health. Although the effects of EDC are likely induced via multiple genomic-based pathways, their non-genomic effects may also be relevant. Furthermore, spermatozoa are transcriptionally inactive cells that can come in direct contact with EDCs in reproductive fluids and secretions and are therefore a good model to address non-genomic effects. This review thus focuses on the non-genomic effects of several important EDCs relevant to mammalian exposure. Notably, EDCs were found to interfere with pre-existing pathways inducing a panoply of deleterious effects to sperm function that included altered intracellular Ca(2) (+) oscillations, induction of oxidative stress, mitochondrial dysfunction, increased DNA damage and decreased sperm motility and viability, among others, potentially jeopardizing male fertility. Although many studies have used non-environmentally relevant concentrations of only one compound for mechanistic studies, it is important to remember that mammals are not exposed to one, but rather to a multitude of environmental EDCs, and synergistic effects may occur. Furthermore, some effects have been detected with single compounds at environmentally relevant concentrations.

  19. Complete genome-wide screening and subtractive genomic approach revealed new virulence factors, potential drug targets against bio-war pathogen Brucella melitensis 16M.

    Science.gov (United States)

    Pradeepkiran, Jangampalli Adi; Sainath, Sri Bhashyam; Kumar, Konidala Kranthi; Bhaskar, Matcha

    2015-01-01

    Brucella melitensis 16M is a Gram-negative coccobacillus that infects both animals and humans. It causes a disease known as brucellosis, which is characterized by acute febrile illness in humans and causes abortions in livestock. To prevent and control brucellosis, identification of putative drug targets is crucial. The present study aimed to identify drug targets in B. melitensis 16M by using a subtractive genomic approach. We used available database repositories (Database of Essential Genes, Kyoto Encyclopedia of Genes and Genomes Automatic Annotation Server, and Kyoto Encyclopedia of Genes and Genomes) to identify putative genes that are nonhomologous to humans and essential for pathogen B. melitensis 16M. The results revealed that among 3 Mb genome size of pathogen, 53 putative characterized and 13 uncharacterized hypothetical genes were identified; further, from Basic Local Alignment Search Tool protein analysis, one hypothetical protein showed a close resemblance (50%) to Silicibacter pomeroyi DUF1285 family protein (2RE3). A further homology model of the target was constructed using MODELLER 9.12 and optimized through variable target function method by molecular dynamics optimization with simulating annealing. The stereochemical quality of the restrained model was evaluated by PROCHECK, VERIFY-3D, ERRAT, and WHATIF servers. Furthermore, structure-based virtual screening was carried out against the predicted active site of the respective protein using the glycerol structural analogs from the PubChem database. We identified five best inhibitors with strong affinities, stable interactions, and also with reliable drug-like properties. Hence, these leads might be used as the most effective inhibitors of modeled protein. The outcome of the present work of virtual screening of putative gene targets might facilitate design of potential drugs for better treatment against brucellosis.

  20. Complete genome-wide screening and subtractive genomic approach revealed new virulence factors, potential drug targets against bio-war pathogen Brucella melitensis 16M

    Science.gov (United States)

    Pradeepkiran, Jangampalli Adi; Sainath, Sri Bhashyam; Kumar, Konidala Kranthi; Bhaskar, Matcha

    2015-01-01

    Brucella melitensis 16M is a Gram-negative coccobacillus that infects both animals and humans. It causes a disease known as brucellosis, which is characterized by acute febrile illness in humans and causes abortions in livestock. To prevent and control brucellosis, identification of putative drug targets is crucial. The present study aimed to identify drug targets in B. melitensis 16M by using a subtractive genomic approach. We used available database repositories (Database of Essential Genes, Kyoto Encyclopedia of Genes and Genomes Automatic Annotation Server, and Kyoto Encyclopedia of Genes and Genomes) to identify putative genes that are nonhomologous to humans and essential for pathogen B. melitensis 16M. The results revealed that among 3 Mb genome size of pathogen, 53 putative characterized and 13 uncharacterized hypothetical genes were identified; further, from Basic Local Alignment Search Tool protein analysis, one hypothetical protein showed a close resemblance (50%) to Silicibacter pomeroyi DUF1285 family protein (2RE3). A further homology model of the target was constructed using MODELLER 9.12 and optimized through variable target function method by molecular dynamics optimization with simulating annealing. The stereochemical quality of the restrained model was evaluated by PROCHECK, VERIFY-3D, ERRAT, and WHATIF servers. Furthermore, structure-based virtual screening was carried out against the predicted active site of the respective protein using the glycerol structural analogs from the PubChem database. We identified five best inhibitors with strong affinities, stable interactions, and also with reliable drug-like properties. Hence, these leads might be used as the most effective inhibitors of modeled protein. The outcome of the present work of virtual screening of putative gene targets might facilitate design of potential drugs for better treatment against brucellosis. PMID:25834405

  1. Complete genome-wide screening and subtractive genomic approach revealed new virulence factors, potential drug targets against bio-war pathogen Brucella melitensis 16M

    Directory of Open Access Journals (Sweden)

    Pradeepkiran JA

    2015-03-01

    Full Text Available Jangampalli Adi Pradeepkiran,1* Sri Bhashyam Sainath,2,3* Konidala Kranthi Kumar,1 Matcha Bhaskar1 1Division of Animal Biotechnology, Department of Zoology, Sri Venkateswara University, Tirupati, India; 2CIMAR/CIIMAR, Centro Interdisciplinar de Investigação Marinha e Ambiental, Universidade do Porto, Rua dos Bragas, Porto, Portugal, 3Department of Biotechnology, Vikrama Simhapuri University, Nellore, Andhra Pradesh, India *These authors contributed equally to this work Abstract: Brucella melitensis 16M is a Gram-negative coccobacillus that infects both animals and humans. It causes a disease known as brucellosis, which is characterized by acute febrile illness in humans and causes abortions in livestock. To prevent and control brucellosis, identification of putative drug targets is crucial. The present study aimed to identify drug targets in B. melitensis 16M by using a subtractive genomic approach. We used available database repositories (Database of Essential Genes, Kyoto Encyclopedia of Genes and Genomes Automatic Annotation Server, and Kyoto Encyclopedia of Genes and Genomes to identify putative genes that are nonhomologous to humans and essential for pathogen B. melitensis 16M. The results revealed that among 3 Mb genome size of pathogen, 53 putative characterized and 13 uncharacterized hypothetical genes were identified; further, from Basic Local Alignment Search Tool protein analysis, one hypothetical protein showed a close resemblance (50% to Silicibacter pomeroyi DUF1285 family protein (2RE3. A further homology model of the target was constructed using MODELLER 9.12 and optimized through variable target function method by molecular dynamics optimization with simulating annealing. The stereochemical quality of the restrained model was evaluated by PROCHECK, VERIFY-3D, ERRAT, and WHATIF servers. Furthermore, structure-based virtual screening was carried out against the predicted active site of the respective protein using the

  2. Use of Modern Chemical Protein Synthesis and Advanced Fluorescent Assay Techniques to Experimentally Validate the Functional Annotation of Microbial Genomes

    Energy Technology Data Exchange (ETDEWEB)

    Kent, Stephen [University of Chicago

    2012-07-20

    The objective of this research program was to prototype methods for the chemical synthesis of predicted protein molecules in annotated microbial genomes. High throughput chemical methods were to be used to make large numbers of predicted proteins and protein domains, based on microbial genome sequences. Microscale chemical synthesis methods for the parallel preparation of peptide-thioester building blocks were developed; these peptide segments are used for the parallel chemical synthesis of proteins and protein domains. Ultimately, it is envisaged that these synthetic molecules would be ‘printed’ in spatially addressable arrays. The unique ability of total synthesis to precision label protein molecules with dyes and with chemical or biochemical ‘tags’ can be used to facilitate novel assay technologies adapted from state-of-the art single molecule fluorescence detection techniques. In the future, in conjunction with modern laboratory automation this integrated set of techniques will enable high throughput experimental validation of the functional annotation of microbial genomes.

  3. Genome mining expands the chemical diversity of the cyanobactin family to include highly modified linear peptides.

    Science.gov (United States)

    Leikoski, Niina; Liu, Liwei; Jokela, Jouni; Wahlsten, Matti; Gugger, Muriel; Calteau, Alexandra; Permi, Perttu; Kerfeld, Cheryl A; Sivonen, Kaarina; Fewer, David P

    2013-08-22

    Ribosomal peptides are produced through the posttranslational modification of short precursor peptides. Cyanobactins are a growing family of cyclic ribosomal peptides produced by cyanobacteria. However, a broad systematic survey of the genetic capacity to produce cyanobactins is lacking. Here we report the identification of 31 cyanobactin gene clusters from 126 genomes of cyanobacteria. Genome mining suggested a complex evolutionary history defined by horizontal gene transfer and rapid diversification of precursor genes. Extensive chemical analyses demonstrated that some cyanobacteria produce short linear cyanobactins with a chain length ranging from three to five amino acids. The linear peptides were N-prenylated and O-methylated on the N and C termini, respectively, and named aeruginosamide and viridisamide. These findings broaden the structural diversity of the cyanobactin family to include highly modified linear peptides with rare posttranslational modifications.

  4. A "genome-to-lead" approach for insecticide discovery: pharmacological characterization and screening of Aedes aegypti D(1-like dopamine receptors.

    Directory of Open Access Journals (Sweden)

    Jason M Meyer

    2012-01-01

    Full Text Available BACKGROUND: Many neglected tropical infectious diseases affecting humans are transmitted by arthropods such as mosquitoes and ticks. New mode-of-action chemistries are urgently sought to enhance vector management practices in countries where arthropod-borne diseases are endemic, especially where vector populations have acquired widespread resistance to insecticides. METHODOLOGY/PRINCIPAL FINDINGS: We describe a "genome-to-lead" approach for insecticide discovery that incorporates the first reported chemical screen of a G protein-coupled receptor (GPCR mined from a mosquito genome. A combination of molecular and pharmacological studies was used to functionally characterize two dopamine receptors (AaDOP1 and AaDOP2 from the yellow fever mosquito, Aedes aegypti. Sequence analyses indicated that these receptors are orthologous to arthropod D(1-like (Gα(s-coupled receptors, but share less than 55% amino acid identity in conserved domains with mammalian dopamine receptors. Heterologous expression of AaDOP1 and AaDOP2 in HEK293 cells revealed dose-dependent responses to dopamine (EC(50: AaDOP1 = 3.1±1.1 nM; AaDOP2 = 240±16 nM. Interestingly, only AaDOP1 exhibited sensitivity to epinephrine (EC(50 = 5.8±1.5 nM and norepinephrine (EC(50 = 760±180 nM, while neither receptor was activated by other biogenic amines tested. Differential responses were observed between these receptors regarding their sensitivity to dopamine agonists and antagonists, level of maximal stimulation, and constitutive activity. Subsequently, a chemical library screen was implemented to discover lead chemistries active at AaDOP2. Fifty-one compounds were identified as "hits," and follow-up validation assays confirmed the antagonistic effect of selected compounds at AaDOP2. In vitro comparison studies between AaDOP2 and the human D(1 dopamine receptor (hD(1 revealed markedly different pharmacological profiles and identified amitriptyline and doxepin as AaDOP2

  5. A comparative genomics screen identifies a Sinorhizobium meliloti 1021 sodM-like gene strongly expressed within host plant nodules

    Directory of Open Access Journals (Sweden)

    Queiroux Clothilde

    2012-05-01

    Full Text Available Abstract Background We have used the genomic data in the Integrated Microbial Genomes system of the Department of Energy’s Joint Genome Institute to make predictions about rhizobial open reading frames that play a role in nodulation of host plants. The genomic data was screened by searching for ORFs conserved in α-proteobacterial rhizobia, but not conserved in closely-related non-nitrogen-fixing α-proteobacteria. Results Using this approach, we identified many genes known to be involved in nodulation or nitrogen fixation, as well as several new candidate genes. We knocked out selected new genes and assayed for the presence of nodulation phenotypes and/or nodule-specific expression. One of these genes, SMc00911, is strongly expressed by bacterial cells within host plant nodules, but is expressed minimally by free-living bacterial cells. A strain carrying an insertion mutation in SMc00911 is not defective in the symbiosis with host plants, but in contrast to expectations, this mutant strain is able to out-compete the S. meliloti 1021 wild type strain for nodule occupancy in co-inoculation experiments. The SMc00911 ORF is predicted to encode a “SodM-like” (superoxide dismutase-like protein containing a rhodanese sulfurtransferase domain at the N-terminus and a chromate-resistance superfamily domain at the C-terminus. Several other ORFs (SMb20360, SMc01562, SMc01266, SMc03964, and the SMc01424-22 operon identified in the screen are expressed at a moderate level by bacteria within nodules, but not by free-living bacteria. Conclusions Based on the analysis of ORFs identified in this study, we conclude that this comparative genomics approach can identify rhizobial genes involved in the nitrogen-fixing symbiosis with host plants, although none of the newly identified genes were found to be essential for this process.

  6. High-Throughput Screening of Chemical Effects on Steroidogenesis Using H295R Human Adrenocortical Carcinoma Cells.

    Science.gov (United States)

    Karmaus, Agnes L; Toole, Colleen M; Filer, Dayne L; Lewis, Kenneth C; Martin, Matthew T

    2016-04-01

    Disruption of steroidogenesis by environmental chemicals can result in altered hormone levels causing adverse reproductive and developmental effects. A high-throughput assay using H295R human adrenocortical carcinoma cells was used to evaluate the effect of 2060 chemical samples on steroidogenesis via high-performance liquid chromatography followed by tandem mass spectrometry quantification of 10 steroid hormones, including progestagens, glucocorticoids, androgens, and estrogens. The study employed a 3 stage screening strategy. The first stage established the maximum tolerated concentration (MTC; ≥ 70% viability) per sample. The second stage quantified changes in hormone levels at the MTC whereas the third stage performed concentration-response (CR) on a subset of samples. At all stages, cells were prestimulated with 10 µM forskolin for 48 h to induce steroidogenesis followed by chemical treatment for 48 h. Of the 2060 chemical samples evaluated, 524 samples were selected for 6-point CR screening, based in part on significantly altering at least 4 hormones at the MTC. CR screening identified 232 chemical samples with concentration-dependent effects on 17β-estradiol and/or testosterone, with 411 chemical samples showing an effect on at least one hormone across the steroidogenesis pathway. Clustering of the concentration-dependent chemical-mediated steroid hormone effects grouped chemical samples into 5 distinct profiles generally representing putative mechanisms of action, including CYP17A1 and HSD3B inhibition. A distinct pattern was observed between imidazole and triazole fungicides suggesting potentially distinct mechanisms of action. From a chemical testing and prioritization perspective, this assay platform provides a robust model for high-throughput screening of chemicals for effects on steroidogenesis.

  7. Developmental neurotoxicity testing: recommendations for developing alternative methods for the screening and prioritization of chemicals.

    Science.gov (United States)

    Crofton, Kevin M; Mundy, William R; Lein, Pamela J; Bal-Price, Anna; Coecke, Sandra; Seiler, Andrea E M; Knaut, Holger; Buzanska, Leonora; Goldberg, Alan

    2011-01-01

    Developmental neurotoxicity testing (DNT) is perceived by many stakeholders to be an area in critical need of alternative methods to current animal testing protocols and guidelines. An immediate goal is to develop test methods that are capable of screening large numbers of chemicals. This document provides recommendations for developing alternative DNT approaches that will generate the type of data required for evaluating and comparing predictive capacity and efficiency across test methods and laboratories. These recommendations were originally drafted to stimulate and focus discussions of alternative testing methods and models for DNT at the TestSmart DNT II meeting (http://caat.jhsph.edu/programs/workshops/dnt2.html) and this document reflects critical feedback from all stakeholders that participated in this meeting. The intent of this document is to serve as a catalyst for engaging the research community in the development of DNT alternatives and it is expected that these recommendations will continue to evolve with the science.

  8. Automated screening for small organic ligands using DNA-encoded chemical libraries.

    Science.gov (United States)

    Decurtins, Willy; Wichert, Moreno; Franzini, Raphael M; Buller, Fabian; Stravs, Michael A; Zhang, Yixin; Neri, Dario; Scheuermann, Jörg

    2016-04-01

    DNA-encoded chemical libraries (DECLs) are collections of organic compounds that are individually linked to different oligonucleotides, serving as amplifiable identification barcodes. As all compounds in the library can be identified by their DNA tags, they can be mixed and used in affinity-capture experiments on target proteins of interest. In this protocol, we describe the screening process that allows the identification of the few binding molecules within the multiplicity of library members. First, the automated affinity selection process physically isolates binding library members. Second, the DNA codes of the isolated binders are PCR-amplified and subjected to high-throughput DNA sequencing. Third, the obtained sequencing data are evaluated using a C++ program and the results are displayed using MATLAB software. The resulting selection fingerprints facilitate the discrimination of binding from nonbinding library members. The described procedures allow the identification of small organic ligands to biological targets from a DECL within 10 d.

  9. Preferential regulation of miRNA targets by environmental chemicals in the human genome

    OpenAIRE

    2011-01-01

    Abstract Background microRNAs (miRNAs) represent a class of small (typically 22 nucleotides in length) non-coding RNAs that can degrade their target mRNAs or block their translation. Recent disease research showed the exposure to some environmental chemicals (ECs) can regulate the expression patterns of miRNAs, which raises the intriguing question of how miRNAs and their targets cope with the exposure to ECs throughout the genome. Results In this study, we comprehensively analyzed the propert...

  10. Penicillium arizonense, a new, genome sequenced fungal species, reveals a high chemical diversity in secreted metabolites

    DEFF Research Database (Denmark)

    Grijseels, Sietske; Nielsen, Jens Christian; Randelovic, Milica;

    2016-01-01

    confirmed the grouping of P. arizonense within section Canescentia. Compared to related species, P. arizonense proved to encode a high number of proteins involved in carbohydrate metabolism, in particular hemicellulases. Mining the genome for genes involved in secondary metabolite biosynthesis resulted...... of biosynthetic gene clusters in P. arizonense responsible for the synthesis of all detected compounds except curvulinic acid. The capacity to produce biomass degrading enzymes and the identification of a high chemical diversity in secreted bioactive secondary metabolites, offers a broad range of potential...

  11. Novel immune-modulator identified by a rapid, functional screen of the parapoxvirus ovis (Orf virus genome

    Directory of Open Access Journals (Sweden)

    McGuire Michael J

    2012-01-01

    Full Text Available Abstract Background The success of new sequencing technologies and informatic methods for identifying genes has made establishing gene product function a critical rate limiting step in progressing the molecular sciences. We present a method to functionally mine genomes for useful activities in vivo, using an unusual property of a member of the poxvirus family to demonstrate this screening approach. Results The genome of Parapoxvirus ovis (Orf virus was sequenced, annotated, and then used to PCR-amplify its open-reading-frames. Employing a cloning-independent protocol, a viral expression-library was rapidly built and arrayed into sub-library pools. These were directly delivered into mice as expressible cassettes and assayed for an immune-modulating activity associated with parapoxvirus infection. The product of the B2L gene, a homolog of vaccinia F13L, was identified as the factor eliciting immune cell accumulation at sites of skin inoculation. Administration of purified B2 protein also elicited immune cell accumulation activity, and additionally was found to serve as an adjuvant for antigen-specific responses. Co-delivery of the B2L gene with an influenza gene-vaccine significantly improved protection in mice. Furthermore, delivery of the B2L expression construct, without antigen, non-specifically reduced tumor growth in murine models of cancer. Conclusion A streamlined, functional approach to genome-wide screening of a biological activity in vivo is presented. Its application to screening in mice for an immune activity elicited by the pathogen genome of Parapoxvirus ovis yielded a novel immunomodulator. In this inverted discovery method, it was possible to identify the adjuvant responsible for a function of interest prior to a mechanistic study of the adjuvant. The non-specific immune activity of this modulator, B2, is similar to that associated with administration of inactivated particles to a host or to a live viral infection. Administration

  12. A Genome-Wide Screen for Interactions Reveals a New Locus on 4p15 Modifying the Effect of Waist-to-Hip Ratio on Total Cholesterol

    NARCIS (Netherlands)

    Surakka, Ida; Isaacs, Aaron; Karssen, Lennart C.; Laurila, Pirkka-Pekka P.; Middelberg, Rita P. S.; Tikkanen, Emmi; Ried, Janina S.; Lamina, Claudia; Mangino, Massimo; Igl, Wilmar; Hottenga, Jouke-Jan; Lagou, Vasiliki; van der Harst, Pim; Mateo Leach, Irene; Esko, Tonu; Kutalik, Zoltan; Wainwright, Nicholas W.; Struchalin, Maksim V.; Sarin, Antti-Pekka; Kangas, Antti J.; Viikari, Jorma S.; Perola, Markus; Rantanen, Taina; Petersen, Ann-Kristin; Soininen, Pasi; Johansson, Asa; Soranzo, Nicole; Heath, Andrew C.; Papamarkou, Theodore; Prokopenko, Inga; Toenjes, Anke; Kronenberg, Florian; Doering, Angela; Rivadeneira, Fernando; Montgomery, Grant W.; Whitfield, John B.; Kahonen, Mika; Lehtimaki, Terho; Freimer, Nelson B.; Willemsen, Gonneke; de Geus, Eco J. C.; Palotie, Aarno; Sandhu, Manj S.; Waterworth, Dawn M.; Metspalu, Andres; Stumvoll, Michael; Uitterlinden, Andre G.; Jula, Antti; Navis, Gerjan; Wijmenga, Cisca; Wolffenbuttel, Bruce H. R.; Taskinen, Marja-Riitta; Ala-Korpela, Mika; Kaprio, Jaakko; Kyvik, Kirsten O.; Boomsma, Dorret I.; Pedersen, Nancy L.; Gyllensten, Ulf; Wilson, James F.; Rudan, Igor; Campbell, Harry; Pramstaller, Peter P.; Spector, Tim D.; Witteman, Jacqueline C. M.; Eriksson, Johan G.; Salomaa, Veikko; Oostra, Ben A.; Raitakari, Olli T.; Wichmann, H. -Erich; Gieger, Christian; Jaervelin, Marjo-Riitta; Martin, Nicholas G.; Hofman, Albert; McCarthy, Mark I.; Peltonen, Leena; van Duijn, Cornelia M.; Aulchenko, Yurii S.; Ripatti, Samuli

    2011-01-01

    Recent genome-wide association (GWA) studies described 95 loci controlling serum lipid levels. These common variants explain similar to 25% of the heritability of the phenotypes. To date, no unbiased screen for gene-environment interactions for circulating lipids has been reported. We screened for v

  13. A genome-wide screen for interactions reveals a new locus on 4p15 modifying the effect of waist-to-hip ratio on total cholesterol

    NARCIS (Netherlands)

    I. Surakka (Ida); A.J. Isaacs (Aaron); L.C. Karssen (Lennart); P.-P.P. Laurila; R.P.S. Middelberg (Rita); E. Tikkanen (Emmi); J.S. Ried (Janina); C. Lamina (Claudia); M. Mangino (Massimo); W. Igl (Wilmar); J.J. Hottenga (Jouke Jan); V. Lagou (Vasiliki); P. van der Harst (Pim); I.M. Leach (Irene Mateo); T. Esko (Tõnu); Z. Kutalik (Zoltán); N.W. Wainwright (Nicholas); M.V. Struchalin (Maksim); A.-P. Sarin; A.J. Kangas (Antti); J. Viikari (Jorma); M. Perola (Markus); T. Rantanen (Taina); A.K. Petersen; P. Soininen (Pasi); A. Johansson (Åsa); N. Soranzo (Nicole); A.C. Heath (Andrew); T. Papamarkou (Theodore); I. Prokopenko (Inga); A. Tönjes (Anke); F. Kronenberg (Florian); A. Döring (Angela); F. Rivadeneira Ramirez (Fernando); G.W. Montgomery (Grant); J.B. Whitfield (John); M. Kähönen (Mika); T. Lehtimäki (Terho); N.B. Freimer (Nelson); G.A.H.M. Willemsen (Gonneke); E.J.C. de Geus (Eco); A. Palotie (Aarno); M.S. Sandhu (Manj); D. Waterworth (Dawn); A. Metspalu (Andres); M. Stumvoll (Michael); A.G. Uitterlinden (André); A. Jula (Antti); G. Navis (Gerjan); C. Wijmenga (Cisca); B.H.R. Wolffenbuttel (Bruce); M.-R. Taskinen; M. Ala-Korpela (Mika); J. Kaprio (Jaakko); K.O. Kyvik (Kirsten Ohm); D.I. Boomsma (Dorret); N.L. Pedersen (Nancy); U. Gyllensten (Ulf); J.F. Wilson (James); I. Rudan (Igor); H. Campbell (Harry); P.P. Pramstaller (Peter Paul); T.D. Spector (Timothy); J.C.M. Witteman (Jacqueline); J.G. Eriksson (Johan); V. Salomaa (Veikko); B.A. Oostra (Ben); O. Raitakari (Olli); H.E. Wichmann (Heinz Erich); C. Gieger (Christian); M.R. Järvelin; N.G. Martin (Nicholas); A. Hofman (Albert); M.I. McCarthy (Mark); Y.S. Aulchenko (Yurii); L. Peltonen (Leena Johanna); P. Tikka-Kleemola (Päivi); S. Ripatti (Samuli)

    2011-01-01

    textabstractRecent genome-wide association (GWA) studies described 95 loci controlling serum lipid levels. These common variants explain ~25% of the heritability of the phenotypes. To date, no unbiased screen for gene-environment interactions for circulating lipids has been reported. We screened for

  14. A Genome-Wide Screen for Interactions Reveals a New Locus on 4p15 Modifying the Effect of Waist-to-Hip Ratio on Total Cholesterol

    DEFF Research Database (Denmark)

    Surakka, I.; Isaacs, A.; Karssen, L. C.;

    2011-01-01

    Recent genome-wide association (GWA) studies described 95 loci controlling serum lipid levels. These common variants explain similar to 25% of the heritability of the phenotypes. To date, no unbiased screen for gene-environment interactions for circulating lipids has been reported. We screened fo...

  15. High-Throughput Chemical Screens Identify Disulfiram as an Inhibitor of Human Glioblastoma Stem Cells

    Science.gov (United States)

    Hothi, Parvinder; Martins, Timothy J.; Chen, LiPing; Deleyrolle, Loic; Yoon, Jae-Geun; Reynolds, Brent; Foltz, Greg

    2012-01-01

    Glioblastoma Multiforme (GBM) continues to have a poor patient prognosis despite optimal standard of care. Glioma stem cells (GSCs) have been implicated as the presumed cause of tumor recurrence and resistance to therapy. With this in mind, we screened a diverse chemical library of 2,000 compounds to identify therapeutic agents that inhibit GSC proliferation and therefore have the potential to extend patient survival. High-throughput screens (HTS) identified 78 compounds that repeatedly inhibited cellular proliferation, of which 47 are clinically approved for other indications and 31 are experimental drugs. Several compounds (such as digitoxin, deguelin, patulin and phenethyl caffeate) exhibited high cytotoxicity, with half maximal inhibitory concentrations (IC50) in the low nanomolar range. In particular, the FDA approved drug for the treatment of alcoholism, disulfiram (DSF), was significantly potent across multiple patient samples (IC50 of 31.1 nM). The activity of DSF was potentiated by copper (Cu), which markedly increased GSC death. DSF–Cu inhibited the chymotrypsin-like proteasomal activity in cultured GSCs, consistent with inactivation of the ubiquitin-proteasome pathway and the subsequent induction of tumor cell death. Given that DSF is a relatively non-toxic drug that can penetrate the blood-brain barrier, we suggest that DSF should be tested (as either a monotherapy or as an adjuvant) in pre-clinical models of human GBM. Data also support targeting of the ubiquitin-proteasome pathway as a therapeutic approach in the treatment of GBM. PMID:23165409

  16. Chemical and metabolomic screens identify novel biomarkers and antidotes for cyanide exposure.

    Science.gov (United States)

    Nath, Anjali K; Roberts, Lee D; Liu, Yan; Mahon, Sari B; Kim, Sonia; Ryu, Justine H; Werdich, Andreas; Januzzi, James L; Boss, Gerry R; Rockwood, Gary A; MacRae, Calum A; Brenner, Matthew; Gerszten, Robert E; Peterson, Randall T

    2013-05-01

    Exposure to cyanide causes a spectrum of cardiac, neurological, and metabolic dysfunctions that can be fatal. Improved cyanide antidotes are needed, but the ideal biological pathways to target are not known. To understand better the metabolic effects of cyanide and to discover novel cyanide antidotes, we developed a zebrafish model of cyanide exposure and scaled it for high-throughput chemical screening. In a screen of 3120 small molecules, we discovered 4 novel antidotes that block cyanide toxicity. The most potent antidote was riboflavin. Metabolomic profiling of cyanide-treated zebrafish revealed changes in bile acid and purine metabolism, most notably by an increase in inosine levels. Riboflavin normalizes many of the cyanide-induced neurological and metabolic perturbations in zebrafish. The metabolic effects of cyanide observed in zebrafish were conserved in a rabbit model of cyanide toxicity. Further, humans treated with nitroprusside, a drug that releases nitric oxide and cyanide ions, display increased circulating bile acids and inosine. In summary, riboflavin may be a novel treatment for cyanide toxicity and prophylactic measure during nitroprusside treatment, inosine may serve as a biomarker of cyanide exposure, and metabolites in the bile acid and purine metabolism pathways may shed light on the pathways critical to reversing cyanide toxicity.

  17. A Chemical Screen Identifies Novel Compounds That Overcome Glial-Mediated Inhibition Of Neuronal Regeneration

    Science.gov (United States)

    Usher, Lynn C.; Johnstone, Andrea; Ertürk, Ali; Hu, Ying; Strikis, Dinara; Wanner, Ina B.; Moorman, Sanne; Lee, Jae-Wook; Min, Jaeki; Ha, Hyung-Ho; Duan, Yuanli; Hoffman, Stanley; Goldberg, Jeffrey L.; Bradke, Frank; Chang, Young-Tae; Lemmon, Vance P.; Bixby, John L.

    2010-01-01

    A major barrier to regeneration of central nervous system (CNS) axons is the presence of growth-inhibitory proteins associated with myelin and the glial scar. To identify chemical compounds with the ability to overcome the inhibition of regeneration, we screened a novel triazine library, based on the ability of compounds to increase neurite outgrowth from cerebellar neurons on inhibitory myelin substrates. The screen produced 4 “hit compounds”, which act with nM potency on several different neuronal types, and on several distinct substrates relevant to glial inhibition. Moreover, the compounds selectively overcome inhibition rather than promote growth in general. The compounds do not affect neuronal cAMP levels, PKC activity, or EGFR activation. Interestingly, one of the compounds alters microtubule dynamics and increases microtubule density in both fibroblasts and neurons. This same compound promotes regeneration of dorsal column axons after acute lesions, and potentiates regeneration of optic nerve axons after nerve crush in vivo. These compounds should provide insight into the mechanisms through which glial-derived inhibitors of regeneration act, and could lead to the development of novel therapies for CNS injury. PMID:20357120

  18. Chemical library screening using a SPR-based inhibition in solution assay: simulations and experimental validation.

    Science.gov (United States)

    Choulier, Laurence; Nominé, Yves; Zeder-Lutz, Gabrielle; Charbonnier, Sebastian; Didier, Bruno; Jung, Marie-Louise; Altschuh, Danièle

    2013-09-17

    We have developed a surface plasmon resonance (SPR)-based inhibition in solution assay (ISA) to search for inhibitors of the medium affinity (KD = 0.8 μM) interaction between an E6-derived peptide (E6peptide) immobilized on the sensor and a PDZ domain (MAGI-1 PDZ1) in the mobile phase. DZ domains are widespread protein-protein interaction modules that recognize the C-terminus of various partners. Simulations indicated that relatively low compound concentrations (10 μM) and limited peptide densities (Rmax < 200 resonance units) should allow the detection of inhibitors with a target affinity close to 100 μM, which was then demonstrated experimentally. ISA screening, carried out on the Prestwick Chemical Library® (1120 compounds), identified 36 compounds that inhibited the interaction by more than 5%. Concentration-dependent ISA, carried out on a subset of 19 potential inhibitors, indicated that 13 of these indeed affected the interaction between MAGI-1 PDZ1 and the E6peptide. No effect was observed for 84 compounds randomly chosen among noninhibitors. One of the four best inhibitors was a peptide binder, and three were PDZ binders with KD in the 10-50 μM range. We propose that a medium (μM) affinity between the target and surface-bound partner is optimal for SPR-based ISA screening.

  19. Discovery of two new inhibitors of Botrytis cinerea chitin synthase by a chemical library screening.

    Science.gov (United States)

    Magellan, Hervé; Boccara, Martine; Drujon, Thierry; Soulié, Marie-Christine; Guillou, Catherine; Dubois, Joëlle; Becker, Hubert F

    2013-09-01

    Chitin synthases polymerize UDP-GlcNAC to form chitin polymer, a key component of fungal cell wall biosynthesis. Furthermore, chitin synthases are desirable targets for fungicides since chitin is absent in plants and mammals. Two potent Botrytis cinerea chitin synthase inhibitors, 2,3,5-tri-O-benzyl-d-ribose (compound 1) and a 2,5-functionalized imidazole (compound 2) were identified by screening a chemical library. We adapted the wheat germ agglutinin (WGA) test for chitin synthase activity detection to allow miniaturization and robotization of the screen. Both identified compounds inhibited chitin synthases in vitro with IC50 values of 1.8 and 10μM, respectively. Compounds 1 and 2 were evaluated for their antifungal activity and were found to be active against B. cinerea BD90 strain with MIC values of 190 and 100μM, respectively. Finally, we discovered that both compounds confer resistance to plant leaves against the attack of the fungus by reducing the propagation of lesions by 37% and 23%, respectively. Based on the inhibitory properties found in different assays, compounds 1 and 2 can be considered as antifungal hit inhibitors of chitin synthase, allowing further optimization of their pharmacological profile to improve their antifungal properties.

  20. Genome-Wide Screen Reveals sec21 Mutants of Saccharomyces cerevisiae Are Methotrexate-Resistant

    Science.gov (United States)

    Wong, Lai H.; Flibotte, Stephane; Sinha, Sunita; Chiang, Jennifer; Giaever, Guri; Nislow, Corey

    2017-01-01

    Drug resistance is a consequence of how most modern medicines work. Drugs exert pressure on cells that causes death or the evolution of resistance. Indeed, highly specific drugs are rendered ineffective by a single DNA mutation. In this study, we apply the drug methotrexate, which is widely used in cancer and rheumatoid arthritis, and perform evolution experiments on Baker’s yeast to ask the different ways in which cells become drug resistant. Because of the conserved nature of biological pathways between yeast and man, our results can inform how the same mechanism may operate to render human cells resistant to treatment. Exposure of cells to small molecules and drug therapies imposes a strong selective pressure. As a result, cells rapidly acquire mutations in order to survive. These include resistant variants of the drug target as well as those that modulate drug transport and detoxification. To systematically explore how cells acquire drug resistance in an unbiased manner, rapid cost-effective approaches are required. Methotrexate, as one of the first rationally designed anticancer drugs, has served as a prototypic example of such acquired resistance. Known methotrexate resistance mechanisms include mutations that increase expression of the dihydrofolate reductase (DHFR) target as well as those that maintain function yet reduce the drug’s binding affinity. Recent evidence suggests that target-independent, epistatic mutations can also result in resistance to methotrexate. Currently, however, the relative contribution of such unlinked resistance mutations is not well understood. To address this issue, we took advantage of Saccharomyces cerevisiae as a model eukaryotic system that combined with whole-genome sequencing and a rapid screening methodology, allowed the identification of causative mutations that modulate resistance to methotrexate. We found a recurrent missense mutation in SEC21 (orthologous to human COPG1), which we confirmed in 10 de novo

  1. Comparison of three marine screening tests and four Oslo and Paris Commission procedures to evaluate toxicity of offshore chemicals

    Energy Technology Data Exchange (ETDEWEB)

    Weideborg, M.; Vik, E.A.; Oefjord, G.D.; Kjoennoe, O. [Aquateam-Norwegian Water Technology Centre A/S, Oslo (Norway)

    1997-02-01

    The results from the screening toxicity tests Artemia salina, Microtox{reg_sign}, and Mitochondria RET test were compared with those obtained from OSPAR (Oslo and Paris Commissions)-authorized procedures for testing of offshore chemicals (Skeletonema costatum, Acartia tonsa, Abra alba, and Corophium volutator). In this study 82 test substances (26 non-water soluble) were included. The Microtox test was found to be the most sensitive of the three screening tests. Microtox and Mitochondria RET test results showed good correlation with results from Acartia and Skeletonema testing, and it was concluded that the Microtox test was a suitable screening test as a base for assessment of further testing, especially regarding water-soluble chemicals. Sensitivity of Artemia salina to the tested chemicals was too low for it to be an appropriate bioassay organism for screening testing. A very good correlation was found between the results obtained with the Skeletonema and Acartia tests. The results indicated no need for more than one of the Skeletonema or Acartia tests if the Skeletonema median effective concentration or Acartia median lethal concentration was greater than 200 mg/L. The sediment-reworker tests (A. Alba or C. volutator) for chemicals that are likely to end up in the sediments (non-water soluble or surfactants) should be performed, independent of results from screening tests and other OSPAR species.

  2. Committee Opinion No. 690 Summary: Carrier Screening in the Age of Genomic Medicine.

    Science.gov (United States)

    2017-03-01

    Carrier screening, whether targeted or expanded, allows individuals to consider their range of reproductive options. Ultimately, the goal of genetic screening is to provide individuals with meaningful information that they can use to guide pregnancy planning based on their personal values. Ethnic-specific, panethnic, and expanded carrier screening are acceptable strategies for prepregnancy and prenatal carrier screening. Because all of these are acceptable strategies, each obstetrician-gynecologist or other health care provider or practice should establish a standard approach that is consistently offered to and discussed with each patient, ideally before pregnancy. Carrier screening will not identify all individuals who are at risk of the screened conditions. Patients should be counseled regarding the residual risk with any test result. Screening for any condition is optional and, after counseling, a patient may decline any or all carrier screening. If a patient requests a screening strategy other than the one used by the obstetrician-gynecologist or other health care provider, the requested test should be made available to her after counseling on its limitations, benefits, and alternatives. Expanded carrier screening does not replace previous risk-based screening recommendations. The determination of the appropriate screening approach for any individual patient should be based on the patient's family history and personal values after counseling. Referral to an obstetrician-gynecologist or other health care provider with genetics expertise should be considered for risk assessment, evaluation, and consideration of diagnostic testing as indicated for any patient with a family history of a genetic condition or concern for a genetic diagnosis.

  3. From genome-wide arrays to tailor-made biomarker readout - Progress towards routine analysis of skin sensitizing chemicals with GARD.

    Science.gov (United States)

    Forreryd, Andy; Zeller, Kathrin S; Lindberg, Tim; Johansson, Henrik; Lindstedt, Malin

    2016-12-01

    Allergic contact dermatitis (ACD) initiated by chemical sensitizers is an important public health concern. To prevent ACD, it is important to identify chemical allergens to limit the use of such compounds in various products. EU legislations, as well as increased mechanistic knowledge of skin sensitization have promoted development of non-animal based approaches for hazard classification of chemicals. GARD is an in vitro testing strategy based on measurements of a genomic biomarker signature. However, current GARD protocols are optimized for identification of predictive biomarker signatures, and not suitable for standardized screening. This study describes improvements to GARD to progress from biomarker discovery into a reliable and cost-effective assay for routine testing. Gene expression measurements were transferred to NanoString nCounter platform, normalization strategy was adjusted to fit serial arrival of testing substances, and a novel strategy to correct batch variations was presented. When challenging GARD with 29 compounds, sensitivity, specificity and accuracy could be estimated to 94%, 83% and 90%, respectively. In conclusion, we present a GARD workflow with improved sample capacity, retained predictive performance, and in a format adapted to standardized screening. We propose that GARD is ready to be considered as part of an integrated testing strategy for skin sensitization.

  4. A systematic prediction of multiple drug-target interactions from chemical, genomic, and pharmacological data.

    Directory of Open Access Journals (Sweden)

    Hua Yu

    Full Text Available In silico prediction of drug-target interactions from heterogeneous biological data can advance our system-level search for drug molecules and therapeutic targets, which efforts have not yet reached full fruition. In this work, we report a systematic approach that efficiently integrates the chemical, genomic, and pharmacological information for drug targeting and discovery on a large scale, based on two powerful methods of Random Forest (RF and Support Vector Machine (SVM. The performance of the derived models was evaluated and verified with internally five-fold cross-validation and four external independent validations. The optimal models show impressive performance of prediction for drug-target interactions, with a concordance of 82.83%, a sensitivity of 81.33%, and a specificity of 93.62%, respectively. The consistence of the performances of the RF and SVM models demonstrates the reliability and robustness of the obtained models. In addition, the validated models were employed to systematically predict known/unknown drugs and targets involving the enzymes, ion channels, GPCRs, and nuclear receptors, which can be further mapped to functional ontologies such as target-disease associations and target-target interaction networks. This approach is expected to help fill the existing gap between chemical genomics and network pharmacology and thus accelerate the drug discovery processes.

  5. CTL epitopes for influenza A including the H5N1 bird flu; genome-, pathogen-, and HLA-wide screening

    DEFF Research Database (Denmark)

    Wang, M.J.; Lamberth, K.; Harndahl, M.

    2007-01-01

    are present in the emerging bird flu isolates. Our study demonstrates that present technology enables a fast global screening for T cell immune epitopes of potential diagnostics and vaccine interest. This technology includes immuno-bioinformatics predictors with the capacity to perform fast genome-, pathogen......-, and HLA-wide searches for immune targets. To exploit this new potential, a coordinated international effort to analyze the precious source of information represented by rare patients, such as the current victims of bird flu, would be essential....

  6. Data Mining Approaches for Genomic Biomarker Development: Applications Using Drug Screening Data from the Cancer Genome Project and the Cancer Cell Line Encyclopedia.

    Directory of Open Access Journals (Sweden)

    David G Covell

    Full Text Available Developing reliable biomarkers of tumor cell drug sensitivity and resistance can guide hypothesis-driven basic science research and influence pre-therapy clinical decisions. A popular strategy for developing biomarkers uses characterizations of human tumor samples against a range of cancer drug responses that correlate with genomic change; developed largely from the efforts of the Cancer Cell Line Encyclopedia (CCLE and Sanger Cancer Genome Project (CGP. The purpose of this study is to provide an independent analysis of this data that aims to vet existing and add novel perspectives to biomarker discoveries and applications. Existing and alternative data mining and statistical methods will be used to a evaluate drug responses of compounds with similar mechanism of action (MOA, b examine measures of gene expression (GE, copy number (CN and mutation status (MUT biomarkers, combined with gene set enrichment analysis (GSEA, for hypothesizing biological processes important for drug response, c conduct global comparisons of GE, CN and MUT as biomarkers across all drugs screened in the CGP dataset, and d assess the positive predictive power of CGP-derived GE biomarkers as predictors of drug response in CCLE tumor cells. The perspectives derived from individual and global examinations of GEs, MUTs and CNs confirm existing and reveal unique and shared roles for these biomarkers in tumor cell drug sensitivity and resistance. Applications of CGP-derived genomic biomarkers to predict the drug response of CCLE tumor cells finds a highly significant ROC, with a positive predictive power of 0.78. The results of this study expand the available data mining and analysis methods for genomic biomarker development and provide additional support for using biomarkers to guide hypothesis-driven basic science research and pre-therapy clinical decisions.

  7. Data Mining Approaches for Genomic Biomarker Development: Applications Using Drug Screening Data from the Cancer Genome Project and the Cancer Cell Line Encyclopedia.

    Science.gov (United States)

    Covell, David G

    2015-01-01

    Developing reliable biomarkers of tumor cell drug sensitivity and resistance can guide hypothesis-driven basic science research and influence pre-therapy clinical decisions. A popular strategy for developing biomarkers uses characterizations of human tumor samples against a range of cancer drug responses that correlate with genomic change; developed largely from the efforts of the Cancer Cell Line Encyclopedia (CCLE) and Sanger Cancer Genome Project (CGP). The purpose of this study is to provide an independent analysis of this data that aims to vet existing and add novel perspectives to biomarker discoveries and applications. Existing and alternative data mining and statistical methods will be used to a) evaluate drug responses of compounds with similar mechanism of action (MOA), b) examine measures of gene expression (GE), copy number (CN) and mutation status (MUT) biomarkers, combined with gene set enrichment analysis (GSEA), for hypothesizing biological processes important for drug response, c) conduct global comparisons of GE, CN and MUT as biomarkers across all drugs screened in the CGP dataset, and d) assess the positive predictive power of CGP-derived GE biomarkers as predictors of drug response in CCLE tumor cells. The perspectives derived from individual and global examinations of GEs, MUTs and CNs confirm existing and reveal unique and shared roles for these biomarkers in tumor cell drug sensitivity and resistance. Applications of CGP-derived genomic biomarkers to predict the drug response of CCLE tumor cells finds a highly significant ROC, with a positive predictive power of 0.78. The results of this study expand the available data mining and analysis methods for genomic biomarker development and provide additional support for using biomarkers to guide hypothesis-driven basic science research and pre-therapy clinical decisions.

  8. Screening of 397 chemicals and development of a quantitative structure-activity relationship model for androgen receptor antagonism

    DEFF Research Database (Denmark)

    Vinggaard, Annemarie; Niemelä, Jay Russell; Wedebye, Eva Bay;

    2008-01-01

    We have screened 397 chemicals for human androgen receptor (AR) antagonism by a sensitive reporter gene assay to generate data for the development of a quantitative structure-activity relationship (QSAR) model. A total of 523 chemicals comprising data on 292 chemicals from our laboratory and data...... by the synthetic androgen R1881. The MultiCASE expert system was used to construct a QSAR model for AR antagonizing potential. A "5 Times, 2-Fold 50% Cross Validation" of the model showed a sensitivity of 64%, a specificity of 84%, and a concordance of 76%. Data for 102 chemicals were generated for an external...... validation of the model resulting in a sensitivity of 57%, a specificity of 98%, and a concordance of 92% of the model. The model was run on a set of 176103 chemicals, and 47% were within the domain of the model. Approximately 8% of chemicals was predicted active for AR antagonism. We conclude...

  9. An evaluation of chemical screening test kits for lead in paint

    Energy Technology Data Exchange (ETDEWEB)

    Oglesby, L.S.

    1996-04-01

    The Residential Lead-Based Paint Hazard Reduction Act (Title X) requires abatement and management of lead-based paint. The purpose of this study was to evaluate three chemical screening test kits using materials and methods from one study and subjecting the results to the statistical analysis of another. The three kits were used to predict the presence of lead in paint at ten weight concentrations from 0.04 to 3.97%. Paint was applied to four wood boards yielding a sample size of 40. Four boards were painted with lead-free paint and used as blanks. All of the boards were tested with the three test kits by an untrained individual having no knowledge of the actual lead content. Sensitivity, specificity, and false positive and negative rates were calculated for the test kit results. The manufactures` detection limits, the observed sensitivity ranged from 1.00 to 0.80, specificity ranged from 1.00 to 0.42, false positive ranged from 0 to 58%, and false negatives ranged from 0 to 20%. At the 0.5% Federal threshold level, the observed sensitivity ranged from 1.00 to 0.94, specificity ranged from 1.00 to 0.5, false positives ranged from 0 to 11.1%, and false negatives ranged from 0 to 20%. The observed false positive and false negative rates for all three kits were found to be significantly lower than those reported in a previous study. These results indicate that the kits perform very well at the Federal threshold, with two of the kits having false negative rates below 12.5% and false positive rates of 3.13%. These results indicate that these two kits would probably be acceptable screening tests for lead in paint.

  10. CRISPR-based screening of genomic island excision events in bacteria.

    Science.gov (United States)

    Selle, Kurt; Klaenhammer, Todd R; Barrangou, Rodolphe

    2015-06-30

    Genomic analysis of Streptococcus thermophilus revealed that mobile genetic elements (MGEs) likely contributed to gene acquisition and loss during evolutionary adaptation to milk. Clustered regularly interspaced short palindromic repeats-CRISPR-associated genes (CRISPR-Cas), the adaptive immune system in bacteria, limits genetic diversity by targeting MGEs including bacteriophages, transposons, and plasmids. CRISPR-Cas systems are widespread in streptococci, suggesting that the interplay between CRISPR-Cas systems and MGEs is one of the driving forces governing genome homeostasis in this genus. To investigate the genetic outcomes resulting from CRISPR-Cas targeting of integrated MGEs, in silico prediction revealed four genomic islands without essential genes in lengths from 8 to 102 kbp, totaling 7% of the genome. In this study, the endogenous CRISPR3 type II system was programmed to target the four islands independently through plasmid-based expression of engineered CRISPR arrays. Targeting lacZ within the largest 102-kbp genomic island was lethal to wild-type cells and resulted in a reduction of up to 2.5-log in the surviving population. Genotyping of Lac(-) survivors revealed variable deletion events between the flanking insertion-sequence elements, all resulting in elimination of the Lac-encoding island. Chimeric insertion sequence footprints were observed at the deletion junctions after targeting all of the four genomic islands, suggesting a common mechanism of deletion via recombination between flanking insertion sequences. These results established that self-targeting CRISPR-Cas systems may direct significant evolution of bacterial genomes on a population level, influencing genome homeostasis and remodeling.

  11. Development and Application of In Vitro Models for Screening Drugs and Environmental Chemicals that Predict Toxicity in Animals and Humans

    Science.gov (United States)

    Development and Application of In Vitro Models for Screening Drugs and Environmental Chemicals that Predict Toxicity in Animals and Humans (Presented by James McKim, Ph.D., DABT, Founder and Chief Science Officer, CeeTox) (5/25/2012)

  12. Screening ToxCast™ Phase I Chemicals in a Mouse Embryonic Stem Cell Adherent Cell Differentiation and Cytotoxicity (ACDC) Assay

    Science.gov (United States)

    An Adherent Cell Differentiation and Cytotoxicity (ACDC) in vitro assay with mouse embryonic stem cells was used to screen the ToxCast Phase I chemical library for effects on cellular differentiation and cell number. The U.S. Environmental Protection Agency (EPA) established the ...

  13. AOP: An R Package For Sufficient Causal Analysis in Pathway-based Screening of Drugs and Chemicals for Adversity

    Science.gov (United States)

    Summary: How can I quickly find the key events in a pathway that I need to monitor to predict that a/an beneficial/adverse event/outcome will occur? This is a key question when using signaling pathways for drug/chemical screening in pharma-cology, toxicology and risk assessment. ...

  14. Economic Evaluations of Pharmacogenetic and Genomic Screening Programs : Update of the Literature

    NARCIS (Netherlands)

    Vegter, Stefan; Jansen, Esther; Postma, Maarten J.; Boersma, Cornelis

    2010-01-01

    Pharmacogenetics and pharmacogenomics show great potential for developing individual treatment modalities to achieve optimal therapy effectiveness. Economic analyses are performed to determine whether pharmacogenetic screening strategies provide good value for money. The current review provides an u

  15. Targeting acetylcholinesterase: identification of chemical leads by high throughput screening, structure determination and molecular modeling.

    Directory of Open Access Journals (Sweden)

    Lotta Berg

    Full Text Available Acetylcholinesterase (AChE is an essential enzyme that terminates cholinergic transmission by rapid hydrolysis of the neurotransmitter acetylcholine. Compounds inhibiting this enzyme can be used (inter alia to treat cholinergic deficiencies (e.g. in Alzheimer's disease, but may also act as dangerous toxins (e.g. nerve agents such as sarin. Treatment of nerve agent poisoning involves use of antidotes, small molecules capable of reactivating AChE. We have screened a collection of organic molecules to assess their ability to inhibit the enzymatic activity of AChE, aiming to find lead compounds for further optimization leading to drugs with increased efficacy and/or decreased side effects. 124 inhibitors were discovered, with considerable chemical diversity regarding size, polarity, flexibility and charge distribution. An extensive structure determination campaign resulted in a set of crystal structures of protein-ligand complexes. Overall, the ligands have substantial interactions with the peripheral anionic site of AChE, and the majority form additional interactions with the catalytic site (CAS. Reproduction of the bioactive conformation of six of the ligands using molecular docking simulations required modification of the default parameter settings of the docking software. The results show that docking-assisted structure-based design of AChE inhibitors is challenging and requires crystallographic support to obtain reliable results, at least with currently available software. The complex formed between C5685 and Mus musculus AChE (C5685•mAChE is a representative structure for the general binding mode of the determined structures. The CAS binding part of C5685 could not be structurally determined due to a disordered electron density map and the developed docking protocol was used to predict the binding modes of this part of the molecule. We believe that chemical modifications of our discovered inhibitors, biochemical and biophysical

  16. [Genomic noncoding sequences and the size of eukaryotic cell nucleus as important factors of gene protection from chemical mutagens].

    Science.gov (United States)

    Minkevich, I G; Patrushev, L I

    2007-01-01

    An improved quantitative model describing a protective function of eukaryotic genomic noncoding sequences was developed. In this new model, two factors affecting gene protection from chemical mutagens are considered: (1) the ratio of the total lengths of coding and noncoding genomic sequences and (2) the volume of the cell nucleus. An increase in the noncoding DNA in the genome reduces the number of mutagen-damaged nucleotides in the coding region, whereas an increase in the volume of the nucleus decreases the flow of mutagens per unit of nuclear volume that attacks its surface.

  17. The genetics of multiple sclerosis: principles, background and updated results of the United Kingdom systematic genome screen.

    Science.gov (United States)

    Chataway, J; Feakes, R; Coraddu, F; Gray, J; Deans, J; Fraser, M; Robertson, N; Broadley, S; Jones, H; Clayton, D; Goodfellow, P; Sawcer, S; Compston, A

    1998-10-01

    Genetic susceptibility to multiple sclerosis is implicated on the basis of classical family studies and phenotype analyses. The only reproducible legacy from the candidate gene approach has been the discovery of population associations with alleles of the major histocompatibility complex. Systematic genome scanning has since been applied using a panel of anonymous markers to identify areas of linkage in co-affected siblings. Here, we describe the principles of genome screening and update the UK survey of multiple sclerosis. This identified 20 regions of potential interest, but in none was there unequivocal linkage. In theory, attempting to replicate these findings in a second set of sibling pair families is the most appropriate way to distinguish true from false positives, but unfortunately the number of families required to do this reliably is prohibitively large. We used three approaches to increase the definition achieved by the screen: (i) the number of sibling pairs typed in an identified region of potential linkage was extended; (ii) the information extraction was increased in an identified region; and (iii) a search was made for missed regions of potential linkage. Each of these approaches has considerable limitations. A chromosome-by-chromosome account is given to direct future searches. Although an additional marker placed distal to the 'hit' on chromosome 14q increased linkage in this area, and typing extra sibling pairs increased linkage on chromosomes 6p and 17q, evidence for linkage was more commonly reduced and no additional regions of interest were found. A further refinement of the genome screen was undertaken by conditioning for the presence of HLA-DR15. This produced a surprising degree of segregation among the regions of interest, which divided into two distinct groups depending on DR15 sharing: the DR15-sharing cohort comprised loci on chromosomal areas 1p, 17q and X; and the DR15-non-sharing cohort was made up of loci on 1cen, 3p, 7p, 14q and

  18. A cell-free testing platform to screen chemicals of potential neurotoxic concern across twenty vertebrate species.

    Science.gov (United States)

    Arini, Adeline; Mittal, Krittika; Dornbos, Peter; Head, Jessica; Rutkiewicz, Jennifer; Basu, Niladri

    2017-06-08

    There is global demand for new in vitro testing tools for ecological risk assessment. The objective of the present study was to apply a set of cell-free neurochemical assays to screen many chemicals across many species in a relatively high-throughput manner. The platform assessed 7 receptors and enzymes that mediate neurotransmission of γ-aminobutyric acid, dopamine, glutamate, and acetylcholine. Each assay was optimized to work across 20 vertebrate species (5 fish, 5 birds, 7 mammalian wildlife, 3 biomedical species including humans). We tested the screening assay platform against 80 chemicals (23 pharmaceuticals and personal care products, 20 metal[loid]s, 22 polycyclic aromatic hydrocarbons and halogenated organic compounds, 15 pesticides). In total, 10 800 species-chemical-assay combinations were tested, and significant differences were found in 4041 cases. All 7 assays were significantly affected by at least one chemical in each species tested. Among the 80 chemicals tested, nearly all resulted in a significant impact on at least one species and one assay. The 5 most active chemicals were prochloraz, HgCl2 , Sn, benzo[a]pyrene, and vinclozolin. Clustering analyses revealed groupings according to chemicals, species, and chemical-assay combinations. The results show that cell-free assays can screen a large number of samples in a short period of time in a cost-effective manner in a range of animals not easily studied using traditional approaches. Strengths and limitations of this approach are discussed, as well as next steps. Environ Toxicol Chem 2017;9999:1-10. © 2017 SETAC. © 2017 SETAC.

  19. A novel chemical screening strategy in zebrafish identifies common pathways in embryogenesis and rhabdomyosarcoma development.

    Science.gov (United States)

    Le, Xiuning; Pugach, Emily K; Hettmer, Simone; Storer, Narie Y; Liu, Jianing; Wills, Airon A; DiBiase, Antony; Chen, Eleanor Y; Ignatius, Myron S; Poss, Kenneth D; Wagers, Amy J; Langenau, David M; Zon, Leonard I

    2013-06-01

    The zebrafish is a powerful genetic model that has only recently been used to dissect developmental pathways involved in oncogenesis. We hypothesized that operative pathways during embryogenesis would also be used for oncogenesis. In an effort to define RAS target genes during embryogenesis, gene expression was evaluated in Tg(hsp70-HRAS(G12V)) zebrafish embryos subjected to heat shock. dusp6 was activated by RAS, and this was used as the basis for a chemical genetic screen to identify small molecules that interfere with RAS signaling during embryogenesis. A KRAS(G12D)-induced zebrafish embryonal rhabdomyosarcoma was then used to assess the therapeutic effects of the small molecules. Two of these inhibitors, PD98059 and TPCK, had anti-tumor activity as single agents in both zebrafish embryonal rhabdomyosarcoma and a human cell line of rhabdomyosarcoma that harbored activated mutations in NRAS. PD98059 inhibited MEK1 whereas TPCK suppressed S6K1 activity; however, the combined treatment completely suppressed eIF4B phosphorylation and decreased translation initiation. Our work demonstrates that the activated pathways in RAS induction during embryogenesis are also important in oncogenesis and that inhibition of these pathways suppresses tumor growth.

  20. Zebrafish chemical screening reveals the impairment of dopaminergic neuronal survival by cardiac glycosides.

    Directory of Open Access Journals (Sweden)

    Yaping Sun

    Full Text Available Parkinson's disease is a neurodegenerative disorder characterized by the prominent degeneration of dopaminergic (DA neurons among other cell types. Here we report a first chemical screen of over 5,000 compounds in zebrafish, aimed at identifying small molecule modulators of DA neuron development or survival. We find that Neriifolin, a member of the cardiac glycoside family of compounds, impairs survival but not differentiation of both zebrafish and mammalian DA neurons. Cardiac glycosides are inhibitors of Na(+/K(+ ATPase activity and widely used for treating heart disorders. Our data suggest that Neriifolin impairs DA neuronal survival by targeting the neuronal enriched Na(+/K(+ ATPase α3 subunit (ATP1A3. Modulation of ionic homeostasis, knockdown of p53, or treatment with antioxidants protects DA neurons from Neriifolin-induced death. These results reveal a previously unknown effect of cardiac glycosides on DA neuronal survival and suggest that it is mediated through ATP1A3 inhibition, oxidative stress, and p53. They also elucidate potential approaches for counteracting the neurotoxicity of this valuable class of medications.

  1. Antibiotic pigment from desert soil actinomycetes; biological activity, purification and chemical screening

    Directory of Open Access Journals (Sweden)

    Selvameenal L

    2009-01-01

    Full Text Available An actinomycete strain, Streptomyces hygroscopicus subsp. ossamyceticus (strain D10 was isolated from Thar Desert soil, Rajasthan during the year 2006 and found to produce a yellow color pigment with antibiotic activity. Crude pigment was produced from strain D10 by solid state fermentation using wheat bran medium followed by extraction with ethyl acetate. The antimicrobial activity of the crude pigment was evaluated against drug resistant pathogens such as methicillin-resistant Staphylococcus aureus, vancomycin-resistant Staphylococcus aureus, extended spectrum b-lactamase producing cultures of Escherichia coli, Pseudomonas aeruginosa and Klebsiella sp. About 420 mg of crude pigment was produced per 10 g of wheat bran medium. In the disc diffusion method the crude ethyl acetate extract showed a minimum of 10 mm inhibition against Klebsiella sp. and maximum of 19 mm of inhibition against Escherichia coli. The crude pigment was partially purified using thin layer chromatography with the solvent system chloroform:methanol (30:70 and the Rf value was calculated as 0.768. Antimicrobial activity of the partially purified compound from thin layer chromatography was determined using the bioautography method. The purified pigment showed minimum of 15 mm inhibition against Klebsiella sp. and a maximum of 23 mm of inhibition against vancomycin-resistant Staphylococcus aureus in the disc diffusion method. Based on the results of chemical screening, the pigment was tentatively identified as group of sugar containing molecules.

  2. Volatile terpenes from actinomycetes: a biosynthetic study correlating chemical analyses to genome data.

    Science.gov (United States)

    Rabe, Patrick; Citron, Christian A; Dickschat, Jeroen S

    2013-11-25

    The volatile terpenes of 24 actinomycetes whose genomes have been sequenced (or are currently being sequenced) were collected by use of a closed-loop stripping apparatus and identified by GC/MS. The analytical data were compared against a phylogenetic analysis of all 192 currently available sequences of bacterial terpene cyclases (excluding geosmin and 2-methylisoborneol synthases). In addition to the several groups of terpenes with known biosynthetic origin, selinadienes were identified as a large group of biosynthetically related sesquiterpenes that are produced by several streptomycetes. The detection of a large number of previously unrecognised side products of known terpene cyclases proved to be particularly important for an in depth understanding of biosynthetic pathways to known terpenes in actinomycetes. Interpretation of the chemical analytical data in the context of the phylogenetic tree of bacterial terpene cyclases pointed to the function of three new enzymes: (E)-β-caryophyllene synthase, selina-3,7(11)-diene synthase and aristolochene synthase.

  3. Expanded roles of leucine-responsive regulatory protein in transcription regulation of the Escherichia coli genome: Genomic SELEX screening of the regulation targets

    Science.gov (United States)

    Saito, Natsumi; Maeda, Michihisa; Tanaka, Kan; Ishihama, Akira

    2015-01-01

    Leucine-responsive regulatory protein (Lrp) is a transcriptional regulator for the genes involved in transport, biosynthesis and catabolism of amino acids in Escherichia coli. In order to identify the whole set of genes under the direct control of Lrp, we performed Genomic SELEX screening and identified a total of 314 Lrp-binding sites on the E. coli genome. As a result, the regulation target of Lrp was predicted to expand from the hitherto identified genes for amino acid metabolism to a set of novel target genes for utilization of amino acids for protein synthesis, including tRNAs, aminoacyl-tRNA synthases and rRNAs. Northern blot analysis indicated alteration of mRNA levels for at least some novel targets, including the aminoacyl-tRNA synthetase genes. Phenotype MicroArray of the lrp mutant indicated significant alteration in utilization of amino acids and peptides, whilst metabolome analysis showed variations in the concentration of amino acids in the lrp mutant. From these two datasets we realized a reverse correlation between amino acid levels and cell growth rate: fast-growing cells contain low-level amino acids, whilst a high level of amino acids exists in slow-growing cells. Taken together, we propose that Lrp is a global regulator of transcription of a large number of the genes involved in not only amino acid transport and metabolism, but also amino acid utilization. PMID:28348809

  4. Screening and breeding of high taxol producing fungi by genome shuffling

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    To apply the fundamental principles of genome shuffling in breeding of taxol-producing fungi, Nodulisporium sylviform was used as starting strain in this work. The procedures of protoplast fusion and genome shuffling were studied. Three hereditarily stable strains with high taxol production were obtained by four cycles of genome shuffling. The qualitative and quantitative analysis of taxol produced was confirmed using thin-layer chromatography (TLC), high performance liquid chromatography (HPLC) and LC-MS. A high taxol producing fungus, Nodulisporium sylviform F4-26, was obtained, which produced 516.37 μg/L taxol. This value is 64.41% higher than that of the starting strain NCEU-1 and 31.52%―44.72% higher than that of the parent strains.

  5. Screening and breeding of high taxol producing fungi by genome shuffling

    Institute of Scientific and Technical Information of China (English)

    ZHAO Kai; PING WenXiang; ZHANG LiNa; LIU Jun; LIN Yan; JIN Tao; ZHOU DongPo

    2008-01-01

    To apply the fundamental principles of genome shuffling in breeding of taxol-producing fungi,Nodulisporium sylviform was used as starting strain in this work. The procedures of protoplast fusion and genome shuffling were studied. Three hereditarily stable strains with high taxol production were obtained by four cycles of genome shuffling. The qualitative and quantitative analysis of taxol produced was confirmed using thin-layer chromatography (TLC), high performance liquid chromatography (HPLC) and LC-MS. A high taxol producing fungus, Nodulisporlum sylviform F4-26, was obtained,which produced 516.37 μg/L taxol. This value is 64.41% higher than that of the starting strain NCEU-1 and 31.52%-44.72% higher than that of the parent strains.

  6. Epigenome: A Biomarker or Screening Tool to Evaluate Health Impact of Cumulative Exposure to Chemical and Non-Chemical Stressors.

    Science.gov (United States)

    Olden, Kenneth; Lin, Yu-Sheng; Bussard, David

    2016-06-01

    Current risk assessment practices and toxicity information are hard to utilize for assessing the health impact of combined or cumulative exposure to multiple chemical and non-chemical stressors encountered in the "real world" environment. Non-chemical stressors such as heat, radiation, noise, humidity, bacterial and viral agents, and social factors, like stress related to violence and socioeconomic position generally cannot be currently incorporated into the risk assessment paradigm. The Science and Decisions report released by the National Research Council (NRC) in 2009 emphasized the need to characterize the effects of multiple stressors, both chemical and non-chemical exposures. One impediment to developing information relating such non-chemical stressors to health effects and incorporating them into cumulative assessment has been the lack of analytical tools to easily and quantitatively monitor the cumulative exposure to combined effects of stressors over the life course.

  7. Tankyrase 1 Inhibitors with Drug-like Properties Identified by Screening a DNA-Encoded Chemical Library.

    Science.gov (United States)

    Samain, Florent; Ekblad, Torun; Mikutis, Gediminas; Zhong, Nan; Zimmermann, Mauro; Nauer, Angela; Bajic, Davor; Decurtins, Willy; Scheuermann, Jörg; Brown, Peter J; Hall, Jonathan; Gräslund, Susanne; Schüler, Herwig; Neri, Dario; Franzini, Raphael M

    2015-06-25

    We describe the synthesis and screening of a DNA-encoded chemical library containing 76230 compounds. In this library, sets of amines and carboxylic acids are directly linked producing encoded compounds with compact structures and drug-like properties. Affinity screening of this library yielded inhibitors of the potential pharmaceutical target tankyrase 1, a poly(ADP-ribose) polymerase. These compounds have drug-like characteristics, and the most potent hit compound (X066/Y469) inhibited tankyrase 1 with an IC50 value of 250 nM.

  8. Genome-Wide Screening of Cytogenetic Abnormalities in Multiple Myeloma Patients Using Array-CGH Technique: A Czech Multicenter Experience

    Directory of Open Access Journals (Sweden)

    Jan Smetana

    2014-01-01

    Full Text Available Characteristic recurrent copy number aberrations (CNAs play a key role in multiple myeloma (MM pathogenesis and have important prognostic significance for MM patients. Array-based comparative genomic hybridization (aCGH provides a powerful tool for genome-wide classification of CNAs and thus should be implemented into MM routine diagnostics. We demonstrate the possibility of effective utilization of oligonucleotide-based aCGH in 91 MM patients. Chromosomal aberrations associated with effect on the prognosis of MM were initially evaluated by I-FISH and were found in 93.4% (85/91. Incidence of hyperdiploidy was 49.5% (45/91; del(13(q14 was detected in 57.1% (52/91; gain(1(q21 occurred in 58.2% (53/91; del(17(p13 was observed in 15.4% (14/91; and t(4;14(p16;q32 was found in 18.6% (16/86. Genome-wide screening using Agilent 44K aCGH microarrays revealed copy number alterations in 100% (91/91. Most common deletions were found at 13q (58.9%, 1p (39.6%, and 8p (31.1%, whereas gain of whole 1q was the most often duplicated region (50.6%. Furthermore, frequent homozygous deletions of genes playing important role in myeloma biology such as TRAF3, BIRC1/BIRC2, RB1, or CDKN2C were observed. Taken together, we demonstrated the utilization of aCGH technique in clinical diagnostics as powerful tool for identification of unbalanced genomic abnormalities with prognostic significance for MM patients.

  9. Screening of potential pseudo att sites of Streptomyces phage ΦC31 integrase in the human genome

    Institute of Scientific and Technical Information of China (English)

    Zhi-peng HU; Lu-sheng CHEN; Cai-yan JIA; Huan-zhang ZHU; Wei WANG; Jiang ZHONG

    2013-01-01

    Aim:ΦC31 integrase mediates site-specific recombination between two short sequences,attP and attB,in phage and bacterial genomes,which is a promising tool in gene regulation-based therapy since the zinc finger structure is probably the DNA recognizing domain that can further be engineered.The aim of this study was to screen potential pseudo att sites of ΦC31 integrase in the human genome,and evaluate the risks of its application in human gene therapy.Methods:TFBS (transcription factor binding sites) were found on the basis of reported pseudo att sites using multiple motif-finding tools,including AlignACE,BioProspector,Consensus,MEME,and Weeder.The human genome with the proposed motif was scanned tc find the potential pseudo att sites of ΦC31 integrase.Results:The possible recognition motif of ΦC31 integrase was identified,which was composed of two co-occurrence conserved elements that were reverse complement to each other flanking the core sequence TTG.In the human genome,a total of 27924 potential pseudo att sites of ΦC31 integrase were found,which were distributed in each human chromosome with high-risk specificity values in the chromosomes 16,17,and 19.When the risks of the sites were evaluate more rigorously,53 hits were discovered,and some of them were just the vital functional genes or regulatory regions,such as ACYP2,AKR1B1,DUSP4,etc.Conclusion:The results provide clues for more comprehensive evaluation of the risks of using ΦC31 integrase in human gene therapy and for drug discovery.

  10. In vitro OECD test methods applied to screen the estrogenic effect of chemicals, used in Korea.

    Science.gov (United States)

    Lee, Hee-Seok; Park, Eun-Jung; Han, Songyi; Oh, Gyeong-Yong; Kim, Min-Hee; Kang, Hui-Seung; Suh, Jin-Hyang; Oh, Jae-Ho; Lee, Kwang-Soo; Hwang, Myung-Sil; Moon, Guiim; Hong, Jin-Hwan; Hwang, In-Gyun

    2016-09-01

    In this study, 27 chemicals found in household products, which became an issue in Korea were screened for the agonistoc and antagonistic effects against human estrogen receptor using official Organization for Economic Cooperation and Development (OECD) in vitro assays, STTA assay using ERα-HeLa-9903 cell line and BG1Luc ER TA assay. In the case of human ER agonist screening by two assays, all tested chemicals did not show agonist effect against ER. In ER antagonist test by BG1Luc ER TA assay, five surfactants α-dodecyl-ω-hydroxypoly(oxyethylene), alcohols C16-18 ethoxylated, nonylphenol, ethoxylated, 3,6,9,12,15,18,21-heptaoxatritriacontan-1-ol, and α-dodecyl-ω-hydroxypoly(oxy-1,2-ethanediyl)) were found to exhibit weak antagonistic activities. The agonist/antagonist effects against human estrogen receptor of various chemicals, used in Korea by OECD test guideline are reported in this study. These results indicated that two OECD in vitro assays will can be applied in Korea by screening of agonistic/antagonistic effects against human ER of various chemicals. Copyright © 2016 Elsevier Ltd. All rights reserved.

  11. Genome-Wide Synthetic Lethal Screens Identify an Interaction Between the Nuclear Envelope Protein, Apq12p, and the Kinetochore in Saccharomyces cerevisiae

    OpenAIRE

    Montpetit, Ben; Thorne, Ken; Barrett, Irene; Andrews, Kim; Jadusingh, Ravi; Hieter, Phil; Measday, Vivien

    2005-01-01

    The maintenance of genome stability is a fundamental requirement for normal cell cycle progression. The budding yeast Saccharomyces cerevisiae is an excellent model to study chromosome maintenance due to its well-defined centromere and kinetochore, the region of the chromosome and associated protein complex, respectively, that link chromosomes to microtubules. To identify genes that are linked to chromosome stability, we performed genome-wide synthetic lethal screens using a series of novel t...

  12. Genome-wide RNAi screen reveals the E3 SUMO-protein ligase gene SIZ1 as a novel determinant of furfural tolerance in Saccharomyces cerevisiae

    OpenAIRE

    Xiao, Han; Zhao, Huimin

    2014-01-01

    Background Furfural is a major growth inhibitor in lignocellulosic hydrolysates and improving furfural tolerance of microorganisms is critical for rapid and efficient fermentation of lignocellulosic biomass. In this study, we used the RNAi-Assisted Genome Evolution (RAGE) method to select for furfural resistant mutants of Saccharomyces cerevisiae, and identified a new determinant of furfural tolerance. Results By using a genome-wide RNAi (RNA-interference) screen in S. cerevisiae for genes in...

  13. Prediction of cancer cell sensitivity to natural products based on genomic and chemical properties.

    Science.gov (United States)

    Yue, Zhenyu; Zhang, Wenna; Lu, Yongming; Yang, Qiaoyue; Ding, Qiuying; Xia, Junfeng; Chen, Yan

    2015-01-01

    Natural products play a significant role in cancer chemotherapy. They are likely to provide many lead structures, which can be used as templates for the construction of novel drugs with enhanced antitumor activity. Traditional research approaches studied structure-activity relationship of natural products and obtained key structural properties, such as chemical bond or group, with the purpose of ascertaining their effect on a single cell line or a single tissue type. Here, for the first time, we develop a machine learning method to comprehensively predict natural products responses against a panel of cancer cell lines based on both the gene expression and the chemical properties of natural products. The results on two datasets, training set and independent test set, show that this proposed method yields significantly better prediction accuracy. In addition, we also demonstrate the predictive power of our proposed method by modeling the cancer cell sensitivity to two natural products, Curcumin and Resveratrol, which indicate that our method can effectively predict the response of cancer cell lines to these two natural products. Taken together, the method will facilitate the identification of natural products as cancer therapies and the development of precision medicine by linking the features of patient genomes to natural product sensitivity.

  14. Prediction of cancer cell sensitivity to natural products based on genomic and chemical properties

    Directory of Open Access Journals (Sweden)

    Zhenyu Yue

    2015-11-01

    Full Text Available Natural products play a significant role in cancer chemotherapy. They are likely to provide many lead structures, which can be used as templates for the construction of novel drugs with enhanced antitumor activity. Traditional research approaches studied structure-activity relationship of natural products and obtained key structural properties, such as chemical bond or group, with the purpose of ascertaining their effect on a single cell line or a single tissue type. Here, for the first time, we develop a machine learning method to comprehensively predict natural products responses against a panel of cancer cell lines based on both the gene expression and the chemical properties of natural products. The results on two datasets, training set and independent test set, show that this proposed method yields significantly better prediction accuracy. In addition, we also demonstrate the predictive power of our proposed method by modeling the cancer cell sensitivity to two natural products, Curcumin and Resveratrol, which indicate that our method can effectively predict the response of cancer cell lines to these two natural products. Taken together, the method will facilitate the identification of natural products as cancer therapies and the development of precision medicine by linking the features of patient genomes to natural product sensitivity.

  15. Protein structure similarity clustering (PSSC) and natural product structure as inspiration sources for drug development and chemical genomics

    NARCIS (Netherlands)

    Dekker, Frank J; Koch, Marcus A; Waldmann, Herbert; Dekker, Frans

    Finding small molecules that modulate protein function is of primary importance in drug development and in the emerging field of chemical genomics. To facilitate the identification of such molecules, we developed a novel strategy making use of structural conservatism found in protein domain

  16. Protein structure similarity clustering (PSSC) and natural product structure as inspiration sources for drug development and chemical genomics

    NARCIS (Netherlands)

    Dekker, Frank J; Koch, Marcus A; Waldmann, Herbert; Dekker, Frans

    2005-01-01

    Finding small molecules that modulate protein function is of primary importance in drug development and in the emerging field of chemical genomics. To facilitate the identification of such molecules, we developed a novel strategy making use of structural conservatism found in protein domain architec

  17. Microwave-assisted chemical insertion: a rapid technique for screening cathodes for Mg-ion batteries

    Energy Technology Data Exchange (ETDEWEB)

    Kaveevivitchai, Watchareeya; Huq, Ashfia; Manthiram, Arumugam

    2016-12-19

    We report an ultrafast microwave-assisted solvothermal method for chemical insertion of Mg2+ ions into host materials using magnesium acetate [Mg(CH3COO)2] as a metal-ion source and diethylene glycol (DEG) as a reducing agent. For instance, up to 3 Mg ions per formula unit of a microporous host framework Mo2.5+yVO9+z could be inserted in as little as 30 min at 170–195 °C in air. This process is superior to the traditional method which involves the use of organometallic reagents, such as di-n-butylmagnesium [(C4H9)2Mg] and magnesium bis(2,6-di-tert-butylphenoxide) [Mg-(O-2,6-But2C6H3)2], and requires an inert atmosphere with extremely long reaction times. Considering the lack of robust electrolytes for Mg-ion batteries, this facile approach can be readily used as a rapid screening technique to identify potential Mg-ion electrode hosts without the necessity of fabricating electrodes and assembling electrochemical cells. Due to the mild reaction conditions, the overall structure and morphology of the Mg-ion inserted products are maintained and the compounds can be used successfully as a cathode in Mg-ion batteries. The combined synchrotron X-ray and neutron diffraction Rietveld analysis reveals the structure of the Mg-inserted compounds and gives an insight into the interactions between the Mg ions and the open-tunnel host framework.

  18. Draft Genome Sequence of Colletotrichum falcatum - A Prelude on Screening of Red Rot Pathogen in Sugarcane.

    Science.gov (United States)

    Viswanathan, Rasappa; Prasanth, Chandrasekaran Naveen; Malathi, Palaniyandi; Sundar, Amalraj Ramesh

    2016-01-01

    Colletotrichum falcatum, a concealed fungal ascomycete causes red rot, which is a serious disease in sugarcane. It infects economically important stalk tissues, considered as store house of sugar in sugarcane. The study is to find genetic complexities of C. falcatum in establishing this as a stalk infecting pathogen and to decipher the unique lifestyle of this pathogen using NGS technology. We report the draft genome of C. falcatum of about 48.16 Mb in size with 12,270 genes. The genome sequences were compared with other fungal species which revealed that C. falcatum is closely related to C. graminicola and C.sublineola the causal organisms of anthracnose in maize and sorghum. These results brought a new revelation to explore the lifestyle of this unique pathogen which is specialized to infect sugarcane stalk tissues in detail.

  19. Simulated Screens of DNA Encoded Libraries: The Potential Influence of Chemical Synthesis Fidelity on Interpretation of Structure-Activity Relationships.

    Science.gov (United States)

    Satz, Alexander L

    2016-07-11

    Simulated screening of DNA encoded libraries indicates that the presence of truncated byproducts complicates the relationship between library member enrichment and equilibrium association constant (these truncates result from incomplete chemical reactions during library synthesis). Further, simulations indicate that some patterns observed in reported experimental data may result from the presence of truncated byproducts in the library mixture and not structure-activity relationships. Potential experimental methods of minimizing the presence of truncates are assessed via simulation; the relationship between enrichment and equilibrium association constant for libraries of differing purities is investigated. Data aggregation techniques are demonstrated that allow for more accurate analysis of screening results, in particular when the screened library contains significant quantities of truncates.

  20. Chemical Genetic Screens for TDP-43 Modifiers and ALS Drug Discovery

    Science.gov (United States)

    2015-03-01

    CRISPR -Cas9 method (Friedland et al., 2013) instead to ensure that transgenic lines have a similar level of expression from the same genomic location...Calarco, J. A. (2013). Heritable genome editing in C. elegans via a CRISPR -Cas9 system. Nat. Meth. 10, 741–743. doi: 10.1038/nmeth.2532 Frøkjaer

  1. Genome-wide RNAi Screen Identifies SEC61A and VCP as Conserved Regulators of Sindbis Virus Entry

    Directory of Open Access Journals (Sweden)

    Debasis Panda

    2013-12-01

    Full Text Available Alphaviruses are a large class of insect-borne human pathogens and little is known about the host-factor requirements for infection. To identify such factors, we performed a genome-wide RNAi screen using model Drosophila cells and validated 94 genes that impacted infection of Sindbis virus (SINV, the prototypical alphavirus. We identified a conserved role for SEC61A and valosin-containing protein (VCP in facilitating SINV entry in insects and mammals. SEC61A and VCP selectively regulate trafficking of the entry receptor NRAMP2, and loss or pharmacological inhibition of these proteins leads to altered NRAMP2 trafficking to lysosomal compartments and proteolytic digestion within lysosomes. NRAMP2 is the major iron transporter in cells, and loss of NRAMP2 attenuates intracellular iron transport. Thus, this study reveals genes and pathways involved in both infection and iron homeostasis that may serve as targets for antiviral therapeutics or for iron-imbalance disorders.

  2. Genome-wide screening of Saccharomyces cerevisiae genes required to foster tolerance towards industrial wheat straw hydrolysates.

    Science.gov (United States)

    Pereira, Francisco B; Teixeira, Miguel C; Mira, Nuno P; Sá-Correia, Isabel; Domingues, Lucília

    2014-12-01

    The presence of toxic compounds derived from biomass pre-treatment in fermentation media represents an important drawback in second-generation bio-ethanol production technology and overcoming this inhibitory effect is one of the fundamental challenges to its industrial production. The aim of this study was to systematically identify, in industrial medium and at a genomic scale, the Saccharomyces cerevisiae genes required for simultaneous and maximal tolerance to key inhibitors of lignocellulosic fermentations. Based on the screening of EUROSCARF haploid mutant collection, 242 and 216 determinants of tolerance to inhibitory compounds present in industrial wheat straw hydrolysate (WSH) and in inhibitor-supplemented synthetic hydrolysate were identified, respectively. Genes associated to vitamin metabolism, mitochondrial and peroxisomal functions, ribosome biogenesis and microtubule biogenesis and dynamics are among the newly found determinants of WSH resistance. Moreover, PRS3, VMA8, ERG2, RAV1 and RPB4 were confirmed as key genes on yeast tolerance and fermentation of industrial WSH.

  3. The promises of genomic screening: building a governance infrastructure. Special issue: genetics and democracy.

    Science.gov (United States)

    Cornel, Martina C; van El, Carla G; Dondorp, Wybo J

    2012-04-01

    New screening possibilities become available at a high rate, both useful and unsound possibilities. All screening programmes do harm, and only few have more advantages than disadvantages at reasonable cost. Horizon scanning is needed to identify those few possibilities with more pros than cons. Attunement is needed between actors involved: scientists developing new high-throughput screening techniques and treatment, health care workers, patients and consumers and governmental agencies. The product of a process of attunement may be a quality mark as a norm for professional conduct, rather than legal measures, as the field is moving fast. As actors may have varying perspectives, a governance structure is needed to develop an agenda that is agreed upon by all or most actors involved. A standing committee might oversee the evaluation of benefits and disadvantages in an integrated approach, taking evidence, economics and ethics into account. A proactive role of governmental agencies is needed to facilitate agenda setting and attunement. Policy making has to be transparent and open to stakeholder engagement.

  4. Generation of chicken Z-chromosome painting probes by microdissection for screening large-insert genomic libraries.

    Science.gov (United States)

    Zimmer, R; King, W A; Verrinder Gibbins, A M

    1997-01-01

    A strategy for rapid generation of chicken sex chromosome-Z painting probes has been developed using microdissection. Whole chromosome painting probes (WCPs) were prepared from 10-15 copies of mitotic metaphase chicken Z chromosomes. The microisolated chromosomes were subjected to PEG/proteinase K treatment in a collection drop to release DNA, which was then amplified using a degenerate oligonucleotide-primed shuttle PCR (DOP-Shuttle-PCR) strategy. Size distributions of the PCR products were analyzed by agarose gel electrophoresis and smears of DNA were revealed that ranged in size from 200-800 bp, without any evidence of preferential amplification. Both specificity and complexity of the probes have been analyzed by Southern blot and fluorescence in situ hybridization (FISH). Non-specific hybridization was efficiently blocked by using chicken competitor DNA. Analysis of the WCPs produced shows that collectively they provide uniform hybridization signals along the entire length of the chicken Z chromosome. To demonstrate one possible application of these complex probes, we screened a large-insert bacterial artificial chromosome (BAC) chicken genomic library to select Z chromosome-specific clones. To address specificity of the selected clones and to physically map them to the Z chromosome, FISH analysis was used. Of the 3 clones initially tested, one clone (C3) carrying a 250-kb insert mapped to the distal portion of the short arm of the chicken Z chromosome. Therefore, this technique has provided appropriate probes for screening large-insert genomic libraries. Further application of these probes includes the analysis of chromosome rearrangements, studies of cases of heteroploidy involving the Z chromosome, positional cloning of Z-linked genes and studies on mechanisms of sex-chromosome evolution in birds.

  5. Genetic screening of new genes responsible for cellular adaptation to hypoxia using a genome-wide shRNA library.

    Science.gov (United States)

    Yoshino, Seiko; Hara, Toshiro; Weng, Jane S; Takahashi, Yuka; Seiki, Motoharu; Sakamoto, Takeharu

    2012-01-01

    Oxygen is a vital requirement for multi-cellular organisms to generate energy and cells have developed multiple compensatory mechanisms to adapt to stressful hypoxic conditions. Such adaptive mechanisms are intricately interconnected with other signaling pathways that regulate cellular functions such as cell growth. However, our understanding of the overall system governing the cellular response to the availability of oxygen remains limited. To identify new genes involved in the response to hypoxic stress, we have performed a genome-wide gene knockdown analysis in human lung carcinoma PC8 cells using an shRNA library carried by a lentiviral vector. The knockdown analysis was performed under both normoxic and hypoxic conditions to identify shRNA sequences enriched or lost in the resulting selected cell populations. Consequently, we identified 56 candidate genes that might contribute to the cellular response to hypoxia. Subsequent individual knockdown of each gene demonstrated that 13 of these have a significant effect upon oxygen-sensitive cell growth. The identification of BCL2L1, which encodes a Bcl-2 family protein that plays a role in cell survival by preventing apoptosis, validates the successful design of our screen. The other selected genes have not previously been directly implicated in the cellular response to hypoxia. Interestingly, hypoxia did not directly enhance the expression of any of the identified genes, suggesting that we have identified a new class of genes that have been missed by conventional gene expression analyses to identify hypoxia response genes. Thus, our genetic screening method using a genome-wide shRNA library and the newly-identified genes represent useful tools to analyze the cellular systems that respond to hypoxic stress.

  6. A Novel High-Throughput Screening System to Evaluate the Behavioral Response of Adult Mosquitoes to Chemicals

    Science.gov (United States)

    2005-01-01

    compact in size, easy to decontam- inate, and requires only a minute quantity of chemical compound. KEY WORDS Assay, screening, behavior, contact...female and male pupae were sorted by size, and 250, determined to be future females, were placed into individual 3.8-liter car- tons. To prepare for a...Oviposition attractants and repellents of mosquitoes: oviposition response of Culex mosquitoes to organic infusions. Environ Entomol 8: 1111-1117. McGovern

  7. Functional toxicogenomic assessment of triclosan in human HepG2 cells using genome-wide CRISPR-Cas9 screen

    Science.gov (United States)

    Thousands of chemicals for which limited toxicological data are available are used and then detected in humans and the environment. Rapid and cost-effective approaches for assessing the toxicological properties of chemicals are needed. We used CRISPR-Cas9 functional genomic scree...

  8. Use of comparative genomics approaches to characterize interspecies differences in response to environmental chemicals: Challenges, opportunities, and research needs

    Energy Technology Data Exchange (ETDEWEB)

    Burgess-Herbert, Sarah L., E-mail: sarah.burgess@alum.mit.edu [American Association for the Advancement of Science (AAAS) Science and Technology Policy Fellow at the US Environmental Protection Agency (EPA), 2009–10 (United States); Euling, Susan Y. [National Center for Environmental Assessment, Office of Research and Development, US Environmental Protection Agency, Washington, DC 20460 (United States)

    2013-09-15

    A critical challenge for environmental chemical risk assessment is the characterization and reduction of uncertainties introduced when extrapolating inferences from one species to another. The purpose of this article is to explore the challenges, opportunities, and research needs surrounding the issue of how genomics data and computational and systems level approaches can be applied to inform differences in response to environmental chemical exposure across species. We propose that the data, tools, and evolutionary framework of comparative genomics be adapted to inform interspecies differences in chemical mechanisms of action. We compare and contrast existing approaches, from disciplines as varied as evolutionary biology, systems biology, mathematics, and computer science, that can be used, modified, and combined in new ways to discover and characterize interspecies differences in chemical mechanism of action which, in turn, can be explored for application to risk assessment. We consider how genetic, protein, pathway, and network information can be interrogated from an evolutionary biology perspective to effectively characterize variations in biological processes of toxicological relevance among organisms. We conclude that comparative genomics approaches show promise for characterizing interspecies differences in mechanisms of action, and further, for improving our understanding of the uncertainties inherent in extrapolating inferences across species in both ecological and human health risk assessment. To achieve long-term relevance and consistent use in environmental chemical risk assessment, improved bioinformatics tools, computational methods robust to data gaps, and quantitative approaches for conducting extrapolations across species are critically needed. Specific areas ripe for research to address these needs are recommended.

  9. Screening of a chemical library by HT-G4-FID for discovery of selective G-quadruplex binders.

    Science.gov (United States)

    Largy, Eric; Saettel, Nicolas; Hamon, Florian; Dubruille, Sylvie; Teulade-Fichou, Marie-Paule

    2012-01-01

    Due to the lack of structural guidelines about G-quadruplex ligands, rational design cannot be the only approach to discover potent G4-ligands. As a complementary approach, screening of chemical library may provide interesting scaffolds known as hits provided that specific tools are available. In this work, the Institut Curie-CNRS chemical library was firstly screened by chemoinformatics methods. Similarity estimations by comparison with reference compounds (Phen-DC3, 360A, MMQ12) provided a set of molecules, which were then evaluated by high-throughput G4-FID (HT-G4-FID) against various G-quadruplex DNA. A full investigation of the most interesting molecules, using the HT-G4-FID assay and molecular modeling, supplied an interesting structure-activity relationship confirming the efficiency of this general approach. Overall, we demonstrated that HT-G4-FID coupled with screening of chemical libraries is a powerful tool to identify new G4-DNA binding scaffolds.

  10. New natural products isolated from Metarhizium robertsii ARSEF 23 by chemical screening and identification of the gene cluster through engineered biosynthesis in Aspergillus nidulans A1145.

    Science.gov (United States)

    Kato, Hiroki; Tsunematsu, Yuta; Yamamoto, Tsuyoshi; Namiki, Takuya; Kishimoto, Shinji; Noguchi, Hiroshi; Watanabe, Kenji

    2016-07-01

    To rapidly identify novel natural products and their associated biosynthetic genes from underutilized and genetically difficult-to-manipulate microbes, we developed a method that uses (1) chemical screening to isolate novel microbial secondary metabolites, (2) bioinformatic analyses to identify a potential biosynthetic gene cluster and (3) heterologous expression of the genes in a convenient host to confirm the identity of the gene cluster and the proposed biosynthetic mechanism. The chemical screen was achieved by searching known natural product databases with data from liquid chromatographic and high-resolution mass spectrometric analyses collected on the extract from a target microbe culture. Using this method, we were able to isolate two new meroterpenes, subglutinols C (1) and D (2), from an entomopathogenic filamentous fungus Metarhizium robertsii ARSEF 23. Bioinformatics analysis of the genome allowed us to identify a gene cluster likely to be responsible for the formation of subglutinols. Heterologous expression of three genes from the gene cluster encoding a polyketide synthase, a prenyltransferase and a geranylgeranyl pyrophosphate synthase in Aspergillus nidulans A1145 afforded an α-pyrone-fused uncyclized diterpene, the expected intermediate of the subglutinol biosynthesis, thereby confirming the gene cluster to be responsible for the subglutinol biosynthesis. These results indicate the usefulness of our methodology in isolating new natural products and identifying their associated biosynthetic gene cluster from microbes that are not amenable to genetic manipulation. Our method should facilitate the natural product discovery efforts by expediting the identification of new secondary metabolites and their associated biosynthetic genes from a wider source of microbes.

  11. The Karyote physico-chemical genomic, proteomic, metabolic cell modeling system.

    Science.gov (United States)

    Ortoleva, P; Berry, E; Brun, Y; Fan, J; Fontus, M; Hubbard, K; Jaqaman, K; Jarymowycz, L; Navid, A; Sayyed-Ahmad, A; Shreif, Z; Stanley, F; Tuncay, K; Weitzke, E; Wu, L-C

    2003-01-01

    Modeling approaches to the dynamics of a living cell are presented that are strongly based on its underlying physical and chemical processes and its hierarchical spatio-temporal organization. Through the inclusion of a broad spectrum of processes and a rigorous analysis of the multiple scale nature of cellular dynamics, we are attempting to advance cell modeling and its applications. The presentation focuses on our cell modeling system, which integrates data archiving and quantitative physico-chemical modeling and information theory to provide a seamless approach to the modeling/data analysis endeavor. Thereby the rapidly growing mess of genomic, proteomic, metabolic, and cell physiological data can be automatically used to develop and calibrate a predictive cell model. The discussion focuses on the Karyote cell modeling system and an introduction to the CellX and VirusX models. The Karyote software system integrates three elements: (1) a model-building and data archiving module that allows one to define a cell type to be modeled through its reaction network, structure, and transport processes as well as to choose the surrounding medium and other parameters of the phenomenon to be modeled; (2) a genomic, proteomic, metabolic cell simulator that solves the equations of metabolic reaction, transcription/translation polymerization and the exchange of molecules between parts of the cell and with the surrounding medium; and (3) an information theory module (ITM) that automates model calibration and development, and integrates a variety of data types with the cell dynamic computations. In Karyote, reactions may be fast (equilibrated) or slow (finite rate), and the special effects of enzymes and other minority species yielding steady-state cycles of arbitrary complexities are accounted for. These features of the dynamics are handled via rigorous multiple scale analysis. A user interface allows for an automated generation and solution of the equations of multiple timescale

  12. Leishmania genome analysis and high-throughput immunological screening identifies tuzin as a novel vaccine candidate against visceral leishmaniasis.

    Science.gov (United States)

    Lakshmi, Bhavana Sethu; Wang, Ruobing; Madhubala, Rentala

    2014-06-24

    Leishmaniasis is a neglected tropical disease caused by Leishmania species. It is a major health concern affecting 88 countries and threatening 350 million people globally. Unfortunately, there are no vaccines and there are limitations associated with the current therapeutic regimens for leishmaniasis. The emerging cases of drug-resistance further aggravate the situation, demanding rapid drug and vaccine development. The genome sequence of Leishmania, provides access to novel genes that hold potential as chemotherapeutic targets or vaccine candidates. In this study, we selected 19 antigenic genes from about 8000 common Leishmania genes based on the Leishmania major and Leishmania infantum genome information available in the pathogen databases. Potential vaccine candidates thus identified were screened using an in vitro high throughput immunological platform developed in the laboratory. Four candidate genes coding for tuzin, flagellar glycoprotein-like protein (FGP), phospholipase A1-like protein (PLA1) and potassium voltage-gated channel protein (K VOLT) showed a predominant protective Th1 response over disease exacerbating Th2. We report the immunogenic properties and protective efficacy of one of the four antigens, tuzin, as a DNA vaccine against Leishmania donovani challenge. Our results show that administration of tuzin DNA protected BALB/c mice against L. donovani challenge and that protective immunity was associated with higher levels of IFN-γ and IL-12 production in comparison to IL-4 and IL-10. Our study presents a simple approach to rapidly identify potential vaccine candidates using the exhaustive information stored in the genome and an in vitro high-throughput immunological platform.

  13. Aneuploidy screening by array comparative genomic hybridization improves success rates of in vitro fertilization: A multicenter Indian study

    Directory of Open Access Journals (Sweden)

    Aditi Kotdawala

    2016-01-01

    Full Text Available Objective: To evaluate the usefulness of preimplantation genetic screening (PGS using array comparative genomic hybridization (aCGH in the Indian population. Materials and Methods: This is a retrospective, multicenter study including 235 PGS cycles following intracytoplasmic sperm injection performed at six different infertility centers from September 2013 to June 2015. Patients were divided as per maternal age in several groups (40 years and as per indication for undergoing PGS. Indications for performing PGS were recurrent miscarriage, repetitive implantation failure, severe male factor, previous trisomic pregnancy, and advanced maternal age (≥35. Day 3 embryo biopsy was performed and analyzed by aCGH followed by day 5 embryo transfer in the same cycle or the following cycle. Outcomes such as pregnancy rates (PRs/transfer, implantation rates, miscarriage rates, percentage of abnormal embryos, and number of embryos with more than one aneuploidy and chaotic patterns were recorded for all the treated subjects based on different age and indication groups. Results: aCGH helped in identifying aneuploid embryos, thus leading to consistent implantation (range: 33.3%-42.9% and PRs per transfer (range: 31.8%-54.9% that were obtained for all the indications in all the age groups, after performing PGS. Conclusion: Aneuploidy is one of the major factors which affect embryo implantation. aCGH can be successfully employed for screening of aneuploid embryos. When euploid embryos are transferred, an increase in PRs can be achieved irrespective of the age or the indication.

  14. Screening of tissue-specific genes and promoters in tomato by comparing genome wide expression profiles of Arabidopsis orthologues.

    Science.gov (United States)

    Lim, Chan Ju; Lee, Ha Yeon; Kim, Woong Bom; Lee, Bok-Sim; Kim, Jungeun; Ahmad, Raza; Kim, Hyun A; Yi, So Young; Hur, Cheol-Goo; Kwon, Suk-Yoon

    2012-07-01

    Constitutive overexpression of transgenes occasionally interferes with normal growth and developmental processes in plants. Thus, the development of tissue-specific promoters that drive transgene expression has become agriculturally important. To identify tomato tissue-specific promoters, tissue-specific genes were screened using a series of in silico-based and experimental procedures, including genome-wide orthologue searches of tomato and Arabidopsis databases, isolation of tissue-specific candidates using an Arabidopsis microarray database, and validation of tissue specificity by reverse transcription-polymerase chain reaction (RT-PCR) analysis and promoter assay. Using these procedures, we found 311 tissue-specific candidate genes and validated 10 tissue-specific genes by RT-PCR. Among these identified genes, histochemical analysis of five isolated promoter::GUS transgenic tomato and Arabidopsis plants revealed that their promoters have different but distinct tissue-specific activities in anther, fruit, and root, respectively. Therefore, it appears these in silico-based screening approaches in addition to the identification of new tissue-specific genes and promoters will be helpful for the further development of tailored crop development.

  15. Genome-wide screening for genes whose deletions confer sensitivity to mutagenic purine base analogs in yeast

    Directory of Open Access Journals (Sweden)

    Kozmin Stanislav G

    2005-06-01

    Full Text Available Abstract Background N-hydroxylated base analogs, such as 6-hydroxylaminopurine (HAP and 2-amino-6-hydroxylaminopurine (AHA, are strong mutagens in various organisms due to their ambiguous base-pairing properties. The systems protecting cells from HAP and related noncanonical purines in Escherichia coli include specialized deoxyribonucleoside triphosphatase RdgB, DNA repair endonuclease V, and a molybdenum cofactor-dependent system. Fewer HAP-detoxification systems have been identified in yeast Saccharomyces cerevisiae and other eukaryotes. Cellular systems protecting from AHA are unknown. In the present study, we performed a genome-wide search for genes whose deletions confer sensitivity to HAP and AHA in yeast. Results We screened the library of yeast deletion mutants for sensitivity to the toxic and mutagenic action of HAP and AHA. We identified novel genes involved in the genetic control of base analogs sensitivity, including genes controlling purine metabolism, cytoskeleton organization, and amino acid metabolism. Conclusion We developed a method for screening the yeast deletion library for sensitivity to the mutagenic and toxic action of base analogs and identified 16 novel genes controlling pathways of protection from HAP. Three of them also protect from AHA.

  16. An siRNA-based functional genomics screen for the identification of regulators of ciliogenesis and ciliopathy genes

    Science.gov (United States)

    Racher, Hilary; Phelps, Ian G.; Toedt, Grischa; Kennedy, Julie; Wunderlich, Kirsten A.; Sorusch, Nasrin; Abdelhamed, Zakia A.; Natarajan, Subaashini; Herridge, Warren; van Reeuwijk, Jeroen; Horn, Nicola; Boldt, Karsten; Parry, David A.; Letteboer, Stef J.F.; Roosing, Susanne; Adams, Matthew; Bell, Sandra M.; Bond, Jacquelyn; Higgins, Julie; Morrison, Ewan E.; Tomlinson, Darren C.; Slaats, Gisela G.; van Dam, Teunis J. P.; Huang, Lijia; Kessler, Kristin; Giessl, Andreas; Logan, Clare V.; Boyle, Evan A.; Shendure, Jay; Anazi, Shamsa; Aldahmesh, Mohammed; Al Hazzaa, Selwa; Hegele, Robert A.; Ober, Carole; Frosk, Patrick; Mhanni, Aizeddin A.; Chodirker, Bernard N.; Chudley, Albert E.; Lamont, Ryan; Bernier, Francois P.; Beaulieu, Chandree L.; Gordon, Paul; Pon, Richard T.; Donahue, Clem; Barkovich, A. James; Wolf, Louis; Toomes, Carmel; Thiel, Christian T.; Boycott, Kym M.; McKibbin, Martin; Inglehearn, Chris F.; Stewart, Fiona; Omran, Heymut; Huynen, Martijn A.; Sergouniotis, Panagiotis I.; Alkuraya, Fowzan S.; Parboosingh, Jillian S.; Innes, A Micheil; Willoughby, Colin E.; Giles, Rachel H.; Webster, Andrew R.; Ueffing, Marius; Blacque, Oliver; Gleeson, Joseph G.; Wolfrum, Uwe; Beales, Philip L.; Gibson, Toby

    2015-01-01

    Defects in primary cilium biogenesis underlie the ciliopathies, a growing group of genetic disorders. We describe a whole genome siRNA-based reverse genetics screen for defects in biogenesis and/or maintenance of the primary cilium, obtaining a global resource. We identify 112 candidate ciliogenesis and ciliopathy genes, including 44 components of the ubiquitin-proteasome system, 12 G-protein-coupled receptors, and three pre-mRNA processing factors (PRPF6, PRPF8 and PRPF31) mutated in autosomal dominant retinitis pigmentosa. The PRPFs localise to the connecting cilium, and PRPF8- and PRPF31-mutated cells have ciliary defects. Combining the screen with exome sequencing data identified recessive mutations in PIBF1/CEP90 and C21orf2/LRRC76 as causes of the ciliopathies Joubert and Jeune syndromes. Biochemical approaches place C21orf2 within key ciliopathy-associated protein modules, offering an explanation for the skeletal and retinal involvement observed in individuals with C21orf2-variants. Our global, unbiased approaches provide insights into ciliogenesis complexity and identify roles for unanticipated pathways in human genetic disease. PMID:26167768

  17. Genes required for growth at high hydrostatic pressure in Escherichia coli K-12 identified by genome-wide screening.

    Science.gov (United States)

    Black, S Lucas; Dawson, Angela; Ward, F Bruce; Allen, Rosalind J

    2013-01-01

    Despite the fact that much of the global microbial biosphere is believed to exist in high pressure environments, the effects of hydrostatic pressure on microbial physiology remain poorly understood. We use a genome-wide screening approach, combined with a novel high-throughput high-pressure cell culture method, to investigate the effects of hydrostatic pressure on microbial physiology in vivo. The Keio collection of single-gene deletion mutants in Escherichia coli K-12 was screened for growth at a range of pressures from 0.1 MPa to 60 MPa. This led to the identification of 6 genes, rodZ, holC, priA, dnaT, dedD and tatC, whose products were required for growth at 30 MPa and a further 3 genes, tolB, rffT and iscS, whose products were required for growth at 40 MPa. Our results support the view that the effects of pressure on cell physiology are pleiotropic, with DNA replication, cell division, the cytoskeleton and cell envelope physiology all being potential failure points for cell physiology during growth at elevated pressure.

  18. A two-host fosmid system for functional screening of (meta)genomic libraries from extreme thermophiles.

    Science.gov (United States)

    Angelov, Angel; Mientus, Markus; Liebl, Susanne; Liebl, Wolfgang

    2009-05-01

    A new cloning system is described, which allows the construction of large-insert fosmid libraries in Escherichia coli and the transfer of the recombinant libraries to the extreme thermophile Thermus thermophilus via natural transformation. Libraries are established in the thermophilic host by site-specific chromosomal insertion of the recombinant fosmids via single crossover or double crossover recombination at the T. thermophilus pyr locus. Comparative screening of a fosmid library constructed from genomic DNA from the thermophilic spirochaete, Spirochaeta thermophila, for clones expressing thermoactive xylanase activity revealed that 50% of the fosmids that conferred xylanase activity upon the corresponding T. thermophilus transformants did not give rise to xylanase-positive E. coli clones, indicating that significantly more S. thermophila genes are functionally expressed in T. thermophilus than in E. coli. The novel T. thermophilus host/vector system may be of value for the construction and functional screening of recombinant DNA libraries from individual thermophilic or extremely thermophilic organisms as well as from complex metagenomes isolated from thermophilic microbial communities.

  19. A whole mitochondrial genome screening in a MELAS patient: A novel mitochondrial tRNA{sup Val} mutation

    Energy Technology Data Exchange (ETDEWEB)

    Mezghani, Najla [Laboratoire de Genetique Moleculaire Humaine, Faculte de Medecine de Sfax, Universite de Sfax (Tunisia); Mnif, Mouna [Service d' endocrinologie, C.H.U. Habib Bourguiba de Sfax (Tunisia); Kacem, Maha [Service de Medecine interne, C.H.U. Fattouma Bourguiba de Monastir (Tunisia); Mkaouar-Rebai, Emna, E-mail: emna_mkaouar@mail2world.com [Laboratoire de Genetique Moleculaire Humaine, Faculte de Medecine de Sfax, Universite de Sfax (Tunisia); Hadj Salem, Ikhlass [Laboratoire de Genetique Moleculaire Humaine, Faculte de Medecine de Sfax, Universite de Sfax (Tunisia); Kallel, Nozha; Charfi, Nadia; Abid, Mohamed [Service d' endocrinologie, C.H.U. Habib Bourguiba de Sfax (Tunisia); Fakhfakh, Faiza [Laboratoire de Genetique Moleculaire Humaine, Faculte de Medecine de Sfax, Universite de Sfax (Tunisia)

    2011-04-22

    Highlights: {yields} We report a young Tunisian patient with clinical features of MELAS syndrome. {yields} Reported mitochondrial mutations were absent after a mutational screening of the whole mtDNA. {yields} We described a novel m.1640A>G mutation in the tRNA{sup Val} gene which was absent in 150 controls. {yields} Mitochondrial deletions and POLG1 gene mutations were absent. {yields} The m.1640A>G mutation could be associated to MELAS syndrome. -- Abstract: Mitochondrial encephalopathy, lactic acidosis and strokelike episodes (MELAS) syndrome is a mitochondrial disorder characterized by a wide variety of clinical presentations and a multisystemic organ involvement. In this study, we report a Tunisian girl with clinical features of MELAS syndrome who was negative for the common m.3243A>G mutation, but also for the reported mitochondrial DNA (mtDNA) mutations and deletions. Screening of the entire mtDNA genome showed several known mitochondrial variants besides to a novel transition m.1640A>G affecting a wobble adenine in the anticodon stem region of the tRNA{sup Val}. This nucleotide was conserved and it was absent in 150 controls suggesting its pathogenicity. In addition, no mutations were found in the nuclear polymerase gamma-1 gene (POLG1). These results suggest further investigation nuclear genes encoding proteins responsible for stability and structural components of the mtDNA or to the oxidative phosphorylation machinery to explain the phenotypic variability in the studied family.

  20. CTL epitopes for influenza A including the H5N1 bird flu; genome-, pathogen-, and HLA-wide screening.

    Science.gov (United States)

    Wang, Mingjun; Lamberth, Kasper; Harndahl, Mikkel; Røder, Gustav; Stryhn, Anette; Larsen, Mette V; Nielsen, Morten; Lundegaard, Claus; Tang, Sheila T; Dziegiel, Morten H; Rosenkvist, Jørgen; Pedersen, Anders E; Buus, Søren; Claesson, Mogens H; Lund, Ole

    2007-04-12

    The purpose of the present study is to perform a global screening for new immunogenic HLA class I (HLA-I) restricted cytotoxic T cell (CTL) epitopes of potential utility as candidates of influenza A-virus diagnostics and vaccines. We used predictions of antigen processing and presentation, the latter encompassing 12 different HLA class I supertypes with >99% population coverage, and searched for conserved epitopes from available influenza A viral protein sequences. Peptides corresponding to 167 predicted peptide-HLA-I interactions were synthesized, tested for peptide-HLA-I interactions in a biochemical assay and for influenza-specific, HLA-I-restricted CTL responses in an IFN-gamma ELISPOT assay. Eighty-nine peptides could be confirmed as HLA-I binders, and 13 could be confirmed as CTL targets. The 13 epitopes, are highly conserved among human influenza A pathogens, and all of these epitopes are present in the emerging bird flu isolates. Our study demonstrates that present technology enables a fast global screening for T cell immune epitopes of potential diagnostics and vaccine interest. This technology includes immuno-bioinformatics predictors with the capacity to perform fast genome-, pathogen-, and HLA-wide searches for immune targets. To exploit this new potential, a coordinated international effort to analyze the precious source of information represented by rare patients, such as the current victims of bird flu, would be essential.

  1. Quantitative genome-wide genetic interaction screens reveal global epistatic relationships of protein complexes in Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Mohan Babu

    2014-02-01

    Full Text Available Large-scale proteomic analyses in Escherichia coli have documented the composition and physical relationships of multiprotein complexes, but not their functional organization into biological pathways and processes. Conversely, genetic interaction (GI screens can provide insights into the biological role(s of individual gene and higher order associations. Combining the information from both approaches should elucidate how complexes and pathways intersect functionally at a systems level. However, such integrative analysis has been hindered due to the lack of relevant GI data. Here we present a systematic, unbiased, and quantitative synthetic genetic array screen in E. coli describing the genetic dependencies and functional cross-talk among over 600,000 digenic mutant combinations. Combining this epistasis information with putative functional modules derived from previous proteomic data and genomic context-based methods revealed unexpected associations, including new components required for the biogenesis of iron-sulphur and ribosome integrity, and the interplay between molecular chaperones and proteases. We find that functionally-linked genes co-conserved among γ-proteobacteria are far more likely to have correlated GI profiles than genes with divergent patterns of evolution. Overall, examining bacterial GIs in the context of protein complexes provides avenues for a deeper mechanistic understanding of core microbial systems.

  2. Combined Rational Design and a High Throughput Screening Platform for Identifying Chemical Inhibitors of a Ras-activating Enzyme*

    Science.gov (United States)

    Evelyn, Chris R.; Biesiada, Jacek; Duan, Xin; Tang, Hong; Shang, Xun; Papoian, Ruben; Seibel, William L.; Nelson, Sandra; Meller, Jaroslaw; Zheng, Yi

    2015-01-01

    The Ras family small GTPases regulate multiple cellular processes, including cell growth, survival, movement, and gene expression, and are intimately involved in cancer pathogenesis. Activation of these small GTPases is catalyzed by a special class of enzymes, termed guanine nucleotide exchange factors (GEFs). Herein, we developed a small molecule screening platform for identifying lead hits targeting a Ras GEF enzyme, SOS1. We employed an ensemble structure-based virtual screening approach in combination with a multiple tier high throughput experimental screen utilizing two complementary fluorescent guanine nucleotide exchange assays to identify small molecule inhibitors of GEF catalytic activity toward Ras. From a library of 350,000 compounds, we selected a set of 418 candidate compounds predicted to disrupt the GEF-Ras interaction, of which dual wavelength GDP dissociation and GTP-loading experimental screening identified two chemically distinct small molecule inhibitors. Subsequent biochemical validations indicate that they are capable of dose-dependently inhibiting GEF catalytic activity, binding to SOS1 with micromolar affinity, and disrupting GEF-Ras interaction. Mutagenesis studies in conjunction with structure-activity relationship studies mapped both compounds to different sites in the catalytic pocket, and both inhibited Ras signaling in cells. The unique screening platform established here for targeting Ras GEF enzymes could be broadly useful for identifying lead inhibitors for a variety of small GTPase-activating GEF reactions. PMID:25825487

  3. Utilizing Chemical Genomics to Identify Cytochrome b as a Novel Drug Target for Chagas Disease.

    Directory of Open Access Journals (Sweden)

    Shilpi Khare

    2015-07-01

    Full Text Available Unbiased phenotypic screens enable identification of small molecules that inhibit pathogen growth by unanticipated mechanisms. These small molecules can be used as starting points for drug discovery programs that target such mechanisms. A major challenge of the approach is the identification of the cellular targets. Here we report GNF7686, a small molecule inhibitor of Trypanosoma cruzi, the causative agent of Chagas disease, and identification of cytochrome b as its target. Following discovery of GNF7686 in a parasite growth inhibition high throughput screen, we were able to evolve a GNF7686-resistant culture of T. cruzi epimastigotes. Clones from this culture bore a mutation coding for a substitution of leucine by phenylalanine at amino acid position 197 in cytochrome b. Cytochrome b is a component of complex III (cytochrome bc1 in the mitochondrial electron transport chain and catalyzes the transfer of electrons from ubiquinol to cytochrome c by a mechanism that utilizes two distinct catalytic sites, QN and QP. The L197F mutation is located in the QN site and confers resistance to GNF7686 in both parasite cell growth and biochemical cytochrome b assays. Additionally, the mutant cytochrome b confers resistance to antimycin A, another QN site inhibitor, but not to strobilurin or myxothiazol, which target the QP site. GNF7686 represents a promising starting point for Chagas disease drug discovery as it potently inhibits growth of intracellular T. cruzi amastigotes with a half maximal effective concentration (EC50 of 0.15 µM, and is highly specific for T. cruzi cytochrome b. No effect on the mammalian respiratory chain or mammalian cell proliferation was observed with up to 25 µM of GNF7686. Our approach, which combines T. cruzi chemical genetics with biochemical target validation, can be broadly applied to the discovery of additional novel drug targets and drug leads for Chagas disease.

  4. Hazard screening of chemical releases and environmental equity analysis of populations proximate to toxic release inventory facilities in Oregon.

    Science.gov (United States)

    Neumann, C M; Forman, D L; Rothlein, J E

    1998-04-01

    A comprehensive approach using hazard screening, demographic analysis, and a geographic information system (GIS) for mapping is employed to address environmental equity issues in Oregon. A media-specific chronic toxicity index [or chronic index (CI)] was used to compare environmental chemical releases reported in the EPA's Toxic Chemical Release Inventory (TRI) database. In 1992, 254 facilities reportedly released more than 40 million pounds of toxic chemicals directly into the environment on-site or transferred them to sewage treatment plants or other off-site facilities for disposal and recycling. For each reported on-site TRI chemical release, a CI based on oral toxicity factors and total mass was calculated. CIs were aggregated on a media-, facility-, and chemical-specific basis. Glycol ethers, nickel, trichloroethylene, chloroform, and manganese were ranked as the top five chemicals released statewide based on total CI. In contrast, based on total mass, methanol, nickel, ammonia, acetone, and toluene were identified as the top five TRI chemicals released in Oregon. TRI facility rankings were related to the demographics and household income of surrounding neighborhoods using bivariate GIS mapping and statistical analysis. TRI facilities were disproportionately located in racial and ethnic minority neighborhoods. They were also located in areas with lower incomes compared to those in the surrounding county. No relationship was observed between the hazard ranking of the TRI facilities overall and socioeconomic characteristics of the community in which they were located.

  5. A genome-wide immunodetection screen in S. cerevisiae uncovers novel genes involved in lysosomal vacuole function and morphology.

    Directory of Open Access Journals (Sweden)

    Florante Ricarte

    Full Text Available Vacuoles of yeast Saccharomyces cerevisiae are functionally analogous to mammalian lysosomes. Both are cellular organelles responsible for macromolecular degradation, ion/pH homeostasis, and stress survival. We hypothesized that undefined gene functions remain at post-endosomal stage of vacuolar events and performed a genome-wide screen directed at such functions at the late endosome and vacuole interface - ENV genes. The immunodetection screen was designed to identify mutants that internally accumulate precursor form of the vacuolar hydrolase carboxypeptidase Y (CPY. Here, we report the uncovering and initial characterizations of twelve ENV genes. The small size of the collection and the lack of genes previously identified with vacuolar events are suggestive of the intended exclusive functional interface of the screen. Most notably, the collection includes four novel genes ENV7, ENV9, ENV10, and ENV11, and three genes previously linked to mitochondrial processes - MAM3, PCP1, PPE1. In all env mutants, vesicular trafficking stages were undisturbed in live cells as assessed by invertase and active α-factor secretion, as well as by localization of the endocytic fluorescent marker FM4-64 to the vacuole. Several mutants exhibit defects in stress survival functions associated with vacuoles. Confocal fluorescence microscopy revealed the collection to be significantly enriched in vacuolar morphologies suggestive of fusion and fission defects. These include the unique phenotype of lumenal vesicles within vacuoles in the novel env9Δ mutant and severely fragmented vacuoles upon deletion of GET4, a gene recently implicated in tail anchored membrane protein insertion. Thus, our results establish new gene functions in vacuolar function and morphology, and suggest a link between vacuolar and mitochondrial events.

  6. An siRNA-based functional genomics screen for the identification of regulators of ciliogenesis and ciliopathy genes

    NARCIS (Netherlands)

    Wheway, Gabrielle; Schmidts, Miriam; Mans, Dorus A; Szymanska, Katarzyna; Nguyen, Thanh-Minh T; Racher, Hilary; Phelps, Ian G; Toedt, Grischa; Kennedy, Julie; Wunderlich, Kirsten A; Sorusch, Nasrin; Abdelhamed, Zakia A; Natarajan, Subaashini; Herridge, Warren; van Reeuwijk, Jeroen; Horn, Nicola; Boldt, Karsten; Parry, David A; Letteboer, Stef J F; Roosing, Susanne; Adams, Matthew; Bell, Sandra M; Bond, Jacquelyn; Higgins, Julie; Morrison, Ewan E; Tomlinson, Darren C; Slaats, Gisela G; van Dam, Teunis J P; Huang, Lijia; Kessler, Kristin; Giessl, Andreas; Logan, Clare V; Boyle, Evan A; Shendure, Jay; Anazi, Shamsa; Aldahmesh, Mohammed; Al Hazzaa, Selwa; Hegele, Robert A; Ober, Carole; Frosk, Patrick; Mhanni, Aizeddin A; Chodirker, Bernard N; Chudley, Albert E; Lamont, Ryan; Bernier, Francois P; Beaulieu, Chandree L; Gordon, Paul; Pon, Richard T; Donahue, Clem; Barkovich, A James; Wolf, Louis; Toomes, Carmel; Thiel, Christian T; Boycott, Kym M; McKibbin, Martin; Inglehearn, Chris F; Stewart, Fiona; Omran, Heymut; Huynen, Martijn A; Sergouniotis, Panagiotis I; Alkuraya, Fowzan S; Parboosingh, Jillian S; Innes, A Micheil; Willoughby, Colin E; Giles, Rachel H|info:eu-repo/dai/nl/173658725; Webster, Andrew R; Ueffing, Marius; Blacque, Oliver; Gleeson, Joseph G; Wolfrum, Uwe; Beales, Philip L; Gibson, Toby; Doherty, Dan; Mitchison, Hannah M; Roepman, Ronald; Johnson, Colin A

    2015-01-01

    Defects in primary cilium biogenesis underlie the ciliopathies, a growing group of genetic disorders. We describe a whole-genome siRNA-based reverse genetics screen for defects in biogenesis and/or maintenance of the primary cilium, obtaining a global resource. We identify 112 candidate ciliogenesis

  7. An siRNA-based functional genomics screen for the identification of regulators of ciliogenesis and ciliopathy genes

    NARCIS (Netherlands)

    Wheway, Gabrielle; Schmidts, Miriam; Mans, Dorus A; Szymanska, Katarzyna; Nguyen, Thanh-Minh T; Racher, Hilary; Phelps, Ian G; Toedt, Grischa; Kennedy, Julie; Wunderlich, Kirsten A; Sorusch, Nasrin; Abdelhamed, Zakia A; Natarajan, Subaashini; Herridge, Warren; van Reeuwijk, Jeroen; Horn, Nicola; Boldt, Karsten; Parry, David A; Letteboer, Stef J F; Roosing, Susanne; Adams, Matthew; Bell, Sandra M; Bond, Jacquelyn; Higgins, Julie; Morrison, Ewan E; Tomlinson, Darren C; Slaats, Gisela G; van Dam, Teunis J P; Huang, Lijia; Kessler, Kristin; Giessl, Andreas; Logan, Clare V; Boyle, Evan A; Shendure, Jay; Anazi, Shamsa; Aldahmesh, Mohammed; Al Hazzaa, Selwa; Hegele, Robert A; Ober, Carole; Frosk, Patrick; Mhanni, Aizeddin A; Chodirker, Bernard N; Chudley, Albert E; Lamont, Ryan; Bernier, Francois P; Beaulieu, Chandree L; Gordon, Paul; Pon, Richard T; Donahue, Clem; Barkovich, A James; Wolf, Louis; Toomes, Carmel; Thiel, Christian T; Boycott, Kym M; McKibbin, Martin; Inglehearn, Chris F; Stewart, Fiona; Omran, Heymut; Huynen, Martijn A; Sergouniotis, Panagiotis I; Alkuraya, Fowzan S; Parboosingh, Jillian S; Innes, A Micheil; Willoughby, Colin E; Giles, Rachel H; Webster, Andrew R; Ueffing, Marius; Blacque, Oliver; Gleeson, Joseph G; Wolfrum, Uwe; Beales, Philip L; Gibson, Toby; Doherty, Dan; Mitchison, Hannah M; Roepman, Ronald; Johnson, Colin A

    2015-01-01

    Defects in primary cilium biogenesis underlie the ciliopathies, a growing group of genetic disorders. We describe a whole-genome siRNA-based reverse genetics screen for defects in biogenesis and/or maintenance of the primary cilium, obtaining a global resource. We identify 112 candidate ciliogenesis

  8. Phylogeny-function analysis of (meta)genomic libraries: screening for expression of ribosomal RNA genes by large-insert library fluorescent in situ hybridization (LIL-FISH)

    NARCIS (Netherlands)

    Leveau, J.H.J.; Gerards, S.; De Boer, W.; Van Veen, J.A.

    2004-01-01

    We assessed the utility of fluorescent in situ hybridization (FISH) in the screening of clone libraries of (meta)genomic or environmental DNA for the presence and expression of bacterial ribosomal RNA (rRNA) genes. To establish proof-of-principle, we constructed a fosmid-based library in Escherichia

  9. In silico prediction and screening of modular crystal structures via a high-throughput genomic approach

    Science.gov (United States)

    Li, Yi; Li, Xu; Liu, Jiancong; Duan, Fangzheng; Yu, Jihong

    2015-09-01

    High-throughput computational methods capable of predicting, evaluating and identifying promising synthetic candidates with desired properties are highly appealing to today's scientists. Despite some successes, in silico design of crystalline materials with complex three-dimensionally extended structures remains challenging. Here we demonstrate the application of a new genomic approach to ABC-6 zeolites, a family of industrially important catalysts whose structures are built from the stacking of modular six-ring layers. The sequences of layer stacking, which we deem the genes of this family, determine the structures and the properties of ABC-6 zeolites. By enumerating these gene-like stacking sequences, we have identified 1,127 most realizable new ABC-6 structures out of 78 groups of 84,292 theoretical ones, and experimentally realized 2 of them. Our genomic approach can extract crucial structural information directly from these gene-like stacking sequences, enabling high-throughput identification of synthetic targets with desired properties among a large number of candidate structures.

  10. The P1 vector system for the preparation and screening of genomic libraries.

    Science.gov (United States)

    Shepherd, N S; Smoller, D

    1994-01-01

    In retrospect, it is remarkable how swiftly the P1 cloning system has progressed in only a few years from a novel cloning system to one now widely used for the production of recombinant libraries and the building of physical maps. As the libraries become larger, better characterized and more widely distributed, we certainly will see a blossoming of research articles and techniques based on the use of P1 recombinant clones. Specifically, we can look forward to scanning P1 clones for expressed sequences (N. Sternberg, personal communication), routine retrofitting of P1 clones with a combination of transposon and P1 transduction techniques (3), the random or loxP-directed (68,69) insertion of P1 clones into host genomes and the subsequent production of transgenic animals (63), a further use of P1 clones in the building of contigs and physical maps, an a higher in vitro cloning efficiency due to the purification of the P1 pacase proteins used during in vitro packaging (70). In summary, P1 bacteriophage cloning is favorably impacting research today and will continue to fill an important niche as a genomic cloning system.

  11. Phenotypic screening of the ToxCast chemical library to classify toxic and therapeutic mechanisms

    Science.gov (United States)

    Addressing the safety aspects of drugs and environmental chemicals has historically been undertaken through animal testing. However, the quantity of chemicals in need of assessment and the challenges of species extrapolation require the development of alternative approaches. Our ...

  12. Predicting Toxic and Therapeutic Mechanisms of the ToxCast Chemical Library by Phenotypic Screening (SOT)

    Science.gov (United States)

    Addressing safety aspects of drugs and environmental chemicals relies extensively on animal testing. However the quantity of chemicals needing assessment and challenges of species extrapolation require development of alternative approaches. Using 8 primary human cell systems (Bio...

  13. Endocrine Disruptor Screening Program (EDSP) Universe of Chemicals and General Validation Principles

    Science.gov (United States)

    This document was developed by the EPA to provide guidance to staff and managers regarding the EDSP universe of chemicals and general validation principles for consideration of computational toxicology tools for chemical prioritization.

  14. High-throughput fluorescence polarization assay for chemical library screening against anti-apoptotic Bcl-2 family member Bfl-1.

    Science.gov (United States)

    Zhai, Dayong; Godoi, Paulo; Sergienko, Eduard; Dahl, Russell; Chan, Xochella; Brown, Brock; Rascon, Justin; Hurder, Andrew; Su, Ying; Chung, Thomas D Y; Jin, Chaofang; Diaz, Paul; Reed, John C

    2012-03-01

    Overexpression of the anti-apoptotic Bcl-2 family proteins occurs commonly in human cancers. Bfl-1 is highly expressed in some types of malignant cells, contributing significantly to tumor cell survival and chemoresistance. Therefore, it would be desirable to have chemical antagonists of Bfl-1. To this end, we devised a fluorescence polarization assay (FPA) using Bfl-1 protein and fluorescein-conjugated Bid BH3 peptide, which was employed for high-throughput screening of chemical libraries. Approximately 66 000 compounds were screened for the ability to inhibit BH3 peptide binding to Bfl-1, yielding 14 reproducible hits with ≥50% displacement. After dose-response analysis and confirmation using a secondary assay based on time-resolved fluorescence resonance energy transfer (TR-FRET), two groups of Bfl-1-specific inhibitors were identified, including chloromaleimide and sulfonylpyrimidine series compounds. FPAs generated for each of the six anti-apoptotic Bcl-2 proteins demonstrated selective binding of both classes of compounds to Bfl-1. Analogs of the sulfonylpyrimidine series were synthesized and compared with the original hit for Bfl-1 binding by both FPAs and TR-FRET assays. The resulting structure-activity relation analysis led to the chemical probe compound CID-2980973 (ML042). Collectively, these findings demonstrate the feasibility of using the HTS assay for discovery of selective chemical inhibitors of Bfl-1.

  15. A Digital PCR-Based Method for Efficient and Highly Specific Screening of Genome Edited Cells.

    Directory of Open Access Journals (Sweden)

    Scott D Findlay

    Full Text Available The rapid adoption of gene editing tools such as CRISPRs and TALENs for research and eventually therapeutics necessitates assays that can rapidly detect and quantitate the desired alterations. Currently, the most commonly used assay employs "mismatch nucleases" T7E1 or "Surveyor" that recognize and cleave heteroduplexed DNA amplicons containing mismatched base-pairs. However, this assay is prone to false positives due to cancer-associated mutations and/or SNPs and requires large amounts of starting material. Here we describe a powerful alternative wherein droplet digital PCR (ddPCR can be used to decipher homozygous from heterozygous mutations with superior levels of both precision and sensitivity. We use this assay to detect knockout inducing alterations to stem cell associated proteins, NODAL and SFRP1, generated using either TALENs or an "all-in-one" CRISPR/Cas plasmid that we have modified for one-step cloning and blue/white screening of transformants. Moreover, we highlight how ddPCR can be used to assess the efficiency of varying TALEN-based strategies. Collectively, this work highlights how ddPCR-based screening can be paired with CRISPR and TALEN technologies to enable sensitive, specific, and streamlined approaches to gene editing and validation.

  16. Genome-wide screen for Mycobacterium tuberculosis genes that regulate host immunity.

    Directory of Open Access Journals (Sweden)

    Aimee M Beaulieu

    Full Text Available In spite of its highly immunogenic properties, Mycobacterium tuberculosis (Mtb establishes persistent infection in otherwise healthy individuals, making it one of the most widespread and deadly human pathogens. Mtb's prolonged survival may reflect production of microbial factors that prevent even more vigorous immunity (quantitative effect or that divert the immune response to a non-sterilizing mode (qualitative effect. Disruption of Mtb genes has produced a list of several dozen candidate immunomodulatory factors. Here we used robotic fluorescence microscopy to screen 10,100 loss-of-function transposon mutants of Mtb for their impact on the expression of promoter-reporter constructs for 12 host immune response genes in a mouse macrophage cell line. The screen identified 364 candidate immunoregulatory genes. To illustrate the utility of the candidate list, we confirmed the impact of 35 Mtb mutant strains on expression of endogenous immune response genes in primary macrophages. Detailed analysis focused on a strain of Mtb in which a transposon disrupts Rv0431, a gene encoding a conserved protein of unknown function. This mutant elicited much more macrophage TNFα, IL-12p40 and IL-6 in vitro than wild type Mtb, and was attenuated in the mouse. The mutant list provides a platform for exploring the immunobiology of tuberculosis, for example, by combining immunoregulatory mutations in a candidate vaccine strain.

  17. Identification of 34 novel proinflammatory proteins in a genome-wide macrophage functional screen.

    Directory of Open Access Journals (Sweden)

    David H Wyllie

    Full Text Available Signal transduction pathways activated by Toll-like Receptors and the IL-1 family of cytokines are fundamental to mounting an innate immune response and thus to clearing pathogens and promoting wound healing. Whilst mechanistic understanding of the regulation of innate signalling pathways has advanced considerably in recent years, there are still a number of critical controllers to be discovered. In order to characterise novel regulators of macrophage inflammation, we have carried out an extensive, cDNA-based forward genetic screen and identified 34 novel activators, based on their ability to induce the expression of cxcl2. Many are physiologically expressed in macrophages, although the majority of genes uncovered in our screen have not previously been linked to innate immunity. We show that expression of particular activators has profound but distinct impacts on LPS-induced inflammatory gene expression, including switch-type, amplifier and sensitiser behaviours. Furthermore, the novel genes identified here interact with the canonical inflammatory signalling network via specific mechanisms, as demonstrated by the use of dominant negative forms of IL1/TLR signalling mediators.

  18. A Digital PCR-Based Method for Efficient and Highly Specific Screening of Genome Edited Cells

    Science.gov (United States)

    Berman, Jennifer R.; Postovit, Lynne-Marie

    2016-01-01

    The rapid adoption of gene editing tools such as CRISPRs and TALENs for research and eventually therapeutics necessitates assays that can rapidly detect and quantitate the desired alterations. Currently, the most commonly used assay employs “mismatch nucleases” T7E1 or “Surveyor” that recognize and cleave heteroduplexed DNA amplicons containing mismatched base-pairs. However, this assay is prone to false positives due to cancer-associated mutations and/or SNPs and requires large amounts of starting material. Here we describe a powerful alternative wherein droplet digital PCR (ddPCR) can be used to decipher homozygous from heterozygous mutations with superior levels of both precision and sensitivity. We use this assay to detect knockout inducing alterations to stem cell associated proteins, NODAL and SFRP1, generated using either TALENs or an “all-in-one” CRISPR/Cas plasmid that we have modified for one-step cloning and blue/white screening of transformants. Moreover, we highlight how ddPCR can be used to assess the efficiency of varying TALEN-based strategies. Collectively, this work highlights how ddPCR-based screening can be paired with CRISPR and TALEN technologies to enable sensitive, specific, and streamlined approaches to gene editing and validation. PMID:27089539

  19. Genomic screen for loci associated with tobacco usage in Mission Indians

    Directory of Open Access Journals (Sweden)

    Wilhelmsen Kirk C

    2006-02-01

    Full Text Available Abstract Background The prevalence of tobacco usage in Native American adults and adolescents is higher than any other racial or ethnic group, yet biological risk and protective factors underlying tobacco use in this ethnic group remain unknown. A genome scan for loci associated with tobacco use phenotypes was performed with data collected from a community sample of Mission Indians residing in Southwest California. Methods A structured diagnostic interview was used to define two tobacco use phenotypes: 1 any regular tobacco usage (smoked daily for one month or more and 2 persistent tobacco usage (smoked at least 10 cigarettes a day for more than one year. Heritability was determined and a linkage analysis was performed, using genotypes for a panel 791 microsatellite polymorphisms, for the two phenotypes using variance component methods implemented in SOLAR. Results Analyses of multipoint variance component LOD scores for the two tobacco use phenotypes revealed two scores that exceeded 2.0 for the regular use phenotype: one on chromosomes 6 and one on 8. Four other loci on chromosomes 1,7,13, and 22 were found with LOD scores between 1.0 and 1.5. Two loci of interest were found on chromosomes 1 and 4 for the persistent use phenotype with LOD scores between 1.3–1.5. Bivariate linkage analysis was conducted at the site on chromosome 4 for persistent tobacco use and an alcohol drinking severity phenotype previously identified at this site. The maximum LOD score for the bivariate analysis for the region was 3.4, however, there was insufficient power to exclude coincident linkage. Conclusion While not providing evidence for linkage to specific chromosomal regions these results identify regions of interest in the genome in this Mission Indian population, for tobacco usage, some of which were identified in previous genome scans of non-native populations. Additionally, these data lend support for the hypothesis that cigarette smoking, alcohol

  20. In vitro and in vivo replication of a chemically synthesized consensus genome of hepatitis B virus genotype B.

    Science.gov (United States)

    Zhang, Zhenhua; Xia, Jianbo; Sun, Binghu; Dai, Yu; Li, Xu; Schlaak, Joerg F; Lu, Mengji

    2015-03-01

    Hepatitis B virus (HBV) genotypes vary in their geographical distribution and virological features. Previous investigations have shown that HBV genotype B is a predominant HBV genotype in China. Studies on HBV concerning different isolates frequently meet the question about the HBV reference strain and its representativeness. Although HBV consensus sequences can be generated easily by sequence alignment, they may not exist in nature or could not usually be isolated from patient samples. Thus, the construction of a consensus HBV genome has been proposed. In this study, an HBV genotype B consensus sequence was established by comparing 42 full-length HBV genotype B sequences and the genome was generated by chemical synthesis. This consensus genome was fully replication competent by in vitro transfection into hepatoma cells. The plasmid pHBV1.3B carrying a 1.3× full-length HBV consensus genome was hydrodynamically injected into Balb/c mice. HBsAg, anti-HBs, HBeAg, anti-HBe, and anti-HBc detection indicated expression and replication of this HBV genome in mice, similar to other HBV isolates. This approach represents a strategy to design and create consensus HBV genomes for future studies.

  1. Expression cloning of different bacterial phosphatase-encoding genes by histochemical screening of genomic libraries onto an indicator medium containing phenolphthalein diphosphate and methyl green.

    Science.gov (United States)

    Riccio, M L; Rossolini, G M; Lombardi, G; Chiesurin, A; Satta, G

    1997-02-01

    A system for expression cloning of bacterial phosphatase-encoding genes has been developed, and its potential has been investigated. The system is based on histochemical screening of bacterial genomic libraries, constructed in an Escherichia coli multicopy plasmid vector, for phosphatase-producing clones using an indicator medium (named TPMG) made of Tryptose-Phosphate agar supplemented with the phosphatase substrate phenolphthalein diphosphate and the stain methyl green. To test the performance of this system, three genomic libraries were constructed from bacterial strains of different species which showed different patterns of phosphatase activity, and were screened using the TPMG medium. Following a partial screening, three different phosphatase-encoding genes (respectively encoding a class A non-specific acid phosphatase, an acid-hexose phosphatase and a non-specific alkaline phosphatase) were shotgun-cloned from the above libraries, indicating that the TPMG-based expression cloning system can be useful for rapid isolation of different bacterial phosphatase-encoding genes.

  2. Rapid and Inexpensive Screening of Genomic Copy Number Variations Using a Novel Quantitative Fluorescent PCR Method

    Directory of Open Access Journals (Sweden)

    Martin Stofanko

    2013-01-01

    Full Text Available Detection of human microdeletion and microduplication syndromes poses significant burden on public healthcare systems in developing countries. With genome-wide diagnostic assays frequently inaccessible, targeted low-cost PCR-based approaches are preferred. However, their reproducibility depends on equally efficient amplification using a number of target and control primers. To address this, the recently described technique called Microdeletion/Microduplication Quantitative Fluorescent PCR (MQF-PCR was shown to reliably detect four human syndromes by quantifying DNA amplification in an internally controlled PCR reaction. Here, we confirm its utility in the detection of eight human microdeletion syndromes, including the more common WAGR, Smith-Magenis, and Potocki-Lupski syndromes with 100% sensitivity and 100% specificity. We present selection, design, and performance evaluation of detection primers using variety of approaches. We conclude that MQF-PCR is an easily adaptable method for detection of human pathological chromosomal aberrations.

  3. An Integrated Genome-Wide Systems Genetics Screen for Breast Cancer Metastasis Susceptibility Genes.

    Directory of Open Access Journals (Sweden)

    Ling Bai

    2016-04-01

    Full Text Available Metastasis remains the primary cause of patient morbidity and mortality in solid tumors and is due to the action of a large number of tumor-autonomous and non-autonomous factors. Here we report the results of a genome-wide integrated strategy to identify novel metastasis susceptibility candidate genes and molecular pathways in breast cancer metastasis. This analysis implicates a number of transcriptional regulators and suggests cell-mediated immunity is an important determinant. Moreover, the analysis identified novel or FDA-approved drugs as potentially useful for anti-metastatic therapy. Further explorations implementing this strategy may therefore provide a variety of information for clinical applications in the control and treatment of advanced neoplastic disease.

  4. Semantic Web for Chemical Genomics – need, how to, and hurdles

    Directory of Open Access Journals (Sweden)

    Talapady Bhat

    2007-08-01

    Full Text Available Semantic Web has been often suggested as the information technology solution to the growing problem in managing the millions of data points generated by modern science such as nanotechnology and high through-put screening for drugs. However, the progress towards this vision envisaged by the W3C has been very limited. Here we discuss –some of the obstacles to the realization of this vision and we make some suggestions as to how one may overcome some of these hurdles? Here we discuss some of these issues and present thoughts on an alternative method to Semantic Web that is less drastic in requirements. This method does not require the use of RDF and Protege, and it works in an environment currently used by the chemical and biological database providers. In our method one attempts to use as many components as possible from the tools already used by the database providers and one brings in far fewer new tools and techniques compared to the method that use RDF or Protégé. Our method uses a standard database environment and web tools rather than the RDF and Protégé to manage user interface and the data is held in a database rather than using RDF. This method shifts the task of building Semantic knowledge-base and ontology from RDF and Protégé to a SQL based database environment.

  5. Peptide reactivity assay using spectrophotometric method for high-throughput screening of skin sensitization potential of chemical haptens.

    Science.gov (United States)

    Jeong, Yun Hyeok; An, Susun; Shin, Kyeho; Lee, Tae Ryong

    2013-02-01

    Haptens must react with cellular proteins to be recognized by antigen presenting cells. Therefore, monitoring reactivity of chemicals with peptide/protein has been considered an in vitro skin sensitization testing method. The reactivity of peptides with chemicals (peptide reactivity) has usually been monitored by chromatographic methods like HPLC or LC/MS, which are robust tools for monitoring common chemical reactions but are rather expensive and time consuming. Here, we examined the possibility of using spectrophotometric methods to monitor peptide reactivity. Two synthetic peptides, Ac-RWAACAA and Ac-RWAAKAA, were reacted with 48 chemicals (34 sensitizers and 14 non-sensitizers). Peptide reactivity was measured by monitoring unreacted peptides with UV-Vis spectrophotometer using 5,5'-dithiobis-2-nitrobenzoic acid as a detection reagent for the free thiol group of cysteine-containing peptide or fluorometer using fluorescamine™ as a detection reagent for the free amine group of lysine-containing peptide. Chemicals were categorized as sensitizers when they induced more than 10% depletion of cysteine-containing peptide or 20% depletion of lysine-containing peptide. The sensitivity, specificity, and accuracy of this method were 82.4%, 85.7%, and 83.3%, respectively. These results demonstrate that spectrophotometric methods can be easy, fast, and high-throughput screening tools for the prediction of the skin sensitization potential of chemical haptens. Copyright © 2012 Elsevier Ltd. All rights reserved.

  6. Data from Tiered High-Throughput Screening Approach to Identify Thyroperoxidase Inhibitors within the ToxCast Phase I and II Chemical Libraries

    Data.gov (United States)

    U.S. Environmental Protection Agency — High-throughput screening for potential thyroid-disrupting chemicals requires a system of assays to capture multiple molecular-initiating events (MIEs) that converge...

  7. Whole Genome Expression Profiling and Signal Pathway Screening of MSCs in Ankylosing Spondylitis

    Directory of Open Access Journals (Sweden)

    Yuxi Li

    2014-01-01

    Full Text Available The pathogenesis of dysfunctional immunoregulation of mesenchymal stem cells (MSCs in ankylosing spondylitis (AS is thought to be a complex process that involves multiple genetic alterations. In this study, MSCs derived from both healthy donors and AS patients were cultured in normal media or media mimicking an inflammatory environment. Whole genome expression profiling analysis of 33,351 genes was performed and differentially expressed genes related to AS were analyzed by GO term analysis and KEGG pathway analysis. Our results showed that in normal media 676 genes were differentially expressed in AS, 354 upregulated and 322 downregulated, while in an inflammatory environment 1767 genes were differentially expressed in AS, 1230 upregulated and 537 downregulated. GO analysis showed that these genes were mainly related to cellular processes, physiological processes, biological regulation, regulation of biological processes, and binding. In addition, by KEGG pathway analysis, 14 key genes from the MAPK signaling and 8 key genes from the TLR signaling pathway were identified as differentially regulated. The results of qRT-PCR verified the expression variation of the 9 genes mentioned above. Our study found that in an inflammatory environment ankylosing spondylitis pathogenesis may be related to activation of the MAPK and TLR signaling pathways.

  8. Tiered High-Throughput Screening Approach to Identify Thyroperoxidase Inhibitors within the ToxCast Phase I and II Chemical Libraries

    Science.gov (United States)

    High-throughput screening (HTS) for potential thyroid–disrupting chemicals requires a system of assays to capture multiple molecular-initiating events (MIEs) that converge on perturbed thyroid hormone (TH) homeostasis. Screening for MIEs specific to TH-disrupting pathways is limi...

  9. Corrosion Screening of EV31A Magnesium and Other Magnesium Alloys using Laboratory-Based Accelerated Corrosion and Electro-Chemical Methods

    Science.gov (United States)

    2014-07-01

    Corrosion Screening of EV31A Magnesium and Other Magnesium Alloys Using Laboratory-Based Accelerated Corrosion and Electro-chemical Methods...originator. Army Research Laboratory Aberdeen Proving Ground, MD 21005-5066 ARL-TR-6899 July 2014 Corrosion Screening of EV31A...Magnesium and Other Magnesium Alloys Using Laboratory-Based Accelerated Corrosion and Electro-chemical Methods Brian E. Placzankis, Joseph P

  10. The ChemScreen project to design a pragmatic alternative approach to predict reproductive toxicity of chemicals.

    Science.gov (United States)

    van der Burg, Bart; Wedebye, Eva Bay; Dietrich, Daniel R; Jaworska, Joanna; Mangelsdorf, Inge; Paune, Eduard; Schwarz, Michael; Piersma, Aldert H; Kroese, E Dinant

    2015-08-01

    There is a great need for rapid testing strategies for reproductive toxicity testing, avoiding animal use. The EU Framework program 7 project ChemScreen aimed to fill this gap in a pragmatic manner preferably using validated existing tools and place them in an innovative alternative testing strategy. In our approach we combined knowledge on critical processes affected by reproductive toxicants with knowledge on the mechanistic basis of such effects. We used in silico methods for prescreening chemicals for relevant toxic effects aiming at reduced testing needs. For those chemicals that need testing we have set up an in vitro screening panel that includes mechanistic high throughput methods and lower throughput assays that measure more integrative endpoints. In silico pharmacokinetic modules were developed for rapid exposure predictions via diverse exposure routes. These modules to match in vitro and in vivo exposure levels greatly improved predictivity of the in vitro tests. As a further step, we have generated examples how to predict reproductive toxicity of chemicals using available data. We have executed formal validations of panel constituents and also used more innovative manners to validate the test panel using mechanistic approaches. We are actively engaged in promoting regulatory acceptance of the tools developed as an essential step towards practical application, including case studies for read-across purposes. With this approach, a significant saving in animal use and associated costs seems very feasible. Copyright © 2015 Elsevier Inc. All rights reserved.

  11. Genome-wide screen for modifiers of ataxin-3 neurodegeneration in Drosophila.

    Directory of Open Access Journals (Sweden)

    Julide Bilen

    2007-10-01

    Full Text Available Spinocerebellar ataxia type-3 (SCA3 is among the most common dominantly inherited ataxias, and is one of nine devastating human neurodegenerative diseases caused by the expansion of a CAG repeat encoding glutamine within the gene. The polyglutamine domain confers toxicity on the protein Ataxin-3 leading to neuronal dysfunction and loss. Although modifiers of polyglutamine toxicity have been identified, little is known concerning how the modifiers function mechanistically to affect toxicity. To reveal insight into spinocerebellar ataxia type-3, we performed a genetic screen in Drosophila with pathogenic Ataxin-3-induced neurodegeneration and identified 25 modifiers defining 18 genes. Despite a variety of predicted molecular activities, biological analysis indicated that the modifiers affected protein misfolding. Detailed mechanistic studies revealed that some modifiers affected protein accumulation in a manner dependent on the proteasome, whereas others affected autophagy. Select modifiers of Ataxin-3 also affected tau, revealing common pathways between degeneration due to distinct human neurotoxic proteins. These findings provide new insight into molecular pathways of polyQ toxicity, defining novel targets for promoting neuronal survival in human neurodegenerative disease.

  12. Molecular and genomic studies of IMMP2L and mutation screening in autism and Tourette syndrome.

    Science.gov (United States)

    Petek, Erwin; Schwarzbraun, Thomas; Noor, Abdul; Patel, Megha; Nakabayashi, Kazuhiko; Choufani, Sanaa; Windpassinger, Christian; Stamenkovic, Mara; Robertson, Mary M; Aschauer, Harald N; Gurling, Hugh M D; Kroisel, Peter M; Wagner, Klaus; Scherer, Stephen W; Vincent, John B

    2007-01-01

    We recently reported the disruption of the inner mitochondrial membrane peptidase 2-like (IMMP2L) gene by a chromosomal breakpoint in a patient with Gilles de la Tourette syndrome (GTS). In the present study we sought to identify genetic variation in IMMP2L, which, through alteration of protein function or level of expression might contribute to the manifestation of GTS. We screened 39 GTS patients, and, due to the localization of IMMP2L in the critical region for the autistic disorder (AD) locus on chromosome 7q (AUTS1), 95 multiplex AD families; however, no coding mutations were found in either GTS or AD patients. In addition, no parental-specific expression of IMMP2L was detected in somatic cell hybrids containing human chromosome 7 and human cell lines carrying a maternal uniparental disomy for chromosome 7 (mUPD7). Despite the fact that no deleterious mutations in IMMPL2 (other than the inverted duplication identified previously) were identified in either GTS or AD, this gene cannot be excluded as a possible rare cause of either disorder.

  13. Genome-Wide Overexpression Screen Identifies Genes Able to Bypass p16-Mediated Senescence in Melanoma.

    Science.gov (United States)

    Lee, Won Jae; Škalamera, Dubravka; Dahmer-Heath, Mareike; Shakhbazov, Konstanin; Ranall, Max V; Fox, Carly; Lambie, Duncan; Stevenson, Alexander J; Yaswen, Paul; Gonda, Thomas J; Gabrielli, Brian

    2017-03-01

    Malignant melanomas often arise from nevi, which result from initial oncogene-induced hyperproliferation of melanocytes that are maintained in a CDKN2A/p16-mediated senescent state. Thus, genes that can bypass this senescence barrier are likely to contribute to melanoma development. We have performed a gain-of-function screen of 17,030 lentivirally expressed human open reading frames (ORFs) in a melanoma cell line containing an inducible p16 construct to identify such genes. Genes known to bypass p16-induced senescence arrest, including the human papilloma virus 18 E7 gene ( HPV18E7), and genes such as the p16-binding CDK6 with expected functions, as well as panel of novel genes, were identified, including high-mobility group box (HMGB) proteins. A number of these were further validated in two other models of p16-induced senescence. Tissue immunohistochemistry demonstrated higher levels of CDK6 in primary melanomas compared with normal skin and nevi. Reduction of CDK6 levels drove melanoma cells expressing functional p16 into senescence, demonstrating its contribution to bypass senescence.

  14. Genome-Wide Screen for Haploinsufficient Cell Size Genes in the Opportunistic Yeast Candida albicans

    Directory of Open Access Journals (Sweden)

    Julien Chaillot

    2017-02-01

    Full Text Available One of the most critical but still poorly understood aspects of eukaryotic cell proliferation is the basis for commitment to cell division in late G1 phase, called Start in yeast and the Restriction Point in metazoans. In all species, a critical cell size threshold coordinates cell growth with cell division and thereby establishes a homeostatic cell size. While a comprehensive survey of cell size genetic determinism has been performed in the saprophytic yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, very little is known in pathogenic fungi. As a number of critical Start regulators are haploinsufficient for cell size, we applied a quantitative analysis of the size phenome, using elutriation-barcode sequencing methodology, to 5639 barcoded heterozygous deletion strains of the opportunistic yeast Candida albicans. Our screen identified conserved known regulators and biological processes required to maintain size homeostasis in the opportunistic yeast C. albicans. We also identified novel C. albicans-specific size genes and provided a conceptual framework for future mechanistic studies. Interestingly, some of the size genes identified were required for fungal pathogenicity suggesting that cell size homeostasis may be elemental to C. albicans fitness or virulence inside the host.

  15. Genome-Wide Screen for Haploinsufficient Cell Size Genes in the Opportunistic Yeast Candida albicans

    Science.gov (United States)

    Chaillot, Julien; Cook, Michael A.; Corbeil, Jacques; Sellam, Adnane

    2016-01-01

    One of the most critical but still poorly understood aspects of eukaryotic cell proliferation is the basis for commitment to cell division in late G1 phase, called Start in yeast and the Restriction Point in metazoans. In all species, a critical cell size threshold coordinates cell growth with cell division and thereby establishes a homeostatic cell size. While a comprehensive survey of cell size genetic determinism has been performed in the saprophytic yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, very little is known in pathogenic fungi. As a number of critical Start regulators are haploinsufficient for cell size, we applied a quantitative analysis of the size phenome, using elutriation-barcode sequencing methodology, to 5639 barcoded heterozygous deletion strains of the opportunistic yeast Candida albicans. Our screen identified conserved known regulators and biological processes required to maintain size homeostasis in the opportunistic yeast C. albicans. We also identified novel C. albicans-specific size genes and provided a conceptual framework for future mechanistic studies. Interestingly, some of the size genes identified were required for fungal pathogenicity suggesting that cell size homeostasis may be elemental to C. albicans fitness or virulence inside the host. PMID:28040776

  16. A DNA minor groove electronegative potential genome map based on photo-chemical probing

    DEFF Research Database (Denmark)

    Lindemose, Søren; Nielsen, Peter Eigil; Hansen, Morten

    2011-01-01

    The double-stranded DNA of the genome contains both sequence information directly relating to the protein and RNA coding as well as functional and structural information relating to protein recognition. Only recently is the importance of DNA shape in this recognition process being fully appreciated...... resolution of any genome, and it is illustrated how such detailed studies of this sequence dependent, inherent property of the DNA may reflect on genome organization, gene expression and chromosomal condensation....

  17. Development of microsatellite markers for common bean (Phaseolus vulgaris L.) based on screening of non-enriched, small-insert genomic libraries.

    Science.gov (United States)

    Blair, Matthew W; Torres, Monica Muñoz; Pedraza, Fabio; Giraldo, Martha C; Buendía, Hector F; Hurtado, Natalia

    2009-09-01

    Microsatellite markers are useful genetic tools for a wide array of genomic analyses although their development is time-consuming and requires the identification of simple sequence repeats (SSRs) from genomic sequences. Screening of non-enriched, small-insert libraries is an effective method of SSR isolation that can give an unbiased picture of motif frequency. Here we adapt high-throughput protocols for the screening of plasmid-based libraries using robotic colony picking and filter preparation. Seven non-enriched genomic libraries from common bean genomic DNA were made by digestion with four frequently cutting restriction enzymes, double digestion with a frequently cutting restriction enzyme and a less frequently cutting restriction enzyme, or sonication. Library quality was compared and three of the small-insert libraries were selected for further analysis. Each library was plated and picked into 384-well plates that were used to create high-density filter arrays of over 18 000 clones each, which were screened with oligonucleotide probes for various SSR motifs. Positive clones were found to have low redundancy. One hundred SSR markers were developed and 80 were tested for polymorphism in a standard parental survey. These microsatellite markers derived from non-SSR-enriched libraries should be useful additions to previous markers developed from enriched libraries.

  18. Human genome-wide RNAi screen identifies an essential role for inositol pyrophosphates in Type-I interferon response.

    Directory of Open Access Journals (Sweden)

    Niyas Kudukkil Pulloor

    2014-02-01

    Full Text Available The pattern recognition receptor RIG-I is critical for Type-I interferon production. However, the global regulation of RIG-I signaling is only partially understood. Using a human genome-wide RNAi-screen, we identified 226 novel regulatory proteins of RIG-I mediated interferon-β production. Furthermore, the screen identified a metabolic pathway that synthesizes the inositol pyrophosphate 1-IP7 as a previously unrecognized positive regulator of interferon production. Detailed genetic and biochemical experiments demonstrated that the kinase activities of IPPK, PPIP5K1 and PPIP5K2 (which convert IP5 to1-IP7 were critical for both interferon induction, and the control of cellular infection by Sendai and influenza A viruses. Conversely, ectopically expressed inositol pyrophosphate-hydrolases DIPPs attenuated interferon transcription. Mechanistic experiments in intact cells revealed that the expression of IPPK, PPIP5K1 and PPIP5K2 was needed for the phosphorylation and activation of IRF3, a transcription factor for interferon. The addition of purified individual inositol pyrophosphates to a cell free reconstituted RIG-I signaling assay further identified 1-IP7 as an essential component required for IRF3 activation. The inositol pyrophosphate may act by β-phosphoryl transfer, since its action was not recapitulated by a synthetic phosphonoacetate analogue of 1-IP7. This study thus identified several novel regulators of RIG-I, and a new role for inositol pyrophosphates in augmenting innate immune responses to viral infection that may have therapeutic applications.

  19. Genomic library screening for viruses from the human dental plaque revealed pathogen-specific lytic phage sequences.

    Science.gov (United States)

    Al-Jarbou, Ahmed Nasser

    2012-01-01

    Bacterial pathogenesis presents an astounding arsenal of virulence factors that allow them to conquer many different niches throughout the course of infection. Principally fascinating is the fact that some bacterial species are able to induce different diseases by expression of different combinations of virulence factors. Nevertheless, studies aiming at screening for the presence of bacteriophages in humans have been limited. Such screening procedures would eventually lead to identification of phage-encoded properties that impart increased bacterial fitness and/or virulence in a particular niche, and hence, would potentially be used to reverse the course of bacterial infections. As the human oral cavity represents a rich and dynamic ecosystem for several upper respiratory tract pathogens. However, little is known about virus diversity in human dental plaque which is an important reservoir. We applied the culture-independent approach to characterize virus diversity in human dental plaque making a library from a virus DNA fraction amplified using a multiple displacement method and sequenced 80 clones. The resulting sequence showed 44% significant identities to GenBank databases by TBLASTX analysis. TBLAST homology comparisons showed that 66% was viral; 18% eukarya; 10% bacterial; 6% mobile elements. These sequences were sorted into 6 contigs and 45 single sequences in which 4 contigs and a single sequence showed significant identity to a small region of a putative prophage in the Corynebacterium diphtheria genome. These findings interestingly highlight the uniqueness of over half of the sequences, whilst the dominance of a pathogen-specific prophage sequences imply their role in virulence.

  20. HIGH-THROUGHPUT CHEMICAL SCREENING USING PROTEIN PROFILING OF FISH PLASMA

    Science.gov (United States)

    Compounds that affect the hormone system, referred to as "endocrine-disrupting chemicals" (EDCs), cause human and animal health problems. It is necessary to test putative EDC chemicals for such deleterious effects, though current testing methodologies are time/animal intensive an...

  1. Using the pea aphid Acrythociphon pisum as a tool for screening biological responses to chemicals and drugs

    Directory of Open Access Journals (Sweden)

    Ledger Terence

    2009-09-01

    Full Text Available Abstract Background Though the biological process of aphid feeding is well documented, no one to date has sought to apply it as a tool to screen the biological responses to chemicals and drugs, in ecotoxicology, genotoxicology and/or for interactions in the cascade of sequential molecular events of embryogenesis. Parthenogenetic insect species present the advantage of an anatomical system composed of multiple germarium/ovarioles in the same mother with all the intermediate maturation stages of embryos from oocyte to first instar larva birth. This could be used as an interesting model to visualize at which step drugs interact with the cell signalling pathway during the ordered developmental process. Findings We designed a simple test for screening drugs by investigating simultaneously zygote mitotic division, the progression of embryo development, cell differentiation at early developmental stages and finally organogenesis and population growth rate. We aimed to analyze the toxicology effects of compounds and/or their interference on cellular signalling by examining at which step of the cascade, from zygote to mature embryo, the developmental process is perturbed. We reasoned that a parthenogenetic founder insect, in which the ovarioles shelter numerous embryos at different developmental stages, would allow us to precisely pinpoint the step of embryogenesis in which chemicals act through specific molecular targets as the known ordered homeobox genes. Conclusion Using this method we report the results of a genotoxicological and demographic analysis of three compound models bearing in common a bromo group: one is integrated as a base analog in DNA synthesis, two others activate permanently kinases. We report that one compound (Br-du altered drastically embryogenesis, which argues in favor of this simple technique as a cheap first screening of chemicals or drugs to be used in a number of genotoxicology applications.

  2. Genome-scale screen for DNA methylation-based detection markers for ovarian cancer.

    Directory of Open Access Journals (Sweden)

    Mihaela Campan

    Full Text Available BACKGROUND: The identification of sensitive biomarkers for the detection of ovarian cancer is of high clinical relevance for early detection and/or monitoring of disease recurrence. We developed a systematic multi-step biomarker discovery and verification strategy to identify candidate DNA methylation markers for the blood-based detection of ovarian cancer. METHODOLOGY/PRINCIPAL FINDINGS: We used the Illumina Infinium platform to analyze the DNA methylation status of 27,578 CpG sites in 41 ovarian tumors. We employed a marker selection strategy that emphasized sensitivity by requiring consistency of methylation across tumors, while achieving specificity by excluding markers with methylation in control leukocyte or serum DNA. Our verification strategy involved testing the ability of identified markers to monitor disease burden in serially collected serum samples from ovarian cancer patients who had undergone surgical tumor resection compared to CA-125 levels. We identified one marker, IFFO1 promoter methylation (IFFO1-M, that is frequently methylated in ovarian tumors and that is rarely detected in the blood of normal controls. When tested in 127 serially collected sera from ovarian cancer patients, IFFO1-M showed post-resection kinetics significantly correlated with serum CA-125 measurements in six out of 16 patients. CONCLUSIONS/SIGNIFICANCE: We implemented an effective marker screening and verification strategy, leading to the identification of IFFO1-M as a blood-based candidate marker for sensitive detection of ovarian cancer. Serum levels of IFFO1-M displayed post-resection kinetics consistent with a reflection of disease burden. We anticipate that IFFO1-M and other candidate markers emerging from this marker development pipeline may provide disease detection capabilities that complement existing biomarkers.

  3. Identification of putative estrogen receptor-mediated endocrine disrupting chemicals using QSAR- and structure-based virtual screening approaches

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Liying; Sedykh, Alexander; Tripathi, Ashutosh [Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC (United States); Zhu, Hao [The Rutgers Center for Computational and Integrative Biology, Rutgers University, Camden, NJ (United States); Department of Chemistry, Rutgers University, Camden, NJ (United States); Afantitis, Antreas; Mouchlis, Varnavas D.; Melagraki, Georgia [NovaMechanics Ltd., Nicosia (Cyprus); Rusyn, Ivan, E-mail: iir@unc.edu [Department of Environmental Sciences and Engineering, University of North Carolina, Chapel Hill, NC (United States); Tropsha, Alexander, E-mail: alex_tropsha@unc.edu [Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC (United States)

    2013-10-01

    Identification of endocrine disrupting chemicals is one of the important goals of environmental chemical hazard screening. We report on the development of validated in silico predictors of chemicals likely to cause estrogen receptor (ER)-mediated endocrine disruption to facilitate their prioritization for future screening. A database of relative binding affinity of a large number of ERα and/or ERβ ligands was assembled (546 for ERα and 137 for ERβ). Both single-task learning (STL) and multi-task learning (MTL) continuous quantitative structure–activity relationship (QSAR) models were developed for predicting ligand binding affinity to ERα or ERβ. High predictive accuracy was achieved for ERα binding affinity (MTL R{sup 2} = 0.71, STL R{sup 2} = 0.73). For ERβ binding affinity, MTL models were significantly more predictive (R{sup 2} = 0.53, p < 0.05) than STL models. In addition, docking studies were performed on a set of ER agonists/antagonists (67 agonists and 39 antagonists for ERα, 48 agonists and 32 antagonists for ERβ, supplemented by putative decoys/non-binders) using the following ER structures (in complexes with respective ligands) retrieved from the Protein Data Bank: ERα agonist (PDB ID: 1L2I), ERα antagonist (PDB ID: 3DT3), ERβ agonist (PDB ID: 2NV7), and ERβ antagonist (PDB ID: 1L2J). We found that all four ER conformations discriminated their corresponding ligands from presumed non-binders. Finally, both QSAR models and ER structures were employed in parallel to virtually screen several large libraries of environmental chemicals to derive a ligand- and structure-based prioritized list of putative estrogenic compounds to be used for in vitro and in vivo experimental validation. - Highlights: • This is the largest curated dataset inclusive of ERα and β (the latter is unique). • New methodology that for the first time affords acceptable ERβ models. • A combination of QSAR and docking enables prediction of affinity and function.

  4. The ChemScreen project to design a pragmatic alternative approach to predict reproductive toxicity of chemicals

    DEFF Research Database (Denmark)

    van der Burg, Bart; Wedebye, Eva Bay; Dietrich, Daniel R.

    2015-01-01

    There is a great need for rapid testing strategies for reproductive toxicity testing, avoiding animal use. The EU Framework program 7 project ChemScreen aimed to fill this gap in a pragmatic manner preferably using validated existing tools and place them in an innovative alternative testing...... in vitro and in vivo exposure levels greatly improved predictivity of the in vitro tests. As a further step, we have generated examples how to predict reproductive toxicity of chemicals using available data. We have executed formal validations of panel constituents and also used more innovative manners...

  5. The ChemScreen project to design a pragmatic alternative approachto predict reproductive toxicity of chemicals

    NARCIS (Netherlands)

    Van der Burg, B.; Wedebye, E.B.; Dietrich, D.R.; Jaworska, J.; Mangelsdorf, I.; Paune, E.; Schwarz, M.; Piersma, A.H.; Kroese, E.D.

    2015-01-01

    There is a great need for rapid testing strategies for reproductive toxicity testing, avoiding animal use.The EU Framework program 7 project ChemScreen aimed to fill this gap in a pragmatic manner prefer-ably using validated existing tools and place them in an innovative alternative testing

  6. The ChemScreen project to design a pragmatic alternative approachto predict reproductive toxicity of chemicals

    NARCIS (Netherlands)

    Van der Burg, B.; Wedebye, E.B.; Dietrich, D.R.; Jaworska, J.; Mangelsdorf, I.; Paune, E.; Schwarz, M.; Piersma, A.H.; Kroese, E.D.

    2015-01-01

    There is a great need for rapid testing strategies for reproductive toxicity testing, avoiding animal use.The EU Framework program 7 project ChemScreen aimed to fill this gap in a pragmatic manner prefer-ably using validated existing tools and place them in an innovative alternative testing strategy

  7. Nuclear domain ‘knock-in’ screen for the evaluation and identification of small molecule enhancers of CRISPR-based genome editing

    OpenAIRE

    Pinder, Jordan; Salsman, Jayme; Dellaire, Graham

    2015-01-01

    CRISPR is a genome-editing platform that makes use of the bacterially-derived endonuclease Cas9 to introduce DNA double-strand breaks at precise locations in the genome using complementary guide RNAs. We developed a nuclear domain knock-in screen, whereby the insertion of a gene encoding the green fluorescent protein variant Clover is inserted by Cas9-mediated homology directed repair (HDR) within the first exon of genes that are required for the structural integrity of subnuclear domains suc...

  8. A genome-wide RNAi screen reveals MAP kinase phosphatases as key ERK pathway regulators during embryonic stem cell differentiation.

    Directory of Open Access Journals (Sweden)

    Shen-Hsi Yang

    Full Text Available Embryonic stem cells and induced pluripotent stem cells represent potentially important therapeutic agents in regenerative medicine. Complex interlinked transcriptional and signaling networks control the fate of these cells towards maintenance of pluripotency or differentiation. In this study we have focused on how mouse embryonic stem cells begin to differentiate and lose pluripotency and, in particular, the role that the ERK MAP kinase and GSK3 signaling pathways play in this process. Through a genome-wide siRNA screen we have identified more than 400 genes involved in loss of pluripotency and promoting the onset of differentiation. These genes were functionally associated with the ERK and/or GSK3 pathways, providing an important resource for studying the roles of these pathways in controlling escape from the pluripotent ground state. More detailed analysis identified MAP kinase phosphatases as a focal point of regulation and demonstrated an important role for these enzymes in controlling ERK activation kinetics and subsequently determining early embryonic stem cell fate decisions.

  9. New Genes Tied to Endocrine, Metabolic, and Dietary Regulation of Lifespan from a Caenorhabditis elegans Genomic RNAi Screen.

    Directory of Open Access Journals (Sweden)

    2005-07-01

    Full Text Available Most of our knowledge about the regulation of aging comes from mutants originally isolated for other phenotypes. To ask whether our current view of aging has been affected by selection bias, and to deepen our understanding of known longevity pathways, we screened a genomic Caenorhabditis elegans RNAi library for clones that extend lifespan. We identified 23 new longevity genes affecting signal transduction, the stress response, gene expression, and metabolism and assigned these genes to specific longevity pathways. Our most important findings are (i that dietary restriction extends C. elegans' lifespan by down-regulating expression of key genes, including a gene required for methylation of many macromolecules, (ii that integrin signaling is likely to play a general, evolutionarily conserved role in lifespan regulation, and (iii that specific lipophilic hormones may influence lifespan in a DAF-16/FOXO-dependent fashion. Surprisingly, of the new genes that have conserved sequence domains, only one could not be associated with a known longevity pathway. Thus, our current view of the genetics of aging has probably not been distorted substantially by selection bias.

  10. Genome-wide screening reveals the emergence and divergence of RTK homologues in basal Metazoan Hydra magnipapillata

    Indian Academy of Sciences (India)

    P C Reddy; Salil S Bidaye; Surendra Ghaskadbi

    2011-06-01

    Receptor tyrosine kinases (RTKs) are key components of cell–cell signalling required for growth and development of multicellular organisms. It is therefore likely that the divergence of RTKs and associated components played a significant role in the evolution of multicellular organisms. We have carried out the present study in hydra, a diploblast, to investigate the divergence of RTKs after parazoa and before emergence of triploblast phyla. The domain-based screening using Hidden Markov Models (HMMs) for RTKs in Genomescan predicted gene models of the Hydra magnipapillata genome resulted in identification of 15 RTKs. These RTKs have been classified into eight families based on domain architecture and homology. Only 5 of these RTKs have been previously reported and a few of these have been partially characterized. A phylogeny-based analysis of these predicted RTKs revealed that seven subtype duplications occurred between `parazoan–eumetazoan split’ and `diploblast–triploblast split’ in animal phyla. These results suggest that most of the RTKs evolved before the radiata–bilateria divergence during animal evolution.

  11. Genome-wide siRNA Screening at Biosafety Level 4 Reveals a Crucial Role for Fibrillarin in Henipavirus Infection

    Science.gov (United States)

    Foo, Chwan Hong; Rootes, Christina L.; Gould, Cathryn M.; Grusovin, Julian; Monaghan, Paul; Lo, Michael K.; Tompkins, S. Mark; Adams, Timothy E.; Lowenthal, John W.; Simpson, Kaylene J.; Stewart, Cameron R.; Bean, Andrew G. D.; Wang, Lin-Fa

    2016-01-01

    Hendra and Nipah viruses (genus Henipavirus, family Paramyxoviridae) are highly pathogenic bat-borne viruses. The need for high biocontainment when studying henipaviruses has hindered the development of therapeutics and knowledge of the viral infection cycle. We have performed a genome-wide siRNA screen at biosafety level 4 that identified 585 human proteins required for henipavirus infection. The host protein with the largest impact was fibrillarin, a nucleolar methyltransferase that was also required by measles, mumps and respiratory syncytial viruses for infection. While not required for cell entry, henipavirus RNA and protein syntheses were greatly impaired in cells lacking fibrillarin, indicating a crucial role in the RNA replication phase of infection. During infection, the Hendra virus matrix protein co-localized with fibrillarin in cell nucleoli, and co-associated as a complex in pulldown studies, while its nuclear import was unaffected in fibrillarin-depleted cells. Mutagenesis studies showed that the methyltransferase activity of fibrillarin was required for henipavirus infection, suggesting that this enzyme could be targeted therapeutically to combat henipavirus infections. PMID:27010548

  12. Genome-wide siRNA Screening at Biosafety Level 4 Reveals a Crucial Role for Fibrillarin in Henipavirus Infection.

    Directory of Open Access Journals (Sweden)

    Celine Deffrasnes

    2016-03-01

    Full Text Available Hendra and Nipah viruses (genus Henipavirus, family Paramyxoviridae are highly pathogenic bat-borne viruses. The need for high biocontainment when studying henipaviruses has hindered the development of therapeutics and knowledge of the viral infection cycle. We have performed a genome-wide siRNA screen at biosafety level 4 that identified 585 human proteins required for henipavirus infection. The host protein with the largest impact was fibrillarin, a nucleolar methyltransferase that was also required by measles, mumps and respiratory syncytial viruses for infection. While not required for cell entry, henipavirus RNA and protein syntheses were greatly impaired in cells lacking fibrillarin, indicating a crucial role in the RNA replication phase of infection. During infection, the Hendra virus matrix protein co-localized with fibrillarin in cell nucleoli, and co-associated as a complex in pulldown studies, while its nuclear import was unaffected in fibrillarin-depleted cells. Mutagenesis studies showed that the methyltransferase activity of fibrillarin was required for henipavirus infection, suggesting that this enzyme could be targeted therapeutically to combat henipavirus infections.

  13. Genome-wide siRNA screen identifies the retromer as a cellular entry factor for human papillomavirus.

    Science.gov (United States)

    Lipovsky, Alex; Popa, Andreea; Pimienta, Genaro; Wyler, Michael; Bhan, Ashima; Kuruvilla, Leena; Guie, Marie-Aude; Poffenberger, Adrian C; Nelson, Christian D S; Atwood, Walter J; DiMaio, Daniel

    2013-04-30

    Despite major advances in our understanding of many aspects of human papillomavirus (HPV) biology, HPV entry is poorly understood. To identify cellular genes required for HPV entry, we conducted a genome-wide screen for siRNAs that inhibited infection of HeLa cells by HPV16 pseudovirus. Many retrograde transport factors were required for efficient infection, including multiple subunits of the retromer, which initiates retrograde transport from the endosome to the trans-Golgi network (TGN). The retromer has not been previously implicated in virus entry. Furthermore, HPV16 capsid proteins arrive in the TGN/Golgi in a retromer-dependent fashion during entry, and incoming HPV proteins form a stable complex with retromer subunits. We propose that HPV16 directly engages the retromer at the early or late endosome and traffics to the TGN/Golgi via the retrograde pathway during cell entry. These results provide important insights into HPV entry, identify numerous potential antiviral targets, and suggest that the role of the retromer in infection by other viruses should be assessed.

  14. A bow-tie genetic architecture for morphogenesis suggested by a genome-wide RNAi screen in Caenorhabditis elegans.

    Directory of Open Access Journals (Sweden)

    Matthew D Nelson

    2011-03-01

    Full Text Available During animal development, cellular morphogenesis plays a fundamental role in determining the shape and function of tissues and organs. Identifying the components that regulate and drive morphogenesis is thus a major goal of developmental biology. The four-celled tip of the Caenorhabditis elegans male tail is a simple but powerful model for studying the mechanism of morphogenesis and its spatiotemporal regulation. Here, through a genome-wide post-embryonic RNAi-feeding screen, we identified 212 components that regulate or participate in male tail tip morphogenesis. We constructed a working hypothesis for a gene regulatory network of tail tip morphogenesis. We found regulatory roles for the posterior Hox genes nob-1 and php-3, the TGF-β pathway, nuclear hormone receptors (e.g. nhr-25, the heterochronic gene blmp-1, and the GATA transcription factors egl-18 and elt-6. The majority of the pathways converge at dmd-3 and mab-3. In addition, nhr-25 and dmd-3/mab-3 regulate each others' expression, thus placing these three genes at the center of a complex regulatory network. We also show that dmd-3 and mab-3 negatively regulate other signaling pathways and affect downstream cellular processes such as vesicular trafficking (e.g. arl-1, rme-8 and rearrangement of the cytoskeleton (e.g. cdc-42, nmy-1, and nmy-2. Based on these data, we suggest that male tail tip morphogenesis is governed by a gene regulatory network with a bow-tie architecture.

  15. IN SILICO SCREENING OF CHEMICAL COMPOUNDS FROM SWEET FLAG (ARACUS CALAMUS L AS α-GLUCOSIDASE INHIBITOR

    Directory of Open Access Journals (Sweden)

    Dewi Yuliana

    2013-03-01

    Full Text Available Research have been conducted screening in silico chemical compound inhibitor α-glucosidase from plants dringo (Acorus calamus L based on the binding site (binding site are owned by some of the compounds obtained respectively from the inhibition of enzyme / receptor (docking using the program Argus Lab. Model of the enzyme α-glucosidase was obtained through the protein data bank with the code 1lwj in the donwload NCBI website. Models of chemical compounds contained in dringo (A. Calamus L obtained through the site Take out "jamu" Knapsack and made in the formula structures of 2D and 3D using the program ACD / Chemsketch. Docking results showed activity in the compound 1-ethenyl-1-methyl-2,4-at (prop-1-en-2-yl Cyclohexane with free energy - 8.04385 kcal / mol, and the compound Isocaespitol with a free energy - 8.28388 kcal / mol.

  16. A quantitative screening-level approach to incorporate chemical exposure and risk/safety into alternative assessment evaluations.

    Science.gov (United States)

    Arnold, Scott M; Greggs, Bill; Goyak, Katy O; Landenberger, Bryce D; Mason, Ann M; Howard, Brett; Zaleski, Rosemary; Howard, Brett; Zaleski, Rosemary T

    2017-03-10

    As the general public and retailers ask for disclosure of chemical ingredients in the marketplace, a number of hazard screening tools were developed to evaluate the so called "greenness" of individual chemical ingredients and/or formulations. The majority of these tools focus only on hazard, often using chemical lists, ignoring the other part of the risk equation: exposure. Using a hazard-only focus can result in regrettable substitutions, changing one chemical ingredient for another that turns out to be more hazardous or shifts the toxicity burden to others. To minimize the incidents of regrettable substitutions, BizNGO describes 'Common Principles' to frame a process for informed substitution. Two of the six principles state reduce hazard and minimize exposure. A number of frameworks have emerged to evaluate and assess alternatives. One framework developed by leading experts under the auspices of the U.S. National Academy of Sciences recommended that hazard and exposure be specifically addressed in the same step when assessing candidate alternatives. For the alternative assessment community, this paper serves as an informational resource for considering exposure in an alternatives assessment using elements of problem formulation; product identity, use, and composition; hazard analysis; exposure analysis; and risk characterization. These conceptual elements build upon practices from government, academia, and industry and are exemplified through two hypothetical case studies demonstrating the questions asked and decisions faced in new product development. These two case studies - inhalation exposure to a generic paint product and environmental exposure to a shampoo rinsed down the drain - demonstrate the criteria, considerations, and methods required to combine exposure models addressing human health and environmental impacts to provide a screening level hazard/exposure (risk) analysis. This paper informs practices for these elements within a comparative risk

  17. Evaluation of food-relevant chemicals in the ToxCast high-throughput screening program

    Data.gov (United States)

    U.S. Environmental Protection Agency — Thousands of chemicals are directly added to or come in contact with food, many of which have undergone little to no toxicological evaluation. The landscape of the...

  18. Screening Chemical Effects on Steroidogenesis in H295R Human Adrenocortical Carcinoma Cells (SOT)

    Science.gov (United States)

    Proper endocrine function requires steroid hormone biosynthesis and metabolism (steroidogenesis). Disruption of steroidogenesis by environmental chemicals can result in altered hormone levels causing adverse reproductive and developmental effects. This study is the first to estab...

  19. Weak Intermolecular Hydrogen Bonds with Fluorine: Detection and Implications for Enzymatic/Chemical Reactions, Chemical Properties, and Ligand/Protein Fluorine NMR Screening.

    Science.gov (United States)

    Dalvit, Claudio; Vulpetti, Anna

    2016-05-23

    It is known that strong hydrogen-bonding interactions play an important role in many chemical and biological systems. However, weak or very weak hydrogen bonds, which are often difficult to detect and characterize, may also be relevant in many recognition and reaction processes. Fluorine serving as a hydrogen-bond acceptor has been the subject of many controversial discussions and there are different opinions about it. It now appears that there is compelling experimental evidence for the involvement of fluorine in weak intramolecular or intermolecular hydrogen bonds. Using established NMR methods, we have previously characterized and measured the strengths of intermolecular hydrogen-bond complexes involving the fluorine moieties CH2 F, CHF2 , and CF3 , and have compared them with the well-known hydrogen-bond complex formed between acetophenone and the strong hydrogen-bond donor p-fluorophenol. We now report evidence for the formation of hydrogen bonds involving fluorine with significantly weaker donors, namely 5-fluoroindole and water. A simple NMR method is proposed for the simultaneous measurement of the strengths of hydrogen bonds between an acceptor and a donor or water. Important implications of these results for enzymatic/chemical reactions involving fluorine, for chemical and physical properties, and for ligand/protein (19) F NMR screening are analyzed through experiments and theoretical simulations.

  20. High-throughput Screening of ToxCast™ Phase I Chemicals in a Mouse Embryonic Stem Cell (mESC) Assay Reveals Disruption of Potential Toxicity Pathways

    Science.gov (United States)

    Little information is available regarding the potential for many commercial chemicals to induce developmental toxicity. The mESC Adherent Cell Differentiation and Cytoxicity (ACDC) assay is a high-throughput screen used to close this data gap. Thus, ToxCast™ Phase I chemicals wer...

  1. A tree based method for the rapid screening of chemical fingerprints

    DEFF Research Database (Denmark)

    Kristensen, Thomas Greve; Nielsen, Jesper; Pedersen, Christian Nørgaard Storm

    2009-01-01

    The fingerprint of a molecule is a bitstring based on its structure, constructed such that structurally similar molecules will have similar fingerprints. Molecular fingerprints can be used in an initial phase for identifying novel drug candidates by screening large databases for molecules...... with fingerprints similar to a query fingerprint. In this paper, we present a method which efficiently finds all fingerprints in a database with Tanimoto coefficient to the query fingerprint above a user defined threshold. The method is based on two novel data structures for rapid screening of large databases......: the kD grid and the Multibit tree. The kD grid is based on splitting the fingerprints into k shorter bitstrings and utilising these to compute bounds on the similarity of the complete bitstrings. The Multibit tree uses hierarchical clustering and similarity within each cluster to compute similar bounds...

  2. Screening of endocrine disrupting chemicals with MELN cells, an ER-transactivation assay combined with cytotoxicity assessment.

    Science.gov (United States)

    Berckmans, P; Leppens, H; Vangenechten, C; Witters, H

    2007-10-01

    There is growing concern that some chemicals can cause endocrine disrupting effects to wild animals and humans. Therefore a rapid and reliable screening assay to assess the activity of endocrine disrupting chemicals (EDCs) is required. These EDCs can act at multiple sites. Most studied mechanism is direct interaction with the hormone receptors, e.g. estrogen receptor. In this study the luciferase reporter gene assay using transgenic human MELN cells was used. Since cytotoxicity of the chemicals can decrease the luminescent signal in the transactivation assays, a cytotoxicity assay must be implemented. Mostly the neutral red (NR) assay is performed in parallel with the estrogenicity assay. To increase the reliability and cost-efficiency of the test, a method to measure estrogenicity and cytotoxicity in the same cell culture plate instead of in parallel plates was developed and evaluated. Therefore the NR-assay was compared with the CytoTox-ONE homogeneous membrane integrity assay. The latter measures LDH (lactate dehydrogenase) leakage based on a fluorometric method. For all compounds tested, the CytoTox-ONE test showed comparable curves and EC50-values to those obtained by the NR-assay. So the CytoTox-ONE kit, which seemed more sensitive than measurements of LDH-leakage based on a colorimetric method, is recommended to test cytotoxicity to MELN cells, with the advantage to use the same cells for ER-transactivation measurements. The chemicals tested in the optimised MELN assay showed estrogenic potencies comparable to those reported for several other transactivation assays.

  3. Quantitative Phenotyping-Based In Vivo Chemical Screening in a Zebrafish Model of Leukemia Stem Cell Xenotransplantation

    Science.gov (United States)

    Zhang, Beibei; Shimada, Yasuhito; Kuroyanagi, Junya; Umemoto, Noriko; Nishimura, Yuhei; Tanaka, Toshio

    2014-01-01

    Zebrafish-based chemical screening has recently emerged as a rapid and efficient method to identify important compounds that modulate specific biological processes and to test the therapeutic efficacy in disease models, including cancer. In leukemia, the ablation of leukemia stem cells (LSCs) is necessary to permanently eradicate the leukemia cell population. However, because of the very small number of LSCs in leukemia cell populations, their use in xenotransplantation studies (in vivo) and the difficulties in functionally and pathophysiologically replicating clinical conditions in cell culture experiments (in vitro), the progress of drug discovery for LSC inhibitors has been painfully slow. In this study, we developed a novel phenotype-based in vivo screening method using LSCs xenotransplanted into zebrafish. Aldehyde dehydrogenase-positive (ALDH+) cells were purified from chronic myelogenous leukemia K562 cells tagged with a fluorescent protein (Kusabira-orange) and then implanted in young zebrafish at 48 hours post-fertilization. Twenty-four hours after transplantation, the animals were treated with one of eight different therapeutic agents (imatinib, dasatinib, parthenolide, TDZD-8, arsenic trioxide, niclosamide, salinomycin, and thioridazine). Cancer cell proliferation, and cell migration were determined by high-content imaging. Of the eight compounds that were tested, all except imatinib and dasatinib selectively inhibited ALDH+ cell proliferation in zebrafish. In addition, these anti-LSC agents suppressed tumor cell migration in LSC-xenotransplants. Our approach offers a simple, rapid, and reliable in vivo screening system that facilitates the phenotype-driven discovery of drugs effective in suppressing LSCs. PMID:24454867

  4. Rapid screening method for determination of Ecstasy and amphetamines in urine samples using gas chromatography-chemical ionisation mass spectrometry.

    Science.gov (United States)

    Pellegrini, M; Rosati, F; Pacifici, R; Zuccaro, R; Romolo, F S; Lopez, A

    2002-04-05

    The need for analytical screening tests more reliable and valid to detect amphetamine and related "designer drugs" in biological samples is becoming critical, due to the increasing diffusion of these drugs on the European illegal market. The most common screening procedures based on immunoassays suffer a number of limitations, including low sensitivity, lack of specificity and limited number of detectable substances. This paper describes a screening method based on gas-chromatography-mass-spectrometry (GC/MS) using positive chemical ionisation (PCI) detection. Methanol was used as reactant gas in the ionisation chamber. Molecular ions of different compounds were monitored, allowing a sensitivity of 5-10 ng/ml with high selectivity. The sensitivity of the method gives positive results in samples taken 48-72 h after intake of one dose of 50-100 mg. The method is simple and rapid. Sample preparation was limited to one liquid-liquid extraction, without any hydrolysis and derivatisation. Hydrolysis is critical to identify metabolites excreted as conjugates. Blank urine samples spiked with known amounts of amphetamine (AM), methylamphetamine (MA), methylenedioxyamphetamine (MDA), methylenedioxymethylamphetamine (MDMA), methylenedioxyethylamphetamine (MDEA) and methylenedioxyphenyl-N-methyl-2-butanamine (MBDB) were analysed. The method was successfully tested on real samples of urine from people, whose use of amphetamine was suspected, and results were compared with results obtained with immunoassays.

  5. Single-Crystalline Silicon Solar Cell with Selective Emitter Formed by Screen Printing and Chemical Etching Method: A Feasibility Study

    Directory of Open Access Journals (Sweden)

    Yen-Po Chen

    2013-01-01

    Full Text Available A new method for fabricating crystalline silicon solar cells with selective emitters is presented. In this method, shallow trenches corresponding to metal contact area are first formed by screen printing and chemical etching, followed by heavy doping over the whole front surface of the silicon wafer. After a polymer mask is pasted by aligned screen-printing to cover the shallow trenches, the silicon wafer is etched such that the heavy doping remains at the shallow trench area, while other areas become lightly doped. With the presented method, two screening printing steps are required for obtaining a selective emitter structure on a solar wafer. Compared with existing etch-back methods, the presented one is believed to be able to easily conform with present industrial process. Experimental results show that optical responses at the short and long wavelengths were both improved by applying the proposed selective emitter technique to fabricate solar cells with an a-Si:H film deposited on the back surface. The selective emitter cell with a-Si:H back surface deposition had improvements of 1.66 mA/cm2 and 1.23% absolute in Jsc and conversion efficiency, respectively, compared to the reference cell that had a homogeneous emitter and no a-Si:H on the back surface.

  6. Low-Cost, Disposable, Flexible and Highly Reproducible Screen Printed SERS Substrates for the Detection of Various Chemicals

    Science.gov (United States)

    Wu, Wei; Liu, Li; Dai, Zhigao; Liu, Juhua; Yang, Shuanglei; Zhou, Li; Xiao, Xiangheng; Jiang, Changzhong; Roy, Vellaisamy A. L.

    2015-05-01

    Ideal SERS substrates for sensing applications should exhibit strong signal enhancement, generate a reproducible and uniform response, and should be able to fabricate in large-scale and low-cost. Herein, we demonstrate low-cost, highly sensitive, disposable and reproducible SERS substrates by means of screen printing Ag nanoparticles (NPs) on a plastic PET (Polyethylene terephthalate) substrates. While there are many complex methods for the fabrication of SERS substrates, screen printing is suitable for large-area fabrication and overcomes the uneven radial distribution. Using as-printed Ag substrates as the SERS platform, detection of various commonly known chemicals have been done. The SERS detection limit of Rhodamine 6G (R6G) is higher than the concentration of 1 × 10-10 M. The relative standard deviation (RSD) value for 784 points on the detection of R6G and Malachite green (MG) is less than 20% revealing a homogeneous SERS distribution and high reproducibility. Moreover, melamine (MA) is detected in fresh liquid-milk without additional pretreatment, which may accelerate the application of rapid on-line detection of MA in liquid milk. Our screen printing method highlights the use of large-scale printing strategies for the fabrication of well-defined functional nanostructures with applications well beyond the field of SERS sensing.

  7. Screening tests for assessing the anaerobic biodegradation of pollutant chemicals in subsurface environments

    Science.gov (United States)

    Suflita, Joseph M.; Concannon, Frank

    1995-01-01

    Screening methods were developed to assess the susceptibility of ground water contaminants to anaerobic biodegradation. One method was an extrapolation of a procedure previously used to measure biodegradation activity in dilute sewage sludge. Aquifer solids and ground water with no additional nutritive media were incubated anaerobically in 160-ml serum bottles containing 250 mg·l−1 carbon of the substrate of interest. This method relied on the detection of gas pressure or methane production in substrateamended microcosms relative to background controls. Other screening procedures involved the consumption of stoichiometrically required amounts of sulfate or nitrate from the same type of incubations. Close agreement was obtained between the measured and calculated amounts of substrate bioconversion based on the measured biogas pressure in methanogenic microcosms. Storage of the microcosms for up to 6 months did not adversely influence the onset or rate of benzoic acid mineralization. The lower detection limits of the methanogenic assay were found to be a function of the size of the microcosm headspace, the mean oxidation state of the substrate carbon, and the method used to correct for background temperature fluctuations. Using these simple screening procedures, biodegradation information of regulatory interest could be generated, including, (i) the length of the adaptation period, (ii) the rate of substrate decay and (iii) the completeness of the bioconversion.

  8. Sulfonamides identified as plant immune-priming compounds in high-throughput chemical screening increase disease resistance in Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Yoshiteru eNoutoshi

    2012-10-01

    Full Text Available Plant activators are agrochemicals that protect crops from diseases by activating the plant immune system. To isolate lead compounds for use as practical plant activators, we screened 2 different chemical libraries composed of various bioactive substances by using an established screening procedure that can selectively identify immune-priming compounds. We identified and characterized a group of sulfonamide compounds—sulfameter, sulfamethoxypyridazine, sulfabenzamide, and sulfachloropyridazine—among the various isolated candidate molecules. These sulfonamide compounds enhanced the avirulent Pseudomonas-induced cell death of Arabidopsis suspension cell cultures and increased disease resistance in Arabidopsis plants against both avirulent and virulent strains of the bacterium. These compounds did not prevent the growth of pathogenic bacteria in minimal liquid media at 200 µM. They also did not induce the expression of defense-related genes in Arabidopsis seedlings, at least not at 24 and 48 h after treatment, suggesting that they do not act as salicylic acid analogs. In addition, although sulfonamides are known to be folate biosynthesis inhibitors, the application of folate did not restore the potentiation effects of the sulfonamides on pathogen-induced cell death. Our data suggest that sulfonamides potentiate Arabidopsis disease resistance by their novel chemical properties.

  9. A functional genomics screen identifies an Importin-α homolog as a regulator of stem cell function and tissue patterning during planarian regeneration

    OpenAIRE

    2015-01-01

    Background Planarians are renowned for their regenerative capacity and are an attractive model for the study of adult stem cells and tissue regeneration. In an effort to better understand the molecular mechanisms underlying planarian regeneration, we performed a functional genomics screen aimed at identifying genes involved in this process in Schmidtea mediterranea. Methods We used microarrays to detect changes in gene expression in regenerating and non-regenerating tissues in planarians rege...

  10. Serum-Free Generation of Multipotent Mesoderm (Kdr-positive) Progenitor Cells in Mouse Embryonic Stem Cells For Functional Genomics Screening

    OpenAIRE

    2012-01-01

    This unit describes a robust protocol for producing multipotent Kdr-expressing mesoderm progenitor cells in serum-free conditions and functional genomics screening using these cells. Kdr-positive cells are known to be able to differentiate into a wide array of mesoderm derivatives including, vascular endothelial cells, cardiomyocytes, hematopietic progenitors and smooth muscle cells. The efficient generation of such progenitor cells is of particular interest because it permits subsequent step...

  11. The carcinoGENOMICS project: Critical selection of model compounds for the development of omics-based in vitro carcinogenicity screening assays

    NARCIS (Netherlands)

    Vinken, M.; Doktorova, T.; Ellinger-Ziegelbauer, H.; Ahr, H.-J.; Lock, E.; Carmichael, P.; Roggen, E.; Delft, J. van; Kleinjans, J.; Castell, J.; Bort, R.; Donato, T.; Ryan, M.; Corvi, R.; Keun, H.; Ebbels, T.; Athersuch, T.; Sansone, S.-A.; Rocca-Serra, P.; Stierum, R.; Jennings, P.; Pfaller, W.; Gmuender, H.; Vanhaecke, T.; Rogiers, V.

    2008-01-01

    Recent changes in the European legislation of chemical-related substances have forced the scientific community to speed up the search for alternative methods that could partly or fully replace animal experimentation. The Sixth Framework Program project carcinoGENOMICS was specifically raised to deve

  12. QSAR Models for Thyroperoxidase Inhibition and Screening of U.S. and EU Chemical Inventories

    DEFF Research Database (Denmark)

    Abildgaard Rosenberg, Sine; D. Watt, Eric; Judson, Richard S.

    2017-01-01

    Thyroperoxidase (TPO) is the enzyme that synthesizes thyroid hormones (THs). TPO inhibition by chemicals can result in decreased TH levels and developmental neurotoxicity, and therefore identification of TPO inhibition is of high relevance in safety evaluation of chemicals. In the present study, we...... to QSAR1. Of the substances predicted within QSAR2’s applicability domain, 8,790 (19.3%) REACH substances and 7,166 (19.0%) U.S. EPA substances, respectively, were predicted to be TPO inhibitors. A case study on butyl hydroxyanisole (BHA), which is extensively used as an antioxidant, was included...

  13. Screening of metagenomic and genomic libraries reveals three classes of bacterial enzymes that overcome the toxicity of acrylate.

    Science.gov (United States)

    Curson, Andrew R J; Burns, Oliver J; Voget, Sonja; Daniel, Rolf; Todd, Jonathan D; McInnis, Kathryn; Wexler, Margaret; Johnston, Andrew W B

    2014-01-01

    Acrylate is produced in significant quantities through the microbial cleavage of the highly abundant marine osmoprotectant dimethylsulfoniopropionate, an important process in the marine sulfur cycle. Acrylate can inhibit bacterial growth, likely through its conversion to the highly toxic molecule acrylyl-CoA. Previous work identified an acrylyl-CoA reductase, encoded by the gene acuI, as being important for conferring on bacteria the ability to grow in the presence of acrylate. However, some bacteria lack acuI, and, conversely, many bacteria that may not encounter acrylate in their regular environments do contain this gene. We therefore sought to identify new genes that might confer tolerance to acrylate. To do this, we used functional screening of metagenomic and genomic libraries to identify novel genes that corrected an E. coli mutant that was defective in acuI, and was therefore hyper-sensitive to acrylate. The metagenomic libraries yielded two types of genes that overcame this toxicity. The majority encoded enzymes resembling AcuI, but with significant sequence divergence among each other and previously ratified AcuI enzymes. One other metagenomic gene, arkA, had very close relatives in Bacillus and related bacteria, and is predicted to encode an enoyl-acyl carrier protein reductase, in the same family as FabK, which catalyses the final step in fatty-acid biosynthesis in some pathogenic Firmicute bacteria. A genomic library of Novosphingobium, a metabolically versatile alphaproteobacterium that lacks both acuI and arkA, yielded vutD and vutE, two genes that, together, conferred acrylate resistance. These encode sequential steps in the oxidative catabolism of valine in a pathway in which, significantly, methacrylyl-CoA is a toxic intermediate. These findings expand the range of bacteria for which the acuI gene encodes a functional acrylyl-CoA reductase, and also identify novel enzymes that can similarly function in conferring acrylate resistance, likely, again

  14. Screening of metagenomic and genomic libraries reveals three classes of bacterial enzymes that overcome the toxicity of acrylate.

    Directory of Open Access Journals (Sweden)

    Andrew R J Curson

    Full Text Available Acrylate is produced in significant quantities through the microbial cleavage of the highly abundant marine osmoprotectant dimethylsulfoniopropionate, an important process in the marine sulfur cycle. Acrylate can inhibit bacterial growth, likely through its conversion to the highly toxic molecule acrylyl-CoA. Previous work identified an acrylyl-CoA reductase, encoded by the gene acuI, as being important for conferring on bacteria the ability to grow in the presence of acrylate. However, some bacteria lack acuI, and, conversely, many bacteria that may not encounter acrylate in their regular environments do contain this gene. We therefore sought to identify new genes that might confer tolerance to acrylate. To do this, we used functional screening of metagenomic and genomic libraries to identify novel genes that corrected an E. coli mutant that was defective in acuI, and was therefore hyper-sensitive to acrylate. The metagenomic libraries yielded two types of genes that overcame this toxicity. The majority encoded enzymes resembling AcuI, but with significant sequence divergence among each other and previously ratified AcuI enzymes. One other metagenomic gene, arkA, had very close relatives in Bacillus and related bacteria, and is predicted to encode an enoyl-acyl carrier protein reductase, in the same family as FabK, which catalyses the final step in fatty-acid biosynthesis in some pathogenic Firmicute bacteria. A genomic library of Novosphingobium, a metabolically versatile alphaproteobacterium that lacks both acuI and arkA, yielded vutD and vutE, two genes that, together, conferred acrylate resistance. These encode sequential steps in the oxidative catabolism of valine in a pathway in which, significantly, methacrylyl-CoA is a toxic intermediate. These findings expand the range of bacteria for which the acuI gene encodes a functional acrylyl-CoA reductase, and also identify novel enzymes that can similarly function in conferring acrylate

  15. Gametocytocidal screen identifies novel chemical classes with Plasmodium falciparum transmission blocking activity.

    Directory of Open Access Journals (Sweden)

    Natalie G Sanders

    Full Text Available Discovery of transmission blocking compounds is an important intervention strategy necessary to eliminate and eradicate malaria. To date only a small number of drugs that inhibit gametocyte development and thereby transmission from the mosquito to the human host exist. This limitation is largely due to a lack of screening assays easily adaptable to high throughput because of multiple incubation steps or the requirement for high gametocytemia. Here we report the discovery of new compounds with gametocytocidal activity using a simple and robust SYBR Green I- based DNA assay. Our assay utilizes the exflagellation step in male gametocytes and a background suppressor, which masks the staining of dead cells to achieve healthy signal to noise ratio by increasing signal of viable parasites and subtracting signal from dead parasites. By determining the contribution of exflagellation to fluorescent signal and using appropriate cutoff values, we were able to screen for gametocytocidal compounds. After assay validation and optimization, we screened an FDA approved drug library of approximately 1500 compounds, as well as the 400 compound MMV malaria box and identified 44 gametocytocidal compounds with sub to low micromolar IC50s. Major classes of compounds with gametocytocidal activity included quaternary ammonium compounds with structural similarity to choline, acridine-like compounds similar to quinacrine and pyronaridine, as well as antidepressant, antineoplastic, and anthelminthic compounds. Top drug candidates showed near complete transmission blocking in membrane feeding assays. This assay is simple, reproducible and demonstrated robust Z-factor values at low gametocytemia levels, making it amenable to HTS for identification of novel and potent gametocytocidal compounds.

  16. Phyto chemical Screening, Antibacterial, Antifungal and Anthelmintic Activity of Morinda citrifolia stem

    Directory of Open Access Journals (Sweden)

    Dr. D. Gopala Krishna

    2013-05-01

    Full Text Available In the present study, the Petroleum Ether and Alcoholic extract of Morinda citrifolia L. (Noni stem were subjected to preliminary screening for Antimicrobial and Aanthelmintic activity. The alcoholic extract exhibited significant Anti bacterial, Antifungal activity, comparable to the standard drug Tetracycline. The Petroleum Ether and Alcoholic extract were evaluated for Anthelmintic activity on adult Indian Earthworms, ‘Pheretima posithuma’. The Alcoholic extract produced more significant Anthelmintic activity than Petroleum ether extract and the activities are comparable with the reference drug Piperazine citrate

  17. Developing, Applying, and Evaluating Models for Rapid Screening of Chemical Exposures

    DEFF Research Database (Denmark)

    Arnot, J.; Shin, H.; Ernstoff, Alexi;

    2015-01-01

    to limited exposure data there is limited information on chemical use patterns and production and emission quantities. These data gaps require the application of mass balance, statistical and quantitative structure-activity relationship (QSAR) models to predict exposure and exposure potential for humans...

  18. Current issues involving screening and identification of chemical contaminants in foods by mass spectrometry

    NARCIS (Netherlands)

    Lehotay, S.J.; Sapozhnikova, Y.; Mol, J.G.J.

    2015-01-01

    Although quantitative analytical methods must be empirically validated prior to their use in a variety of applications, including regulatory monitoring of chemical adulterants in foods, validation of qualitative method performance for the analytes and matrices of interest is frequently ignored, or g

  19. Current issues involving screening and identification of chemical contaminants in foods by mass spectrometry

    NARCIS (Netherlands)

    Lehotay, S.J.; Sapozhnikova, Y.; Mol, J.G.J.

    2015-01-01

    Although quantitative analytical methods must be empirically validated prior to their use in a variety of applications, including regulatory monitoring of chemical adulterants in foods, validation of qualitative method performance for the analytes and matrices of interest is frequently ignored, or

  20. Chip Technologies for Screening Chemical and Biological Agents Against Plant-Parasitic Nematodes.

    Science.gov (United States)

    Beeman, Augustine Q; Njus, Zach L; Pandey, Santosh; Tylka, Gregory L

    2016-12-01

    Plant-parasitic nematodes cause substantial damage to agricultural crops worldwide. Long-term management of these pests requires novel strategies to reduce infection of host plants. Disruption of nematode chemotaxis to root systems has been proposed as a potential management approach, and novel assays are needed to test the chemotactic behavior of nematodes against a wide range of synthetic chemicals and root exudates. Two microfluidic chips were developed that measure the attraction or repulsion of nematodes to chemicals ("chemical chip") and young plant roots ("root chip"). The chip designs allowed for chemical concentration gradients to be maintained up to 24 h, the nematodes to remain physically separate from the chemical reservoirs, and for images of nematode populations to be captured using either a microscope or a flatbed scanner. In the experiments using the chemical chips, seven ionic solutions were tested on second-stage juveniles (J2s) of Meloidogyne incognita and Heterodera glycines. Results were consistent with previous reports of repellency of M. incognita to a majority of the ionic solutions, including NH4NO3, KNO3, KCl, MgCl2, and CaCl2. H. glycines was found to be attracted to both NH4NO3 and KNO3, which has not been reported previously. A software program was written to aid in monitoring the location of nematodes at regular time intervals using the root chip. In experiments with the root chip, H. glycines J2s were attracted to roots of 3-day-old, susceptible (cultivar Williams 82) soybean seedlings, and attraction of H. glycines to susceptible soybean was similar across the length of the root. Attraction to resistant (cultivar Jack) soybean seedlings relative to the water only control was inconsistent across runs, and H. glycines J2s were not preferentially attracted to the roots of resistant or susceptible cultivars when both were placed on opposite sides of the same root chip. The chips developed allow for direct tests of plant

  1. Modelling physico-chemical properties of (benzo)triazoles, and screening for environmental partitioning.

    Science.gov (United States)

    Bhhatarai, B; Gramatica, P

    2011-01-01

    (Benzo)triazoles are distributed throughout the environment, mainly in water compartments, because of their wide use in industry where they are employed in pharmaceutical, agricultural and deicing products. They are hazardous chemicals that adversely affect humans and other non-target species, and are on the list of substances of very high concern (SVHC) in the new European regulation of chemicals - REACH (Registration, Evaluation, Authorization and Restriction of Chemical substances). Thus there is a vital need for further investigations to understand the behavior of these compounds in biota and the environment. In such a scenario, physico-chemical properties like aqueous solubility, hydrophobicity, vapor pressure and melting point can be useful. However, the limited availability and the high cost of lab testing prevents the acquisition of necessary experimental data that industry must submit for the registration of these chemicals. In such cases a preliminary analysis can be made using Quantitative Structure-Property Relationships (QSPR) models. For such an analysis, we propose Multiple Linear Regression (MLR) models based on theoretical molecular descriptors selected by Genetic Algorithm (GA). Training and prediction sets were prepared a priori by splitting the available experimental data, which were then used to derive statistically robust and predictive (both internally and externally) models. These models, after verification of their structural applicability domain (AD), were used to predict the properties of a total of 351 compounds, including those in the REACH preregistration list. Finally, Principal Component Analysis was applied to the predictions to rank the environmental partitioning properties (relevant for leaching and volatility) of new and untested (benzo)triazoles within the AD of each model. Our study using this approach highlighted compounds dangerous for the aquatic compartment. Similar analyses using predictions obtained by the EPI Suite and

  2. A Tree Based Method for the Rapid Screening of Chemical Fingerprints

    Science.gov (United States)

    Kristensen, Thomas G.; Nielsen, Jesper; Pedersen, Christian N. S.

    The fingerprint of a molecule is a bitstring based on its structure, constructed such that structurally similar molecules will have similar fingerprints. Molecular fingerprints can be used in an initial phase for identifying novel drug candidates by screening large databases for molecules with fingerprints similar to a query fingerprint. In this paper, we present a method which efficiently finds all fingerprints in a database with Tanimoto coefficient to the query fingerprint above a user defined threshold. The method is based on two novel data structures for rapid screening of large databases: the kD grid and the Multibit tree. The kD grid is based on splitting the fingerprints into k shorter bitstrings and utilising these to compute bounds on the similarity of the complete bitstrings. The Multibit tree uses hierarchical clustering and similarity within each cluster to compute similar bounds. We have implemented our method and tested it on a large data set from the industry. Our experiments show that our method yields a three-fold speed-up over previous methods.

  3. Evaluation of a partial genome screening of two asthma susceptibility regions using bayesian network based bayesian multilevel analysis of relevance.

    Directory of Open Access Journals (Sweden)

    Ildikó Ungvári

    Full Text Available Genetic studies indicate high number of potential factors related to asthma. Based on earlier linkage analyses we selected the 11q13 and 14q22 asthma susceptibility regions, for which we designed a partial genome screening study using 145 SNPs in 1201 individuals (436 asthmatic children and 765 controls. The results were evaluated with traditional frequentist methods and we applied a new statistical method, called bayesian network based bayesian multilevel analysis of relevance (BN-BMLA. This method uses bayesian network representation to provide detailed characterization of the relevance of factors, such as joint significance, the type of dependency, and multi-target aspects. We estimated posteriors for these relations within the bayesian statistical framework, in order to estimate the posteriors whether a variable is directly relevant or its association is only mediated.With frequentist methods one SNP (rs3751464 in the FRMD6 gene provided evidence for an association with asthma (OR = 1.43(1.2-1.8; p = 3×10(-4. The possible role of the FRMD6 gene in asthma was also confirmed in an animal model and human asthmatics.In the BN-BMLA analysis altogether 5 SNPs in 4 genes were found relevant in connection with asthma phenotype: PRPF19 on chromosome 11, and FRMD6, PTGER2 and PTGDR on chromosome 14. In a subsequent step a partial dataset containing rhinitis and further clinical parameters was used, which allowed the analysis of relevance of SNPs for asthma and multiple targets. These analyses suggested that SNPs in the AHNAK and MS4A2 genes were indirectly associated with asthma. This paper indicates that BN-BMLA explores the relevant factors more comprehensively than traditional statistical methods and extends the scope of strong relevance based methods to include partial relevance, global characterization of relevance and multi-target relevance.

  4. A genome-wide siRNA screen in mammalian cells for regulators of S6 phosphorylation.

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    Angela Papageorgiou

    Full Text Available mTOR complex1, the major regulator of mRNA translation in all eukaryotic cells, is strongly activated in most cancers. We performed a genome-wide RNAi screen in a human cancer cell line, seeking genes that regulate S6 phosphorylation, readout of mTORC1 activity. Applying a stringent selection, we retrieved nearly 600 genes wherein at least two RNAis gave significant reduction in S6-P. This cohort contains known regulators of mTOR complex 1 and is significantly enriched in genes whose depletion affects the proliferation/viability of the large set of cancer cell lines in the Achilles database in a manner paralleling that caused by mTOR depletion. We next examined the effect of RNAi pools directed at 534 of these gene products on S6-P in TSC1 null mouse embryo fibroblasts. 76 RNAis reduced S6 phosphorylation significantly in 2 or 3 replicates. Surprisingly, among this cohort of genes the only elements previously associated with the maintenance of mTORC1 activity are two subunits of the vacuolar ATPase and the CUL4 subunit DDB1. RNAi against a second set of 84 targets reduced S6-P in only one of three replicates. However, an indication that this group also bears attention is the presence of rpS6KB1 itself, Rac1 and MAP4K3, a protein kinase that supports amino acid signaling to rpS6KB1. The finding that S6 phosphorylation requires a previously unidentified, functionally diverse cohort of genes that participate in fundamental cellular processes such as mRNA translation, RNA processing, DNA repair and metabolism suggests the operation of feedback pathways in the regulation of mTORC1 operating through novel mechanisms.

  5. Genome-wide screening and identification of new Trypanosoma cruzi antigens with potential application for chronic Chagas disease diagnosis.

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    João Luís Reis-Cunha

    Full Text Available The protozoan Trypanosoma cruzi is the etiologic agent of Chagas disease, an infection that afflicts approximately 8 million people in Latin America. Diagnosis of chronic Chagas disease is currently based on serological tests because this condition is usually characterized by high anti-T. cruzi IgG titers and low parasitemia. The antigens used in these assays may have low specificity due to cross reactivity with antigens from related parasite infections, such as leishmaniasis, and low sensitivity caused by the high polymorphism among T. cruzi strains. Therefore, the identification of new T. cruzi-specific antigens that are conserved among the various parasite discrete typing units (DTUs is still required. In the present study, we have explored the hybrid nature of the T. cruzi CL Brener strain using a broad genome screening approach to select new T. cruzi antigens that are conserved among the different parasite DTUs and that are absent in other trypanosomatid species. Peptide arrays containing the conserved antigens with the highest epitope prediction scores were synthesized, and the reactivity of the peptides were tested by immunoblot using sera from C57BL/6 mice chronically infected with T. cruzi strains from the TcI, TcII or TcVI DTU. The two T. cruzi proteins that contained the most promising peptides were expressed as recombinant proteins and tested in ELISA experiments with sera from chagasic patients with distinct clinical manifestations: those infected with T. cruzi from different DTUs and those with cutaneous or visceral leishmaniasis. These proteins, named rTc_11623.20 and rTc_N_10421.310, exhibited 94.83 and 89.66% sensitivity, 98.2 and 94.6% specificity, respectively, and a pool of these 2 proteins exhibited 96.55% sensitivity and 98.18% specificity. This work led to the identification of two new antigens with great potential application in the diagnosis of chronic Chagas disease.

  6. Effects of immunostimulation on social behavior, chemical communication and genome-wide gene expression in honey bee workers (Apis mellifera

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    Richard Freddie-Jeanne

    2012-10-01

    Full Text Available Abstract Background Social insects, such as honey bees, use molecular, physiological and behavioral responses to combat pathogens and parasites. The honey bee genome contains all of the canonical insect immune response pathways, and several studies have demonstrated that pathogens can activate expression of immune effectors. Honey bees also use behavioral responses, termed social immunity, to collectively defend their hives from pathogens and parasites. These responses include hygienic behavior (where workers remove diseased brood and allo-grooming (where workers remove ectoparasites from nestmates. We have previously demonstrated that immunostimulation causes changes in the cuticular hydrocarbon profiles of workers, which results in altered worker-worker social interactions. Thus, cuticular hydrocarbons may enable workers to identify sick nestmates, and adjust their behavior in response. Here, we test the specificity of behavioral, chemical and genomic responses to immunostimulation by challenging workers with a panel of different immune stimulants (saline, Sephadex beads and Gram-negative bacteria E. coli. Results While only bacteria-injected bees elicited altered behavioral responses from healthy nestmates compared to controls, all treatments resulted in significant changes in cuticular hydrocarbon profiles. Immunostimulation caused significant changes in expression of hundreds of genes, the majority of which have not been identified as members of the canonical immune response pathways. Furthermore, several new candidate genes that may play a role in cuticular hydrocarbon biosynthesis were identified. Effects of immune challenge expression of several genes involved in immune response, cuticular hydrocarbon biosynthesis, and the Notch signaling pathway were confirmed using quantitative real-time PCR. Finally, we identified common genes regulated by pathogen challenge in honey bees and other insects. Conclusions These results demonstrate that

  7. Screening of chemicals with anti-estrogenic activity using in vitro and in vivo vitellogenin induction responses in zebrafish (Danio rerio).

    Science.gov (United States)

    Sun, Liwei; Wen, Liling; Shao, Xiaolu; Qian, Haifeng; Jin, Yuanxiang; Liu, Weiping; Fu, Zhengwei

    2010-02-01

    Growing concern over possible adverse effects of endocrine-disrupting chemicals (EDCs) has driven the development of associated screening methods. The use of the vitellogenin (VTG) induction response in cultured teleost hepatocytes has been suggested as an in vitro screening assay for EDCs. However, current data do not sufficiently support this assay in the routine screening of chemicals. This study established and validated the use of primary cultured hepatocytes from zebrafish to screen chemicals for anti-estrogenic activities. Here we measured the transcript levels of selected hepatic estrogen-response genes, including vtg1, vtg2 and eralpha. Two model anti-estrogens, letrozole (LET), an aromatase inhibitor, and tamoxifen (TAM), a competitive estrogen receptor, were selected as representative chemicals. Additionally, comparisons between in vitro and in vivo assays were performed. As expected, there were concentration-dependent decreases for all three genes in the liver of female zebrafish exposed to LET in vivo for 72h. Similar responses were observed in males. As for in vitro testing, no discernable alterations in the gene transcripts were found in hepatocytes from males or females. In the case of TAM, exposure for 72h caused transcriptional reduction of hepatic estrogen-response genes in females in vivo and in vitro. In males, low concentrations of TAM resulted in increased expression of genes, while the expression decreased slightly at higher concentrations. Since these observations were in agreement with the pharmaceutical properties of two tested chemicals, the primary hepatocyte culture could be a promising tool for screening suspected EDCs.

  8. Development of a stable cell line with an intact PGC-1α/ERRα axis for screening environmental chemicals

    Energy Technology Data Exchange (ETDEWEB)

    Teng, Christina T., E-mail: teng1@niehs.nih.gov [DNTP, BioMolecular Screening Branch, Division, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709 (United States); Beames, Burton; Alex Merrick, B. [DNTP, BioMolecular Screening Branch, Division, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709 (United States); Martin, Negin; Romeo, Charles [DIR, Viral Core Lab, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709 (United States); Jetten, Anton M. [DIR Laboratory of Respiratory Biology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709 (United States)

    2014-02-07

    Highlights: • We developed a stable cell line with intact PGC-1α/ERRα axis. • The ERRα repressor, XCT790, down regulates this pathway. • Phytoestrogen, genisten stimulates this pathway. - Abstract: The estrogen-related receptor α (ERRα) and the peroxisome proliferator-activated receptor γ (PPARγ) coactivator 1α (PGC-1α) play critical roles in the control of several physiological functions, including the regulation of genes involved in energy homeostasis. However, little is known about the ability of environmental chemicals to disrupt or modulate this important bioenergetics pathway in humans. The goal of this study was to develop a cell-based assay system with an intact PGC-1α/ERRα axis that could be used as a screening assay for detecting such chemicals. To this end, we successfully generated several stable cell lines expressing PGC-1α and showed that the reporter driven by the native ERRα hormone response unit (AAB-Luc) is active in these cell lines and that the activation is PGC-1α-dependent. Furthermore, we show that this activation can be blocked by the ERRα selective inverse agonist, XCT790. In addition, we find that genistein and bisphenol A further stimulate the reporter activity, while kaempferol has minimal effect. These cell lines will be useful for identifying environmental chemicals that modulate this important pathway.

  9. Applicability of passive sampling to bioanalytical screening of bioaccumulative chemicals in marine wildlife.

    Science.gov (United States)

    Jin, Ling; Gaus, Caroline; van Mourik, Louise; Escher, Beate I

    2013-07-16

    Quantification of bioaccumulative contaminants in biota is time and cost-intensive and the required extensive cleanup steps make it selective toward targeted chemical groups. Therefore tissue extracts prepared for chemical analysis are not amenable to assess the combined effects of unresolved complex mixtures. Passive equilibrium sampling with polydimethylsiloxane (PDMS) has the potential for unbiased sampling of mixtures, and the PDMS extracts can be directly dosed into cell-based bioassays. The passive sampling approach was tested by exposing PDMS to lipid-rich tissue (dugong blubber; 85% lipid) spiked with a known mixture of hydrophobic contaminants (five congeners of tetra- to octachloro-dibenzo-p-dioxins). The equilibrium was attained within 24 h. Lipid-PDMS partition coefficients (Klip-PDMS) ranged from 20 to 38, were independent of hydrophobicity, and within the range of those previously measured for organochlorine compounds. To test if passive sampling can be combined with bioanalysis without the need for chemical cleanup, spiked blubber-PDMS extracts were dosed into the CAFLUX bioassay, which specifically targets dioxin-like chemicals. Small quantities of lipids coextracted by the PDMS were found to affect the kinetics in the regularly applied 24-h bioassay; however, this effect was eliminated by a longer exposure period (72 h). The validated method was applied to 11 unspiked dugong blubber samples with known (native) dioxin concentrations. These results provide the first proof of concept for linking passive sampling of lipid-rich tissue with cell-based bioassays, and could be further extended to other lipid rich species and a wider range of bioanalytical end points.

  10. Germanium doping of GaN by metalorganic chemical vapor deposition for polarization screening applications

    KAUST Repository

    Young, N.G.

    2016-10-01

    We demonstrate n-type doping of GaN with Ge by MOCVD at high concentrations that are necessary to fully screen the polarization fields in c-plane InGaN/GaN quantum wells. Hall measurements show linear Ge incorporation with dopant flow rate and carrier concentrations exceeding 1×10 cm. GaN:Ge layers exhibit excellent electron mobility, high conductivity, and contact resistivity comparable to the best unannealed contacts to Si-doped GaN. However, the surface morphology begins to degrade with Ge concentrations above 1×10 cm, resulting in severe step bunching and a network of plateaus and trenches, even in layers as thin as 10 nm.

  11. NeuroChip: a microfluidic electrophysiological device for genetic and chemical biology screening of Caenorhabditis elegans adult and larvae.

    Science.gov (United States)

    Hu, Chunxiao; Dillon, James; Kearn, James; Murray, Caitriona; O'Connor, Vincent; Holden-Dye, Lindy; Morgan, Hywel

    2013-01-01

    Genetic and chemical biology screens of C. elegans have been of enormous benefit in providing fundamental insight into neural function and neuroactive drugs. Recently the exploitation of microfluidic devices has added greater power to this experimental approach providing more discrete and higher throughput phenotypic analysis of neural systems. Here we make a significant addition to this repertoire through the design of a semi-automated microfluidic device, NeuroChip, which has been optimised for selecting worms based on the electrophysiological features of the pharyngeal neural network. We demonstrate this device has the capability to sort mutant from wild-type worms based on high definition extracellular electrophysiological recordings. NeuroChip resolves discrete differences in excitatory, inhibitory and neuromodulatory components of the neural network from individual animals. Worms may be fed into the device consecutively from a reservoir and recovered unharmed. It combines microfluidics with integrated electrode recording for sequential trapping, restraining, recording, releasing and recovering of C. elegans. Thus mutant worms may be selected, recovered and propagated enabling mutagenesis screens based on an electrophysiological phenotype. Drugs may be rapidly applied during the recording thus permitting compound screening. For toxicology, this analysis can provide a precise description of sub-lethal effects on neural function. The chamber has been modified to accommodate L2 larval stages showing applicability for small size nematodes including parasitic species which otherwise are not tractable to this experimental approach. We also combine NeuroChip with optogenetics for targeted interrogation of the function of the neural circuit. NeuroChip thus adds a new tool for exploitation of C. elegans and has applications in neurogenetics, drug discovery and neurotoxicology.

  12. Designing and screening of universal drug from neem (Azadirachta indica) and standard drug chemicals against influenza virus nucleoprotein.

    Science.gov (United States)

    Ahmad, Aftab; Javed, Muhammad Rizwan; Rao, Abdul Qayyum; Husnain, Tayyab

    2016-12-16

    Different strains of influenza virus are affecting a large number of people worldwide. Many synthetic antiviral medicines are available for influenza virus in the market. But still there is a need for the development of universal drugs against these strains of influenza virus. For this purpose conserved residues within the influenza virus nucleoprotein have been retrieved. The drugs, previously known to have antiviral properties, were screened to identify the best candidate universal drug against Influenza virus strains. Compounds from leaf extracts of neem, were also screened to identify the natural drugs without side effects. Molecular docking identified three potential compounds (Nimbaflavone, Rutin, and Hyperoside) having perfect binding with reported conserved residues (ASP302, SER50) of influenza virus nucleoprotein that is involved in the binding of drugs. Further analysis showed Hyperoside as a universal drug against various influenza strains. Some chemical drugs were also evaluated through screening against nucleoprotein. The results showed six drugs (OMS, CBX, LGH, Naproxen, BMS-883559, and BMS-885838) which were interacting with same conserved residues (ASP302, TYR52, SER50, GLY288, SER376, and ARG99) as were found in the case of neem phytochemicals. Hyperoside from neem leaf extract along with drugs LGH, Naproxen, BMS-885838, and BMS-883559 showed best interactions with conserved residues of nucleoprotein. The compound Hyperoside from neem leaf extract along with drugs LGH, Naproxen, BMS-885838, and BMS-883559 showed best interactions with conserved residues of nucleoprotein. So these compounds have been identified for their potential against influenza strains to be utilized as a universal drug.

  13. NeuroChip: a microfluidic electrophysiological device for genetic and chemical biology screening of Caenorhabditis elegans adult and larvae.

    Directory of Open Access Journals (Sweden)

    Chunxiao Hu

    Full Text Available Genetic and chemical biology screens of C. elegans have been of enormous benefit in providing fundamental insight into neural function and neuroactive drugs. Recently the exploitation of microfluidic devices has added greater power to this experimental approach providing more discrete and higher throughput phenotypic analysis of neural systems. Here we make a significant addition to this repertoire through the design of a semi-automated microfluidic device, NeuroChip, which has been optimised for selecting worms based on the electrophysiological features of the pharyngeal neural network. We demonstrate this device has the capability to sort mutant from wild-type worms based on high definition extracellular electrophysiological recordings. NeuroChip resolves discrete differences in excitatory, inhibitory and neuromodulatory components of the neural network from individual animals. Worms may be fed into the device consecutively from a reservoir and recovered unharmed. It combines microfluidics with integrated electrode recording for sequential trapping, restraining, recording, releasing and recovering of C. elegans. Thus mutant worms may be selected, recovered and propagated enabling mutagenesis screens based on an electrophysiological phenotype. Drugs may be rapidly applied during the recording thus permitting compound screening. For toxicology, this analysis can provide a precise description of sub-lethal effects on neural function. The chamber has been modified to accommodate L2 larval stages showing applicability for small size nematodes including parasitic species which otherwise are not tractable to this experimental approach. We also combine NeuroChip with optogenetics for targeted interrogation of the function of the neural circuit. NeuroChip thus adds a new tool for exploitation of C. elegans and has applications in neurogenetics, drug discovery and neurotoxicology.

  14. New agents with potential leishmanicidal activity identified by virtual screening of chemical databases: New agents with potential leishmanicidal activity

    Directory of Open Access Journals (Sweden)

    Juan Rebollo

    2013-04-01

    Full Text Available Introduction and Objectives: Leishmaniosis, a disease caused by a protozoan parasite, remains a serious public health problem threatening about 350 million people around the world, of which 12 million are believed to be currently infected (WHO 2010. To date, there are no vaccines against the species of parasites and the treatment is based only on chemotherapy with toxic-, expensive- and inefficient- drugs. There is an urgent need for better drugs against Leishmania, the etiological agent of the disease. The main anti-leishmanial drug used in Colombia is meglumineantimoniate [chemical name according to the International Union of Pure and Applied Chemistry (IUPAC: Hydroxy-dioxostiborane; (2R,3R,4R,5S- 6-methylaminohexane-1,2,3,4,5-pentol, (C7H17NO5], which is not efficient in the treatment of infections caused by Leishmania braziliensis, the most prevalent specie in the Caribbean coast of Colombia. Methods: We performed an in silico virtual screening of several datasets including ChemBridge and Pubchem. We virtually screened a total of 28.755 compounds against a 3D model of 6-phosphoglucono -lactonase (6-PGL from Leishmania braziliensis to identify novel inhibitors.Molecular docking of databases was performed using the software Sybyl 8.0 and AutoDockVina. Results: The initial virtual screening using a structure-based method identified 10 compounds, which were later tested with AutodockVina and classified according to their docking scores. Conclusions: These novel and potential inhibitors constitute new drug candidates that must be biologically tested to define their value as an alternative chemotherapeutic agent in the treatment of these protozoan infections. Salud UIS 2013; 45 (1: 33-40

  15. Quantitative phenotyping-based in vivo chemical screening in a zebrafish model of leukemia stem cell xenotransplantation.

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    Beibei Zhang

    Full Text Available Zebrafish-based chemical screening has recently emerged as a rapid and efficient method to identify important compounds that modulate specific biological processes and to test the therapeutic efficacy in disease models, including cancer. In leukemia, the ablation of leukemia stem cells (LSCs is necessary to permanently eradicate the leukemia cell population. However, because of the very small number of LSCs in leukemia cell populations, their use in xenotransplantation studies (in vivo and the difficulties in functionally and pathophysiologically replicating clinical conditions in cell culture experiments (in vitro, the progress of drug discovery for LSC inhibitors has been painfully slow. In this study, we developed a novel phenotype-based in vivo screening method using LSCs xenotransplanted into zebrafish. Aldehyde dehydrogenase-positive (ALDH+ cells were purified from chronic myelogenous leukemia K562 cells tagged with a fluorescent protein (Kusabira-orange and then implanted in young zebrafish at 48 hours post-fertilization. Twenty-four hours after transplantation, the animals were treated with one of eight different therapeutic agents (imatinib, dasatinib, parthenolide, TDZD-8, arsenic trioxide, niclosamide, salinomycin, and thioridazine. Cancer cell proliferation, and cell migration were determined by high-content imaging. Of the eight compounds that were tested, all except imatinib and dasatinib selectively inhibited ALDH+ cell proliferation in zebrafish. In addition, these anti-LSC agents suppressed tumor cell migration in LSC-xenotransplants. Our approach offers a simple, rapid, and reliable in vivo screening system that facilitates the phenotype-driven discovery of drugs effective in suppressing LSCs.

  16. Facile high-throughput forward chemical genetic screening by in situ monitoring of glucuronidase-based reporter gene expression in Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Vivek eHalder

    2015-01-01

    Full Text Available The use of biologically active small molecules to perturb biological functions holds enormous potential for investigating complex signaling networks. However, in contrast to animal systems, the search for and application of chemical tools for basic discovery in the plant sciences, generally referred to as ‘chemical genetics’, has only recently gained momentum. In addition to cultured cells, the well-characterized, small-sized model plant Arabidopsis thaliana is suitable for cultivation in microplates, which allows employing diverse cell- or phenotype-based chemical screens. In such screens, a chemical’s bioactivity is typically assessed either through scoring its impact on morphological traits or quantifying molecular attributes such as enzyme or reporter activities. Here, we describe a facile forward chemical screening methodology for intact Arabidopsis seedlings harboring the β-glucuronidase (GUS reporter by directly quantifying GUS activity in situ with 4-methylumbelliferyl-β-D-glucuronide (4-MUG as substrate. The quantitative nature of this screening assay has an obvious advantage over the also convenient histochemical GUS staining method, as it allows application of statistical procedures and unbiased hit selection based on threshold values as well as distinction between compounds with strong or weak bioactivity. At the same time, the in situ bioassay is very convenient requiring less effort and time for sample handling in comparison to the conventional quantitative in vitro GUS assay using 4-MUG, as validated with several Arabidopsis lines harboring different GUS reporter constructs. To demonstrate that the developed assays is particularly suitable for large-scale screening projects, we performed a pilot screen for chemical activators or inhibitors of salicylic acid-mediated defense signaling using the Arabidopsis PR1p::GUS line. Importantly, the screening methodology provided here can be adopted for any inducible GUS reporter line.

  17. Identification of three classes of heteroaromatic compounds with activity against intracellular Trypanosoma cruzi by chemical library screening.

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    Esther Bettiol

    Full Text Available The development of new drugs against Chagas disease is a priority since the currently available medicines have toxic effects, partial efficacy and are targeted against the acute phase of disease. At present, there is no drug to treat the chronic stage. In this study, we have optimized a whole cell-based assay for high throughput screening of compounds that inhibit infection of mammalian cells by Trypanosoma cruzi trypomastigotes. A 2000-compound chemical library was screened using a recombinant T. cruzi (Tulahuen strain expressing beta-galactosidase. Three hits were selected for their high activity against T. cruzi and low toxicity to host cells in vitro: PCH1, NT1 and CX1 (IC(50: 54, 190 and 23 nM, respectively. Each of these three compounds presents a different mechanism of action on intracellular proliferation of T. cruzi amastigotes. CX1 shows strong trypanocidal activity, an essential characteristic for the development of drugs against the chronic stage of Chagas disease where parasites are found intracellular in a quiescent stage. NT1 has a trypanostatic effect, while PCH1 affects parasite division. The three compounds also show high activity against intracellular T. cruzi from the Y strain and against the related kinetoplastid species Leishmania major and L. amazonensis. Characterization of the anti-T. cruzi activity of molecules chemically related to the three library hits allowed the selection of two compounds with IC(50 values of 2 nM (PCH6 and CX2. These values are approximately 100 times lower than those of the medicines used in patients against T. cruzi. These results provide new candidate molecules for the development of treatments against Chagas disease and leishmaniasis.

  18. Screening of adulterants in powdered foods and ingredients using line-scan Raman chemical imaging

    Science.gov (United States)

    Qin, Jianwei; Chao, Kuanglin; Kim, Moon S.

    2015-05-01

    A newly developed line-scan Raman imaging system using a 785 nm line laser was used to authenticate powdered foods and ingredients. The system was used to collect hyperspectral Raman images in a wavenumber range of 102-2865 cm-1 from three representative food powders mixed with selected adulterants with a concentration of 0.5%, including milk and melamine, flour and benzoyl peroxide, and starch and maleic anhydride. An acoustic mixer was used to create food adulterant mixtures. All the mixed samples were placed in sample holders with a surface area of 50 mm×50 mm. Spectral and image processing algorithms were developed based on single-band images at unique Raman peaks of the individual adulterants. Chemical images were created to show identification, spatial distribution, and morphological features of the adulterant particles mixed in the food powders. The potential of estimating mass concentrations of the adulterants using the percentages of the adulterant pixels in the chemical images was also demonstrated.

  19. Phytochemical screening and chemical variability in volatile oils of aerial parts of Morinda morindoides.

    Science.gov (United States)

    Kiazolu, J Boima; Intisar, Azeem; Zhang, Lingyi; Wang, Yun; Zhang, Runsheng; Wu, Zhongping; Zhang, Weibing

    2016-10-01

    Morinda morindoides is an important Liberian traditional medicine for the treatment of malaria, fever, worms etc. The plant was subjected to integrated approaches including phytochemical screening and gas chromatography mass spectrometry (GC-MS) analyses. Phytochemical investigation of the powdered plant revealed the presence of phenolics, tannins, flavonoids, saponins, terpenes, steroidal compounds and volatile oil. Steam distillation followed by GC-MS resulted in the identification of 47 volatiles in its aerial parts: 28 were in common including various bioactive volatiles. Major constituents of leaves were phytol (43.63%), palmitic acid (8.55%) and geranyl linalool (6.95%) and stem were palmitic acid (14.95%), eicosane (9.67%) and phytol (9.31%), and hence, a significant difference in the percentage composition of aerial parts was observed. To study seasonal changes, similarity analysis was carried out by calculating correlation coefficient (r) and vector angle cosine (z) that were more than 0.91 for stem-to-stem and leaf-to-leaf batches indicating considerable consistency.

  20. A chemical screen probing the relationship between mitochondrial content and cell size.

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    Toshimori Kitami

    Full Text Available The cellular content of mitochondria changes dynamically during development and in response to external stimuli, but the underlying mechanisms remain obscure. To systematically identify molecular probes and pathways that control mitochondrial abundance, we developed a high-throughput imaging assay that tracks both the per cell mitochondrial content and the cell size in confluent human umbilical vein endothelial cells. We screened 28,786 small molecules and observed that hundreds of small molecules are capable of increasing or decreasing the cellular content of mitochondria in a manner proportionate to cell size, revealing stereotyped control of these parameters. However, only a handful of compounds dissociate this relationship. We focus on one such compound, BRD6897, and demonstrate through secondary assays that it increases the cellular content of mitochondria as evidenced by fluorescence microscopy, mitochondrial protein content, and respiration, even after rigorous correction for cell size, cell volume, or total protein content. BRD6897 increases uncoupled respiration 1.6-fold in two different, non-dividing cell types. Based on electron microscopy, BRD6897 does not alter the percent of cytoplasmic area occupied by mitochondria, but instead, induces a striking increase in the electron density of existing mitochondria. The mechanism is independent of known transcriptional programs and is likely to be related to a blockade in the turnover of mitochondrial proteins. At present the molecular target of BRD6897 remains to be elucidated, but if identified, could reveal an important additional mechanism that governs mitochondrial biogenesis and turnover.

  1. A Forward Chemical Screen Using Zebrafish Embryos with Novel 2-Substituted 2H-Chromene Derivatives

    Science.gov (United States)

    Torregroza, Ingrid; Evans, Todd; Das, Bhaskar C.

    2011-01-01

    We synthesized 2-substituted 2H-chromene derivatives from salicylaldehyde using potassium vinylic borates in the presence of secondary amines. Our goal was to generate novel compounds that might modulate transforming growth factor-β signaling, based on limited rational design. Potassium vinyl trifluoroborates react with salicylaldehydes at 80 °C in the presence of a secondary amine and produce 2-substituted 2H-chromene derivatives with a 70–90% yield. A small library of these compounds, predicted to potentially interact with transforming growth factor-β receptors, was screened for bioactivity in living zebrafish embryos. We found that the related compounds differentially affect development, and demonstrate one compound that produces severe body axis alterations in early embryogenesis and at lower doses affects specifically cardiovascular development. This compound modulates specifically a Smad-independent transforming growth factor-β-regulated mitogen-activated protein kinase pathway, namely p-SAPK/JNK. These compounds, as suggested by our biological assays, may prove useful to manipulate developmental programs and develop therapeutic tools. PMID:19207470

  2. DART-MS as a preliminary screening method for "herbal incense": chemical analysis of synthetic cannabinoids.

    Science.gov (United States)

    Lesiak, Ashton D; Musah, Rabi A; Domin, Marek A; Shepard, Jason R E

    2014-03-01

    Direct analysis in real time mass spectrometry (DART-MS) served as a method for rapid high-throughput screening of six commercially available "Spice" products, detecting various combinations of five synthetic cannabinoids. Direct analysis in real time is an ambient ionization process that, along with high mass accuracy time-of-flight (TOF)-MS to 0.0001 Da, was employed to establish the presence of cannabinoids. Mass spectra were acquired by simply suspending a small portion of sample between the ion source and the mass spectrometer inlet. The ability to test minute amounts of sample is a major advantage when very limited amounts of evidentiary material are available. In addition, reports are widespread regarding the testing backlogs that now exist because of the large influx of designer drugs. This method circumvents time-consuming sample extraction, derivatization, chromatographic, and other sample preparative steps required for analysis by more conventional mass spectrometric methods. Accordingly, the synthetic cannabinoids AM-2201, JWH-122, JWH-203, JWH-210, and RCS-4 were identified in commercially available herbal Spice products, singly and in tandem, at concentrations within the range of 4-141 mg/g of material. Direct analysis in real time mass spectrometry decreases the time necessary to triage analytical evidence, and therefore, it has the potential to contribute to backlog reduction and more timely criminal prosecution.

  3. Screening of phyto-chemical constituents, trace metals and antimicrobial efficiency of Cissus vitiginea

    Directory of Open Access Journals (Sweden)

    V. Subramani

    2014-06-01

    Full Text Available The present study focused on the phytochemical constituents, antimicrobial activity and trace metal concentrations of the Cissus vitiginea plant leaves which were collected from the Tiruchirappalli district, southern India. Preliminary phytochemical screening of leaves extracts revealed the presence of the bioactive compounds, such as steroids, triterpenoids, glycosides, sugar, alkaloids, flavonoids, tannins, amino acid, and coumarin in the leaves. The bacterial and fungal strains were tested for antimicrobial sensitivity against C. vitiginea using the disc diffusion method. The methanol extracts of the plant leaves exhibited the higher zone of inhibition against bacterial strains than fungal strains. The trace metal concentrations were analyzed form the powered plant leaves by 797 VA Computrace voltametry, Metrohm. The average concentrations of Cd, Cr, Cu, Fe, Ni, Pb and Zn were 0.05, BDL, 018, 0.38, BDL, BDL and 0.48 mg kg-1, respectively. The bioactive compounds responsible for these antimicrobial activities could be isolated and identified to develop a new drug of pharmaceutical interest.

  4. Nuclear domain 'knock-in' screen for the evaluation and identification of small molecule enhancers of CRISPR-based genome editing.

    Science.gov (United States)

    Pinder, Jordan; Salsman, Jayme; Dellaire, Graham

    2015-10-30

    CRISPR is a genome-editing platform that makes use of the bacterially-derived endonuclease Cas9 to introduce DNA double-strand breaks at precise locations in the genome using complementary guide RNAs. We developed a nuclear domain knock-in screen, whereby the insertion of a gene encoding the green fluorescent protein variant Clover is inserted by Cas9-mediated homology directed repair (HDR) within the first exon of genes that are required for the structural integrity of subnuclear domains such as the nuclear lamina and promyelocytic leukemia nuclear bodies (PML NBs). Using this approach, we compared strategies for enhancing CRISPR-mediated HDR, focusing on known genes and small molecules that impact non-homologous end joining (NHEJ) and homologous recombination (HR). Ultimately, we identified the small molecule RS-1 as a potent enhancer of CRISPR-based genome editing, enhancing HDR 3- to 6-fold depending on the locus and transfection method. We also characterized U2OS human osteosarcoma cells expressing Clover-tagged PML and demonstrate that this strategy generates cell lines with PML NBs that are structurally and functionally similar to bodies in the parental cell line. Thus, the nuclear domain knock-in screen that we describe provides a simple means of rapidly evaluating methods and small molecules that have the potential to enhance Cas9-mediated HDR.

  5. Preliminary Phytochemical Screening and Physico-Chemical Parameters of Artemisia absinthium and Artemisia annua.

    Directory of Open Access Journals (Sweden)

    Praveen Kumar Ashok

    2013-03-01

    Full Text Available The family Asteraceae or compositae known as the ester, daisy or sunflower family is the largest family of flowering plants. Artemisia is a large diverse genus of plants with between 100 to 150 species belonging to the family asteraceae (compositae. It comprises hardy herbs and shrubs known for their volatile oils. They grow in temperate climate of the northern hemisphere and southern hemisphere usually in dry or, semidry habitats. The collected herbs were authenticated, dried and extracted to calculate the percentage of yield. Phytochemical studies of the Hexane and alcoholic extracts showed the presence of various phytoconstituents i.e. carbohydrate, saponins, phytosterol, proteins and amino acid, tannin, phenolic compounds and flavonoids. It was observed that all the extracts show more important chemical constituents for various pharmacological activities. The determination of these characters will aid future investigators in their Pharmacological analysis of this species.

  6. Wet Chemical Synthesis and Screening of Thick Porous Oxide Films for Resistive Gas Sensing Applications

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    Wilhelm F. Maier

    2006-11-01

    Full Text Available A method of wet chemical synthesis suitable for high throughput and combinatorial applications has been developed for the synthesis of porous resistive thick-film gas sensors. This method is based on the robot-controlled application of unstable metal oxide suspensions on an array of 64 inter-digital electrodes positioned on an Al2O3 substrate. SnO2, WO3, ZrO2, TiO2, CeO2, In2O3 and Bi2O3 were chosen as base oxides, and were optimised by doping or mixed oxide formation. The parallel synthesis of mixed oxide sensors is illustrated by representative examples. The electrical characteristics and the sensor performance of the films were measured by high-throughput impedance spectroscopy while supplying various test gases (H2, CO, NO, NO2, propene. Data collection, data mining techniques applied and the best potential sensor materials discovered are presented.

  7. Approaches to advancing quantitative human health risk assessment of environmental chemicals in the post-genomic era

    Energy Technology Data Exchange (ETDEWEB)

    Chiu, Weihsueh A., E-mail: chiu.weihsueh@epa.gov [National Center for Environmental Assessment, U.S. Environmental Protection Agency, Washington DC, 20460 (United States); Euling, Susan Y.; Scott, Cheryl Siegel; Subramaniam, Ravi P. [National Center for Environmental Assessment, U.S. Environmental Protection Agency, Washington DC, 20460 (United States)

    2013-09-15

    The contribution of genomics and associated technologies to human health risk assessment for environmental chemicals has focused largely on elucidating mechanisms of toxicity, as discussed in other articles in this issue. However, there is interest in moving beyond hazard characterization to making more direct impacts on quantitative risk assessment (QRA) — i.e., the determination of toxicity values for setting exposure standards and cleanup values. We propose that the evolution of QRA of environmental chemicals in the post-genomic era will involve three, somewhat overlapping phases in which different types of approaches begin to mature. The initial focus (in Phase I) has been and continues to be on “augmentation” of weight of evidence — using genomic and related technologies qualitatively to increase the confidence in and scientific basis of the results of QRA. Efforts aimed towards “integration” of these data with traditional animal-based approaches, in particular quantitative predictors, or surrogates, for the in vivo toxicity data to which they have been anchored are just beginning to be explored now (in Phase II). In parallel, there is a recognized need for “expansion” of the use of established biomarkers of susceptibility or risk of human diseases and disorders for QRA, particularly for addressing the issues of cumulative assessment and population risk. Ultimately (in Phase III), substantial further advances could be realized by the development of novel molecular and pathway-based biomarkers and statistical and in silico models that build on anticipated progress in understanding the pathways of human diseases and disorders. Such efforts would facilitate a gradual “reorientation” of QRA towards approaches that more directly link environmental exposures to human outcomes.

  8. Possibilities of chemical weed control in Lupinus albus and Lupinus luteus-screening of herbicides.

    Science.gov (United States)

    Dewitte, K; Latré, J; Haesaert, G

    2006-01-01

    Weed control in sweet lupins is still a problem. Especially the phytotoxicity of herbicides in sweet lupins is not enough studied. Therefore a screening with 16 selected herbicides and 4 lupin varieties has been set up. During the growing season 2005, 10 of the tested herbicides were applied in pre-emergence, 6 in post-emergence. Pre-emergence: Most of the active matters tested in pre-emergence were not phytotoxic for lupins. Pendimethalin (1000 g/ha), linuron (500 g/ha), chlorotoluron (1500 g/ha), prosulfocarb (2400 g/ha), clomazone (72 g/ha), isoxaben (100 g/ha), metamitron (1050 g/ha) and dimethenamid-P (720 g/ha) were applied without causing any significant phytotoxic symptoms. Only the lupins treated with aclonifen (1200 g/ha) showed a significant growth inhibition, 3 weeks after treatment. Significantly more chlorosis was noticed when the lupins were treated with aclonifen or with diflufenican, in preemergence. Post-emergence: In post-emergence, diflufenican (50 g/ha) did not cause any crop damage. Florasulam (5 g/ha) caused almost 100% necrosis in L. albus as well as in L. luteus. Bentazon (652 g/ha), thifensulfuron-methyl (15 g/ha) and metribuzin (175 g/ha) caused obvious necrosis and growth inhibition of the crop. The growth inhibition was significantly more severe for lupins treated with bentazon than if they were treated with thifensulfuron-methyl or metribuzin. Three weeks after treatment, clomazone (90 g/ha) and diflufenican (50 g/ha), did not cause any crop injury at all. The results indicated an interesting range of active matters which can be applied in pre-emergence, but weed control in post-emergence stays difficult.

  9. An inducible krasV12 transgenic zebrafish model for liver tumorigenesis and chemical drug screening

    Directory of Open Access Journals (Sweden)

    Anh Tuan Nguyen

    2012-01-01

    Because Ras signaling is frequently activated by major hepatocellular carcinoma etiological factors, a transgenic zebrafish constitutively expressing the krasV12 oncogene in the liver was previously generated by our laboratory. Although this model depicted and uncovered the conservation between zebrafish and human liver tumorigenesis, the low tumor incidence and early mortality limit its use for further studies of tumor progression and inhibition. Here, we employed a mifepristone-inducible transgenic system to achieve inducible krasV12 expression in the liver. The system consisted of two transgenic lines: the liver-driver line had a liver-specific fabp10 promoter to produce the LexPR chimeric transactivator, and the Ras-effector line contained a LexA-binding site to control EGFP-krasV12 expression. In double-transgenic zebrafish (driver-effector embryos and adults, we demonstrated mifepristone-inducible EGFP-krasV12 expression in the liver. Robust and homogeneous liver tumors developed in 100% of double-transgenic fish after 1 month of induction and the tumors progressed from hyperplasia by 1 week post-treatment (wpt to carcinoma by 4 wpt. Strikingly, liver tumorigenesis was found to be ‘addicted’ to Ras signaling for tumor maintenance, because mifepristone withdrawal led to tumor regression via cell death in transgenic fish. We further demonstrated the potential use of the transparent EGFP-krasV12 larvae in inhibitor treatments to suppress Ras-driven liver tumorigenesis by targeting its downstream effectors, including the Raf-MEK-ERK and PI3K-AKT-mTOR pathways. Collectively, this mifepristone-inducible and reversible krasV12 transgenic system offers a novel model for understanding hepatocarcinogenesis and a high-throughput screening platform for anti-cancer drugs.

  10. Yeast Toxicogenomics: genome-wide responses to chemical stresses with impact in Environmental Health, Pharmacology and Biotechnology

    Directory of Open Access Journals (Sweden)

    Sandra Costa dos Santos

    2012-04-01

    Full Text Available The emerging transdisciplinary field of Toxicogenomics aims to study the cell response to a given toxicant at the genome, transcriptome, proteome and metabolome levels. This approach is expected to provide earlier and more sensitive biomarkers of toxicological responses and help in the delineation of regulatory risk assessment. The use of model organisms to gather such genomic information, through the exploitation of Omics and Bioinformatics approaches and tools, together with more focused molecular and cellular biology studies are rapidly increasing our understanding and providing an integrative view on how cells interact with their environment. The use of the model eukaryote Saccharomyces cerevisiae in the field of Toxicogenomics is discussed in this review. Despite the limitations intrinsic to the use of such a simple single cell experimental model, S. cerevisiae appears to be very useful as a first screening tool, limiting the use of animal models. Moreover, it is also one of the most interesting systems to obtain a truly global understanding of the toxicological response and resistance mechanisms, being in the frontline of systems biology research and developments. The impact of the knowledge gathered in the yeast model, through the use of Toxicogenomics approaches, is highlighted here by its use in prediction of toxicological outcomes of exposure to pesticides and pharmaceutical drugs, but also by its impact in biotechnology, namely in the development of more robust crops and in the improvement of yeast strains as cell factories.

  11. Potential of gas chromatography-atmospheric pressure chemical ionization-tandem mass spectrometry for screening and quantification of hexabromocyclododecane.

    Science.gov (United States)

    Sales, Carlos; Portolés, Tania; Sancho, Juan Vicente; Abad, Esteban; Ábalos, Manuela; Sauló, Jordi; Fiedler, Heidelore; Gómara, Belén; Beltrán, Joaquim

    2016-01-01

    A fast method for the screening and quantification of hexabromocyclododecane (sum of all isomers) by gas chromatography using a triple quadrupole mass spectrometer with atmospheric pressure chemical ionization (GC-APCI-QqQ) is proposed. This novel procedure makes use of the soft atmospheric pressure chemical ionization source, which results in less fragmentation of the analyte than by conventional electron impact (EI) and chemical ionization (CI) sources, favoring the formation of the [M - Br](+) ion and, thus, enhancing sensitivity and selectivity. Detection was based on the consecutive loses of HBr from the [M - Br](+) ion to form the specific [M - H5Br6](+) and [M - H4Br5](+) ions, which were selected as quantitation (Q) and qualification (q) transitions, respectively. Parameters affecting ionization and MS/MS detection were studied. Method performance was also evaluated; calibration curves were found linear from 1 pg/μL to 100 pg/μL for the total HBCD concentration; instrumental detection limit was estimated to be 0.10 pg/μL; repeatability and reproducibility, expressed as relative standard deviation, were better than 7% in both cases. The application to different real samples [polyurethane foam disks (PUFs), food, and marine samples] pointed out a rapid way to identify and allow quantification of this compound together with a number of polybrominated diphenyl ethers (BDE congeners 28, 47, 66, 85, 99, 100, 153, 154, 183, 184, 191, 196, 197, and 209) and two other novel brominated flame retardants [i.e., decabromodiphenyl ethane (DBDPE) and 1,2-bis(2,4,6-tribromophenoxy)ethane (BTBPE)] because of their presence in the same fraction when performing the usual sample treatment.

  12. A screen for F1 hybrid male rescue reveals no major-effect hybrid lethality loci in the Drosophila melanogaster autosomal genome.

    Science.gov (United States)

    Cuykendall, Tawny N; Satyaki, P; Ji, Shuqing; Clay, Derek M; Edelman, Nathaniel B; Kimchy, Alexandra; Li, Ling-Hei; Nuzzo, Erin A; Parekh, Neil; Park, Suna; Barbash, Daniel A

    2014-10-27

    Hybrid sons between Drosophila melanogaster females and D. simulans males die as 3rd instar larvae. Two genes, D. melanogaster Hybrid male rescue (Hmr) on the X chromosome, and D. simulans Lethal hybrid rescue (Lhr) on chromosome II, interact to cause this lethality. Loss-of-function mutations in either gene suppress lethality, but several pieces of evidence suggest that additional factors are required for hybrid lethality. Here we screen the D. melanogaster autosomal genome by using the Bloomington Stock Center Deficiency kit to search for additional regions that can rescue hybrid male lethality. Our screen is designed to identify putative hybrid incompatibility (HI) genes similar to Hmr and Lhr which, when removed, are dominant suppressors of lethality. After screening 89% of the autosomal genome, we found no regions that rescue males to the adult stage. We did, however, identify several regions that rescue up to 13% of males to the pharate adult stage. This weak rescue suggests the presence of multiple minor-effect HI loci, but we were unable to map these loci to high resolution, presumably because weak rescue can be masked by genetic background effects. We attempted to test one candidate, the dosage compensation gene male specific lethal-3 (msl-3), by using RNA interference with short hairpin microRNA constructs targeted specifically against D. simulans msl-3 but failed to achieve knockdown, in part due to off-target effects. We conclude that the D. melanogaster autosomal genome likely does not contain additional major-effect HI loci. We also show that Hmr is insufficient to fully account for the lethality associated with the D. melanogaster X chromosome, suggesting that additional X-linked genes contribute to hybrid lethality. Copyright © 2014 Cuykendall et al.

  13. Serum-Free Generation of Multipotent Mesoderm (Kdr-positive) Progenitor Cells in Mouse Embryonic Stem Cells For Functional Genomics Screening

    Science.gov (United States)

    McKeithan, Wesley; Colas, Alexandre; Bushway, Paul J.; Ray, Saugata; Mercola, Mark

    2013-01-01

    This unit describes a robust protocol for producing multipotent Kdr-expressing mesoderm progenitor cells in serum-free conditions and functional genomics screening using these cells. Kdr-positive cells are known to be able to differentiate into a wide array of mesoderm derivatives including, vascular endothelial cells, cardiomyocytes, hematopietic progenitors and smooth muscle cells. The efficient generation of such progenitor cells is of particular interest because it permits subsequent steps in cardiovascular development to be analyzed in detail, including deciphering the mechanisms that direct differentiation. The oligonucleotide transfection protocol used to functionally screen siRNA and microRNA libraries is a powerful tool to reveal networks of genes, signaling proteins and microRNAs that control the diversification of cardiovascular lineages from multipotent progenitors. The discussion addresses technical limitations, troubleshooting and potential applications. PMID:23154934

  14. Ex vivo genome-wide RNAi screening of the Drosophila Toll signaling pathway elicited by a larva-derived tissue extract.

    Science.gov (United States)

    Kanoh, Hirotaka; Kuraishi, Takayuki; Tong, Li-Li; Watanabe, Ryo; Nagata, Shinji; Kurata, Shoichiro

    2015-11-13

    Damage-associated molecular patterns (DAMPs), so-called "danger signals," play important roles in host defense and pathophysiology in mammals and insects. In Drosophila, the Toll pathway confers damage responses during bacterial infection and improper cell-fate control. However, the intrinsic ligands and signaling mechanisms that potentiate innate immune responses remain unknown. Here, we demonstrate that a Drosophila larva-derived tissue extract strongly elicits Toll pathway activation via the Toll receptor. Using this extract, we performed ex vivo genome-wide RNAi screening in Drosophila cultured cells, and identified several signaling factors that are required for host defense and antimicrobial-peptide expression in Drosophila adults. These results suggest that our larva-derived tissue extract contains active ingredients that mediate Toll pathway activation, and the screening data will shed light on the mechanisms of damage-related Toll pathway signaling in Drosophila.

  15. A genome-wide screen for interactions reveals a new locus on 4p15 modifying the effect of waist-to-hip ratio on total cholesterol.

    Directory of Open Access Journals (Sweden)

    Ida Surakka

    2011-10-01

    Full Text Available Recent genome-wide association (GWA studies described 95 loci controlling serum lipid levels. These common variants explain ∼25% of the heritability of the phenotypes. To date, no unbiased screen for gene-environment interactions for circulating lipids has been reported. We screened for variants that modify the relationship between known epidemiological risk factors and circulating lipid levels in a meta-analysis of genome-wide association (GWA data from 18 population-based cohorts with European ancestry (maximum N = 32,225. We collected 8 further cohorts (N = 17,102 for replication, and rs6448771 on 4p15 demonstrated genome-wide significant interaction with waist-to-hip-ratio (WHR on total cholesterol (TC with a combined P-value of 4.79×10(-9. There were two potential candidate genes in the region, PCDH7 and CCKAR, with differential expression levels for rs6448771 genotypes in adipose tissue. The effect of WHR on TC was strongest for individuals carrying two copies of G allele, for whom a one standard deviation (sd difference in WHR corresponds to 0.19 sd difference in TC concentration, while for A allele homozygous the difference was 0.12 sd. Our findings may open up possibilities for targeted intervention strategies for people characterized by specific genomic profiles. However, more refined measures of both body-fat distribution and metabolic measures are needed to understand how their joint dynamics are modified by the newly found locus.

  16. Phylogeny-function analysis of (meta)genomic libraries: screening for expression of ribosomal RNA genes by large-insert library fluorescent in situ hybridization (LIL-FISH).

    Science.gov (United States)

    Leveau, Johan H J; Gerards, Saskia; de Boer, Wietse; van Veen, Johannes A

    2004-09-01

    We assessed the utility of fluorescent in situ hybridization (FISH) in the screening of clone libraries of (meta)genomic or environmental DNA for the presence and expression of bacterial ribosomal RNA (rRNA) genes. To establish proof-of-principle, we constructed a fosmid-based library in Escherichia coli of large-sized genomic DNA fragments of the mycophagous soil bacterium Collimonas fungivorans, and hybridized 768 library clones with the Collimonas-specific fluorescent probe CTE998-1015. Critical to the success of this approach (which we refer to as large-insert library FISH or LIL-FISH) was the ability to induce fosmid copy number, the exponential growth status of library clones in the FISH assay and the use of a simple pooling strategy to reduce the number of hybridizations. Twelve out of 768 E. coli clones were suspected to harbour and express Collimonas 16S rRNA genes based on their hybridization to CTE998-1015. This was confirmed by the finding that all 12 clones were also identified in an independent polymerase chain reaction-based screening of the same 768 clones using a primer set for the specific detection of Collimonas 16S ribosomal DNA (rDNA). Fosmids isolated from these clones were grouped by restriction analysis into two distinct contigs, confirming that C. fungivorans harbours at least two 16S rRNA genes. For one contig, representing 1-2% of the genome, the nucleotide sequence was determined, providing us with a narrow but informative view of Collimonas genome structure and content.

  17. On-line derivatization gas chromatography with furan chemical ionization tandem mass spectrometry for screening of amphetamines in urine.

    Science.gov (United States)

    Tzing, Shin-Hwa; Ghule, Anil; Liu, Jen-Yu; Ling, Yong-Chien

    2006-12-22

    A simple alternative method with minimal sample pretreatment is investigated for screening of amphetamines in small volume (using only 20 microL) of urine sample. The method is sensitive and selective. The method uses gas chromatography (GC) direct sample introduction (DSI) for on-line derivatization (acylation) of amphetamines to improve sensitivity. Furan as chemical ionization (CI) reagent in conjunction with tandem mass spectrometry (MS/MS) is used to improve selectivity. Low background with sharp protonated molecular ion peaks of analytes is the evidence of improvement in sensitivity and selectivity. Blank urine samples spiked with known amounts of amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxymethamphetamine and 3,4-methylenedioxyethylamphetamine is analyzed. Selected ion monitoring of the characteristic product ions (m/z 119+136+150+163) using furan CI-MS/MS in positive ion mode is used for quantification. Limits of detection (LOD) between 0.4 and 1.0 ng mL(-1) and limits of quantitation (LOQ) between 1.0 and 2.0 ng mL(-1) are established. Linear response over the range of 1-1000 ng mL(-1) (r(2)>0.997) is observed for all analytes, except for methamphetamine (2.0-1000 ng mL(-1)). Good accuracy between 86 and 113% and precision ranging from 4 to 18% is obtained. The method is also tested on real samples of urine from suspected drug abusers. This method could be used for screening and determination of amphetamines in urine samples, however needs additional work for full validation.

  18. The international validation of bio- and chemical-anlaytical screening methods for dioxins and dioxin-like PCBs: the DIFFERENCE project rounds 1 and 2

    NARCIS (Netherlands)

    Loco, van J.; Leeuwen, van S.P.J.; Roos, P.; Carbonnelle, S.; Boer, de J.; Goeyens, L.; Beernaert, H.

    2004-01-01

    The European research project DIFFERENCE is focussed on the development, optimisation and validation of screening methods for dioxin analysis, including bio-analytical and chemical techniques (CALUX, GC-LRMS/MS, GC x GC-ECD) and on the optimisation and validation of new extraction and clean-up

  19. Validation of a qualitative screening method for pesticides in fruits and vegetables by gas chromatography quadrupole-time of flight mass spectrometry with atmospheric pressure chemical ionization

    NARCIS (Netherlands)

    Portoles, T.; Mol, J.G.J.; Sancho, J.V.; Lopez, F.J.; Hernandez, F.

    2014-01-01

    A wide-scope screening method was developed for the detection of pesticides in fruit and vegetables. The method was based on gas chromatography coupled to a hybrid quadrupole time-of-flight mass spectrometer with an atmospheric pressure chemical ionization source (GC-(APCI)QTOF MS). A non-target acq

  20. Photothermal and photoacoustic methods for mapping surface absorbance: Adaptation for screening chemical and biomolecular libraries

    Science.gov (United States)

    Koebel, Matthias M.

    Photoacoustic and photothermal methods are useful tools for the analysis of solid state samples and thin films. Both techniques may be used to study surface absorber distributions on surfaces. In the photoacoustic experiment, light absorption at a solid/air interface launches a pressure wave which propagates through the air. The acoustic wave is detected by deflection of a probe laser beam. For non-parallel orientation of the probe beam with respect to the sample surface, acoustic waves launched from individual absorber features travel different distances before they intersect with the probe beam. This allows temporal encoding of the spatial distribution of surface absorbers. An experimental demonstration of this novel photoacoustic of detection scheme is presented. In the photothermal experiment, detection is based on production of a temperature change at the sample surface following light absorption. Thermal diffusion generates temperature gradients in the solid sample and the adjacent fluid layer. The resulting refractive index gradient in the adjacent fluid medium is measured by deflection of a probe laser beam. Using the transverse photothermal deflection spectroscopy (t-PDS) method, two dimensional absorber distribution maps of a flat sample surface can be recorded. A number of colored thin polymer film are used to characterize the sensitivity in air and a value of 7.5 · 10-6 W is found. Gold nanoparticles are excellent optical absorber labels for biological and biochemical binding assays. The synthesis and characterization of gold nanoparticles of different sizes and surface chemical functionalities is presented. A novel readout method for protein microarrays based on photothermal detection of nanoparticle labeled proteins is described. Protein microarrays are developed with functionalized gold nanoparticles and analyzed using t-PDS. The observed coloration intensity performance depends on the intrinsic nature of the target protein. Neutravidin produces the

  1. Development of a bioassay to screen for chemicals mimicking the anti-aging effects of calorie restriction

    Energy Technology Data Exchange (ETDEWEB)

    Chiba, Takuya, E-mail: takuya@nagasaki-u.ac.jp [Department of Investigative Pathology, Graduate School of Biomedical Sciences, Nagasaki University, 1-12-4 Sakamoto, Nagasaki 852-8523 (Japan); Tsuchiya, Tomoshi [Division of Surgical Oncology, Graduate School of Biomedical Sciences, Nagasaki University, 1-7-1 Sakamoto, Nagasaki 852-8501 (Japan); Komatsu, Toshimitsu; Mori, Ryoichi; Hayashi, Hiroko [Department of Investigative Pathology, Graduate School of Biomedical Sciences, Nagasaki University, 1-12-4 Sakamoto, Nagasaki 852-8523 (Japan); Shimano, Hitoshi [Department of Internal Medicine (Endocrinology and Metabolism), Graduate School of Comprehensive Human Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba Ibaraki 305-8575 (Japan); Spindler, Stephen R. [Department of Biochemistry, Room 5478, Boyce Hall, University of California - Riverside, Riverside, CA 92521 (United States); Shimokawa, Isao [Department of Investigative Pathology, Graduate School of Biomedical Sciences, Nagasaki University, 1-12-4 Sakamoto, Nagasaki 852-8523 (Japan)

    2010-10-15

    Research highlights: {yields} We identified four sequence motifs lying upstream of putative pro-longevity genes. {yields} One of these motifs binds to HNF-4{alpha}. {yields} HNF-4{alpha}/PGC-1{alpha} could up-regulate the transcription of a reporter gene linked to this motif. {yields} The reporter system described here could be used to screen candidate anti-aging molecules. -- Abstract: Suppression of the growth hormone/insulin-like growth factor-I pathway in Ames dwarf (DF) mice, and caloric restriction (CR) in normal mice extends lifespan and delays the onset of age-related disorders. In combination, these interventions have an additive effect on lifespan in Ames DF mice. Therefore, common signaling pathways regulated by DF and CR could have additive effects on longevity. In this study, we tried to identity the signaling mechanism and develop a system to assess pro-longevity status in cells and mice. We previously identified genes up-regulated in the liver of DF and CR mice by DNA microarray analysis. Motif analysis of the upstream sequences of those genes revealed four major consensus sequence motifs, which have been named dwarfism and calorie restriction-responsive elements (DFCR-REs). One of the synthesized sequences bound to hepatocyte nuclear factor-4{alpha} (HNF-4{alpha}), an important transcription factor involved in liver metabolism. Furthermore, using this sequence information, we developed a highly sensitive bioassay to identify chemicals mimicking the anti-aging effects of CR. When the reporter construct, containing an element upstream of a secreted alkaline phosphatase (SEAP) gene, was co-transfected with HNF-4{alpha} and its regulator peroxisome proliferator-activated receptor (PPAR) {gamma} coactivator-1{alpha} (PGC-1{alpha}), SEAP activity was increased compared with untransfected controls. Moreover, transient transgenic mice established using this construct showed increased SEAP activity in CR mice compared with ad libitum-fed mice. These data

  2. Draft genome sequence and chemical profiling of Fusarium langsethiae, an emerging producer of type A trichothecenes.

    Science.gov (United States)

    Lysøe, Erik; Frandsen, Rasmus J N; Divon, Hege H; Terzi, Valeria; Orrù, Luigi; Lamontanara, Antonella; Kolseth, Anna-Karin; Nielsen, Kristian F; Thrane, Ulf

    2016-03-16

    Fusarium langsethiae is a widespread pathogen of small grain cereals, causing problems with T-2 and HT-2 toxin contamination in grains every year. In an effort to better understand the biology of this fungus, we present a draft genome sequence of F. langsethiae Fl201059 isolated from oats in Norway. The assembly was fragmented, but reveals a genome of approximately 37.5 Mb, with a GC content around 48%, and 12,232 predicted protein-coding genes. Focusing on secondary metabolism we identified candidate genes for 12 polyketide synthases, 13 non-ribosomal peptide synthetases, and 22 genes for terpene/isoprenoid biosynthesis. Some of these were found to be unique compared to sequence databases. The identified putative Tri5 cluster was highly syntenic to the cluster reported in F. sporotrichioides. Fusarium langsethiae Fl201059 produces a high number of secondary metabolites on Yeast Extract Sucrose (YES) agar medium, dominated by type A trichothecenes. Interestingly we found production of glucosylated HT-2 toxin (Glu-HT-2), previously suggested to be formed by the host plant and not by the fungus itself. In greenhouse inoculations of F. langsethiae Fl201059 on barley and oats, we detected the type A trichothecenes: neosolaniol, HT-2 toxin, T-2 toxin, Glu-HT-2 and numerous derivatives of these.

  3. Effect of Cu supplementation on genomic instability in chemically-induced mammary carcinogenesis in the rat

    Directory of Open Access Journals (Sweden)

    Bobrowska Barbara

    2011-12-01

    Full Text Available Abstract Backround The aim of the present study was to assess the effect of dietary supplementation (copper or copper and resveratrol on the intensity of carcinogenesis and the frequency of microsatellite instability in a widely used model of mammary carcinogenesis induced in the rat by treatment with 7,12-dimethylbenz[a]anthracene (DMBA. Methods DNA was extracted from rat mammary cancers and normal tisues, amplified by PCR, using different polymorphic DNA markers and the reaction products were analyzed for microsatellite instability. Results It was found that irrespectively of the applied diet there was no inhibition of mammary carcinogenesis in the rats due to DMBA. Besides, in the groups supplemented with Cu (II or Cu (II and resveratrol the tumor formation was clearly accelerated. Unlike the animals that were fed with standard diet, the supplemented rats were characterized by the loss of heterozygosity of microsatellite D3Mgh9 in cancer tumors (by respectively 50 and 40%. When the animals received Cu (II and resveratrol supplemented diet the occurrence of genomic instability was additionally found in their livers in the case of microsatellite D1Mgh6 (which was stable in the animals without dietary supplementation. Conclusions Identification of the underlying mechanisms by which dietary factors affect genomic stability might prove useful in the treatment of mammary cancer as well as in the incorporation of dietary factors into mammary cancer prevention strategies.

  4. BYSTANDER EFFECTS, GENOMIC INSTABILITY, ADAPTIVE RESPONSE AND CANCER RISK ASSESSMENT FOR RADIATION AND CHEMICAL EXPOSURES

    Science.gov (United States)

    There is an increased interest in utilizing mechanistic data in support of the cancer risk assessment process for ionizing radiation and environmental chemical exposures. In this regard the use of biologically based dose-response models is particularly advocated. The aim is to pr...

  5. High-throughput screen for the chemical inhibitors of antiapoptotic bcl-2 family proteins by multiplex flow cytometry.

    Science.gov (United States)

    Curpan, Ramona F; Simons, Peter C; Zhai, Dayong; Young, Susan M; Carter, Mark B; Bologa, Cristian G; Oprea, Tudor I; Satterthwait, Arnold C; Reed, John C; Edwards, Bruce S; Sklar, Larry A

    2011-10-01

    The human Bcl-2 family includes six antiapoptotic members (Bcl-2, Bcl-B, Bcl-W, Bcl-X(L), Bfl-1, and Mcl-1) and many proapoptotic members, wherein a balance between the two determines cell life or death in many physiological and disease contexts. Elevated expression of various antiapoptotic Bcl-2 members is commonly observed in cancers, and chemical inhibitors of these proteins have been shown to promote apoptosis of malignant cells in culture, in animal models, and in human clinical trials. All six antiapoptotic members bind a helix from the proapoptotic family member Bim, thus quenching Bim's apoptotic signal. Here, we describe the use of a multiplex, high-throughput flow cytometry assay for the discovery of small molecule modulators that disrupt the interaction between the antiapoptotic members of the Bcl-2 family and Bim. The six antiapoptotic Bcl-2 family members were expressed as glutathione-S-transferase fusion proteins and bound individually to six glutathione bead sets, with each set having a different intensity of red fluorescence. A fluorescein-conjugated Bcl-2 homology region 3 (BH3) peptide from Bim was employed as a universal ligand. Flow cytometry measured the amount of green peptide bound to each bead set in a given well, with inhibitory compounds resulting in a decrease of green fluorescence on one or more bead set(s). Hits and cheminformatically selected analogs were retested in a dose-response series, resulting in three "active" compounds for Bcl-B. These three compounds were validated by fluorescence polarization and isothermal titration calorimetry. We discuss some of the lessons learned about screening a chemical library provided by the National Institutes of Health Small Molecule Repository (∼195,000 compounds) using high-throughput flow cytometry.

  6. Effects of genetic mutations and chemical exposures on Caenorhabditis elegans feeding: evaluation of a novel, high-throughput screening assay.

    Directory of Open Access Journals (Sweden)

    Windy A Boyd

    Full Text Available BACKGROUND: Government agencies have defined a need to reduce, refine or replace current mammalian-based bioassays with testing methods that use alternative species. Invertebrate species, such as Caenorhabditis elegans, provide an attractive option because of their short life cycles, inexpensive maintenance, and high degree of evolutionary conservation with higher eukaryotes. The C. elegans pharynx is a favorable model for studying neuromuscular function, and the effects of chemicals on neuromuscular activity, i.e., feeding. Current feeding methodologies, however, are labor intensive and only semi-quantitative. METHODOLOGY/PRINCIPAL FINDINGS: Here a high-throughput assay is described that uses flow cytometry to measure C. elegans feeding by determining the size and intestinal fluorescence of hundreds of nematodes after exposure to fluorescent-labeled microspheres. This assay was validated by quantifying fluorescence in feeding-defective C. elegans (eat mutants, and by exposing wild-type nematodes to the neuroactive compounds, serotonin and arecoline. The eat mutations previously determined to cause slow pumping rates exhibited the lowest feeding levels with our assay. Concentration-dependent increases in feeding levels after serotonin exposures were dependent on food availability, while feeding levels decreased in arecoline-exposed nematodes regardless of the presence of food. The effects of the environmental contaminants, cadmium chloride and chlorpyrifos, on wild-type C. elegans feeding were then used to demonstrate an application of the feeding assay. Cadmium exposures above 200 microM led to a sharp drop in feeding levels. Feeding of chlorpyrifos-exposed nematodes decreased in a concentration-dependent fashion with an EC(50 of 2 microM. CONCLUSIONS/SIGNIFICANCE: The C. elegans fluorescence microsphere feeding assay is a rapid, reliable method for the assessment of neurotoxic effects of pharmaceutical drugs, industrial chemicals or

  7. Protein structure similarity clustering (PSSC) and natural product structure as inspiration sources for drug development and chemical genomics.

    Science.gov (United States)

    Dekker, Frank J; Koch, Marcus A; Waldmann, Herbert

    2005-06-01

    Finding small molecules that modulate protein function is of primary importance in drug development and in the emerging field of chemical genomics. To facilitate the identification of such molecules, we developed a novel strategy making use of structural conservatism found in protein domain architecture and natural product inspired compound library design. Domains and proteins identified as being structurally similar in their ligand-sensing cores are grouped in a protein structure similarity cluster (PSSC). Natural products can be considered as evolutionary pre-validated ligands for multiple proteins and therefore natural products that are known to interact with one of the PSSC member proteins are selected as guiding structures for compound library synthesis. Application of this novel strategy for compound library design provided enhanced hit rates in small compound libraries for structurally similar proteins.

  8. Genomic and chemical insights into biosurfactant production by the mangrove-derived strain Bacillus safensis CCMA-560.

    Science.gov (United States)

    Domingos, Daniela Ferreira; de Faria, Andreia Fonseca; de Souza Galaverna, Renan; Eberlin, Marcos Nogueira; Greenfield, Paul; Zucchi, Tiago Domingues; Melo, Itamar Soares; Tran-Dinh, Nai; Midgley, David; de Oliveira, Valéria Maia

    2015-04-01

    Many Bacillus species can produce biosurfactant, although most of the studies on lipopeptide production by this genus have been focused on Bacillus subtilis. Surfactants are broadly used in pharmaceutical, food and petroleum industry, and biological surfactant shows some advantages over the chemical surfactants, such as less toxicity, production from renewable, cheaper feedstocks and development of novel recombinant hyperproducer strains. This study is aimed to unveil the biosurfactant metabolic pathway and chemical composition in Bacillus safensis strain CCMA-560. The whole genome of the CCMA-560 strain was previously sequenced, and with the aid of bioinformatics tools, its biosurfactant metabolic pathway was compared to other pathways of closely related species. Fourier transform infrared (FTIR) and high-resolution TOF mass spectrometry (MS) were used to characterize the biosurfactant molecule. B. safensis CCMA-560 metabolic pathway is similar to other Bacillus species; however, some differences in amino acid incorporation were observed, and chemical analyses corroborated the genetic results. The strain CCMA-560 harbours two genes flanked by srfAC and srfAD not present in other Bacillus spp., which can be involved in the production of the analogue gramicidin. FTIR and MS showed that B. safensis CCMA-560 produces a mixture of at least four lipopeptides with seven amino acids incorporated and a fatty acid chain with 14 carbons, which makes this molecule similar to the biosurfactant of Bacillus pumilus, namely, pumilacidin. This is the first report on the biosurfactant production by B. safensis, encompassing the investigation of the metabolic pathway and chemical characterization of the biosurfactant molecule.

  9. Genome-wide Mapping of Cellular Protein-RNA Interactions Enabled by Chemical Crosslinking

    Institute of Scientific and Technical Information of China (English)

    Xiaoyu Li; Jinghui Song; Chengqi Yi

    2014-01-01

    RNA-protein interactions influence many biological processes. Identifying the binding sites of RNA-binding proteins (RBPs) remains one of the most fundamental and important chal-lenges to the studies of such interactions. Capturing RNA and RBPs via chemical crosslinking allows stringent purification procedures that significantly remove the non-specific RNA and protein interactions. Two major types of chemical crosslinking strategies have been developed to date, i.e., UV-enabled crosslinking and enzymatic mechanism-based covalent capture. In this review, we com-pare such strategies and their current applications, with an emphasis on the technologies themselves rather than the biology that has been revealed. We hope such methods could benefit broader audi-ence and also urge for the development of new methods to study RNA RBP interactions.

  10. Screening estrogenic activities of chemicals or mixtures in vivo using transgenic (cyp19a1b-GFP zebrafish embryos.

    Directory of Open Access Journals (Sweden)

    François Brion

    Full Text Available The tg(cyp19a1b-GFP transgenic zebrafish expresses GFP (green fluorescent protein under the control of the cyp19a1b gene, encoding brain aromatase. This gene has two major characteristics: (i it is only expressed in radial glial progenitors in the brain of fish and (ii it is exquisitely sensitive to estrogens. Based on these properties, we demonstrate that natural or synthetic hormones (alone or in binary mixture, including androgens or progestagens, and industrial chemicals induce a concentration-dependent GFP expression in radial glial progenitors. As GFP expression can be quantified by in vivo imaging, this model presents a very powerful tool to screen and characterize compounds potentially acting as estrogen mimics either directly or after metabolization by the zebrafish embryo. This study also shows that radial glial cells that act as stem cells are direct targets for a large panel of endocrine disruptors, calling for more attention regarding the impact of environmental estrogens and/or certain pharmaceuticals on brain development. Altogether these data identify this in vivo bioassay as an interesting alternative to detect estrogen mimics in hazard and risk assessment perspective.

  11. Screening estrogenic activities of chemicals or mixtures in vivo using transgenic (cyp19a1b-GFP) zebrafish embryos.

    Science.gov (United States)

    Brion, François; Le Page, Yann; Piccini, Benjamin; Cardoso, Olivier; Tong, Sok-Keng; Chung, Bon-chu; Kah, Olivier

    2012-01-01

    The tg(cyp19a1b-GFP) transgenic zebrafish expresses GFP (green fluorescent protein) under the control of the cyp19a1b gene, encoding brain aromatase. This gene has two major characteristics: (i) it is only expressed in radial glial progenitors in the brain of fish and (ii) it is exquisitely sensitive to estrogens. Based on these properties, we demonstrate that natural or synthetic hormones (alone or in binary mixture), including androgens or progestagens, and industrial chemicals induce a concentration-dependent GFP expression in radial glial progenitors. As GFP expression can be quantified by in vivo imaging, this model presents a very powerful tool to screen and characterize compounds potentially acting as estrogen mimics either directly or after metabolization by the zebrafish embryo. This study also shows that radial glial cells that act as stem cells are direct targets for a large panel of endocrine disruptors, calling for more attention regarding the impact of environmental estrogens and/or certain pharmaceuticals on brain development. Altogether these data identify this in vivo bioassay as an interesting alternative to detect estrogen mimics in hazard and risk assessment perspective.

  12. Phenotypic chemical screening using a zebrafish neural crest EMT reporter identifies retinoic acid as an inhibitor of epithelial morphogenesis

    Directory of Open Access Journals (Sweden)

    Laura Jimenez

    2016-04-01

    Full Text Available The epithelial-to-mesenchymal transition (EMT is a highly conserved morphogenetic program essential for embryogenesis, regeneration and cancer metastasis. In cancer cells, EMT also triggers cellular reprogramming and chemoresistance, which underlie disease relapse and decreased survival. Hence, identifying compounds that block EMT is essential to prevent or eradicate disseminated tumor cells. Here, we establish a whole-animal-based EMT reporter in zebrafish for rapid drug screening, called Tg(snai1b:GFP, which labels epithelial cells undergoing EMT to produce sox10-positive neural crest (NC cells. Time-lapse and lineage analysis of Tg(snai1b:GFP embryos reveal that cranial NC cells delaminate from two regions: an early population delaminates adjacent to the neural plate, whereas a later population delaminates from within the dorsal neural tube. Treating Tg(snai1b:GFP embryos with candidate small-molecule EMT-inhibiting compounds identified TP-0903, a multi-kinase inhibitor that blocked cranial NC cell delamination in both the lateral and medial populations. RNA sequencing (RNA-Seq analysis and chemical rescue experiments show that TP-0903 acts through stimulating retinoic acid (RA biosynthesis and RA-dependent transcription. These studies identify TP-0903 as a new therapeutic for activating RA in vivo and raise the possibility that RA-dependent inhibition of EMT contributes to its prior success in eliminating disseminated cancer cells.

  13. Phenotypic chemical screening using a zebrafish neural crest EMT reporter identifies retinoic acid as an inhibitor of epithelial morphogenesis.

    Science.gov (United States)

    Jimenez, Laura; Wang, Jindong; Morrison, Monique A; Whatcott, Clifford; Soh, Katherine K; Warner, Steven; Bearss, David; Jette, Cicely A; Stewart, Rodney A

    2016-04-01

    The epithelial-to-mesenchymal transition (EMT) is a highly conserved morphogenetic program essential for embryogenesis, regeneration and cancer metastasis. In cancer cells, EMT also triggers cellular reprogramming and chemoresistance, which underlie disease relapse and decreased survival. Hence, identifying compounds that block EMT is essential to prevent or eradicate disseminated tumor cells. Here, we establish a whole-animal-based EMT reporter in zebrafish for rapid drug screening, calledTg(snai1b:GFP), which labels epithelial cells undergoing EMT to producesox10-positive neural crest (NC) cells. Time-lapse and lineage analysis ofTg(snai1b:GFP)embryos reveal that cranial NC cells delaminate from two regions: an early population delaminates adjacent to the neural plate, whereas a later population delaminates from within the dorsal neural tube. TreatingTg(snai1b:GFP)embryos with candidate small-molecule EMT-inhibiting compounds identified TP-0903, a multi-kinase inhibitor that blocked cranial NC cell delamination in both the lateral and medial populations. RNA sequencing (RNA-Seq) analysis and chemical rescue experiments show that TP-0903 acts through stimulating retinoic acid (RA) biosynthesis and RA-dependent transcription. These studies identify TP-0903 as a new therapeutic for activating RAin vivoand raise the possibility that RA-dependent inhibition of EMT contributes to its prior success in eliminating disseminated cancer cells. © 2016. Published by The Company of Biologists Ltd.

  14. Automated cell analysis tool for a genome-wide RNAi screen with support vector machine based supervised learning

    Science.gov (United States)

    Remmele, Steffen; Ritzerfeld, Julia; Nickel, Walter; Hesser, Jürgen

    2011-03-01

    RNAi-based high-throughput microscopy screens have become an important tool in biological sciences in order to decrypt mostly unknown biological functions of human genes. However, manual analysis is impossible for such screens since the amount of image data sets can often be in the hundred thousands. Reliable automated tools are thus required to analyse the fluorescence microscopy image data sets usually containing two or more reaction channels. The herein presented image analysis tool is designed to analyse an RNAi screen investigating the intracellular trafficking and targeting of acylated Src kinases. In this specific screen, a data set consists of three reaction channels and the investigated cells can appear in different phenotypes. The main issue of the image processing task is an automatic cell segmentation which has to be robust and accurate for all different phenotypes and a successive phenotype classification. The cell segmentation is done in two steps by segmenting the cell nuclei first and then using a classifier-enhanced region growing on basis of the cell nuclei to segment the cells. The classification of the cells is realized by a support vector machine which has to be trained manually using supervised learning. Furthermore, the tool is brightness invariant allowing different staining quality and it provides a quality control that copes with typical defects during preparation and acquisition. A first version of the tool has already been successfully applied for an RNAi-screen containing three hundred thousand image data sets and the SVM extended version is designed for additional screens.

  15. Editor's Highlight: Screening ToxCast Prioritized Chemicals for PPARG Function in a Human Adipose-Derived Stem Cell Model of Adipogenesis.

    Science.gov (United States)

    Foley, Briana; Doheny, Daniel L; Black, Michael B; Pendse, Salil N; Wetmore, Barbara A; Clewell, Rebecca A; Andersen, Melvin E; Deisenroth, Chad

    2017-01-01

    The developmental origins of obesity hypothesis posits a multifaceted contribution of factors to the fetal origins of obesity and metabolic disease. Adipocyte hyperplasia in gestation and early childhood may result in predisposition for obesity later in life. Rodent in vitro and in vivo studies indicate that some chemicals may directly affect adipose progenitor cell differentiation, but the human relevance of these findings is unclear. The nuclear receptor peroxisome proliferator-activated receptor gamma (PPARG) is the master regulator of adipogenesis. Human adipose-derived stem cells (hASC) isolated from adipose tissue express endogenous isoforms of PPARG and represent a biologically relevant cell-type for evaluating activity of PPARG ligands. Here, a multi-endpoint approach based on a phenotypic adipogenesis assay was applied to screen a set of 60 chemical compounds identified in ToxCast Phase I as PPARG active (49) or inactive (11). Chemicals showing activity in the adipogenesis screen were further evaluated in a series of 4 orthogonal assays representing 7 different key events in PPARG-dependent adipogenesis, including gene transcription, protein expression, and adipokine secretion. An siRNA screen was also used to evaluate PPARG-dependence of the adipogenesis phenotype. A universal concentration-response design enabled inter-assay comparability and implementation of a weight-of-evidence approach for bioactivity classification. Collectively, a total of 14/49 (29%) prioritized chemicals were identified with moderate-to-strong activity for human adipogenesis. These results provide the first integrated screening approach of prioritized ToxCast chemicals in a human stem cell model of adipogenesis and provide insight into the capacity of PPARG-activating chemicals to modulate early life programming of adipose tissue.

  16. Editor’s Highlight: Screening ToxCast Prioritized Chemicals for PPARG Function in a Human Adipose-Derived Stem Cell Model of Adipogenesis

    Science.gov (United States)

    Foley, Briana; Doheny, Daniel L.; Black, Michael B.; Pendse, Salil N.; Wetmore, Barbara A.; Clewell, Rebecca A.; Andersen, Melvin E.; Deisenroth, Chad

    2017-01-01

    The developmental origins of obesity hypothesis posits a multifaceted contribution of factors to the fetal origins of obesity and metabolic disease. Adipocyte hyperplasia in gestation and early childhood may result in predisposition for obesity later in life. Rodent in vitro and in vivo studies indicate that some chemicals may directly affect adipose progenitor cell differentiation, but the human relevance of these findings is unclear. The nuclear receptor peroxisome proliferator-activated receptor gamma (PPARG) is the master regulator of adipogenesis. Human adipose-derived stem cells (hASC) isolated from adipose tissue express endogenous isoforms of PPARG and represent a biologically relevant cell-type for evaluating activity of PPARG ligands. Here, a multi-endpoint approach based on a phenotypic adipogenesis assay was applied to screen a set of 60 chemical compounds identified in ToxCast Phase I as PPARG active (49) or inactive (11). Chemicals showing activity in the adipogenesis screen were further evaluated in a series of 4 orthogonal assays representing 7 different key events in PPARG-dependent adipogenesis, including gene transcription, protein expression, and adipokine secretion. An siRNA screen was also used to evaluate PPARG-dependence of the adipogenesis phenotype. A universal concentration-response design enabled inter-assay comparability and implementation of a weight-of-evidence approach for bioactivity classification. Collectively, a total of 14/49 (29%) prioritized chemicals were identified with moderate-to-strong activity for human adipogenesis. These results provide the first integrated screening approach of prioritized ToxCast chemicals in a human stem cell model of adipogenesis and provide insight into the capacity of PPARG-activating chemicals to modulate early life programming of adipose tissue. PMID:27664422

  17. Ligand Discovery for a Peptide-Binding GPCR by Structure-Based Screening of Fragment- and Lead-Like Chemical Libraries.

    Science.gov (United States)

    Ranganathan, Anirudh; Heine, Philipp; Rudling, Axel; Plückthun, Andreas; Kummer, Lutz; Carlsson, Jens

    2017-03-17

    Peptide-recognizing G protein-coupled receptors (GPCRs) are promising therapeutic targets but often resist drug discovery efforts. Determination of crystal structures for peptide-binding GPCRs has provided opportunities to explore structure-based methods in lead development. Molecular docking screens of two chemical libraries, containing either fragment- or lead-like compounds, against a neurotensin receptor 1 crystal structure allowed for a comparison between different drug development strategies for peptide-binding GPCRs. A total of 2.3 million molecules were screened computationally, and 25 fragments and 27 leads that were top-ranked in each library were selected for experimental evaluation. Of these, eight fragments and five leads were confirmed as ligands by surface plasmon resonance. The hit rate for the fragment screen (32%) was thus higher than for the lead-like library (19%), but the affinities of the fragments were ∼100-fold lower. Both screens returned unique scaffolds and demonstrated that a crystal structure of a stabilized peptide-binding GPCR can guide the discovery of small-molecule agonists. The complementary advantages of exploring fragment- and lead-like chemical space suggest that these strategies should be applied synergistically in structure-based screens against challenging GPCR targets.

  18. Zebrafish small molecule screens: Taking the phenotypic plunge

    Directory of Open Access Journals (Sweden)

    Charles H. Williams

    2016-01-01

    Full Text Available Target based chemical screens are a mainstay of modern drug discovery, but the effectiveness of this reductionist approach is being questioned in light of declines in pharmaceutical R & D efficiency. In recent years, phenotypic screens have gained increasing acceptance as a complementary/alternative approach to early drug discovery. We discuss the various model organisms used in phenotypic screens, with particular focus on zebrafish, which has emerged as a leading model of in vivo phenotypic screens. Additionally, we anticipate therapeutic opportunities, particularly in orphan disease space, in the context of rapid advances in human Mendelian genetics, electronic health record (EHR-enabled genome–phenome associations, and genome editing.

  19. Cas-Database: web-based genome-wide guide RNA library design for gene knockout screens using CRISPR-Cas9.

    Science.gov (United States)

    Park, Jeongbin; Kim, Jin-Soo; Bae, Sangsu

    2016-07-01

    CRISPR-derived RNA guided endonucleases (RGENs) have been widely used for both gene knockout and knock-in at the level of single or multiple genes. RGENs are now available for forward genetic screens at genome scale, but single guide RNA (sgRNA) selection at this scale is difficult. We develop an online tool, Cas-Database, a genome-wide gRNA library design tool for Cas9 nucleases from Streptococcus pyogenes (SpCas9). With an easy-to-use web interface, Cas-Database allows users to select optimal target sequences simply by changing the filtering conditions. Furthermore, it provides a powerful way to select multiple optimal target sequences from thousands of genes at once for the creation of a genome-wide library. Cas-Database also provides a web application programming interface (web API) for advanced bioinformatics users. Free access at http://www.rgenome.net/cas-database/ sangsubae@hanyang.ac.kr or jskim01@snu.ac.kr Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press.

  20. A Novel Pan-Genome Reverse Vaccinology Approach Employing a Negative-Selection Strategy for Screening Surface-Exposed Antigens against leptospirosis

    Science.gov (United States)

    Zeng, LingBing; Wang, Dongliang; Hu, NiYa; Zhu, Qing; Chen, Kaishen; Dong, Ke; Zhang, Yan; Yao, YuFeng; Guo, XiaoKui; Chang, Yung-Fu; Zhu, YongZhang

    2017-01-01

    Reverse vaccinology (RV) has been widely used for screening of surface-exposed proteins (PSEs) of important pathogens, including outer membrane proteins (OMPs), and extracellular proteins (ECPs) as potential vaccine candidates. In this study, we applied a novel RV negative strategy and a pan-genome analysis for screening of PSEs from 17 L. interrogans strains covering 11 predominately epidemic serovars and 17 multilocus typing (MLST) sequence types (STs) worldwide. Our results showed, for instance, out of a total of 633 predicted PSEs in strain 56601, 92.8% were OMPs or ECPs (588/633). Among the 17 strains, 190 core PSEs, 913 dispensable PSEs and 861 unique PSEs were identified. Of the 190 PSEs, 121 were further predicted to be highly antigenic and thus may serve as potential vaccine candidates against leptospirosis. With the exception of LipL45, OmpL1, and LigB, the majority of the 121 PSEs were newly identified antigens. For example, hypothetical proteins BatC, LipL71, and the OmpA family proteins sharing many common features, such as surface-exposed localization, universal conservation, and eliciting strong antibody responses in patients, are regarded as the most promising vaccine antigens. Additionally, a wide array of potential virulence factors among the predicted PSEs including TonB-dependent receptor, sphingomyelinase 2, leucine-rich repeat protein, and 4 neighboring hypothetical proteins were identified as potential antigenicity, and deserve further investigation. Our results can contribute to the prediction of suitable antigens as potential vaccine candidates against leptospirosis and also provide further insights into mechanisms of leptospiral pathogenicity. In addition, our novel negative-screening strategy combined with pan-genome analysis can be a routine RV method applied to numerous other pathogens.

  1. Discovery of novel targets for multi-epitope vaccines: Screening of HIV-1 genomes using association rule mining

    Directory of Open Access Journals (Sweden)

    Piontkivska Helen

    2009-07-01

    Full Text Available Abstract Background Studies have shown that in the genome of human immunodeficiency virus (HIV-1 regions responsible for interactions with the host's immune system, namely, cytotoxic T-lymphocyte (CTL epitopes tend to cluster together in relatively conserved regions. On the other hand, "epitope-less" regions or regions with relatively low density of epitopes tend to be more variable. However, very little is known about relationships among epitopes from different genes, in other words, whether particular epitopes from different genes would occur together in the same viral genome. To identify CTL epitopes in different genes that co-occur in HIV genomes, association rule mining was used. Results Using a set of 189 best-defined HIV-1 CTL/CD8+ epitopes from 9 different protein-coding genes, as described by Frahm, Linde & Brander (2007, we examined the complete genomic sequences of 62 reference HIV sequences (including 13 subtypes and sub-subtypes with approximately 4 representative sequences for each subtype or sub-subtype, and 18 circulating recombinant forms. The results showed that despite inclusion of recombinant sequences that would be expected to break-up associations of epitopes in different genes when two different genomes are recombined, there exist particular combinations of epitopes (epitope associations that occur repeatedly across the world-wide population of HIV-1. For example, Pol epitope LFLDGIDKA is found to be significantly associated with epitopes GHQAAMQML and FLKEKGGL from Gag and Nef, respectively, and this association rule is observed even among circulating recombinant forms. Conclusion We have identified CTL epitope combinations co-occurring in HIV-1 genomes including different subtypes and recombinant forms. Such co-occurrence has important implications for design of complex vaccines (multi-epitope vaccines and/or drugs that would target multiple HIV-1 regions at once and, thus, may be expected to overcome challenges

  2. A genome-wide RNAi screen in Caenorhabditis elegans identifies the nicotinic acetylcholine receptor subunit ACR-7 as an antipsychotic drug target.

    Directory of Open Access Journals (Sweden)

    Taixiang Saur

    Full Text Available We report a genome-wide RNA interference (RNAi screen for Suppressors of Clozapine-induced Larval Arrest (scla genes in Caenorhabditis elegans, the first genetic suppressor screen for antipsychotic drug (APD targets in an animal. The screen identifies 40 suppressors, including the α-like nicotinic acetylcholine receptor (nAChR homolog acr-7. We validate the requirement for acr-7 by showing that acr-7 knockout suppresses clozapine-induced larval arrest and that expression of a full-length translational GFP fusion construct rescues this phenotype. nAChR agonists phenocopy the developmental effects of clozapine, while nAChR antagonists partially block these effects. ACR-7 is strongly expressed in the pharynx, and clozapine inhibits pharyngeal pumping. acr-7 knockout and nAChR antagonists suppress clozapine-induced inhibition of pharyngeal pumping. These findings suggest that clozapine activates ACR-7 channels in pharyngeal muscle, leading to tetanus of pharyngeal muscle with consequent larval arrest. No APDs are known to activate nAChRs, but a number of studies indicate that α7-nAChR agonists may prove effective for the treatment of psychosis. α-like nAChR signaling is a mechanism through which clozapine may produce its therapeutic and/or toxic effects in humans, a hypothesis that could be tested following identification of the mammalian ortholog of C. elegans acr-7.

  3. Identification of hepta-histidine as a candidate drug for Huntington’s disease by in silico-in vitro- in vivo-integrated screens of chemical libraries

    Science.gov (United States)

    Imamura, Tomomi; Fujita, Kyota; Tagawa, Kazuhiko; Ikura, Teikichi; Chen, Xigui; Homma, Hidenori; Tamura, Takuya; Mao, Ying; Taniguchi, Juliana Bosso; Motoki, Kazumi; Nakabayashi, Makoto; Ito, Nobutoshi; Yamada, Kazunori; Tomii, Kentaro; Okano, Hideyuki; Kaye, Julia; Finkbeiner, Steven; Okazawa, Hitoshi

    2016-09-01

    We identified drug seeds for treating Huntington’s disease (HD) by combining in vitro single molecule fluorescence spectroscopy, in silico molecular docking simulations, and in vivo fly and mouse HD models to screen for inhibitors of abnormal interactions between mutant Htt and physiological Ku70, an essential DNA damage repair protein in neurons whose function is known to be impaired by mutant Htt. From 19,468 and 3,010,321 chemicals in actual and virtual libraries, fifty-six chemicals were selected from combined in vitro-in silico screens; six of these were further confirmed to have an in vivo effect on lifespan in a fly HD model, and two chemicals exerted an in vivo effect on the lifespan, body weight and motor function in a mouse HD model. Two oligopeptides, hepta-histidine (7H) and Angiotensin III, rescued the morphological abnormalities of primary neurons differentiated from iPS cells of human HD patients. For these selected drug seeds, we proposed a possible common structure. Unexpectedly, the selected chemicals enhanced rather than inhibited Htt aggregation, as indicated by dynamic light scattering analysis. Taken together, these integrated screens revealed a new pathway for the molecular targeted therapy of HD.

  4. Identification of hepta-histidine as a candidate drug for Huntington’s disease by in silico-in vitro- in vivo-integrated screens of chemical libraries

    Science.gov (United States)

    Imamura, Tomomi; Fujita, Kyota; Tagawa, Kazuhiko; Ikura, Teikichi; Chen, Xigui; Homma, Hidenori; Tamura, Takuya; Mao, Ying; Taniguchi, Juliana Bosso; Motoki, Kazumi; Nakabayashi, Makoto; Ito, Nobutoshi; Yamada, Kazunori; Tomii, Kentaro; Okano, Hideyuki; Kaye, Julia; Finkbeiner, Steven; Okazawa, Hitoshi

    2016-01-01

    We identified drug seeds for treating Huntington’s disease (HD) by combining in vitro single molecule fluorescence spectroscopy, in silico molecular docking simulations, and in vivo fly and mouse HD models to screen for inhibitors of abnormal interactions between mutant Htt and physiological Ku70, an essential DNA damage repair protein in neurons whose function is known to be impaired by mutant Htt. From 19,468 and 3,010,321 chemicals in actual and virtual libraries, fifty-six chemicals were selected from combined in vitro-in silico screens; six of these were further confirmed to have an in vivo effect on lifespan in a fly HD model, and two chemicals exerted an in vivo effect on the lifespan, body weight and motor function in a mouse HD model. Two oligopeptides, hepta-histidine (7H) and Angiotensin III, rescued the morphological abnormalities of primary neurons differentiated from iPS cells of human HD patients. For these selected drug seeds, we proposed a possible common structure. Unexpectedly, the selected chemicals enhanced rather than inhibited Htt aggregation, as indicated by dynamic light scattering analysis. Taken together, these integrated screens revealed a new pathway for the molecular targeted therapy of HD. PMID:27653664

  5. Genomic mechanisms of stress tolerance for the industrial yeast Saccharomyces cerevisiae against the major chemical classes of inhibitors derived from lignocellulosic biomass conversion

    Science.gov (United States)

    Scientists at ARS developed tolerant industrial yeast that is able to reduce major chemical classes of inhibitors into less toxic or none toxic compounds while producing ethanol. Using genomic studies, we defined mechanisms of in situ detoxification involved in novel gene functions, vital cofactor r...

  6. Variation in fish mercury concentrations in streams of the Adirondack region, New York: A simplified screening approach using chemical metrics

    Science.gov (United States)

    Burns, Douglas A.; Riva-Murray, Karen

    2018-01-01

    Simple screening approaches for the neurotoxicant methylmercury (MeHg) in aquatic ecosystems may be helpful in risk assessments of natural resources. We explored the development of such an approach in the Adirondack Mountains of New York, USA, a region with high levels of MeHg bioaccumulation. Thirty-six perennial streams broadly representative of 1st and 2nd order streams in the region were sampled during summer low flow and analyzed for several solutes and for Hg concentrations in fish. Several landscape and chemical metrics that are typically strongly related to MeHg concentrations in aquatic biota were explored for strength of association with fish Hg concentrations. Data analyses were based on site mean length-normalized and standardized Hg concentrations (assumed to be dominantly MeHg) in whole juvenile and adult Brook Trout Salvelinus fontinalis, Creek Chub Semotilus atromaculatus, Blacknose Dace Rhinichthys atratulus, and Central Mudminnow Umbra limi, as well as on multi-species z-scores. Surprisingly, none of the landscape metrics was related significantly to regional variation in fish Hg concentrations or to z-scores across the study streams. In contrast, several chemical metrics including dissolved organic carbon (DOC) concentrations, sulfate concentrations (SO42−), pH, ultra-violet absorbance (UV254), and specific ultra-violet absorbance were significantly related to regional variation in fish Hg concentrations. A cluster analysis based on DOC, SO42−, and pH identified three distinct groups of streams: (1) high DOC, acidic streams, (2) moderate DOC, slightly acidic streams, and (3) low DOC circum-neutral streams with relatively high SO42−. Preliminary analysis indicated no significant difference in fish Hg z-scores between the moderate and high DOC groups, so these were combined for further analysis. The resulting two groups showed strong differences (p 6.9 mg/L, SO42− 0.31 cm−1 were tested as thresholds to identify Adirondack

  7. Characterization of pellicle inhibition in Gluconacetobacter xylinus 53582 by a small molecule, pellicin, identified by a chemical genetics screen.

    Directory of Open Access Journals (Sweden)

    Janice L Strap

    Full Text Available Pellicin ([2E]-3-phenyl-1-[2,3,4,5-tetrahydro-1,6-benzodioxocin-8-yl]prop-2-en-1-one was identified in a chemical genetics screen of 10,000 small molecules for its ability to completely abolish pellicle production in Gluconacetobacter xylinus. Cells grown in the presence of pellicin grew 1.5 times faster than untreated cells. Interestingly, growth in pellicin also caused G. xylinus cells to elongate. Measurement of cellulose synthesis in vitro showed that cellulose synthase activity was not directly inhibited by pellicin. Rather, when cellulose synthase activity was measured in cells that were pre-treated with the compound, the rate of cellulose synthesis increased eight-fold over that observed for untreated cells. This phenomenon was also apparent in the rapid production of cellulose when cells grown in the presence of pellicin were washed and transferred to media lacking the inhibitor. The rate at which cellulose was produced could not be accounted for by growth of the organism. Pellicin was not detected when intracellular contents were analyzed. Furthermore, it was found that pellicin exerts its effect extracellularly by interfering with the crystallization of pre-cellulosic tactoidal aggregates. This interference of the crystallization process resulted in enhanced production of cellulose II as evidenced by the ratio of acid insoluble to acid soluble product in in vitro assays and confirmed in vivo by scanning electron microscopy and powder X-ray diffraction. The relative crystallinity index, RCI, of pellicle produced by untreated G. xylinus cultures was 70% while pellicin-grown cultures had RCI of 38%. Mercerized pellicle of untreated cells had RCI of 42%, which further confirms the mechanism of action of pellicin as an inhibitor of the cellulose I crystallization process. Pellicin is a useful tool for the study of cellulose biosynthesis in G. xylinus.

  8. Pharmacoeconomic evaluations of pharmacogenetic and genomic screening programmes : A systematic review on content and adherence to guidelines

    NARCIS (Netherlands)

    Vegter, Stefan; Boersma, Cornelis; Rozenbaum, Mark; Wilffert, Bob; Navis, Gerjan; Postma, Maarten J.

    2008-01-01

    The fields of pharmacogenetics and pharmacogenomics have become important practical tools to progress goals in medical and pharmaceutical research and development. As more screening tests are being developed, with some already used in clinical practice, consideration of cost-effectiveness implicatio

  9. A genome-wide screen for spatially restricted expression patterns identifies transcription factors that regulate glial development

    NARCIS (Netherlands)

    Fu, H.; Cai, J.; Clevers, H.; Fast, E.; Gray, S.; Greenberg, R.; Jain, M.K.; Ma, Q.; Qiu, M.; Rowitch, D.H.; Taylor, C.; Stiles, C.D.

    2009-01-01

    Forward genetic screens in genetically accessible invertebrate organisms such as Drosophila melanogaster have shed light on transcription factors that specify formation of neurons in the vertebrate CNS. However, invertebrate models have, to date, been uninformative with respect to genes that specify

  10. Plant-symbiotic fungi as chemical engineers: multi-genome analysis of the clavicipitaceae reveals dynamics of alkaloid loci.

    Directory of Open Access Journals (Sweden)

    Christopher L Schardl

    Full Text Available The fungal family Clavicipitaceae includes plant symbionts and parasites that produce several psychoactive and bioprotective alkaloids. The family includes grass symbionts in the epichloae clade (Epichloë and Neotyphodium species, which are extraordinarily diverse both in their host interactions and in their alkaloid profiles. Epichloae produce alkaloids of four distinct classes, all of which deter insects, and some-including the infamous ergot alkaloids-have potent effects on mammals. The exceptional chemotypic diversity of the epichloae may relate to their broad range of host interactions, whereby some are pathogenic and contagious, others are mutualistic and vertically transmitted (seed-borne, and still others vary in pathogenic or mutualistic behavior. We profiled the alkaloids and sequenced the genomes of 10 epichloae, three ergot fungi (Claviceps species, a morning-glory symbiont (Periglandula ipomoeae, and a bamboo pathogen (Aciculosporium take, and compared the gene clusters for four classes of alkaloids. Results indicated a strong tendency for alkaloid loci to have conserved cores that specify the skeleton structures and peripheral genes that determine chemical variations that are known to affect their pharmacological specificities. Generally, gene locations in cluster peripheries positioned them near to transposon-derived, AT-rich repeat blocks, which were probably involved in gene losses, duplications, and neofunctionalizations. The alkaloid loci in the epichloae had unusual structures riddled with large, complex, and dynamic repeat blocks. This feature was not reflective of overall differences in repeat contents in the genomes, nor was it characteristic of most other specialized metabolism loci. The organization and dynamics of alkaloid loci and abundant repeat blocks in the epichloae suggested that these fungi are under selection for alkaloid diversification. We suggest that such selection is related to the variable life histories

  11. Plant-symbiotic fungi as chemical engineers: multi-genome analysis of the clavicipitaceae reveals dynamics of alkaloid loci.

    Science.gov (United States)

    Schardl, Christopher L; Young, Carolyn A; Hesse, Uljana; Amyotte, Stefan G; Andreeva, Kalina; Calie, Patrick J; Fleetwood, Damien J; Haws, David C; Moore, Neil; Oeser, Birgitt; Panaccione, Daniel G; Schweri, Kathryn K; Voisey, Christine R; Farman, Mark L; Jaromczyk, Jerzy W; Roe, Bruce A; O'Sullivan, Donal M; Scott, Barry; Tudzynski, Paul; An, Zhiqiang; Arnaoudova, Elissaveta G; Bullock, Charles T; Charlton, Nikki D; Chen, Li; Cox, Murray; Dinkins, Randy D; Florea, Simona; Glenn, Anthony E; Gordon, Anna; Güldener, Ulrich; Harris, Daniel R; Hollin, Walter; Jaromczyk, Jolanta; Johnson, Richard D; Khan, Anar K; Leistner, Eckhard; Leuchtmann, Adrian; Li, Chunjie; Liu, JinGe; Liu, Jinze; Liu, Miao; Mace, Wade; Machado, Caroline; Nagabhyru, Padmaja; Pan, Juan; Schmid, Jan; Sugawara, Koya; Steiner, Ulrike; Takach, Johanna E; Tanaka, Eiji; Webb, Jennifer S; Wilson, Ella V; Wiseman, Jennifer L; Yoshida, Ruriko; Zeng, Zheng

    2013-01-01

    The fungal family Clavicipitaceae includes plant symbionts and parasites that produce several psychoactive and bioprotective alkaloids. The family includes grass symbionts in the epichloae clade (Epichloë and Neotyphodium species), which are extraordinarily diverse both in their host interactions and in their alkaloid profiles. Epichloae produce alkaloids of four distinct classes, all of which deter insects, and some-including the infamous ergot alkaloids-have potent effects on mammals. The exceptional chemotypic diversity of the epichloae may relate to their broad range of host interactions, whereby some are pathogenic and contagious, others are mutualistic and vertically transmitted (seed-borne), and still others vary in pathogenic or mutualistic behavior. We profiled the alkaloids and sequenced the genomes of 10 epichloae, three ergot fungi (Claviceps species), a morning-glory symbiont (Periglandula ipomoeae), and a bamboo pathogen (Aciculosporium take), and compared the gene clusters for four classes of alkaloids. Results indicated a strong tendency for alkaloid loci to have conserved cores that specify the skeleton structures and peripheral genes that determine chemical variations that are known to affect their pharmacological specificities. Generally, gene locations in cluster peripheries positioned them near to transposon-derived, AT-rich repeat blocks, which were probably involved in gene losses, duplications, and neofunctionalizations. The alkaloid loci in the epichloae had unusual structures riddled with large, complex, and dynamic repeat blocks. This feature was not reflective of overall differences in repeat contents in the genomes, nor was it characteristic of most other specialized metabolism loci. The organization and dynamics of alkaloid loci and abundant repeat blocks in the epichloae suggested that these fungi are under selection for alkaloid diversification. We suggest that such selection is related to the variable life histories of the

  12. The international validation of chemical and biological screening methods for dioxins and dl-PCB's. The DIFFERENCE project round 3

    Energy Technology Data Exchange (ETDEWEB)

    Loco, J. van; Carbonelle, S.; Goeyens, L.; Beernaert, H. [Scientific Institute for Public Health (Belgium); Leeuwen, S.P.J. van [Netherlands Institute for Fisheries Research, IJmuiden (Netherlands). Animal Sciences Group

    2004-09-15

    The European research project DIFFERENCE focuses on the development, optimisation and validation of screening methods for dioxin analysis, including bio-analytical and chemical screening techniques (CALUX, GC-LRMS/MS, GCxGC-ECD) and on the optimisation and validation of new extraction and cleanup procedures. The performance of these techniques is assessed in an international validation study and the results are compared with the reference technique GC-HRMS. The purpose of the validation study is to ensure that the bio-analytical and chemical analytical screening methods for dioxins and dioxin-like PCBs (dl-PCBs) respond to the EU criteria. Screening methods are used to distinguish between compliant and non-compliant samples. The requirements for analytical methods for the official control of dioxins and dioxin-like PCBs in food and feeding stuffs are laid down in the EU commission directives 2002/69/EC and 2002/70/EC. The analytical procedures must have a high sensitivity, a low limit of detection and a high accuracy. This international validation protocol, which is based on the International Harmonized Protocol for Proficiency Testing, will provide information about the accuracy (trueness and precision), ruggedness, detection capability and selectivity of the bio and chemical analytical screening methods in three rounds. The first round focussed on the goodness-of-fit of the calibration curve and on the accuracy of the methods. In round 2 the detection capability and selectivity are assessed. The results of the first 2 rounds of the project have been reported by Van Loco et al. In round 3, the accuracy and robustness of the methods are evaluated on several samples of different origin. This paper summarizes some of the results of round 3 of the validation study.

  13. Epigenetic siRNA and Chemical Screens Identify SETD8 Inhibition as a Therapeutic Strategy for p53 Activation in High-Risk Neuroblastoma.

    Science.gov (United States)

    Veschi, Veronica; Liu, Zhihui; Voss, Ty C; Ozbun, Laurent; Gryder, Berkley; Yan, Chunhua; Hu, Ying; Ma, Anqi; Jin, Jian; Mazur, Sharlyn J; Lam, Norris; Souza, Barbara K; Giannini, Giuseppe; Hager, Gordon L; Arrowsmith, Cheryl H; Khan, Javed; Appella, Ettore; Thiele, Carol J

    2017-01-09

    Given the paucity of druggable mutations in high-risk neuroblastoma (NB), we undertook chromatin-focused small interfering RNA and chemical screens to uncover epigenetic regulators critical for the differentiation block in high-risk NB. High-content Opera imaging identified 53 genes whose loss of expression led to a decrease in NB cell proliferation and 16 also induced differentiation. From these, the secondary chemical screen identified SETD8, the H4(K20me1) methyltransferase, as a druggable NB target. Functional studies revealed that SETD8 ablation rescued the pro-apoptotic and cell-cycle arrest functions of p53 by decreasing p53(K382me1), leading to activation of the p53 canonical pathway. In pre-clinical xenograft NB models, genetic or pharmacological (UNC0379) SETD8 inhibition conferred a significant survival advantage, providing evidence for SETD8 as a therapeutic target in NB. Published by Elsevier Inc.

  14. 化学基因组学和化学蛋白质组学用于细胞自噬研究%Application of chemical genomics and chemical proteomics methods in research of autophagy mechanism

    Institute of Scientific and Technical Information of China (English)

    罗继壮; 苗俊英

    2012-01-01

    化学基因组学和化学蛋白质组学作为后基因组时代的新技术,是以化学小分了为工具,对细胞的生理过程进行精确干扰,研究有机体和细胞的功能,同时也是新药开发的重要手段.本文综述了化学基因组学和化学蛋白质组学在自噬相关靶点的特异性小分子的发现,及小分子在自噬机理研究中的应用.%As new technologies in post-genome era, chemical genomics and chemical proteomics utilize small molecules as tools to accurate interference the physiological processes of cells and conduct researches on functions of organisms and cells. Meanwhile, they are important techniques for the development of new drugs. This review includes the progress for the application of chemical genomics and chemical proteomics tools on the discovery of small molecule chemicals specific for autophagy related targets and inducers of autophagy, as well as the application of the small molecules to the autophagy mechanism research.

  15. US Army Public Health Command’s (Prov) (Formly USACHPPM) Process to Screen Chemicals in Support of DoD’s CMRM Emerging Contaminants Program

    Science.gov (United States)

    2010-06-01

    US Army Public Health Command’s (Prov) (Formly USACHPPM) Process to Screen Chemicals in Support of DoD’s CMRM Emerging Contaminants Program...CMRM Emerging Contaminants Program 5a. CONTRACT NUMBER 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) 5d. PROJECT NUMBER 5e. TASK NUMBER...Occupational Health • DoD Instruction 5000.2 Operation of the Defense Acquisition System • DoD Instruction 4715.18 Emerging Contaminants (EC

  16. The Broad Institute: Screening for Dependencies in Cancer Cell Lines Using Small Molecules | Office of Cancer Genomics

    Science.gov (United States)

    Using cancer cell-line profiling, we established an ongoing resource to identify, as comprehensively as possible, the drug-targetable dependencies that specific genomic alterations impart on human cancers. We measured the sensitivity of hundreds of genetically characterized cancer cell lines to hundreds of small-molecule probes and drugs that have highly selective interactions with their targets, and that collectively modulate many distinct nodes in cancer cell circuitry.

  17. Genome-wide screen identifies new candidate genes associated with artemisinin susceptibility in Plasmodium falciparum in Kenya

    OpenAIRE

    Borrmann, Steffen; Straimer, Judith; Mwai, Leah; Abdi, Abdirahman; Rippert, Anja; Okombo, John; Muriithi, Steven; Sasi, Philip; Kortok, Moses Mosobo; LOWE, BRETT; Campino, Susana; Assefa, Samuel; Auburn, Sarah; Manske, Magnus; Maslen, Gareth

    2013-01-01

    Early identification of causal genetic variants underlying antimalarial drug resistance could provide robust epidemiological tools for timely public health interventions. Using a novel natural genetics strategy for mapping novel candidate genes we analyzed >75,000 high quality single nucleotide polymorphisms selected from high-resolution whole-genome sequencing data in 27 isolates of Plasmodium falciparum. We identified genetic variants associated with susceptibility to dihydroartemisinin tha...

  18. Screening of repetitive motifs inside the genome of the flat oyster (Ostrea edulis): Transposable elements and short tandem repeats.

    Science.gov (United States)

    Vera, Manuel; Bello, Xabier; Álvarez-Dios, Jose-Antonio; Pardo, Belen G; Sánchez, Laura; Carlsson, Jens; Carlsson, Jeanette E L; Bartolomé, Carolina; Maside, Xulio; Martinez, Paulino

    2015-12-01

    The flat oyster (Ostrea edulis) is one of the most appreciated molluscs in Europe, but its production has been greatly reduced by the parasite Bonamia ostreae. Here, new generation genomic resources were used to analyse the repetitive fraction of the oyster genome, with the aim of developing molecular markers to face this main oyster production challenge. The resulting oyster database, consists of two sets of 10,318 and 7159 unique contigs (4.8 Mbp and 6.8 Mbp in total length) representing the oyster's genome (WG) and haemocyte transcriptome (HT), respectively. A total of 1083 sequences were identified as TE-derived, which corresponded to 4.0% of WG and 1.1% of HT. They were clustered into 142 homology groups, most of which were assigned to the Penelope order of retrotransposons, and to the Helitron and TIR DNA-transposons. Simple repeats and rRNA pseudogenes, also made a significant contribution to the oyster's genome (0.5% and 0.3% of WG and HT, respectively).The most frequent short tandem repeats identified in WG were tetranucleotide motifs while trinucleotide motifs were in HT. Forty identified microsatellite loci, 20 from each database, were selected for technical validation. Success was much lower among WG than HT microsatellites (15% vs 55%), which could reflect higher variation in anonymous regions interfering with primer annealing. All microsatellites developed adjusted to Hardy-Weinberg proportions and represent a useful tool to support future breeding programmes and to manage genetic resources of natural flat oyster beds.

  19. CHEMICALS

    CERN Document Server

    Medical Service

    2002-01-01

    It is reminded that all persons who use chemicals must inform CERN's Chemistry Service (TIS-GS-GC) and the CERN Medical Service (TIS-ME). Information concerning their toxicity or other hazards as well as the necessary individual and collective protection measures will be provided by these two services. Users must be in possession of a material safety data sheet (MSDS) for each chemical used. These can be obtained by one of several means : the manufacturer of the chemical (legally obliged to supply an MSDS for each chemical delivered) ; CERN's Chemistry Service of the General Safety Group of TIS ; for chemicals and gases available in the CERN Stores the MSDS has been made available via EDH either in pdf format or else via a link to the supplier's web site. Training courses in chemical safety are available for registration via HR-TD. CERN Medical Service : TIS-ME :73186 or service.medical@cern.ch Chemistry Service : TIS-GS-GC : 78546

  20. Genome-wide CRISPR-Cas9 Screens Reveal Loss of Redundancy between PKMYT1 and WEE1 in Glioblastoma Stem-like Cells

    Directory of Open Access Journals (Sweden)

    Chad M. Toledo

    2015-12-01

    Full Text Available To identify therapeutic targets for glioblastoma (GBM, we performed genome-wide CRISPR-Cas9 knockout (KO screens in patient-derived GBM stem-like cells (GSCs and human neural stem/progenitors (NSCs, non-neoplastic stem cell controls, for genes required for their in vitro growth. Surprisingly, the vast majority GSC-lethal hits were found outside of molecular networks commonly altered in GBM and GSCs (e.g., oncogenic drivers. In vitro and in vivo validation of GSC-specific targets revealed several strong hits, including the wee1-like kinase, PKMYT1/Myt1. Mechanistic studies demonstrated that PKMYT1 acts redundantly with WEE1 to inhibit cyclin B-CDK1 activity via CDK1-Y15 phosphorylation and to promote timely completion of mitosis in NSCs. However, in GSCs, this redundancy is lost, most likely as a result of oncogenic signaling, causing GBM-specific lethality.

  1. Genome-wide CRISPR-Cas9 Screens Reveal Loss of Redundancy between PKMYT1 and WEE1 in Glioblastoma Stem-like Cells.

    Science.gov (United States)

    Toledo, Chad M; Ding, Yu; Hoellerbauer, Pia; Davis, Ryan J; Basom, Ryan; Girard, Emily J; Lee, Eunjee; Corrin, Philip; Hart, Traver; Bolouri, Hamid; Davison, Jerry; Zhang, Qing; Hardcastle, Justin; Aronow, Bruce J; Plaisier, Christopher L; Baliga, Nitin S; Moffat, Jason; Lin, Qi; Li, Xiao-Nan; Nam, Do-Hyun; Lee, Jeongwu; Pollard, Steven M; Zhu, Jun; Delrow, Jeffery J; Clurman, Bruce E; Olson, James M; Paddison, Patrick J

    2015-12-22

    To identify therapeutic targets for glioblastoma (GBM), we performed genome-wide CRISPR-Cas9 knockout (KO) screens in patient-derived GBM stem-like cells (GSCs) and human neural stem/progenitors (NSCs), non-neoplastic stem cell controls, for genes required for their in vitro growth. Surprisingly, the vast majority GSC-lethal hits were found outside of molecular networks commonly altered in GBM and GSCs (e.g., oncogenic drivers). In vitro and in vivo validation of GSC-specific targets revealed several strong hits, including the wee1-like kinase, PKMYT1/Myt1. Mechanistic studies demonstrated that PKMYT1 acts redundantly with WEE1 to inhibit cyclin B-CDK1 activity via CDK1-Y15 phosphorylation and to promote timely completion of mitosis in NSCs. However, in GSCs, this redundancy is lost, most likely as a result of oncogenic signaling, causing GBM-specific lethality.

  2. A Class of Diacylglycerol Acyltransferase 1 Inhibitors Identified by a Combination of Phenotypic High-throughput Screening, Genomics, and Genetics.

    Science.gov (United States)

    Tschapalda, Kirsten; Zhang, Ya-Qin; Liu, Li; Golovnina, Kseniya; Schlemper, Thomas; Eichmann, Thomas O; Lal-Nag, Madhu; Sreenivasan, Urmila; McLenithan, John; Ziegler, Slava; Sztalryd, Carole; Lass, Achim; Auld, Douglas; Oliver, Brian; Waldmann, Herbert; Li, Zhuyin; Shen, Min; Boxer, Matthew B; Beller, Mathias

    2016-06-01

    Excess lipid storage is an epidemic problem in human populations. Thus, the identification of small molecules to treat or prevent lipid storage-related metabolic complications is of great interest. Here we screened >320.000 compounds for their ability to prevent a cellular lipid accumulation phenotype. We used fly cells because the multifarious tools available for this organism should facilitate unraveling the mechanism-of-action of active small molecules. Of the several hundred lipid storage inhibitors identified in the primary screen we concentrated on three structurally diverse and potent compound classes active in cells of multiple species (including human) and negligible cytotoxicity. Together with Drosophila in vivo epistasis experiments, RNA-Seq expression profiles suggested that the target of one of the small molecules was diacylglycerol acyltransferase 1 (DGAT1), a key enzyme in the production of triacylglycerols and prominent human drug target. We confirmed this prediction by biochemical and enzymatic activity tests.

  3. A Class of Diacylglycerol Acyltransferase 1 Inhibitors Identified by a Combination of Phenotypic High-throughput Screening, Genomics, and Genetics

    Directory of Open Access Journals (Sweden)

    Kirsten Tschapalda

    2016-06-01

    Full Text Available Excess lipid storage is an epidemic problem in human populations. Thus, the identification of small molecules to treat or prevent lipid storage-related metabolic complications is of great interest. Here we screened >320.000 compounds for their ability to prevent a cellular lipid accumulation phenotype. We used fly cells because the multifarious tools available for this organism should facilitate unraveling the mechanism-of-action of active small molecules. Of the several hundred lipid storage inhibitors identified in the primary screen we concentrated on three structurally diverse and potent compound classes active in cells of multiple species (including human and negligible cytotoxicity. Together with Drosophila in vivo epistasis experiments, RNA-Seq expression profiles suggested that the target of one of the small molecules was diacylglycerol acyltransferase 1 (DGAT1, a key enzyme in the production of triacylglycerols and prominent human drug target. We confirmed this prediction by biochemical and enzymatic activity tests.

  4. A Drosophila Genome-Wide Screen Identifies Regulators of Steroid Hormone Production and Developmental Timing

    DEFF Research Database (Denmark)

    Thomas Danielsen, E.; E. Møller, Morten; Yamanaka, Naoki;

    2016-01-01

    and developmental timing. Here, we use our screen as a resource to identify mechanisms regulating intracellular levels of cholesterol, a substrate for steroidogenesis. We identify a conserved fatty acid elongase that underlies a mechanism that adjusts cholesterol trafficking and steroidogenesis with nutrition...... are regulated by TOR and feedback signaling that couples steroidogenesis with growth and ensures proper maturation timing. These results reveal genes regulating steroidogenesis during development that likely modulate disease mechanisms....

  5. A Genome-wide CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) Screen Identifies NEK7 as an Essential Component of NLRP3 Inflammasome Activation.

    Science.gov (United States)

    Schmid-Burgk, Jonathan L; Chauhan, Dhruv; Schmidt, Tobias; Ebert, Thomas S; Reinhardt, Julia; Endl, Elmar; Hornung, Veit

    2016-01-01

    Inflammasomes are high molecular weight protein complexes that assemble in the cytosol upon pathogen encounter. This results in caspase-1-dependent pro-inflammatory cytokine maturation, as well as a special type of cell death, known as pyroptosis. The Nlrp3 inflammasome plays a pivotal role in pathogen defense, but at the same time, its activity has also been implicated in many common sterile inflammatory conditions. To this effect, several studies have identified Nlrp3 inflammasome engagement in a number of common human diseases such as atherosclerosis, type 2 diabetes, Alzheimer disease, or gout. Although it has been shown that known Nlrp3 stimuli converge on potassium ion efflux upstream of Nlrp3 activation, the exact molecular mechanism of Nlrp3 activation remains elusive. Here, we describe a genome-wide CRISPR/Cas9 screen in immortalized mouse macrophages aiming at the unbiased identification of gene products involved in Nlrp3 inflammasome activation. We employed a FACS-based screen for Nlrp3-dependent cell death, using the ionophoric compound nigericin as a potassium efflux-inducing stimulus. Using a genome-wide guide RNA (gRNA) library, we found that targeting Nek7 rescued macrophages from nigericin-induced lethality. Subsequent studies revealed that murine macrophages deficient in Nek7 displayed a largely blunted Nlrp3 inflammasome response, whereas Aim2-mediated inflammasome activation proved to be fully intact. Although the mechanism of Nek7 functioning upstream of Nlrp3 yet remains elusive, these studies provide a first genetic handle of a component that specifically functions upstream of Nlrp3. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. Construction of Genomic Fosmid Library of Brassicajuncea and Screening of Cytological Markers for B-genome Chromosomes%芥菜Fosmid文库构建及B基因组细胞学标记的筛选利用

    Institute of Scientific and Technical Information of China (English)

    彭元凤; 孟德璇; 黄玉碧; 王桂香

    2012-01-01

    构建了由60000个克隆组成的芥菜无偏倚Fosmid文库,该文库外源片段插入率为100%,外源DNA平均插入长度为32kb,文库覆盖率约为芥菜基因组的1.8倍。利用不同来源的分子标记筛选文库,得到的阳性单克隆经荧光原位杂交(FISH)鉴定后,获得两类B基因组细胞学标记,一类在所有染色体上都有信号,另一类仅在一对染色体上有信号。%In this study, an unbiased Fosmid library of Brassica juncea was established, which consisted of 60 000 clones with 100% inserting frequency. In the constructed library, the size of average inserts was approximately 32 kb, corresponding to 1.8 genome equivalents. Subsequently, two types of B-genome cytological markers were identified by means of library screening and chromosome fluorescence in situ hybridization (FISH) . One type was that the signal located on all chromosomes, and the other one was that the signal was detected only on one pair of chromosomes.

  7. Final Technical Report for GO15052 Intematix: Combinatorial Synthesis and High Throughput Screening of Effective Catalysts for Chemical Hydrides

    Energy Technology Data Exchange (ETDEWEB)

    Melman, Jonathan [Intematix Corporation, Fremont, CA (United States)

    2017-02-22

    The objectives of this project are: to discover cost-effective catalysts for release of hydrogen from chemical hydrogen storage systems; and to discover cost-effective catalysts for the regeneration of spent chemical hydrogen storage materials.

  8. Screening of antibiotics and chemical analysis of penicillin residue in fresh milk and traditional dairy products in Oyo state, Nigeria

    Science.gov (United States)

    Olatoye, Isaac Olufemi; Daniel, Oluwayemisi Folashade; Ishola, Sunday Ayobami

    2016-01-01

    Background and Aim: There are global public health and economic concerns on chemical residues in food of animal origin. The use of antibiotics in dairy cattle for the treatment of diseases such as mastitis has contributed to the presence of residues in dairy products. Penicillin residues as low as 1 ppb can lead to allergic reactions and shift of resistance patterns in microbial population as well as interfere with the processing of several dairy products. Antibiotic monitoring is an essential quality control measure in safe milk production. This study was aimed at determining antibiotic residue contamination and the level of penicillin in dairy products from Fulani cattle herds in Oyo State. Materials and Methods: The presence of antibiotic residues in 328 samples of fresh milk, 180 local cheese (wara), and 90 fermented milk (nono) from Southwest, Nigeria were determined using Premi® test kit (R-Biopharm AG, Germany) followed by high-performance liquid chromatography analysis of penicillin-G residue. Results: Antibiotic residues were obtained in 40.8%, 24.4% and 62.3% fresh milk, wara and nono, respectively. Penicillin-G residue was also detected in 41.1% fresh milk, 40.2% nono and 24.4% wara at mean concentrations of 15.22±0.61, 8.24±0.50 and 7.6±0.60 μg/L with 39.3%, 36.7% and 21.1%, respectively, containing penicillin residue above recommended Codex maximum residue limit (MRL) of 5 μg/L in dairy. There was no significant difference between the mean penicillin residues in all the dairy products in this study. Conclusion: The results are of food safety concern since the bulk of the samples and substantial quantities of dairy products in Oyo state contained violative levels of antibiotic residues including penicillin residues in concentrations above the MRL. This could be due to indiscriminate and unregulated administration of antibiotics to dairy cattle. Regulatory control of antibiotic use, rapid screening of milk and dairy farmers’ extension education

  9. Screening of antibiotics and chemical analysis of penicillin residue in fresh milk and traditional dairy products in Oyo state, Nigeria

    Directory of Open Access Journals (Sweden)

    Isaac Olufemi Olatoye

    2016-09-01

    Full Text Available Background and Aim: There are global public health and economic concerns on chemical residues in food of animal origin. The use of antibiotics in dairy cattle for the treatment of diseases such as mastitis has contributed to the presence of residues in dairy products. Penicillin residues as low as 1 ppb can lead to allergic reactions and shift of resistance patterns in microbial population as well as interfere with the processing of several dairy products. Antibiotic monitoring is an essential quality control measure in safe milk production. This study was aimed at determining antibiotic residue contamination and the level of penicillin in dairy products from Fulani cattle herds in Oyo State. Materials and Methods: The presence of antibiotic residues in 328 samples of fresh milk, 180 local cheese (wara, and 90 fermented milk (nono from Southwest, Nigeria were determined using Premi® test kit (R-Biopharm AG, Germany followed by high-performance liquid chromatography analysis of penicillin-G residue. Results: Antibiotic residues were obtained in 40.8%, 24.4% and 62.3% fresh milk, wara and nono, respectively. Penicillin-G residue was also detected in 41.1% fresh milk, 40.2% nono and 24.4% wara at mean concentrations of 15.22±0.61, 8.24±0.50 and 7.6±0.60 μg/L with 39.3%, 36.7% and 21.1%, respectively, containing penicillin residue above recommended Codex maximum residue limit (MRL of 5 μg/L in dairy. There was no significant difference between the mean penicillin residues in all the dairy products in this study. Conclusion: The results are of food safety concern since the bulk of the samples and substantial quantities of dairy products in Oyo state contained violative levels of antibiotic residues including penicillin residues in concentrations above the MRL. This could be due to indiscriminate and unregulated administration of antibiotics to dairy cattle. Regulatory control of antibiotic use, rapid screening of milk and dairy farmers

  10. Identification of three wheat globulin genes by screening a Triticum aestivum BAC genomic library with cDNA from a diabetes-associated globulin

    Directory of Open Access Journals (Sweden)

    MacFarlane Amanda J

    2009-07-01

    Full Text Available Abstract Background Exposure to dietary wheat proteins in genetically susceptible individuals has been associated with increased risk for the development of Type 1 diabetes (T1D. Recently, a wheat protein encoded by cDNA WP5212 has been shown to be antigenic in mice, rats and humans with autoimmune T1D. To investigate the genomic origin of the identified wheat protein cDNA, a hexaploid wheat genomic library from Glenlea cultivar was screened. Results Three unique wheat globulin genes, Glo-3A, Glo3-B and Glo-3C, were identified. We describe the genomic structure of these genes and their expression pattern in wheat seeds. The Glo-3A gene shared 99% identity with the cDNA of WP5212 at the nucleotide and deduced amino acid level, indicating that we have identified the gene(s encoding wheat protein WP5212. Southern analysis revealed the presence of multiple copies of Glo-3-like sequences in all wheat samples, including hexaploid, tetraploid and diploid species wheat seed. Aleurone and embryo tissue specificity of WP5212 gene expression, suggested by promoter region analysis, which demonstrated an absence of endosperm specific cis elements, was confirmed by immunofluorescence microscopy using anti-WP5212 antibodies. Conclusion Taken together, the results indicate that a diverse group of globulins exists in wheat, some of which could be associated with the pathogenesis of T1D in some susceptible individuals. These data expand our knowledge of specific wheat globulins and will enable further elucidation of their role in wheat biology and human health.

  11. Using genome-wide CRISPR library screening with library resistant DCK to find new sources of Ara-C drug resistance in AML.

    Science.gov (United States)

    Kurata, Morito; Rathe, Susan K; Bailey, Natashay J; Aumann, Natalie K; Jones, Justine M; Veldhuijzen, G Willemijn; Moriarity, Branden S; Largaespada, David A

    2016-11-03

    Acute myeloid leukemia (AML) can display de novo or acquired resistance to cytosine arabinoside (Ara-C), a primary component of induction chemotherapy. To identify genes capable of independently imposing Ara-C resistance, we applied a genome-wide CRISPR library to human U937 cells and exposed to them to Ara-C. Interestingly, all drug resistant clones contained guide RNAs for DCK. To avoid DCK gene modification, gRNA resistant DCK cDNA was created by the introduction of silent mutations. The CRISPR screening was repeated using the gRNA resistant DCK, and loss of SLC29A was identified as also being capable of conveying Ara-C drug resistance. To determine if loss of Dck results in increased sensitivity to other drugs, we conducted a screen of 446 FDA approved drugs using two Dck-defective BXH-2 derived murine AML cell lines and their Ara-C sensitive parental lines. Both cell lines showed an increase in sensitivity to prednisolone. Guide RNA resistant cDNA rescue was a legitimate strategy and multiple DCK or SLC29A deficient human cell clones were established with one clone becoming prednisolone sensitive. Dck-defective leukemic cells may become prednisolone sensitive indicating prednisolone may be an effective adjuvant therapy in some cases of DCK-negative AML.

  12. Using genome-wide CRISPR library screening with library resistant DCK to find new sources of Ara-C drug resistance in AML

    Science.gov (United States)

    Kurata, Morito; Rathe, Susan K.; Bailey, Natashay J.; Aumann, Natalie K.; Jones, Justine M.; Veldhuijzen, G. Willemijn; Moriarity, Branden S.; Largaespada, David A.

    2016-01-01

    Acute myeloid leukemia (AML) can display de novo or acquired resistance to cytosine arabinoside (Ara-C), a primary component of induction chemotherapy. To identify genes capable of independently imposing Ara-C resistance, we applied a genome-wide CRISPR library to human U937 cells and exposed to them to Ara-C. Interestingly, all drug resistant clones contained guide RNAs for DCK. To avoid DCK gene modification, gRNA resistant DCK cDNA was created by the introduction of silent mutations. The CRISPR screening was repeated using the gRNA resistant DCK, and loss of SLC29A was identified as also being capable of conveying Ara-C drug resistance. To determine if loss of Dck results in increased sensitivity to other drugs, we conducted a screen of 446 FDA approved drugs using two Dck-defective BXH-2 derived murine AML cell lines and their Ara-C sensitive parental lines. Both cell lines showed an increase in sensitivity to prednisolone. Guide RNA resistant cDNA rescue was a legitimate strategy and multiple DCK or SLC29A deficient human cell clones were established with one clone becoming prednisolone sensitive. Dck-defective leukemic cells may become prednisolone sensitive indicating prednisolone may be an effective adjuvant therapy in some cases of DCK-negative AML. PMID:27808171

  13. A genome-wide screen in Saccharomyces cerevisiae reveals altered transport as a mechanism of resistance to the anticancer drug bleomycin.

    Science.gov (United States)

    Aouida, Mustapha; Pagé, Nicolas; Leduc, Anick; Peter, Matthias; Ramotar, Dindial

    2004-02-01

    The potent DNA damaging agent bleomycin (BLM) is highly effective for treating various cancers, although, in certain individuals, the development of cellular resistance to the drug can severely diminish its antineoplastic properties. We performed two independent genome-wide screens using a Saccharomyces cerevisiae mutant collection to isolate variants exhibiting either sensitivity or resistance to BLM. This procedure reproducibly identified a relatively large collection of 231 BLM-hypersensitive mutants, representing genes belonging to diverse functional groups. In contrast, only five BLM-resistant mutants could be recovered by our screens. Among these latter mutants, three were deleted for genes involved in plasma membrane transport, including the L-carnitine transporter Agp2, as well as the kinases Ptk2 and Sky1, which are involved in regulating polyamine transport. We further showed that Agp2 acts as a transporter of BLM and that overexpression of this transporter significantly enhances BLM-induced cell killing. Our data strongly implicate membrane transport as a key determinant in BLM resistance in yeast. This finding is critical, given that very little is known about BLM transport in human cells. Indeed, characterization of analogous mechanisms in humans may ultimately lead to enhancement of the antitumor properties of BLM.

  14. Genome-Wide CRISPR-Cas9 Screen Identifies MicroRNAs That Regulate Myeloid Leukemia Cell Growth

    OpenAIRE

    Wallace, Jared; Hu, Ruozhen; Mosbruger, Timothy L.; Dahlem, Timothy J.; Stephens, W. Zac; Rao, Dinesh S.; Round, June L.; O’Connell, Ryan M.

    2016-01-01

    Mammalian microRNA expression is dysregulated in human cancer. However, the functional relevance of many microRNAs in the context of tumor biology remains unclear. Using CRISPR-Cas9 technology, we performed a global loss-of-function screen to simultaneously test the functions of individual microRNAs and protein-coding genes during the growth of a myeloid leukemia cell line. This approach identified evolutionarily conserved human microRNAs that suppress or promote cell growth, revealing that m...

  15. Recent advances in genome-based polyketide discovery.

    Science.gov (United States)

    Helfrich, Eric J N; Reiter, Silke; Piel, Jörn

    2014-10-01

    Polyketides are extraordinarily diverse secondary metabolites of great pharmacological value and with interesting ecological functions. The post-genomics era has led to fundamental changes in natural product research by inverting the workflow of secondary metabolite discovery. As opposed to traditional bioactivity-guided screenings, genome mining is an in silico method to screen and analyze sequenced genomes for natural product biosynthetic gene clusters. Since genes for known compounds can be recognized at the early computational stage, genome mining presents an opportunity for dereplication. This review highlights recent progress in bioinformatics, pathway engineering and chemical analytics to extract the biosynthetic secrets hidden in the genome of both well-known natural product sources as well as previously neglected bacteria.

  16. Whole-Genome Expression Analysis and Signal Pathway Screening of Synovium-Derived Mesenchymal Stromal Cells in Rheumatoid Arthritis

    Directory of Open Access Journals (Sweden)

    Jingyi Hou

    2016-01-01

    Full Text Available Synovium-derived mesenchymal stromal cells (SMSCs may play an important role in the pathogenesis of rheumatoid arthritis (RA and show promise for therapeutic applications in RA. In this study, a whole-genome microarray analysis was used to detect differential gene expression in SMSCs from RA patients and healthy donors (HDs. Our results showed that there were 4828 differentially expressed genes in the RA group compared to the HD group; 3117 genes were upregulated, and 1711 genes were downregulated. A Gene Ontology analysis showed significantly enriched terms of differentially expressed genes in the biological process, cellular component, and molecular function domains. A Kyoto Encyclopedia of Genes and Genomes analysis showed that the MAPK signaling and rheumatoid arthritis pathways were upregulated and that the p53 signaling pathway was downregulated in RA SMSCs. Quantitative real-time polymerase chain reaction was applied to verify the expression variations of the partial genes mentioned above, and a western blot analysis was used to determine the expression levels of p53, p-JNK, p-ERK, and p-p38. Our study found that differentially expressed genes in the MAPK signaling, rheumatoid arthritis, and p53 signaling pathways may help to explain the pathogenic mechanism of RA and lead to therapeutic RA SMSC applications.

  17. Genome-wide screen identifies new candidate genes associated with artemisinin susceptibility in Plasmodium falciparum in Kenya

    Science.gov (United States)

    Borrmann, Steffen; Straimer, Judith; Mwai, Leah; Abdi, Abdirahman; Rippert, Anja; Okombo, John; Muriithi, Steven; Sasi, Philip; Kortok, Moses Mosobo; Lowe, Brett; Campino, Susana; Assefa, Samuel; Auburn, Sarah; Manske, Magnus; Maslen, Gareth; Peshu, Norbert; Kwiatkowski, Dominic P.; Marsh, Kevin; Nzila, Alexis; Clark, Taane G.

    2013-01-01

    Early identification of causal genetic variants underlying antimalarial drug resistance could provide robust epidemiological tools for timely public health interventions. Using a novel natural genetics strategy for mapping novel candidate genes we analyzed >75,000 high quality single nucleotide polymorphisms selected from high-resolution whole-genome sequencing data in 27 isolates of Plasmodium falciparum. We identified genetic variants associated with susceptibility to dihydroartemisinin that implicate one region on chromosome 13, a candidate gene on chromosome 1 (PFA0220w, a UBP1 ortholog) and others (PFB0560w, PFB0630c, PFF0445w) with putative roles in protein homeostasis and stress response. There was a strong signal for positive selection on PFA0220w, but not the other candidate loci. Our results demonstrate the power of full-genome sequencing-based association studies for uncovering candidate genes that determine parasite sensitivity to artemisinins. Our study provides a unique reference for the interpretation of results from resistant infections. PMID:24270944

  18. DEVELOPMENT OF A SCREENING APPROACH TO DETECT THYROID DISRUPTING CHEMICALS THAT INHIBIT THE HUMAN SODIUM IODIDE SYMPORTER (NIS)

    Data.gov (United States)

    U.S. Environmental Protection Agency — Data pertaining to a NIS-expressing cell line, hNIS-HEK293T-EPA, and its screening capabilities for determining inhibitors of NIS-mediated iodide uptake. This...

  19. Can Genomic Amplification of Human Telomerase Gene and C-MYC in Liquid-Based Cytological Specimens Be Used as a Method for Opportunistic Cervical Cancer Screening?

    Science.gov (United States)

    Gao, Kun; Eurasian, Menglan; Zhang, Jieqing; Wei, Yuluan; Zheng, Qian; Ye, Hongtao; Li, Li

    2015-01-01

    To evaluate the effectiveness of five methods including the ThinPrep cytological test (TCT), liquid-based cytology, the human papillomavirus (HPV) test, detection of the TERC and C-MYC genes and visual inspection with acetic acid/Lugol's iodine (VIA/VILI) for opportunistic cervical cancer screening, and to explore whether genomic amplification of the human telomerase gene and C-MYC in liquid-based cytological specimens can be used as a method for opportunistic cervical cancer screening. Data were collected prospectively from 1,010 consecutive patients who visited the gynecology clinic and agreed to participate in opportunistic cervical cancer screening at our institution from November 2010 to July 2011. The five methods mentioned above were used for the screening in all cases. The histopathological diagnosis served as the gold standard for the evaluation. A comparison between the five screening methods for the diagnosis of high-grade cervical intraepithelial neoplasia (CIN II and III) was performed for their sensitivity, specificity, false-positive rate, false-negative rate, accuracy rate, positive likelihood ratio and negative likelihood ratio. A comprehensive comparison of the different combination programs for screening was performed according to the analysis of the receiver operating characteristic (ROC) curve area. The accuracy of the five screening methods for the diagnosis of high-grade CIN (CIN II and III) was compared in the different age groups. A joint model for the diagnosis using different combinations of the five methods was developed according to the analysis by the SAS 8.0 software. The model was used to evaluate the accuracy of the different combination programs for the diagnosis of high-grade CIN, and the results were confirmed by the histopathological examination. The sensitivity and specificity of the single screen method (TCT, HPV test, detection of the TERC and C-MYC genes, and VIA/VILI method) for CIN II was 80.9, 70.2, 72.3, 76.6, and 72

  20. Rapid screening and identification of chemical hazards in surface and drinking water using high resolution mass spectrometry and a case-control filter.

    Science.gov (United States)

    Kaserzon, Sarit L; Heffernan, Amy L; Thompson, Kristie; Mueller, Jochen F; Gomez Ramos, Maria Jose

    2017-09-01

    Access to clean, safe drinking water poses a serious challenge to regulators, and requires analytical strategies capable of rapid screening and identification of potentially hazardous chemicals, specifically in situations when threats to water quality or security require rapid investigations and potential response. This study describes a fast and efficient chemical hazard screening strategy for characterising trace levels of polar organic contaminants in water matrices, based on liquid chromatography high resolution mass spectrometry with post-acquisition 'case-control' data processing. This method allowed for a rapid response time of less than 24 h for the screening of target, suspect and non-target unknown chemicals via direct injection analysis, and a second, more sensitive analysis option requiring sample pre-concentration. The method was validated by fortifying samples with a range of pesticides, pharmaceuticals and personal care products (n = 46); with >90% of target compounds positively screened in samples at 1 ng mL(-1), and 46% at 0.1 ng mL(-1) when analysed via direct injection. To simulate a contamination event samples were fortified with compounds not present in the commercial library (designated 'non-target compounds'; fipronil and fenitrothion), tentatively identified at 0.2 and 1 ng mL(-1), respectively; and a compound not included in any known commercial library or public database (designated 'unknown' compounds; 8Cl(-) perfluorooctanesulfonic acid), at 0.8 ng mL(-1). The method was applied to two 'real-case' scenarios: (1) the assessment of drinking water safety during a high-profile event in Brisbane, Australia; and (2) to screen treated, re-circulated drinking water and pre-treated (raw) water. The validated workflow was effective for rapid prioritisation and screening of suspect and non-target potential hazards at trace levels, and could be applied to a wide range of matrices and investigations where comparison of organic contaminants

  1. Discovery of a novel neuroprotective compound, AS1219164, by high-throughput chemical screening of a newly identified apoptotic gene marker.

    Science.gov (United States)

    Yamazaki, Takao; Muramoto, Masakazu; Okitsu, Osamu; Morikawa, Noriyuki; Kita, Yasuhiro

    2011-11-01

    We have reported that tacrolimus (FK506), an immunosuppressive drug, and diclofenac, a non-steroidal anti-inflammatory drug, possess different modes of neuroprotective action. FK506 suppresses only thapsigargin-induced apoptosis in neuroblastoma SH-SY5Y cells while diclofenac reverses tunicamycin-induced as well as thapsigargin-induced apoptosis. The aim of this study is to discover novel compounds that exert neuroprotective properties by using the transcriptional response of a newly identified gene, which was regulated by both FK506 and diclofenac, as a surrogate screening marker in high-throughput chemical screening and characterize the compounds in comparison with FK506 and diclofenac. Using a microarray with 4504 human cDNAs and quantitative RT-PCR, two genes as apoptotic markers, transmembrane protein 100 (TMEM100) and limb-bud and heart (LBH), were identified because the thapsigargin-induced elevations in their mRNA levels were reversed by both FK506 and diclofenac. A luciferase reporter assay with a TMEM100 promoter region was applied to high-throughput chemical screening. AS1219164, {3-[(E)-2-{5-[(E)-2-pyridin-4-ylvinyl]pyridin-3-yl} vinyl]aniline}, suppressed thapsigargin-induced transactivation of the TMEM100 gene and reversed thapsigargin-induced increases in TMEM100 and LBH mRNA levels in SH-SY5Y cells, similar to the effects of FK506 and diclofenac. Furthermore, AS1219164 protected against SH-SY5Y cell death induced by four apoptotic agents including thapsigargin, similar to diclofenac, but was more potent than diclofenac, while FK506 only showed protective effects against thapsigargin-induced cell death. In conclusion, a novel neuroprotecitve compound, AS1219164, was discovered by high-throughput chemical screening using a reporter assay with the TMEM100 gene promoter regulated by both FK506 and diclofenac. Reporter assay using the promoter region of a gene under pharmacological and physiological transcriptional regulation would be well suit for use

  2. [CHANGING OF PHYSICO-CHEMICAL PARAMETERS OF NON-CONTACT (ELECTROCHEMICAL) ACTIVATED DRINKING WATER IS ASSOCIATED WITH INDUCTION OF GENOMIC INSTABILITY OF CULTIVATED HUMAN BLOOD LYMPHOCYTES].

    Science.gov (United States)

    Zatsepina, O V; Ingel, F I

    2016-01-01

    In the article there are presented data which are the fragment of large multidisciplinary study of genetic safety of non-contact electrochemically activated water (NAW). The aim of this study was the analysis of the relation of impacts of genomic instability (micronucleus test with cytochalasin B) detected in human blood cells, cultured in medias prepared on the base of these NAWs, with physical and chemical properties of these NaWs. In experiments there were used catholytes and anolytes obtained by activation of osmotic, tap and dining bottled water As a result of such activation, all waters were shown to acquire the ability to induce genomic instability in cellular cultures. Notably in cell cultures on catholytes and anolytes these effects differed between themselves and have been associated with different physical and chemical properties of the NAWs.

  3. Functional Genomic Screen Identifies Klebsiella pneumoniae Factors Implicated in Blocking Nuclear Factor κB (NF-κB) Signaling.

    Science.gov (United States)

    Tomás, Anna; Lery, Leticia; Regueiro, Verónica; Pérez-Gutiérrez, Camino; Martínez, Verónica; Moranta, David; Llobet, Enrique; González-Nicolau, Mar; Insua, Jose L; Tomas, Juan M; Sansonetti, Philippe J; Tournebize, Régis; Bengoechea, José A

    2015-07-03

    Klebsiella pneumoniae is an etiologic agent of community-acquired and nosocomial pneumonia. It has been shown that K. pneumoniae infections are characterized by reduced early inflammatory response. Recently our group has shown that K. pneumoniae dampens the activation of inflammatory responses by antagonizing the activation of the NF-κB canonical pathway. Our results revealed that K. pneumoniae capsule polysaccharide (CPS) was necessary but not sufficient to attenuate inflammation. To identify additional Klebsiella factors required to dampen inflammation, we standardized and applied a high-throughput gain-of-function screen to examine a Klebsiella transposon mutant library. We identified 114 mutants that triggered the activation of NF-κB. Two gene ontology categories accounted for half of the loci identified in the screening: metabolism and transport genes (32% of the mutants) and envelope-related genes (17%). Characterization of the mutants revealed that the lack of the enterobactin siderophore was linked to a reduced CPS expression, which in turn underlined the NF-κB activation induced by the mutant. The lipopolysaccharide (LPS) O-polysaccharide and the pullulanase (PulA) type 2 secretion system (T2SS) are required for full effectiveness of the immune evasion. Importantly, these factors do not play a redundant role. The fact that LPS O-polysaccharide and T2SS mutant-induced responses were dependent on TLR2-TLR4-MyD88 activation suggested that LPS O-polysaccharide and PulA perturbed Toll-like receptor (TLR)-dependent recognition of K. pneumoniae. Finally, we demonstrate that LPS O-polysaccharide and pulA mutants are attenuated in the pneumonia mouse model. We propose that LPS O-polysaccharide and PulA T2SS could be new targets for the design of new antimicrobials. Increasing TLR-governed defense responses might provide also selective alternatives for the management of K. pneumoniae pneumonia.

  4. Top Value Added Chemicals from Biomass - Volume I, Results of Screening for Potential Candidates from Sugars and Synthesis Gas

    Energy Technology Data Exchange (ETDEWEB)

    None

    2004-08-01

    This report identifies twelve building block chemicals that can be produced from sugars via biological or chemical conversions. The twelve building blocks can be subsequently converted to a number of high-value bio-based chemicals or materials. Building block chemicals, as considered for this analysis, are molecules with multiple functional groups that possess the potential to be transformed into new families of useful molecules. The twelve sugar-based building blocks are 1,4-diacids (succinic, fumaric and malic), 2,5-furan dicarboxylic acid, 3-hydroxy propionic acid, aspartic acid, glucaric acid, glutamic acid, itaconic acid, levulinic acid, 3-hydroxybutyrolactone, glycerol, sorbitol, and xylitol/arabinitol.

  5. The role of acoustic screens in distribution of technogenic magnetic particles and chemical pollution in roadside soil

    Science.gov (United States)

    Wawer, Małgorzata; Magiera, Tadeusz; Szuszkiewicz, Marcin

    2015-04-01

    Roads constructed nowadays should by all means be functional for their motorized users but at the same time their effect on the environment ought to be limited to the minimum. Despite the existence of various methods for preventing from negative influence of roads on the environment, there is still lack of adequate techniques to monitor and reduce the spreading of roadside pollution in the air and soils. The aim of the study was to assess the influence of acoustic screens on spreading and deposition of solid pollutants deriving from car emissions, based on their quantitative and qualitative analysis. During this study, measurements of magnetic susceptibility and analyses of heavy metals as well as Pt and Rh contents in soil and plant samples (Taraxacum officinale, Plantago major, Parthenocissus quinquefolia) collected near different kinds of acoustic screens ("green walls", Plexiglass, sawdust concrete, steel panels and earth embankments) have been done. Previous investigations showed showed that most of traffic emission is deposited in the close vicinity of the roads (up to 10 m) and the level of contamination decreased with increasing distance from the road edge. However, the results of this project indicate that, in the area where the acoustic screens are located, this distribution is disturbed and the additional enrichment of heavy metals in soil about 10 - 15 m behind screens is observed. Spatial distribution of heavy metal contents in soil samples corresponds to its magnetic susceptibility values. High contents of Fe, Zn, Mn and Pb was observed next to acoustic screens made of sawdust concrete and steel panels. Additionally, concentration of Zn in soil samples collected close to these screens exceeded threshold value. Analyses of plants showed that the highest content of examined elements and highest values of magnetic susceptibility were recorded near road edge. What is more, samples of Parthenocissus quinquefolia collected at height 0.2 m were characterized

  6. Reliability analysis of the Ahringer Caenorhabditis elegans RNAi feeding library: a guide for genome-wide screens

    Directory of Open Access Journals (Sweden)

    Lu Yiming

    2011-03-01

    Full Text Available Abstract Background The Ahringer C. elegans RNAi feeding library prepared by cloning genomic DNA fragments has been widely used in genome-wide analysis of gene function. However, the library has not been thoroughly validated by direct sequencing, and there are potential errors, including: 1 mis-annotation (the clone with the retired gene name should be remapped to the actual target gene; 2 nonspecific PCR amplification; 3 cross-RNAi; 4 mis-operation such as sample loading error, etc. Results Here we performed a reliability analysis on the Ahringer C. elegans RNAi feeding library, which contains 16,256 bacterial strains, using a bioinformatics approach. Results demonstrated that most (98.3% of the bacterial strains in the library are reliable. However, we also found that 2,851 (17.54% bacterial strains need to be re-annotated even they are reliable. Most of these bacterial strains are the clones having the retired gene names. Besides, 28 strains are grouped into unreliable category and 226 strains are marginal because of probably expressing unrelated double-stranded RNAs (dsRNAs. The accuracy of the prediction was further confirmed by direct sequencing analysis of 496 bacterial strains. Finally, a freely accessible database named CelRNAi (http://biocompute.bmi.ac.cn/CelRNAi/ was developed as a valuable complement resource for the feeding RNAi library by providing the predicted information on all bacterial strains. Moreover, submission of the direct sequencing result or any other annotations for the bacterial strains to the database are allowed and will be integrated into the CelRNAi database to improve the accuracy of the library. In addition, we provide five candidate primer sets for each of the unreliable and marginal bacterial strains for users to construct an alternative vector for their own RNAi studies. Conclusions Because of the potential unreliability of the Ahringer C. elegans RNAi feeding library, we strongly suggest the user examine

  7. Genome Wide Screening of Candidate Genes for Improving Piglet Birth Weight Using High and Low Estimated Breeding Value Populations

    Science.gov (United States)

    Zhang, Lifan; Zhou, Xiang; Michal, Jennifer J.; Ding, Bo; Li, Rui; Jiang, Zhihua

    2014-01-01

    Birth weight is an economically important trait in pig production because it directly impacts piglet growth and survival rate. In the present study, we performed a genome wide survey of candidate genes and pathways associated with individual birth weight (IBW) using the Illumina PorcineSNP60 BeadChip on 24 high (HEBV) and 24 low estimated breeding value (LEBV) animals. These animals were selected from a reference population of 522 individuals produced by three sires and six dam lines, which were crossbreds with multiple breeds. After quality-control, 43,257 SNPs (single nucleotide polymorphisms), including 42,243 autosomal SNPs and 1,014 SNPs on chromosome X, were used in the data analysis. A total of 27 differentially selected regions (DSRs), including 1 on Sus scrofa chromosome 1 (SSC1), 1 on SSC4, 2 on SSC5, 4 on SSC6, 2 on SSC7, 5 on SSC8, 3 on SSC9, 1 on SSC14, 3 on SSC18, and 5 on SSCX, were identified to show the genome wide separations between the HEBV and LEBV groups for IBW in piglets. A DSR with the most number of significant SNPs (including 7 top 0.1% and 31 top 5% SNPs) was located on SSC6, while another DSR with the largest genetic differences in FST was found on SSC18. These regions harbor known functionally important genes involved in growth and development, such as TNFRSF9 (tumor necrosis factor receptor superfamily member 9), CA6 (carbonic anhydrase VI) and MDFIC (MyoD family inhibitor domain containing). A DSR rich in imprinting genes appeared on SSC9, which included PEG10 (paternally expressed 10), SGCE (sarcoglycan, epsilon), PPP1R9A (protein phosphatase 1, regulatory subunit 9A) and ASB4 (ankyrin repeat and SOCS box containing 4). More importantly, our present study provided evidence to support six quantitative trait loci (QTL) regions for pig birth weight, six QTL regions for average birth weight (ABW) and three QTL regions for litter birth weight (LBW) reported previously by other groups. Furthermore, gene ontology analysis with 183 genes

  8. PROLIFERATION AS A KEY EVENT IN DEVELOPMENTAL TOXICITY: "CHEMICAL SCREENING IN HUMAN NEURAL STEM CELLS USING HIGH CONTENT IMAGING

    Science.gov (United States)

    New toxicity testing approaches will rely on in vitro assays to assess chemical effects at the cellular and molecular level. Cell proliferation is imperative to normal development, and chemical disruption of this process can be detrimental to the organism. As part of an effort to...

  9. High-throughput screening of chemical effects on steroidogenesis using H295R human adrenocortical carcinoma cells

    Science.gov (United States)

    Disruption of steroidogenesis by environmental chemicals can result in altered hormone levels causing adverse reproductive and developmental effects. A high-throughput assay using H295R human adrenocortical carcinoma cells was used to evaluate the effect of 2,060 chemical samples...

  10. Prioritizing Environmental Chemicals for Obesity and Diabetes Outcomes Research: A Screening Approach Using ToxCast High Throughput Data

    Science.gov (United States)

    Background: Diabetes and obesity are major threats to public health in the US and abroad. Understanding the role chemicals in our environment play in the development of these conditions is an emerging issue in environmental health, although identifying and prioritizing chemicals ...

  11. A genome-wide screen for promoter methylation in lung cancer identifies novel methylation markers for multiple malignancies.

    Directory of Open Access Journals (Sweden)

    David S Shames

    2006-12-01

    Full Text Available BACKGROUND: Promoter hypermethylation coupled with loss of heterozygosity at the same locus results in loss of gene function in many tumor cells. The "rules" governing which genes are methylated during the pathogenesis of individual cancers, how specific methylation profiles are initially established, or what determines tumor type-specific methylation are unknown. However, DNA methylation markers that are highly specific and sensitive for common tumors would be useful for the early detection of cancer, and those required for the malignant phenotype would identify pathways important as therapeutic targets. METHODS AND FINDINGS: In an effort to identify new cancer-specific methylation markers, we employed a high-throughput global expression profiling approach in lung cancer cells. We identified 132 genes that have 5' CpG islands, are induced from undetectable levels by 5-aza-2'-deoxycytidine in multiple non-small cell lung cancer cell lines, and are expressed in immortalized human bronchial epithelial cells. As expected, these genes were also expressed in normal lung, but often not in companion primary lung cancers. Methylation analysis of a subset (45/132 of these promoter regions in primary lung cancer (n = 20 and adjacent nonmalignant tissue (n = 20 showed that 31 genes had acquired methylation in the tumors, but did not show methylation in normal lung or peripheral blood cells. We studied the eight most frequently and specifically methylated genes from our lung cancer dataset in breast cancer (n = 37, colon cancer (n = 24, and prostate cancer (n = 24 along with counterpart nonmalignant tissues. We found that seven loci were frequently methylated in both breast and lung cancers, with four showing extensive methylation in all four epithelial tumors. CONCLUSIONS: By using a systematic biological screen we identified multiple genes that are methylated with high penetrance in primary lung, breast, colon, and prostate cancers. The cross

  12. Genomics-based screening of differentially expressed genes in the brains of mice exposed to silver nanoparticles via inhalation

    Science.gov (United States)

    Lee, Hye-Young; Choi, You-Jin; Jung, Eun-Jung; Yin, Hu-Quan; Kwon, Jung-Taek; Kim, Ji-Eun; Im, Hwang-Tae; Cho, Myung-Haing; Kim, Ju-Han; Kim, Hyun-Young; Lee, Byung-Hoon

    2010-06-01

    Silver nanoparticles (AgNP) are among the fastest growing product categories in the nanotechnology industry. Despite the importance of AgNP in consumer products and clinical applications, relatively little is known regarding AgNP toxicity and its associated risks. We investigated the effects of AgNP on gene expression in the mouse brain using Affymetrix Mouse Genome Arrays. C57BL/6 mice were exposed to AgNP (geometric mean diameter, 22.18 ± 1.72 nm; 1.91 × 107 particles/cm3) for 6 h/day, 5 days/week using the nose-only exposure system for 2 weeks. Total RNA isolated from the cerebrum and cerebellum was subjected to hybridization. From over 39,000 probe sets, 468 genes in the cerebrum and 952 genes in the cerebellum were identified as AgNP-responsive (one-way analysis of variance; p cerebrum and 144 genes in the cerebellum. AgNP exposure modulated the expression of several genes associated with motor neuron disorders, neurodegenerative disease, and immune cell function, indicating potential neurotoxicity and immunotoxicity associated with AgNP exposure. Real-time PCR data for five genes analyzed from whole blood showed good correlation with the observed changes in the brain. Following rigorous validation and substantiation, these genes may assist in the development of surrogate markers for AgNP exposure and/or toxicity.

  13. Top Value Added Chemicals From Biomass: I. Results of Screening for Potential Candidates from Sugars and Synthesis Gas

    Energy Technology Data Exchange (ETDEWEB)

    Werpy, Todd A.; Holladay, John E.; White, James F.

    2004-11-01

    This report identifies twelve building block chemicals that can be produced from sugars via biological or chemical conversions. The twelve building blocks can be subsequently converted to a number of high-value bio-based chemicals or materials. Building block chemicals, as considered for this analysis, are molecules with multiple functional groups that possess the potential to be transformed into new families of useful molecules. The twelve sugar-based building blocks are 1,4-diacids (succinic, fumaric and malic), 2,5-furan dicarboxylic acid, 3-hydroxy propionic acid, aspartic acid, glucaric acid, glutamic acid, itaconic acid, levulinic acid, 3-hydroxybutyrolactone, glycerol, sorbitol, and xylitol/arabinitol. In addition to building blocks, the report outlines the central technical barriers that are preventing the widespread use of biomass for products and chemicals.

  14. Genome-Wide CRISPR-Cas9 Screen Identifies MicroRNAs That Regulate Myeloid Leukemia Cell Growth.

    Science.gov (United States)

    Wallace, Jared; Hu, Ruozhen; Mosbruger, Timothy L; Dahlem, Timothy J; Stephens, W Zac; Rao, Dinesh S; Round, June L; O'Connell, Ryan M

    2016-01-01

    Mammalian microRNA expression is dysregulated in human cancer. However, the functional relevance of many microRNAs in the context of tumor biology remains unclear. Using CRISPR-Cas9 technology, we performed a global loss-of-function screen to simultaneously test the functions of individual microRNAs and protein-coding genes during the growth of a myeloid leukemia cell line. This approach identified evolutionarily conserved human microRNAs that suppress or promote cell growth, revealing that microRNAs are extensively integrated into the molecular networks that control tumor cell physiology. miR-155 was identified as a top microRNA candidate promoting cellular fitness, which we confirmed with two distinct miR-155-targeting CRISPR-Cas9 lentiviral constructs. Further, we performed anti-correlation functional profiling to predict relevant microRNA-tumor suppressor gene or microRNA-oncogene interactions in these cells. This analysis identified miR-150 targeting of p53, a connection that was experimentally validated. Taken together, our study describes a powerful genetic approach by which the function of individual microRNAs can be assessed on a global level, and its use will rapidly advance our understanding of how microRNAs contribute to human disease.

  15. A genome-wide screen identifies a single β-defensin gene cluster in the chicken: implications for the origin and evolution of mammalian defensins

    Directory of Open Access Journals (Sweden)

    Xiao Yanjing

    2004-08-01

    Full Text Available Abstract Background Defensins comprise a large family of cationic antimicrobial peptides that are characterized by the presence of a conserved cysteine-rich defensin motif. Based on the spacing pattern of cysteines, these defensins are broadly divided into five groups, namely plant, invertebrate, α-, β-, and θ-defensins, with the last three groups being mostly found in mammalian species. However, the evolutionary relationships among these five groups of defensins remain controversial. Results Following a comprehensive screen, here we report that the chicken genome encodes a total of 13 different β-defensins but with no other groups of defensins being discovered. These chicken β-defensin genes, designated as Gallinacin 1–13, are clustered densely within a 86-Kb distance on the chromosome 3q3.5-q3.7. The deduced peptides vary from 63 to 104 amino acid residues in length sharing the characteristic defensin motif. Based on the tissue expression pattern, 13 β-defensin genes can be divided into two subgroups with Gallinacin 1–7 being predominantly expressed in bone marrow and the respiratory tract and the remaining genes being restricted to liver and the urogenital tract. Comparative analysis of the defensin clusters among chicken, mouse, and human suggested that vertebrate defensins have evolved from a single β-defensin-like gene, which has undergone rapid duplication, diversification, and translocation in various vertebrate lineages during evolution. Conclusions We conclude that the chicken genome encodes only β-defensin sequences and that all mammalian defensins are evolved from a common β-defensin-like ancestor. The α-defensins arose from β-defensins by gene duplication, which may have occurred after the divergence of mammals from other vertebrates, and θ-defensins have arisen from α-defensins specific to the primate lineage. Further analysis of these defensins in different vertebrate lineages will shed light on the mechanisms of

  16. Genome-wide functional genetic screen with the anticancer agent AMPI-109 identifies PRL-3 as an oncogenic driver in triple-negative breast cancers.

    Science.gov (United States)

    Gari, Hamid H; Gearheart, Christy M; Fosmire, Susan; DeGala, Gregory D; Fan, Zeying; Torkko, Kathleen C; Edgerton, Susan M; Lucia, M Scott; Ray, Rahul; Thor, Ann D; Porter, Christopher C; Lambert, James R

    2016-03-29

    Triple-negative breast cancers (TNBC) are among the most aggressive and heterogeneous cancers with a high propensity to invade, metastasize and relapse. Here, we demonstrate that the anticancer compound, AMPI-109, is selectively efficacious in inhibiting proliferation and inducing apoptosis of multiple TNBC subtype cell lines as assessed by activation of pro-apoptotic caspases-3 and 7, PARP cleavage and nucleosomal DNA fragmentation. AMPI-109 had little to no effect on growth in the majority of non-TNBC cell lines examined. We therefore utilized AMPI-109 in a genome-wide shRNA screen in the TNBC cell line, BT-20, to investigate the utility of AMPI-109 as a tool in helping to identify molecular alterations unique to TNBC. Our screen identified the oncogenic phosphatase, PRL-3, as a potentially important driver of TNBC growth, migration and invasion. Through stable lentiviral knock downs and transfection with catalytically impaired PRL-3 in TNBC cells, loss of PRL-3 expression, or functionality, led to substantial growth inhibition. Moreover, AMPI-109 treatment, downregulation of PRL-3 expression or impairment of PRL-3 activity reduced TNBC cell migration and invasion. Histological evaluation of human breast cancers revealed PRL-3 was significantly, though not exclusively, associated with the TNBC subtype and correlated positively with regional and distant metastases, as well as 1 and 3 year relapse free survival. Collectively, our study is proof-of-concept that AMPI-109, a selectively active agent against TNBC cell lines, can be used as a molecular tool to uncover unique drivers of disease progression, such as PRL-3, which we show promotes oncogenic phenotypes in TNBC cells.

  17. The conditional nature of genetic interactions: the consequences of wild-type backgrounds on mutational interactions in a genome-wide modifier screen.

    Directory of Open Access Journals (Sweden)

    Sudarshan Chari

    Full Text Available The phenotypic outcome of a mutation cannot be simply mapped onto the underlying DNA variant. Instead, the phenotype is a function of the allele, the genetic background in which it occurs and the environment where the mutational effects are expressed. While the influence of genetic background on the expressivity of individual mutations is recognized, its consequences on the interactions between genes, or the genetic network they form, is largely unknown. The description of genetic networks is essential for much of biology; yet if, and how, the topologies of such networks are influenced by background is unknown. Furthermore, a comprehensive examination of the background dependent nature of genetic interactions may lead to identification of novel modifiers of biological processes. Previous work in Drosophila melanogaster demonstrated that wild-type genetic background influences the effects of an allele of scalloped (sd, with respect to both its principal consequence on wing development and its interactions with a mutation in optomotor blind. In this study we address whether the background dependence of mutational interactions is a general property of genetic systems by performing a genome wide dominant modifier screen of the sd(E3 allele in two wild-type genetic backgrounds using molecularly defined deletions. We demonstrate that ~74% of all modifiers of the sd(E3 phenotype are background-dependent due in part to differential sensitivity to genetic perturbation. These background dependent interactions include some with qualitative differences in the phenotypic outcome, as well as instances of sign epistasis. This suggests that genetic interactions are often contingent on genetic background, with flexibility in genetic networks due to segregating variation in populations. Such background dependent effects can substantially alter conclusions about how genes influence biological processes, the potential for genetic screens in alternative wild

  18. Possible roles of vacuolar H+-ATPase and mitochondrial function in tolerance to air-drying stress revealed by genome-wide screening of Saccharomyces cerevisiae deletion strains.

    Science.gov (United States)

    Shima, Jun; Ando, Akira; Takagi, Hiroshi

    2008-03-01

    Yeasts used in bread making are exposed to air-drying stress during dried yeast production processes. To clarify the genes required for air-drying tolerance, we performed genome-wide screening using the complete deletion strain collection of diploid Saccharomyces cerevisiae. The screening identified 278 gene deletions responsible for air-drying sensitivity. These genes were classified based on their cellular function and on the localization of their gene products. The results showed that the genes required for air-drying tolerance were frequently involved in mitochondrial functions and in connection with vacuolar H(+)-ATPase, which plays a role in vacuolar acidification. To determine the role of vacuolar acidification in air-drying stress tolerance, we monitored intracellular pH. The results showed that intracellular acidification was induced during air-drying and that this acidification was amplified in a deletion mutant of the VMA2 gene encoding a component of vacuolar H(+)-ATPase, suggesting that vacuolar H(+)-ATPase helps maintain intracellular pH homeostasis, which is affected by air-drying stress. To determine the effects of air-drying stress on mitochondria, we analysed the mitochondrial membrane potential under air-drying stress conditions using MitoTracker. The results showed that mitochondria were extremely sensitive to air-drying stress, suggesting that a mitochondrial function is required for tolerance to air-drying stress. We also analysed the correlation between oxidative-stress sensitivity and air-drying-stress sensitivity. The results suggested that oxidative stress is a critical determinant of sensitivity to air-drying stress, although ROS-scavenging systems are not necessary for air-drying stress tolerance. (c) 2008 John Wiley & Sons, Ltd.

  19. QSAR model for human pregnane X receptor (PXR) binding: Screening of environmental chemicals and correlations with genotoxicity, endocrine disruption and teratogenicity

    Energy Technology Data Exchange (ETDEWEB)

    Dybdahl, Marianne, E-mail: mdyb@food.dtu.dk; Nikolov, Nikolai G.; Wedebye, Eva Bay; Jónsdóttir, Svava Ósk; Niemelä, Jay R.

    2012-08-01

    The pregnane X receptor (PXR) has a key role in regulating the metabolism and transport of structurally diverse endogenous and exogenous compounds. Activation of PXR has the potential to initiate adverse effects, causing drug–drug interactions, and perturbing normal physiological functions. Therefore, identification of PXR ligands would be valuable information for pharmaceutical and toxicological research. In the present study, we developed a quantitative structure–activity relationship (QSAR) model for the identification of PXR ligands using data based on a human PXR binding assay. A total of 631 molecules, representing a variety of chemical structures, constituted the training set of the model. Cross-validation of the model showed a sensitivity of 82%, a specificity of 85%, and a concordance of 84%. The developed model provided knowledge about molecular descriptors that may influence the binding of molecules to PXR. The model was used to screen a large inventory of environmental chemicals, of which 47% was found to be within domain of the model. Approximately 35% of the chemicals within domain were predicted to be PXR ligands. The predicted PXR ligands were found to be overrepresented among chemicals predicted to cause adverse effects, such as genotoxicity, teratogenicity, estrogen receptor activation and androgen receptor antagonism compared to chemicals not causing these effects. The developed model may be useful as a tool for predicting potential PXR ligands and for providing mechanistic information of toxic effects of chemicals. -- Highlights: ► Global QSAR model for the identification of PXR ligands was developed. ► Molecular descriptors that may influence PXR binding were identified. ► 35% of a large set of environmental chemicals were predicted to be PXR ligands. ► Predicted PXR binding was associated with various adverse effects.

  20. Genomics-based screening of differentially expressed genes in the brains of mice exposed to silver nanoparticles via inhalation

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Hye-Young; Choi, You-Jin; Jung, Eun-Jung; Yin, Hu-Quan [Seoul National University, College of Pharmacy and Research Institute of Pharmaceutical Sciences (Korea, Republic of); Kwon, Jung-Taek; Kim, Ji-Eun; Im, Hwang-Tae; Cho, Myung-Haing [Seoul National University, College of Veterinary Medicine (Korea, Republic of); Kim, Ju-Han [Seoul National University, College of Medicine (Korea, Republic of); Kim, Hyun-Young [Occupational Safety and Health Research Institute, Chemical Safety and Health Research Center (Korea, Republic of); Lee, Byung-Hoon, E-mail: lee@snu.ac.k [Seoul National University, College of Pharmacy and Research Institute of Pharmaceutical Sciences (Korea, Republic of)

    2010-06-15

    Silver nanoparticles (AgNP) are among the fastest growing product categories in the nanotechnology industry. Despite the importance of AgNP in consumer products and clinical applications, relatively little is known regarding AgNP toxicity and its associated risks. We investigated the effects of AgNP on gene expression in the mouse brain using Affymetrix Mouse Genome Arrays. C57BL/6 mice were exposed to AgNP (geometric mean diameter, 22.18 {+-} 1.72 nm; 1.91 x 10{sup 7} particles/cm{sup 3}) for 6 h/day, 5 days/week using the nose-only exposure system for 2 weeks. Total RNA isolated from the cerebrum and cerebellum was subjected to hybridization. From over 39,000 probe sets, 468 genes in the cerebrum and 952 genes in the cerebellum were identified as AgNP-responsive (one-way analysis of variance; p < 0.05). The largest groups of gene products affected by AgNP exposure included 73 genes in the cerebrum and 144 genes in the cerebellum. AgNP exposure modulated the expression of several genes associated with motor neuron disorders, neurodegenerative disease, and immune cell function, indicating potential neurotoxicity and immunotoxicity associated with AgNP exposure. Real-time PCR data for five genes analyzed from whole blood showed good correlation with the observed changes in the brain. Following rigorous validation and substantiation, these genes may assist in the development of surrogate markers for AgNP exposure and/or toxicity.

  1. Acanthocephalan fish parasites (Rhadinorhynchidae Lühe, 1912) as potential biomarkers: Molecular-chemical screening by pyrolysis-field ionization mass spectrometry

    Science.gov (United States)

    Kleinertz, S.; Eckhardt, K.-U.; Theisen, S.; Palm, H. W.; Leinweber, P.

    2016-07-01

    The present study represents the first molecular-chemical screening by pyrolysis-field ionization mass spectrometry applied on fish parasites. A total of 71 fishes from Balinese fish markets, 36 Auxis rochei (Risso, 1810) and 35 A. thazard (Lacepède, 1800), were studied for their acanthocephalan parasites. This is the first record of Rhadinorhynchus zhukovi in Balinese waters, Indonesia, and we describe for the first time A. rochei and A. thazard as R. zhukovi hosts. Using this method, small scale variations within the chemical compounds of acanthocephalans could be detected. Using this methodology it will be possible to generate additional, pollutant specific information from aquatic habitats in future with the potential of a new bioindicator application for parasite/host origin and/or environmental pollution.

  2. Impact of human genome initiative-derived technology on genetic testing, screening and counseling: Cultural, ethical and legal issues

    Energy Technology Data Exchange (ETDEWEB)

    Trottier, R.W.; Hodgin, F.C.; Imara, M.; Phoenix, D.; Lybrook, S. (Morehouse Coll., Atlanta, GA (United States). School of Medicine); Crandall, L.A.; Moseley, R.E.; Armotrading, D. (Florida Univ., Gainesville, FL (United States). Coll. of Medicine)

    1993-01-01

    Genetic medical services provided by the Georgia Division of Public Health in two northern and two central districts are compared to services provided in a district in which a tertiary care facility is located. Genetics outreach public health nurses play key roles in Georgia's system of Children's Health Services Genetics Program, including significant roles as counselors and information sources on special needs social services and support organizations. Unique features of individual health districts, (e.g., the changing face of some rural communities in ethnocultural diversity and socioeconomic character), present new challenges to current and future genetics services delivery. Preparedness as to educational needs of both health professionals and the lay population is of foremost concern in light of the ever expanding knowledge and technology in medical genetics. Perspectives on genetics and an overview of services offered by a local private sector counselor are included for comparison to state supported services. The nature of the interactions which transpire between private and public genetic services resources in Georgia will be described. A special focus of this research includes issues associated with sickle cell disease newborn screening service delivery process in Georgia, with particular attention paid to patient follow-up and transition to primary care. Of particular interest to this focus is the problem of loss to follow-up in the current system. Critical factors in education and counseling of sickle cell patients and the expectations of expanding roles of primary care physicians are discussed. The Florida approach to the delivery of genetic services contrasts to the Georgia model by placing more emphasis on a consultant-specialist team approach.

  3. An epigenetic genome-wide screen identifies SPINT2 as a novel tumor suppressor gene in pediatric medulloblastoma.

    Science.gov (United States)

    Kongkham, Paul N; Northcott, Paul A; Ra, Young Shin; Nakahara, Yukiko; Mainprize, Todd G; Croul, Sidney E; Smith, Christian A; Taylor, Michael D; Rutka, James T

    2008-12-01

    Medulloblastoma (MB) is a malignant cerebellar tumor that occurs primarily in children. The hepatocyte growth factor (HGF)/MET pathway has an established role in both normal cerebellar development as well as the development and progression of human brain tumors, including MB. To identify novel tumor suppressor genes involved in MB pathogenesis, we performed an epigenome-wide screen in MB cell lines, using 5-aza-2'deoxycytidine to identify genes aberrantly silenced by promoter hypermethylation. Using this technique, we identified an inhibitor of HGF/MET signaling, serine protease inhibitor kunitz-type 2 (SPINT2/HAI-2), as a putative tumor suppressor silenced by promoter methylation in MB. In addition, based on single nucleotide polymorphism array analysis in primary MB samples, we identified hemizygous deletions targeting the SPINT2 locus in addition to gains on chromosome 7 encompassing the HGF and MET loci. SPINT2 gene expression was down-regulated and MET expression was up-regulated in 73.2% and 45.5% of tumors, respectively, by quantitative real-time PCR. SPINT2 promoter methylation was detected in 34.3% of primary MBs examined by methylation-specific PCR. SPINT2 reexpression in MB cell lines reduced proliferative capacity, anchorage independent growth, cell motility in vitro, and increased overall survival times in vivo in a xenograft model (P<0.0001). Taken together, these data support the role of SPINT2 as a putative tumor suppressor gene in MB, and further implicate dysregulation of the HGF/MET signaling pathway in the pathogenesis of MB.

  4. Genome-Wide Screening Identifies Six Genes That Are Associated with Susceptibility to Escherichia coli Microcin PDI.

    Science.gov (United States)

    Zhao, Zhe; Eberhart, Lauren J; Orfe, Lisa H; Lu, Shao-Yeh; Besser, Thomas E; Call, Douglas R

    2015-10-01

    The microcin PDI inhibits a diverse group of pathogenic Escherichia coli strains. Coculture of a single-gene knockout library (BW25113; n=3,985 mutants) against a microcin PDI-producing strain (E. coli 25) identified six mutants that were not susceptible (ΔatpA, ΔatpF, ΔdsbA, ΔdsbB, ΔompF, and ΔompR). Complementation of these genes restored susceptibility in all cases, and the loss of susceptibility was confirmed through independent gene knockouts in E. coli O157:H7 Sakai. Heterologous expression of E. coli ompF conferred susceptibility to Salmonella enterica and Yersinia enterocolitica strains that are normally unaffected by microcin PDI. The expression of chimeric OmpF and site-directed mutagenesis revealed that the K47G48N49 region within the first extracellular loop of E. coli OmpF is a putative binding site for microcin PDI. OmpR is a transcriptional regulator for ompF, and consequently loss of susceptibility by the ΔompR strain most likely is related to this function. Deletion of AtpA and AtpF, as well as AtpE and AtpH (missed in the original library screen), resulted in the loss of susceptibility to microcin PDI and the loss of ATP synthase function. Coculture of a susceptible strain in the presence of an ATP synthase inhibitor resulted in a loss of susceptibility, confirming that a functional ATP synthase complex is required for microcin PDI activity. In trans expression of ompF in the ΔdsbA and ΔdsbB strains did not restore a susceptible phenotype, indicating that these proteins are probably involved with the formation of disulfide bonds for OmpF or microcin PDI.

  5. Validation of a qualitative screening method for pesticides in fruits and vegetables by gas chromatography quadrupole-time of flight mass spectrometry with atmospheric pressure chemical ionization

    Energy Technology Data Exchange (ETDEWEB)

    Portolés, T. [Research Institute for Pesticides and Water, University Jaume I, 12071 Castellón (Spain); RIKILT Institute of Food Safety, Wageningen University and Research Centre, Akkermaalsbos 2, 6708 WB Wageningen (Netherlands); Mol, J.G.J. [RIKILT Institute of Food Safety, Wageningen University and Research Centre, Akkermaalsbos 2, 6708 WB Wageningen (Netherlands); Sancho, J.V.; López, Francisco J. [Research Institute for Pesticides and Water, University Jaume I, 12071 Castellón (Spain); Hernández, F., E-mail: hernandf@uji.es [Research Institute for Pesticides and Water, University Jaume I, 12071 Castellón (Spain)

    2014-08-01

    Highlights: • Applicability of GC-(APCI)QTOF MS as new tool for wide-scope screening of pesticides in fruits and vegetables demonstrated. • Validation of screening method according to SANCO/12571/2013. • Detection of the pesticides based on the presence of M+·/MH+ in most cases. • Screening detection limit 0.01 mg kg{sup −1} for 77% of the pesticides investigated. • Successful identification at 0.01 mg kg{sup −1} for 70% of the pesticides/matrix combinations. - Abstract: A wide-scope screening method was developed for the detection of pesticides in fruit and vegetables. The method was based on gas chromatography coupled to a hybrid quadrupole time-of-flight mass spectrometer with an atmospheric pressure chemical ionization source (GC-(APCI)QTOF MS). A non-target acquisition was performed through two alternating scan events: one at low collision energy and another at a higher collision energy ramp (MS{sup E}). In this way, both protonated molecule and/or molecular ion together with fragment ions were obtained in a single run. Validation was performed according to SANCO/12571/2013 by analysing 20 samples (10 different commodities in duplicate), fortified with a test set of 132 pesticides at 0.01, 0.05 and 0.20 mg kg{sup −1}. For screening, the detection was based on one diagnostic ion (in most cases the protonated molecule). Overall, at the 0.01 mg kg{sup −1} level, 89% of the 2620 fortifications made were detected. The screening detection limit for individual pesticides was 0.01 mg kg{sup −1} for 77% of the pesticides investigated. The possibilities for identification according to the SANCO criteria, requiring two ions with a mass accuracy ≤±5 ppm and an ion-ratio deviation ≤±30%, were investigated. At the 0.01 mg kg{sup −1} level, identification was possible for 70% of the pesticides detected during screening. This increased to 87% and 93% at the 0.05 and 0.20 mg kg{sup −1} level, respectively. Insufficient sensitivity for the second

  6. Formation of hydrogen sulfide from cysteine in Saccharomyces cerevisiae BY4742: genome wide screen reveals a central role of the vacuole.

    Directory of Open Access Journals (Sweden)

    Gal Winter

    Full Text Available Discoveries on the toxic effects of cysteine accumulation and, particularly, recent findings on the many physiological roles of one of the products of cysteine catabolism, hydrogen sulfide (H2S, are highlighting the importance of this amino acid and sulfur metabolism in a range of cellular activities. It is also highlighting how little we know about this critical part of cellular metabolism. In the work described here, a genome-wide screen using a deletion collection of Saccharomyces cerevisiae revealed a surprising set of genes associated with this process. In addition, the yeast vacuole, not previously associated with cysteine catabolism, emerged as an important compartment for cysteine degradation. Most prominent among the vacuole-related mutants were those involved in vacuole acidification; we identified each of the eight subunits of a vacuole acidification sub-complex (V1 of the yeast V-ATPase as essential for cysteine degradation. Other functions identified included translation, RNA processing, folate-derived one-carbon metabolism, and mitochondrial iron-sulfur homeostasis. This work identified for the first time cellular factors affecting the fundamental process of cysteine catabolism. Results obtained significantly contribute to the understanding of this process and may provide insight into the underlying cause of cysteine accumulation and H2S generation in eukaryotes.

  7. Array-based genome-wide RNAi screening to identify shRNAs that enhance p53-related apoptosis in human cancer cells.

    Science.gov (United States)

    Idogawa, Masashi; Ohashi, Tomoko; Sugisaka, Jun; Sasaki, Yasushi; Suzuki, Hiromu; Tokino, Takashi

    2014-09-15

    p53 transduction is a potentially effective cancer therapy but does not result in a good therapeutic response in all human cancers due to resistance to apoptosis. To discover factors that overcome resistance to p53-induced apoptosis, we attempted to identify RNAi sequences that enhance p53-induced apoptosis. We screened a genome-wide lentiviral shRNA library in liver cancer Huh-7 and pancreatic cancer Panc-1 cells, both of which resist p53-induced apoptosis. After the infection of adenovirus expressing p53 or LacZ as a control, shRNA-treated populations were analyzed by microarray. We identified shRNAs that were significantly decreased in p53-infected cells compared with control cells. Among these shRNAs, shRNA-58335 was markedly decreased in both cancer cell lines tested. shRNA-58335 enhanced p53-related apoptosis in vitro and augmented the inhibitory effect of adenoviral p53 transduction on tumor growth in vivo. Furthermore, the enhanced apoptotic response by shRNA-58335 was also confirmed by treatment with PRIMA-1, which reactivates mutant p53, instead of adenoviral p53 transduction. We found that shRNA-58335 evokes the apoptotic response following p53 transduction or functional restoration of p53 with a small molecule drug in cancer cells resistant to p53-induced apoptosis. The combination of p53 restoration and RNAi-based drugs is expected to be a promising novel cancer therapy.

  8. A genome-wide RNAi screen reveals that mRNA decapping restricts bunyaviral replication by limiting the pools of Dcp2-accessible targets for cap-snatching

    Science.gov (United States)

    Hopkins, Kaycie C.; McLane, Laura M.; Maqbool, Tariq; Panda, Debasis; Gordesky-Gold, Beth; Cherry, Sara

    2013-01-01

    Bunyaviruses are an emerging group of medically important viruses, many of which are transmitted from insects to mammals. To identify host factors that impact infection, we performed a genome-wide RNAi screen in Drosophila and identified 131 genes that impacted infection of the mosquito-transmitted bunyavirus Rift Valley fever virus (RVFV). Dcp2, the catalytic component of the mRNA decapping machinery, and two decapping activators, DDX6 and LSM7, were antiviral against disparate bunyaviruses in both insect cells and adult flies. Bunyaviruses 5′ cap their mRNAs by “cap-snatching” the 5′ ends of poorly defined host mRNAs. We found that RVFV cap-snatches the 5′ ends of Dcp2 targeted mRNAs, including cell cycle-related genes. Loss of Dcp2 allows increased viral transcription without impacting viral mRNA stability, while ectopic expression of Dcp2 impedes viral transcription. Furthermore, arresting cells in late S/early G2 led to increased Dcp2 mRNA targets and increased RVFV replication. Therefore, RVFV competes for the Dcp2-accessible mRNA pool, which is dynamically regulated and can present a bottleneck for viral replication. PMID:23824541

  9. Identification of striated muscle activator of Rho signaling (STARS) as a novel calmodulin target by a newly developed genome-wide screen.

    Science.gov (United States)

    Furuya, Yusui; Denda, Miwako; Sakane, Kyohei; Ogusu, Tomoko; Takahashi, Sumio; Magari, Masaki; Kanayama, Naoki; Morishita, Ryo; Tokumitsu, Hiroshi

    2016-07-01

    To search for novel target(s) of the Ca(2+)-signaling transducer, calmodulin (CaM), we performed a newly developed genome-wide CaM interaction screening of 19,676 GST-fused proteins expressed in human. We identified striated muscle activator of Rho signaling (STARS) as a novel CaM target and characterized its CaM binding ability and found that the Ca(2+)/CaM complex interacted stoichiometrically with the N-terminal region (Ala13-Gln35) of STARS in vitro as well as in living cells. Mutagenesis studies identified Ile20 and Trp33 as the essential hydrophobic residues in CaM anchoring. Furthermore, the CaM binding deficient mutant (Ile20Ala, Trp33Ala) of STARS further enhanced its stimulatory effect on SRF-dependent transcriptional activation. These results suggest a connection between Ca(2+)-signaling via excitation-contraction coupling and the regulation of STARS-mediated gene expression in muscles.

  10. Genome-wide screen of cell-cycle regulators in normal and tumor cells identifies a differential response to nucleosome depletion.

    Science.gov (United States)

    Sokolova, Maria; Turunen, Mikko; Mortusewicz, Oliver; Kivioja, Teemu; Herr, Patrick; Vähärautio, Anna; Björklund, Mikael; Taipale, Minna; Helleday, Thomas; Taipale, Jussi

    2017-01-17

    To identify cell cycle regulators that enable cancer cells to replicate DNA and divide in an unrestricted manner, we performed a parallel genome-wide RNAi screen in normal and cancer cell lines. In addition to many shared regulators, we found that tumor and normal cells are differentially sensitive to loss of the histone genes transcriptional regulator CASP8AP2. In cancer cells, loss of CASP8AP2 leads to a failure to synthesize sufficient amount of histones in the S-phase of the cell cycle, resulting in slowing of individual replication forks. Despite this, DNA replication fails to arrest, and tumor cells progress in an elongated S-phase that lasts several days, finally resulting in death of most of the affected cells. In contrast, depletion of CASP8AP2 in normal cells triggers a response that arrests viable cells in S-phase. The arrest is dependent on p53, and preceded by accumulation of markers of DNA damage, indicating that nucleosome depletion is sensed in normal cells via a DNA-damage -like response that is defective in tumor cells.

  11. Genome-wide screen for salmonella genes required for long-term systemic infection of the mouse.

    Directory of Open Access Journals (Sweden)

    2006-02-01

    Full Text Available A microarray-based negative selection screen was performed to identify Salmonella enterica serovar Typhimurium (serovar Typhimurium genes that contribute to long-term systemic infection in 129X1/SvJ (Nramp1(r mice. A high-complexity transposon-mutagenized library was used to infect mice intraperitoneally, and the selective disappearance of mutants was monitored after 7, 14, 21, and 28 d postinfection. One hundred and eighteen genes were identified to contribute to serovar Typhimurium infection of the spleens of mice by 28 d postinfection. The negatively selected mutants represent many known aspects of Salmonella physiology and pathogenesis, although the majority of the identified genes are of putative or unknown function. Approximately 30% of the negatively selected genes correspond to horizontally acquired regions such as those within Salmonella pathogenicity islands (SPI 1-5, prophages (Gifsy-1 and -2 and remnant, and the pSLT virulence plasmid. In addition, mutations in genes responsible for outer membrane structure and remodeling, such as LPS- and PhoP-regulated and fimbrial genes, were also selected against. Competitive index experiments demonstrated that the secreted SPI2 effectors SseK2 and SseJ as well as the SPI4 locus are attenuated relative to wild-type bacteria during systemic infection. Interestingly, several SPI1-encoded type III secretion system effectors/translocases are required by serovar Typhimurium to establish and, unexpectedly, to persist systemically, challenging the present description of Salmonella pathogenesis. Moreover, we observed a progressive selection against serovar Typhimurium mutants based upon the duration of the infection, suggesting that different classes of genes may be required at distinct stages of infection. Overall, these data indicate that Salmonella long-term systemic infection in the mouse requires a diverse repertoire of virulence factors. This diversity of genes presumably reflects the fact that

  12. A chemical genetic screen uncovers a small molecule enhancer of the N-acylethanolamine degrading enzyme, fatty acid amide hydrolase, in Arabidopsis

    Science.gov (United States)

    Khan, Bibi Rafeiza; Faure, Lionel; Chapman, Kent D.; Blancaflor, Elison B.

    2017-01-01

    N-Acylethanolamines (NAEs) are a group of fatty acid amides that play signaling roles in diverse physiological processes in eukaryotes. Fatty acid amide hydrolase (FAAH) degrades NAE into ethanolamine and free fatty acid to terminate its signaling function. In animals, chemical inhibitors of FAAH have been used for therapeutic treatment of pain and as tools to probe deeper into biochemical properties of FAAH. In a chemical genetic screen for small molecules that dampened the inhibitory effect of N-lauroylethanolamine (NAE 12:0) on Arabidopsis thaliana seedling growth, we identified 6-(2-methoxyphenyl)-1,3-dimethyl-5-phenyl-1H-pyrrolo[3,4-d]pyrimidine-2,4(3 H,6 H)-dione (or MDPD). MDPD alleviated the growth inhibitory effects of NAE 12:0, in part by enhancing the enzymatic activity of Arabidopsis FAAH (AtFAAH). In vitro, biochemical assays showed that MDPD enhanced the apparent Vmax of AtFAAH but did not alter the affinity of AtFAAH for its NAE substrates. Structural analogs of MDPD did not affect AtFAAH activity or dampen the inhibitory effect of NAE 12:0 on seedling growth indicating that MDPD is a specific synthetic chemical activator of AtFAAH. Collectively, our study demonstrates the feasibility of using an unbiased chemical genetic approach to identify new pharmacological tools for manipulating FAAH- and NAE-mediated physiological processes in plants. PMID:28112243

  13. Genomic mechanisms of stress tolerance for the industrial yeast Saccharomyces cerevisiae against major chemical classes of inhibitors

    Science.gov (United States)

    Numerous toxic chemical compounds liberated from lignocellulosic biomass pretreatment inhibit subsequent microbial fermentation that pose a significant challenge to a sustainable and renewable bio-based fermentation industry. Toxin removal procedures by physical or chemical means are essentially imp...

  14. Identification of Important Chemical Features of 11β-Hydroxysteroid Dehydrogenase Type1 Inhibitors: Application of Ligand Based Virtual Screening and Density Functional Theory

    Directory of Open Access Journals (Sweden)

    Keun Woo Lee

    2012-04-01

    Full Text Available 11ß-Hydroxysteroid dehydrogenase type1 (11ßHSD1 regulates the conversion from inactive cortisone to active cortisol. Increased cortisol results in diabetes, hence quelling the activity of 11ßHSD1 has been thought of as an effective approach for the treatment of diabetes. Quantitative hypotheses were developed and validated to identify the critical chemical features with reliable geometric constraints that contribute to the inhibition of 11ßHSD1 function. The best hypothesis, Hypo1, which contains one-HBA; one-Hy-Ali, and two-RA features, was validated using Fischer’s randomization method, a test and a decoy set. The well validated, Hypo1, was used as 3D query to perform a virtual screening of three different chemical databases. Compounds selected by Hypo1 in the virtual screening were filtered by applying Lipinski’s rule of five, ADMET, and molecular docking. Finally, five hit compounds were selected as virtual novel hit molecules for 11ßHSD1 based on their electronic properties calculated by Density functional theory.

  15. Direct probe atmospheric pressure photoionization/atmospheric pressure chemical ionization high-resolution mass spectrometry for fast screening of flame retardants and plasticizers in products and waste.

    Science.gov (United States)

    Ballesteros-Gómez, A; Brandsma, S H; de Boer, J; Leonards, P E G

    2014-04-01

    In this study, we develop fast screening methods for flame retardants and plasticizers in products and waste based on direct probe (DP) atmospheric pressure photoionization (APPI) and atmospheric pressure chemical ionization (APCI) coupled to a high-resolution (HR) time-of-flight mass spectrometer. DP-APPI is reported for the first time in this study, and DP-APCI that has been scarcely exploited is optimized