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Sample records for activity-based functional screening

  1. Activity-Based Metagenomic Screening and Biochemical Characterization of Bovine Ruminal Protozoan Glycoside Hydrolases▿†

    PubMed Central

    Findley, Seth D.; Mormile, Melanie R.; Sommer-Hurley, Andrea; Zhang, Xue-Cheng; Tipton, Peter; Arnett, Krista; Porter, James H.; Kerley, Monty; Stacey, Gary

    2011-01-01

    The rumen, the foregut of herbivorous ruminant animals such as cattle, functions as a bioreactor to process complex plant material. Among the numerous and diverse microbes involved in ruminal digestion are the ruminal protozoans, which are single-celled, ciliated eukaryotic organisms. An activity-based screen was executed to identify genes encoding fibrolytic enzymes present in the metatranscriptome of a bovine ruminal protozoan-enriched cDNA expression library. Of the four novel genes identified, two were characterized in biochemical assays. Our results provide evidence for the effective use of functional metagenomics to retrieve novel enzymes from microbial populations that cannot be maintained in axenic cultures. PMID:21948825

  2. Identification of selective inhibitors of uncharacterized enzymes by high-throughput screening with fluorescent activity-based probes.

    PubMed

    Bachovchin, Daniel A; Brown, Steven J; Rosen, Hugh; Cravatt, Benjamin F

    2009-04-01

    High-throughput screening to discover small-molecule modulators of enzymes typically relies on highly tailored substrate assays, which are not available for poorly characterized enzymes. Here we report a general, substrate-free method for identifying inhibitors of uncharacterized enzymes. The assay measures changes in the kinetics of covalent active-site labeling with broad-spectrum, fluorescent probes in the presence of inhibitors by monitoring the fluorescence polarization signal. We show that this technology is applicable to enzymes from at least two mechanistic classes, regardless of their degree of functional annotation, and can be coupled with secondary proteomic assays that use competitive activity-based profiling to rapidly determine the specificity of screening hits. Using this method, we identify the bioactive alkaloid emetine as a selective inhibitor of the uncharacterized cancer-associated hydrolase RBBP9. Furthermore, we show that the detoxification enzyme GSTO1, also implicated in cancer, is inhibited by several electrophilic compounds found in public libraries, some of which display high selectivity for this protein. PMID:19329999

  3. Enzyme family-specific and activity-based screening of chemical libraries using enzyme microarrays.

    PubMed

    Funeriu, Daniel P; Eppinger, Jörg; Denizot, Lucile; Miyake, Masato; Miyake, Jun

    2005-05-01

    The potential of protein microarrays in high-throughput screening (HTS) still remains largely unfulfilled, essentially because of the difficulty of extracting meaningful, quantitative data from such experiments. In the particular case of enzyme microarrays, low-molecular-weight fluorescent affinity labels (FALs) can function as ideally suited activity probes of the microarrayed enzymes. FALs form covalent bonds with enzymes in an activity-dependent manner and therefore can be used to characterize enzyme activity at each enzyme's address, as predetermined by the microarraying process. Relying on this principle, we introduce herein thematic enzyme microarrays (TEMA). In a kinetic setup we used TEMAs to determine the full set of kinetic constants and the reaction mechanism between the microarrayed enzymes (the theme of the microarray) and a family-wide FAL. Based on this kinetic understanding, in an HTS setup we established the practical and theoretical methodology for quantitative, multiplexed determination of the inhibition profile of compounds from a chemical library against each microarrayed enzyme. Finally, in a validation setup, K(i)(app) values and inhibitor profiles were confirmed and refined. PMID:15821728

  4. An optimized micro-assay of myosin II ATPase activity based on the molybdenum blue method and its application in screening natural product inhibitors.

    PubMed

    Chen, Hong-Lin; Zhao, Jing; Zhang, Guan-Jun; Kou, Jun-Ping; Yu, Bo-Yang

    2016-06-01

    Myosin II plays multiple roles in physiological and pathological functions through its ATPase activity. The present study was designed to optimize a micro-assay of myosin II ATPase activity based on molybdenum blue method, using a known myosin II ATPase inhibitor, blebbistatin. Several parameters were observed in the enzymatic reaction procedure, including the concentrations of the substrate (ATP) and calcium chloride, pH, and the reaction and incubation times. The proportion of coloration agent was also investigated. The sensitivity of this assay was compared with the malachite green method and bioluminescence method. Additionally, 20 natural compounds were studied for myosin II ATPase inhibitory activity using the optimized method. Our results showed that ATP at the concentration of 5 mmol·L(-1) and ammonium molybdate : stannous chloride at the ratio of 15 : 1 could greatly improve the sensitivity of this method. The IC50 of blebbistatin obtained by this method was consistent with literature. Compound 8 was screened with inhibitory activity on myosin II ATPase. The optimized method showed similar accuracy, lower detecting limit, and wider linear range, which could be a promising approach to screening myosin II ATPase inhibitors in vitro. PMID:27473959

  5. CRISPR Screens to Discover Functional Noncoding Elements.

    PubMed

    Wright, Jason B; Sanjana, Neville E

    2016-09-01

    A major challenge in genomics is to identify functional elements in the noncoding genome. Recently, pooled clustered regularly interspersed palindromic repeat (CRISPR) mutagenesis screens of noncoding regions have emerged as a novel method for finding elements that impact gene expression and phenotype/disease-relevant biological processes. Here we review and compare different approaches for high-throughput dissection of noncoding elements. PMID:27423542

  6. Application of Activity-Based Protein Profiling to Study Enzyme Function in Adipocytes

    PubMed Central

    Galmozzi, Andrea; Dominguez, Eduardo; Cravatt, Benjamin F.; Saez, Enrique

    2014-01-01

    Activity-Based Protein Profiling (ABPP) is a chemical proteomics approach that utilizes small-molecule probes to determine the functional state of enzymes directly in native systems. ABPP probes selectively label active enzymes, but not their inactive forms, facilitating the characterization of changes in enzyme activity that occur without alterations in protein levels. ABPP can be a tool superior to conventional gene expression and proteomic profiling methods to discover new enzymes active in adipocytes, and to detect differences in the activity of characterized enzymes that may be associated with disorders of adipose tissue function. ABPP probes have been developed that react selectively with most members of specific enzyme classes. Here, using as an example the serine hydrolase family that includes many enzymes with critical roles in adipocyte physiology, we describe methods to apply ABPP analysis to the study of adipocyte enzymatic pathways. PMID:24529438

  7. Novel inhibitors for PRMT1 discovered by high-throughput screening using activity-based fluorescence polarization.

    PubMed

    Dillon, Myles B C; Bachovchin, Daniel A; Brown, Steven J; Finn, M G; Rosen, Hugh; Cravatt, Benjamin F; Mowen, Kerri A

    2012-07-20

    Protein arginine methyltransferases (PRMTs) catalyze the posttranslational methylation of arginine using S-adenosylmethionine (SAM) as a methyl-donor. The PRMT family is widely expressed and has been implicated in biological functions such as RNA splicing, transcriptional control, signal transduction, and DNA repair. Therefore, specific inhibitors of individual PRMTs have potentially significant research and therapeutic value. In particular, PRMT1 is responsible for >85% of arginine methyltransferase activity, but currently available inhibitors of PRMT1 lack specificity, efficacy, and bioavailability. To address this limitation, we developed a high-throughput screening assay for PRMT1 that utilizes a hyper-reactive cysteine within the active site, which is lacking in almost all other PRMTs. This assay, which monitors the kinetics of the fluorescence polarization signal increase upon PRMT1 labeling by a rhodamine-containing cysteine-reactive probe, successfully identified two novel inhibitors selective for PRMT1 over other SAM-dependent methyltransferases. PMID:22506763

  8. A substrate-free activity-based protein profiling screen for the discovery of selective PREPL inhibitors

    PubMed Central

    Lone, Anna Mari; Bachovchin, Daniel A.; Westwood, David; Speers, Anna E.; Spicer, Timothy P.; Fernandez-Vega, Virneliz; Chase, Peter; Hodder, Peter S.; Rosen, Hugh; Cravatt, Benjamin F.; Saghatelian, Alan

    2011-01-01

    Peptidases play vital roles in physiology through the biosynthesis, degradation, and regulation of peptides. Prolyl endopeptidase-like (PREPL) is a newly described member of the prolyl peptidase family, with significant homology to mammalian prolyl endopeptidase (PEP) and the bacterial peptidase oligopeptidase B (OPDB). The biochemistry and biology of PREPL is of fundamental interest due to this enzyme’s homology to the biomedically important prolyl peptidases and its localization in the central nervous system (CNS). Furthermore, genetic studies of patients suffering from hypotonia-cystinuria syndrome (HCS) have revealed a deletion of a portion of the genome that includes the PREPL gene. HCS symptoms thought to be caused by lack of PREPL include neuromuscular and mild cognitive deficits. A number of complementary approaches, ranging from biochemistry to genetics, will be required to understand the biochemical, cellular, physiological, and pathological mechanisms regulated by PREPL. We are particularly interested in investigating physiological substrates and pathways controlled by PREPL. Here, we use a fluorescence polarization activity-based protein profiling (fluopol-ABPP) assay to discover selective small-molecule inhibitors of PREPL. Fluopol-ABPP is a substrate-free approach that is ideally suited for studying serine hydrolases for which no substrates are known, such as PREPL. After screening over 300,000 compounds using fluopol-ABPP, we employed a number of secondary assays to confirm assay hits and characterize a group of 3-oxo-1-phenyl-2,3,5,6,7,8-hexahydroisoquinoline-4-carbonitrile and 1-alkyl-3-oxo-3,5,6,7-tetrahydro-2H-cyclopenta[c]pyridine-4-carbonitrile PREPL inhibitors that are able to block PREPL activity in cells. Moreover, when administered to mice, 1-isobutyl-3-oxo-3,5,6,7-tetrahydro-2H-cyclopenta[c]pyridine-4-carbonitrile distributes to the brain, indicating that it crosses the blood-brain barrier, and may be useful for in vivo studies. The

  9. A substrate-free activity-based protein profiling screen for the discovery of selective PREPL inhibitors.

    PubMed

    Lone, Anna Mari; Bachovchin, Daniel A; Westwood, David B; Speers, Anna E; Spicer, Timothy P; Fernandez-Vega, Virneliz; Chase, Peter; Hodder, Peter S; Rosen, Hugh; Cravatt, Benjamin F; Saghatelian, Alan

    2011-08-01

    Peptidases play vital roles in physiology through the biosynthesis, degradation, and regulation of peptides. Prolyl endopeptidase-like (PREPL) is a newly described member of the prolyl peptidase family, with significant homology to mammalian prolyl endopeptidase and the bacterial peptidase oligopeptidase B. The biochemistry and biology of PREPL are of fundamental interest due to this enzyme's homology to the biomedically important prolyl peptidases and its localization in the central nervous system. Furthermore, genetic studies of patients suffering from hypotonia-cystinuria syndrome (HCS) have revealed a deletion of a portion of the genome that includes the PREPL gene. HCS symptoms thought to be caused by lack of PREPL include neuromuscular and mild cognitive deficits. A number of complementary approaches, ranging from biochemistry to genetics, will be required to understand the biochemical, cellular, physiological, and pathological mechanisms regulated by PREPL. We are particularly interested in investigating physiological substrates and pathways controlled by PREPL. Here, we use a fluorescence polarization activity-based protein profiling (fluopol-ABPP) assay to discover selective small-molecule inhibitors of PREPL. Fluopol-ABPP is a substrate-free approach that is ideally suited for studying serine hydrolases for which no substrates are known, such as PREPL. After screening over 300,000 compounds using fluopol-ABPP, we employed a number of secondary assays to confirm assay hits and characterize a group of 3-oxo-1-phenyl-2,3,5,6,7,8-hexahydroisoquinoline-4-carbonitrile and 1-alkyl-3-oxo-3,5,6,7-tetrahydro-2H-cyclopenta[c]pyridine-4-carbonitrile PREPL inhibitors that are able to block PREPL activity in cells. Moreover, when administered to mice, 1-isobutyl-3-oxo-3,5,6,7-tetrahydro-2H-cyclopenta[c]pyridine-4-carbonitrile distributes to the brain, indicating that it may be useful for in vivo studies. The application of fluopol-ABPP has led to the first reported

  10. In vivo imaging and biochemical characterization of protease function using fluorescent activity-based probes

    PubMed Central

    Edgington, Laura E.; Bogyo, Matthew

    2013-01-01

    Activity-based probes (ABPs) are reactive small molecules that covalently bind to active enzymes. When tagged with a fluorophore, ABPs serve as powerful tools to investigate enzymatic activity across a wide variety of applications. In this article, we will provide detailed protocols for using fluorescent ABPs to biochemically characterize the activity of proteases in vitro. Furthermore, we will describe how these probes can be applied to image protease activity in live animals and tissues along with subsequent analysis by histology, flow cytometry, and SDS-PAGE. PMID:23788323

  11. Activity Based High-Throughput Screening for Novel O-GlcNAc Transferase Substrates Using a Dynamic Peptide Microarray.

    PubMed

    Shi, Jie; Sharif, Suhela; Ruijtenbeek, Rob; Pieters, Roland J

    2016-01-01

    O-GlcNAcylation is a reversible and dynamic protein post-translational modification in mammalian cells. The O-GlcNAc cycle is catalyzed by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). O-GlcNAcylation plays important role in many vital cellular events including transcription, cell cycle regulation, stress response and protein degradation, and altered O-GlcNAcylation has long been implicated in cancer, diabetes and neurodegenerative diseases. Recently, numerous approaches have been developed to identify OGT substrates and study their function, but there is still a strong demand for highly efficient techniques. Here we demonstrated the utility of the peptide microarray approach to discover novel OGT substrates and study its specificity. Interestingly, the protein RBL-2, which is a key regulator of entry into cell division and may function as a tumor suppressor, was identified as a substrate for three isoforms of OGT. Using peptide Ala scanning, we found Ser 420 is one possible O-GlcNAc site in RBL-2. Moreover, substitution of Ser 420, on its own, inhibited OGT activity, raising the possibility of mechanism-based development for selective OGT inhibitors. This approach will prove useful for both discovery of novel OGT substrates and studying OGT specificity. PMID:26960196

  12. Activity Based High-Throughput Screening for Novel O-GlcNAc Transferase Substrates Using a Dynamic Peptide Microarray

    PubMed Central

    Shi, Jie; Sharif, Suhela; Ruijtenbeek, Rob; Pieters, Roland J.

    2016-01-01

    O-GlcNAcylation is a reversible and dynamic protein post-translational modification in mammalian cells. The O-GlcNAc cycle is catalyzed by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). O-GlcNAcylation plays important role in many vital cellular events including transcription, cell cycle regulation, stress response and protein degradation, and altered O-GlcNAcylation has long been implicated in cancer, diabetes and neurodegenerative diseases. Recently, numerous approaches have been developed to identify OGT substrates and study their function, but there is still a strong demand for highly efficient techniques. Here we demonstrated the utility of the peptide microarray approach to discover novel OGT substrates and study its specificity. Interestingly, the protein RBL-2, which is a key regulator of entry into cell division and may function as a tumor suppressor, was identified as a substrate for three isoforms of OGT. Using peptide Ala scanning, we found Ser 420 is one possible O-GlcNAc site in RBL-2. Moreover, substitution of Ser 420, on its own, inhibited OGT activity, raising the possibility of mechanism-based development for selective OGT inhibitors. This approach will prove useful for both discovery of novel OGT substrates and studying OGT specificity. PMID:26960196

  13. Functional Metagenomics: Construction and High-Throughput Screening of Fosmid Libraries for Discovery of Novel Carbohydrate-Active Enzymes.

    PubMed

    Ufarté, Lisa; Bozonnet, Sophie; Laville, Elisabeth; Cecchini, Davide A; Pizzut-Serin, Sandra; Jacquiod, Samuel; Demanèche, Sandrine; Simonet, Pascal; Franqueville, Laure; Veronese, Gabrielle Potocki

    2016-01-01

    Activity-based metagenomics is one of the most efficient approaches to boost the discovery of novel biocatalysts from the huge reservoir of uncultivated bacteria. In this chapter, we describe a highly generic procedure of metagenomic library construction and high-throughput screening for carbohydrate-active enzymes. Applicable to any bacterial ecosystem, it enables the swift identification of functional enzymes that are highly efficient, alone or acting in synergy, to break down polysaccharides and oligosaccharides. PMID:26791508

  14. Phenotypic lentivirus screens to identify functional single domain antibodies.

    PubMed

    Schmidt, Florian I; Hanke, Leo; Morin, Benjamin; Brewer, Rebeccah; Brusic, Vesna; Whelan, Sean P J; Ploegh, Hidde L

    2016-01-01

    Manipulation of proteins is key in assessing their in vivo function. Although genetic ablation is straightforward, reversible and specific perturbation of protein function remains a challenge. Single domain antibody fragments, such as camelid-derived VHHs, can serve as inhibitors or activators of intracellular protein function, but functional testing of identified VHHs is laborious. To address this challenge, we have developed a lentiviral screening approach to identify VHHs that elicit a phenotype when expressed intracellularly. We identified 19 antiviral VHHs that protect human A549 cells from lethal infection with influenza A virus (IAV) or vesicular stomatitis virus (VSV), respectively. Both negative-sense RNA viruses are vulnerable to VHHs uniquely specific for their respective nucleoproteins. Antiviral VHHs prevented nuclear import of viral ribonucleoproteins or mRNA transcription, respectively, and may provide clues for novel antiviral reagents. In principle, the screening approach described here should be applicable to identify inhibitors of any pathogen or biological pathway. PMID:27573105

  15. 49 CFR 1546.407 - Training, testing, and knowledge of individuals who perform screening functions.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... who perform screening functions. 1546.407 Section 1546.407 Transportation Other Regulations Relating... Carrier Conducts Screening § 1546.407 Training, testing, and knowledge of individuals who perform screening functions. (a) Training required. Before performing screening functions, an individual must...

  16. 49 CFR 1544.407 - Training, testing, and knowledge of individuals who perform screening functions.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... who perform screening functions. 1544.407 Section 1544.407 Transportation Other Regulations Relating... Qualifications When the Aircraft Operator Performs Screening § 1544.407 Training, testing, and knowledge of individuals who perform screening functions. (a) Training required. Before performing screening functions,...

  17. Functional imaging of proteases: recent advances in the design and application of substrate-based and activity-based probes.

    PubMed

    Edgington, Laura E; Verdoes, Martijn; Bogyo, Matthew

    2011-12-01

    Proteases are enzymes that cleave peptide bonds in protein substrates. This process can be important for regulated turnover of a target protein but it can also produce protein fragments that then perform other functions. Because the last few decades of protease research have confirmed that proteolysis is an essential regulatory process in both normal physiology and in multiple disease-associated conditions, there has been an increasing interest in developing methods to image protease activity. Proteases are also considered to be one of the few 'druggable' classes of proteins and therefore a large number of small molecule based inhibitors of proteases have been reported. These compounds serve as a starting point for the design of probes that can be used to target active proteases for imaging applications. Currently, several classes of fluorescent probes have been developed to visualize protease activity in live cells and even whole organisms. The two primary classes of protease probes make use of either peptide/protein substrates or covalent inhibitors that produce a fluorescent signal when bound to an active protease target. This review outlines some of the most recent advances in the design of imaging probes for proteases. In particular, it highlights the strengths and weaknesses of both substrate-based and activity-based probes and their applications for imaging cysteine proteases that are important biomarkers for multiple human diseases. PMID:22098719

  18. Strain screening, fermentation, separation, and encapsulation for production of nattokinase functional food.

    PubMed

    Wei, Xuetuan; Luo, Mingfang; Xie, Yuchun; Yang, Liangrong; Li, Haojian; Xu, Lin; Liu, Huizhou

    2012-12-01

    This study presents a novel and integrated preparation technology for nattokinase functional food, including strain screening, fermentation, separation, and encapsulation. To rapidly screen a nattokinase-productive strain, PCR-based screening method was combined with fibrinolytic activity-based method, and a high productive strain, Bacillus subtilis LSSE-22, was isolated from Chinese soybean paste. Reduction of poly-γ-glutamic acid (γ-PGA) concentration may contribute to separation of nattokinase and reduction of late-onset anaphylaxis risk. Chickpeas were confirmed as the favorable substrate for enhancement of nattokinase production and reduction of γ-PGA yield. Using cracked chickpeas, the nattokinase activity reached 356.25 ± 17.18 FU/g (dry weight), which is much higher than previous reports. To further reduce γ-PGA concentration, ethanol fractional extraction and precipitation were applied for separation of nattokinase. By extraction with 50 % and precipitation with 75 % ethanol solution, 4,000.58 ± 192.98 FU/g of nattokinase powders were obtained, and the activity recovery reached 89 ± 1 %, while γ-PGA recovery was reduced to 21 ± 2 %. To improve the nattokinase stability at acidic pH condition, the nattokinase powders were encapsulated, and then coated with methacrylic acid-ethyl acrylate copolymer. After encapsulation, the nattokinase was protected from being denatured under various acid conditions, and pH-responsible controlled release at simulated intestinal fluid was realized. PMID:22987066

  19. Functional metagenomic screen reveals new and diverse microbial rhodopsins

    PubMed Central

    Pushkarev, Alina; Béjà, Oded

    2016-01-01

    Ion-translocating retinylidene rhodopsins are widely distributed among marine and freshwater microbes. The translocation is light-driven, contributing to the production of biochemical energy in diverse microbes. Until today, most microbial rhodopsins had been detected using bioinformatics based on homology to other rhodopsins. In the past decade, there has been increased interest in microbial rhodopsins in the field of optogenetics since microbial rhodopsins were found to be most useful in vertebrate neuronal systems. Here we report on a functional metagenomic assay for detecting microbial rhodopsins. Using an array of narrow pH electrodes and light-emitting diode illumination, we were able to screen a metagenomic fosmid library to detect diverse marine proteorhodopsins and an actinorhodopsin based solely on proton-pumping activity. Our assay therefore provides a rather simple phenotypic means to enrich our understanding of microbial rhodopsins without any prior knowledge of the genomic content of the environmental entities screened. PMID:26894445

  20. Functional metagenomic screen reveals new and diverse microbial rhodopsins.

    PubMed

    Pushkarev, Alina; Béjà, Oded

    2016-09-01

    Ion-translocating retinylidene rhodopsins are widely distributed among marine and freshwater microbes. The translocation is light-driven, contributing to the production of biochemical energy in diverse microbes. Until today, most microbial rhodopsins had been detected using bioinformatics based on homology to other rhodopsins. In the past decade, there has been increased interest in microbial rhodopsins in the field of optogenetics since microbial rhodopsins were found to be most useful in vertebrate neuronal systems. Here we report on a functional metagenomic assay for detecting microbial rhodopsins. Using an array of narrow pH electrodes and light-emitting diode illumination, we were able to screen a metagenomic fosmid library to detect diverse marine proteorhodopsins and an actinorhodopsin based solely on proton-pumping activity. Our assay therefore provides a rather simple phenotypic means to enrich our understanding of microbial rhodopsins without any prior knowledge of the genomic content of the environmental entities screened. PMID:26894445

  1. An efficient RNA interference screening strategy for gene functional analysis

    PubMed Central

    2012-01-01

    Background RNA interference (RNAi) is commonly applied in genome-scale gene functional screens. However, a one-on-one RNAi analysis that targets each gene is cost-ineffective and laborious. Previous studies have indicated that siRNAs can also affect RNAs that are near-perfectly complementary, and this phenomenon has been termed an off-target effect. This phenomenon implies that it is possible to silence several genes simultaneously with a carefully designed siRNA. Results We propose a strategy that is combined with a heuristic algorithm to design suitable siRNAs that can target multiple genes and a group testing method that would reduce the number of required RNAi experiments in a large-scale RNAi analysis. To verify the efficacy of our strategy, we used the Orchid expressed sequence tag data as a case study to screen the putative transcription factors that are involved in plant disease responses. According to our computation, 94 qualified siRNAs were sufficient to examine all of the predicated 229 transcription factors. In addition, among the 94 computer-designed siRNAs, an siRNA that targets both TF15 (a previously identified transcription factor that is involved in the plant disease-response pathway) and TF21 was introduced into orchids. The experimental results showed that this siRNA can simultaneously silence TF15 and TF21, and application of our strategy successfully confirmed that TF15 is involved in plant defense responses. Interestingly, our second-round analysis, which used an siRNA specific to TF21, indicated that TF21 is a previously unidentified transcription factor that is related to plant defense responses. Conclusions Our computational results showed that it is possible to screen all genes with fewer experiments than would be required for the traditional one-on-one RNAi screening. We also verified that our strategy is capable of identifying genes that are involved in a specific phenotype. PMID:22988976

  2. Functional screening of an asthma QTL in YAC transgenic mice

    SciTech Connect

    Symula, Derek J.; Frazer, Kelly A.; Ueda, Yukihiko; Denefle, Patrice; Stevens, Mary E.; Wang, Zhi-En; Locksley, Richard; Rubin, Edward M.

    1999-07-02

    While large numbers of quantitative trait loci (QTLs) contributing to genetically complex conditions have been discovered, few causative genes have been identified. This is mainly due to the large size of QTLs and the subtle connection between genotype and quantitative phenotype associated with these conditions. While large numbers of quantitative trait loci (QTLs) contributing to genetically complex conditions have been discovered, few causative genes have been identified. This is mainly due to the large size of QTLs and the subtle connection between genotype and quantitative phenotype associated with these conditions. To screen for genes contributing to an asthma QTL mapped to human chromosome 5q33, the authors characterized a panel of large-insert 5q31 transgenics based on studies demonstrating that altering gene dosage frequently affects quantitative phenotypes normally influenced by that gene. This panel of human YAC transgenics, propagating a one megabase interva2048 chromosome 5q31 containing 23 genes, was screened for quantitative changes in several asthma-associated phenotypes. Multiple independent transgenic lines with altered IgE response to antigen treatment shared a 180 kb region containing 5 genes, including human interleukin 4 (IL4) and interleukin 13 (IL13), which induce IgE class switching in B cells5. Further analysis of these mice and mice transgenic for only murine Il4 and Il13 demonstrated that moderate changes in murine Il4 and Il13 expression affect asthma-associated phenotypes in vivo. This functional screen of large-insert transgenics enabled them to sift through multiple genes in the 5q3 asthma QTL without prior consideration of assumed individual gene function and identify genes that influence the QTL phenotype in vivo.

  3. Array-based functional screening of heparin glycans.

    PubMed

    Puvirajesinghe, Tania M; Ahmed, Yassir A; Powell, Andrew K; Fernig, David G; Guimond, Scott E; Turnbull, Jeremy E

    2012-05-25

    Array methodologies have become powerful tools for interrogation of glycan-protein interactions but have critically lacked the ability to generate cell response data. Here, we report the development of a slide-based array method exemplified by measurement of activation of fibroblast growth factor signaling by heparin saccharides. Heparan sulfate-deficient Swiss 3T3 cells were overlaid onto an aminosilane-coated slide surface onto which heparin saccharides had been spotted and immobilized. The cells were transiently stimulated with FGF2 and immunofluorescence measured to assess downstream ERK1/2 phosphorylation. Activation of this signaling pathway response was restricted to cells exposed to heparin saccharides competent to activate FGF2 signaling. Differential activation of the overlaid cells by different-sized heparin saccharides was demonstrated by quantitative measurement of fluorescence intensity. This "glycobioarray" platform has significant potential as a generic tool for functional glycomics screening. PMID:22633407

  4. A Functional Genomic Yeast Screen to Identify Pathogenic Bacterial Proteins

    PubMed Central

    Slagowski, Naomi L; Kramer, Roger W; Morrison, Monica F; LaBaer, Joshua; Lesser, Cammie F

    2008-01-01

    Many bacterial pathogens promote infection and cause disease by directly injecting into host cells proteins that manipulate eukaryotic cellular processes. Identification of these translocated proteins is essential to understanding pathogenesis. Yet, their identification remains limited. This, in part, is due to their general sequence uniqueness, which confounds homology-based identification by comparative genomic methods. In addition, their absence often does not result in phenotypes in virulence assays limiting functional genetic screens. Translocated proteins have been observed to confer toxic phenotypes when expressed in the yeast Saccharomyces cerevisiae. This observation suggests that yeast growth inhibition can be used as an indicator of protein translocation in functional genomic screens. However, limited information is available regarding the behavior of non-translocated proteins in yeast. We developed a semi-automated quantitative assay to monitor the growth of hundreds of yeast strains in parallel. We observed that expression of half of the 19 Shigella translocated proteins tested but almost none of the 20 non-translocated Shigella proteins nor ∼1,000 Francisella tularensis proteins significantly inhibited yeast growth. Not only does this study establish that yeast growth inhibition is a sensitive and specific indicator of translocated proteins, but we also identified a new substrate of the Shigella type III secretion system (TTSS), IpaJ, previously missed by other experimental approaches. In those cases where the mechanisms of action of the translocated proteins are known, significant yeast growth inhibition correlated with the targeting of conserved cellular processes. By providing positive rather than negative indication of activity our assay complements existing approaches for identification of translocated proteins. In addition, because this assay only requires genomic DNA it is particularly valuable for studying pathogens that are difficult to

  5. Interrater and intrarater reliability of the functional movement screen.

    PubMed

    Smith, Craig A; Chimera, Nicole J; Wright, Nicholas J; Warren, Meghan

    2013-04-01

    The purpose of this study was to investigate interrater and intrarater reliability of the Functional Movement Screen (FMS) with real-time administration with raters of different educational background and experience. The FMS was assessed with real-time administration in healthy injury-free men and women and included a certified FMS rater for comparison with other raters. A relatively new tool, the FMS, was developed to screen 7 individual movement patterns to classify subjects' injury risk. Previous reliability studies have been published with only one investigating intrarater reliability. These studies had limitations in study design and clinical applicability such as the use of only video to rate or the use of raters without comparison to a certified FMS rater. Raters (n = 4) with varying degrees of FMS experience and educational levels underwent a 2-hour FMS training session. Subjects (n = 19) were rated during 2 sessions, 1 week apart, using standard FMS protocol and equipment. Interrater reliability was good for session 1 (intraclass correlation coefficient [ICC] = 0.89) and for session 2 (ICC = 0.87). The individual FMS movements showed hurdle step as the least reliable (ICC = 0.30 for session 1 and 0.35 for session 2), whereas the most reliable was shoulder mobility (ICC = 0.98 for session 1 and 0.96 for session 2). Intrarater reliability was good for all raters (ICC = 0.81-0.91), with similar ICC regardless of education or previous experience with FMS. The results showed that the FMS could be consistently scored by people with varying degrees of experience with the FMS after a 2-hour training session. Intrarater reliability was not increased with FMS certification. PMID:22692121

  6. Efficacy of the functional movement screen: a review.

    PubMed

    Kraus, Kornelius; Schütz, Elisabeth; Taylor, William R; Doyscher, Ralf

    2014-12-01

    The aim of this review was to evaluate and synthesize the scientific literature of the functional movement screen (FMS)-driven research for scientists and strength and conditioning specialists. An additional purpose was to optimize the methodological quality of prospective studies. Relevant research was identified through using a manual and electronically database search. Thirty-four articles met the inclusion criteria and were read, abstracted, and coded for this review. The publications were classified into different stages of Bishops Applied Research Model for the Sport Sciences (ARMSS). Thirteen descriptive studies explored the main tasks in test development like factor structure, objectivity, and reliability. They can be classified to the second stage of Bishops Model (ARMSS stage 2). Twelve studies covered ability of FMS to predict sporting performance and injury risk (ARMSS stages 3 and 4). Seven studies investigated the effectiveness of the FMS in designing programs (ARMSS stages 6 and 8). In addition, 2 assessed norming data. On the descriptive level, results suggest that the FMS is a reliable screen, if the rater is educated and has solid experience (>100 trials). Factor analysis describes the FMS as a unitary construct, which is an argument against the FMS total score. Studies clearly illustrate its limited ability to predict athletic performance. On the contrary, to predict injury risk in team sports, the FMS total score is supported by moderate scientific evidence. The majority of the FMS based intervention programs showed an improvement on general motor quality. However, a randomized trial does not confirm that results. Hence, to implement the findings on field, a critical strength and conditioning specialist is crucial. PMID:24918299

  7. Functional Genome Screening to Elucidate the Colistin Resistance Mechanism

    PubMed Central

    Kumar, Mohit; Gupta, Ashutosh; Sahoo, Rajesh Kumar; Jena, Jayanti; Debata, Nagen Kumar; Subudhi, Enketeswara

    2016-01-01

    Antibiogram profile of 1590 clinical bacterial isolates based on thirteen different antimicrobial compounds showed that 1.6% of the bacterial isolates are multidrug resistant. Distribution pattern based on 16S rRNA sequence analysis showed that Pseudomonas aeruginosa constituted the largest group (83.6%) followed by Burkholderia pseudomallei sp. A191 (5.17%), Staphylococcus sp. A261 (3.45%). Among the various antibiotics used, colistin appeared to be the most effective against the Gram negative bacteria. Burkholderia pseudomallei sp. A191 and Pseudomonas aeruginosa sp. A111 showed resistance to 1500 μg/ml and 750 μg/ml of colistin respectively which constitutes 7.7% of the bacterial population. A functional genomics strategy was employed to discover the molecular support for colistin resistance in Burkholderia pseudomallei sp. A191. A pUC plasmid-based genomic expression library was constructed with an estimated library size of 2.1 × 107bp. Five colistin resistant clones were obtained after functional screening of the library. Analysis of DNA sequence of five colistin resistant clones showed homology to two component regularity systems (TCRS) encoding for a histidine kinase (mrgS) and its regulatory component (mrgR). Cross complementation assay showed that mutations in mrgS were sufficient enough to confer colistin resistant phenotype in a sensitive strain. PMID:26988670

  8. 49 CFR 1544.407 - Training, testing, and knowledge of individuals who perform screening functions.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... CIVIL AVIATION SECURITY AIRCRAFT OPERATOR SECURITY: AIR CARRIERS AND COMMERCIAL OPERATORS Screener... required training. This paragraph does not prohibit the performance of screening functions during...

  9. 49 CFR 1544.407 - Training, testing, and knowledge of individuals who perform screening functions.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... CIVIL AVIATION SECURITY AIRCRAFT OPERATOR SECURITY: AIR CARRIERS AND COMMERCIAL OPERATORS Screener... required training. This paragraph does not prohibit the performance of screening functions during...

  10. 49 CFR 1544.407 - Training, testing, and knowledge of individuals who perform screening functions.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... CIVIL AVIATION SECURITY AIRCRAFT OPERATOR SECURITY: AIR CARRIERS AND COMMERCIAL OPERATORS Screener... required training. This paragraph does not prohibit the performance of screening functions during...

  11. Pooled shRNA Screening in Mammalian Cells as a Functional Genomic Discovery Platform.

    PubMed

    Jastrzebski, Katarzyna; Evers, Bastiaan; Beijersbergen, Roderick L

    2016-01-01

    Functional genomic screens using shRNA technology are a great tool in biomedical research. As more labs gain access to the necessary reagents and technology to perform such screens, some may lack in-depth knowledge on the difficulties often encountered. With this protocol, we aim to point out the most important caveats of performing shRNA based screens and provide a streamlined workflow that can be easily adapted to meet the specific needs of any particular screening project. PMID:27581284

  12. Multiplexed Activity-based Protein Profiling of the Human Pathogen Aspergillus fumigatus Reveals Large Functional Changes upon Exposure to Human Serum

    SciTech Connect

    Wiedner, Susan D.; Burnum, Kristin E.; Pederson, Leeanna M.; Anderson, Lindsey N.; Fortuin, Suereta; Chauvigne-Hines, Lacie M.; Shukla, Anil K.; Ansong, Charles; Panisko, Ellen A.; Smith, Richard D.; Wright, Aaron T.

    2012-08-03

    Environmental and metabolic adaptability is critical for survival of the fungal human pathogen Aspergillus fumigatus in the immunocompromised lung. We employed an activity-based protein profiling (ABPP) approach utilizing a new aryl vinyl sulfonate probe and a serine hydrolase probe combined with quantitative LC-MS based accurate mass and time (AMT) tag proteomics for the identification of functional pathway adaptation of A. fumigatus to environmental variability relevant to pulmonary Invasive Aspergillosis. When the fungal pathogen was grown with human serum, metabolism and energy processes were markedly decreased compared to no serum culture. Additionally, functional pathways associated with amino acid and protein biosynthesis were limited as the fungus scavenged from the serum to obtain essential nutrients. Our approach revealed significant metabolic adaptation by A. fumigatus, and provides direct insight into this pathogen’s ability to survive and proliferate.

  13. Unsupervised spatio-temporal detection of brain functional activation based on hidden Markov multiple event sequence models

    NASA Astrophysics Data System (ADS)

    Faisan, Sylvain; Thoraval, Laurent; Armspach, Jean-Paul; Heitz, Fabrice; Foucher, Jack

    2005-04-01

    This paper presents a novel, completely unsupervised fMRI brain mapping approach that addresses the three problems of hemodynamic response function (HRF) shape variability, neural event timing, and fMRI response linearity. To make it robust, the method takes into account spatial and temporal information directly into the core of the activation detection process. In practice, activation detection is formulated in terms of temporal alignment between the sequence of hemodynamic response onsets (HROs) detected in the fMRI signal at υ and in the spatial neighbourhood of υ, and the sequence of "off-on" transitions observed in the input blocked stimulation paradigm (when considering epoch-related fMRI data), or the sequence of stimuli of the event-based paradigm (when considering event-related fMRI data). This multiple event sequence alignment problem, which comes under multisensor data fusion, is solved within the probabilistic framework of hidden Markov multiple event sequence models (HMMESMs), a special class of hidden Markov models. Results obtained on real and synthetic data compete with those obtained with the popular statistical parametric mapping (SPM) approach, but without necessitating any prior definition of the expected activation patterns, the HMMESM mapping approach being completely unsupervised.

  14. Virus-Host Interactions: From Unbiased Genetic Screens to Function.

    PubMed

    Ramage, Holly; Cherry, Sara

    2015-11-01

    Deciphering the many interactions that occur between a virus and host cell over the course of infection is paramount to understanding mechanisms of pathogenesis and to the future development of antiviral therapies. Over the past decade, researchers have started to understand these complicated relationships through the development of methodologies, including advances in RNA interference, proteomics, and the development of genetic tools such as haploid cell lines, allowing high-throughput screening to identify critical contact points between virus and host. These advances have produced a wealth of data regarding host factors hijacked by viruses to promote infection, as well as antiviral factors responsible for subverting viral infection. This review highlights findings from virus-host screens and discusses our thoughts on the direction of screening strategies moving forward. PMID:26958926

  15. Assessing the Effectiveness of Functional Genetic Screens for the Identification of Bioactive Metabolites

    PubMed Central

    Penesyan, Anahit; Ballestriero, Francesco; Daim, Malak; Kjelleberg, Staffan; Thomas, Torsten; Egan, Suhelen

    2012-01-01

    A common limitation for the identification of novel activities from functional (meta) genomic screens is the low number of active clones detected relative to the number of clones screened. Here we demonstrate that constructing libraries with strains known to produce bioactives can greatly enhance the screening efficiency, by increasing the “hit-rate” and unmasking multiple activities from the same bacterial source. PMID:23271424

  16. Application of Microarray and Functional-Based Screening Methods for the Detection of Antimicrobial Resistance Genes in the Microbiomes of Healthy Humans

    PubMed Central

    Card, Roderick M.; Warburton, Philip J.; MacLaren, Nikki; Mullany, Peter; Allan, Elaine; Anjum, Muna F.

    2014-01-01

    The aim of this study was to screen for the presence of antimicrobial resistance genes within the saliva and faecal microbiomes of healthy adult human volunteers from five European countries. Two non-culture based approaches were employed to obviate potential bias associated with difficult to culture members of the microbiota. In a gene target-based approach, a microarray was employed to screen for the presence of over 70 clinically important resistance genes in the saliva and faecal microbiomes. A total of 14 different resistance genes were detected encoding resistances to six antibiotic classes (aminoglycosides, β-lactams, macrolides, sulphonamides, tetracyclines and trimethoprim). The most commonly detected genes were erm(B), blaTEM, and sul2. In a functional-based approach, DNA prepared from pooled saliva samples was cloned into Escherichia coli and screened for expression of resistance to ampicillin or sulphonamide, two of the most common resistances found by array. The functional ampicillin resistance screen recovered genes encoding components of a predicted AcrRAB efflux pump. In the functional sulphonamide resistance screen, folP genes were recovered encoding mutant dihydropteroate synthase, the target of sulphonamide action. The genes recovered from the functional screens were from the chromosomes of commensal species that are opportunistically pathogenic and capable of exchanging DNA with related pathogenic species. Genes identified by microarray were not recovered in the activity-based screen, indicating that these two methods can be complementary in facilitating the identification of a range of resistance mechanisms present within the human microbiome. It also provides further evidence of the diverse reservoir of resistance mechanisms present in bacterial populations in the human gut and saliva. In future the methods described in this study can be used to monitor changes in the resistome in response to antibiotic therapy. PMID:24466089

  17. Multiplexed Activity-based Protein Profiling of the Human Pathogen Aspergillus fumigatus Reveals Large Functional Changes upon Exposure to Human Serum*

    PubMed Central

    Wiedner, Susan D.; Burnum, Kristin E.; Pederson, LeeAnna M.; Anderson, Lindsey N.; Fortuin, Suereta; Chauvigné-Hines, Lacie M.; Shukla, Anil K.; Ansong, Charles; Panisko, Ellen A.; Smith, Richard D.; Wright, Aaron T.

    2012-01-01

    Environmental adaptability is critical for survival of the fungal human pathogen Aspergillus fumigatus in the immunocompromised host lung. We hypothesized that exposure of the fungal pathogen to human serum would lead to significant alterations to the organism's physiology, including metabolic activity and stress response. Shifts in functional pathway and corresponding enzyme reactivity of A. fumigatus upon exposure to the human host may represent much needed prognostic indicators of fungal infection. To address this, we employed a multiplexed activity-based protein profiling (ABPP) approach coupled to quantitative mass spectrometry-based proteomics to measure broad enzyme reactivity of the fungus cultured with and without human serum. ABPP showed a shift from aerobic respiration to ethanol fermentation and utilization over time in the presence of human serum, which was not observed in serum-free culture. Our approach provides direct insight into this pathogen's ability to survive, adapt, and proliferate. Additionally, our multiplexed ABPP approach captured a broad swath of enzyme reactivity and functional pathways and provides a method for rapid assessment of the A. fumigatus response to external stimuli. PMID:22865858

  18. Antibacterial enzymes from the functional screening of metagenomic libraries hosted in Ralstonia metallidurans

    PubMed Central

    Iqbal, Hala A.; Craig, Jeffrey W.; Brady, Sean F.

    2014-01-01

    Phenotype-based screening of bacterial metagenomic libraries provides an avenue for the discovery of novel genes, enzymes and metabolites that have a variety of potential clinical and industrial uses. Here we report the identification of a functionally diverse collection of antibacterially active enzymes from the phenotypic screening of 700,000 cosmid clones prepared from Arizona soil DNA and hosted in Ralstonia metallidurans. Environmental DNA clones surrounded by zones of growth inhibition in a bacterial overlay assay were found, through bioinformatics and functional analyses, to encode enzymes with predicted peptidase, lipase and glycolytic activities conferring antibiosis. The antibacterial activities observed in our R. metallidurans-based assay could not be replicated with the same clones in screens using Escherichia coli as a heterologous host, suggesting that the large-scale screening of metagenomic libraries for antibiosis using phylogenetically diverse hosts should be a productive strategy for identifying enzymes with functionally diverse antibacterial activities. PMID:24661178

  19. Functional-based screening methods for lipases, esterases, and phospholipases in metagenomic libraries.

    PubMed

    Reyes-Duarte, Dolores; Ferrer, Manuel; García-Arellano, Humberto

    2012-01-01

    The use of metagenomic techniques for enzyme discovery constitutes a powerful approach. Functional screens, in contrast to sequence homology search, enable us to select enzymes based on their activity. It is noteworthy that they additionally guarantee the identification of genes coding for enzymes that exhibited no sequence similarity to known counterparts from public databases and that even do not match any putative catalytic residues, involved in the selected catalytic function. Therefore, this strategy not only provides new enzymes for new biotechnological applications, but also allows functional assignment of many proteins, found in abundance in the databases, currently designated as "hypothetical" or "conserved hypothetical" proteins. In the past decade, there has been an exponential increase in the design of functional screening programmes, the majority of them established for hydrolases and oxidoreductases. Here, functional screening methods that guarantee the greatest enzyme diversity, for mining esterases and lipases, are described. PMID:22426714

  20. The screening effects of the screened exchange hybrid functional in surface systems: A case study on the CO/Pt(111) problem

    NASA Astrophysics Data System (ADS)

    Li, H.; Gillen, R.; Robertson, J.

    2016-06-01

    The screened exchange (sX) hybrid functional has been widely used in computational material science. Although it has widely been studied in bulk systems, less is known about its functional behavior in surface systems which are crucial to many technologies such as materials synthesis and nano-electronic devices. Assessing the screening dependent functional behaviors in the surface systems is therefore important for its application in such systems. In this work, we investigate the screening effects of the sX in CO adsorption on Pt(111) surface. The differences between the sX and Heyd-Scuseria-Ernzerhof (HSE06) hybrid functionals, and the effects of screening parameters are studied. The screening has two effects: first, the HOMO-LUMO gap is screening dependent. This affects the site preference most significantly. In this work, atop adsorption of CO/Pt(111) is predicted by the hybrid functionals with screened exchange potential. The sX(1.44) gives the largest HOMO-LUMO gap for the isolated CO molecule. The adsorption energy difference between the atop and fcc site is also the largest by the sX(1.44) which is explained by the reduced metal d states to the CO 2π* state back-donation, with stronger effect for the fcc adsorption than for the atop adsorption; second, the adsorption energy is screening dependent. This can be seen by comparing the sX(2.38) and HSE06 which have different screening strengths. They show similar surface band structures for the CO adsorption but different adsorption energies, which is explained by the stronger CO 5σ state to the metal d states donation or the effectively screened Pauli repulsion. This work underlines the screening strength as a main difference between sX and HSE06, as well as an important hybrid functional parameter for surface calculation.

  1. Screening and Functional Analyses of Nilaparvata lugens Salivary Proteome.

    PubMed

    Huang, Hai-Jian; Liu, Cheng-Wen; Huang, Xiao-Hui; Zhou, Xiang; Zhuo, Ji-Chong; Zhang, Chuan-Xi; Bao, Yan-Yuan

    2016-06-01

    Most phloem-feeding insects secrete gelling and watery saliva during the feeding process. However, the functions of salivary proteins are poorly understood. In this study, our purpose was to reveal the components and functions of saliva in a rice sap-sucking insect pest, Nilaparvata lugens. The accomplishment of the whole genome and transcriptome sequencing in N. lugens would be helpful for elucidating the gene information and expression specificity of the salivary proteins. In this study, we have, for the first time, identified the abundant protein components from gelling and watery saliva in a monophagous sap-sucking insect species through shotgun proteomic detection combined with the genomic and transcriptomic analysis. Eight unknown secreted proteins were limited to N. lugens, indicating species-specific saliva components. A group of annexin-like proteins first identified in the secreted saliva displayed different domain structure and expression specificity with typical insect annexins. Nineteen genes encoding five annexin-like proteins, six salivaps (salivary glands-specific proteins with unknown function), seven putative enzymes, and a mucin-like protein showed salivary gland-specific expression pattern, suggesting their importance in the physiological mechanisms of salivary gland and saliva in this insect species. RNA interference revealed that salivap-3 is a key protein factor in forming the salivary sheath, while annexin-like5 and carbonic anhydrase are indispensable for N. lugens survival. These novel findings will greatly help to clarify the detailed functions of salivary proteins in the physiological process of N. lugens and elucidate the interaction mechanisms between N. lugens and the rice plant, which could provide important targets for the future management of rice pests. PMID:27142481

  2. Pose prediction and virtual screening performance of GOLD scoring functions in a standardized test.

    PubMed

    Liebeschuetz, John W; Cole, Jason C; Korb, Oliver

    2012-06-01

    The performance of all four GOLD scoring functions has been evaluated for pose prediction and virtual screening under the standardized conditions of the comparative docking and scoring experiment reported in this Edition. Excellent pose prediction and good virtual screening performance was demonstrated using unmodified protein models and default parameter settings. The best performing scoring function for both pose prediction and virtual screening was demonstrated to be the recently introduced scoring function ChemPLP. We conclude that existing docking programs already perform close to optimally in the cognate pose prediction experiments currently carried out and that more stringent pose prediction tests should be used in the future. These should employ cross-docking sets. Evaluation of virtual screening performance remains problematic and much remains to be done to improve the usefulness of publically available active and decoy sets for virtual screening. Finally we suggest that, for certain target/scoring function combinations, good enrichment may sometimes be a consequence of 2D property recognition rather than a modelling of the correct 3D interactions. PMID:22371207

  3. [High-throughput functional screening using CRISPR/Cas9 system].

    PubMed

    Wang, Gancheng; Ming, Ma; Ye, Yanzhen; Xi, Jianzhong

    2016-05-01

    High-throughput screening, a powerful tool for the discovery of functionally important genes responsible for certain phenotypes, is performed according to loss-of-function or gain-of-function strategies. RNAi technology or knockout approaches have been widely used in high throughput screening due to their advantages of ease use, low cost and so on. However, imcomplete knockdown activity and off-target effect hindered their utility. More recently, CRISPR/Cas9 technology is becoming a robust tool for genome editing in diverse cells or animals, since it could generate a gene mutation in a target-specific manner. In this review, we first summarize the characterization of CRISPR/Cas9 and make comparison with traditional genetic tools, then describe recent achievements of genetic screen in several model organisms using CRISPR/Cas9, finally discuss on its future challenges and opportunities. PMID:27232487

  4. The cognitive effects of trauma: reversal of alpha function and the formation of a beta screen.

    PubMed

    Brown, Lawrence J

    2005-04-01

    Following a brief review of Freud's writings on trauma, the author discusses relevant theories of Bion, and in particular the concepts of the alpha function and the beta screen. A clinical example is presented in which the patient's relatively recent trauma in adulthood had become fused with prior related experiences, leading to a propensity for repeated enactments in analysis and a failure to learn from experience. Drawing on the analyst's alpha function, the patient was gradually able to use mentalization to transform her rigidly structured traumatic organization. The author highlights the roles of dreams/dream associations and of screen memories in the patient's analysis. PMID:15889686

  5. Functional screen identifies regulators of murine hematopoietic stem cell repopulation.

    PubMed

    Holmfeldt, Per; Ganuza, Miguel; Marathe, Himangi; He, Bing; Hall, Trent; Kang, Guolian; Moen, Joseph; Pardieck, Jennifer; Saulsberry, Angelica C; Cico, Alba; Gaut, Ludovic; McGoldrick, Daniel; Finkelstein, David; Tan, Kai; McKinney-Freeman, Shannon

    2016-03-01

    Understanding the molecular regulation of hematopoietic stem and progenitor cell (HSPC) engraftment is paramount to improving transplant outcomes. To discover novel regulators of HSPC repopulation, we transplanted >1,300 mice with shRNA-transduced HSPCs within 24 h of isolation and transduction to focus on detecting genes regulating repopulation. We identified 17 regulators of HSPC repopulation: Arhgef5, Armcx1, Cadps2, Crispld1, Emcn, Foxa3, Fstl1, Glis2, Gprasp2, Gpr56, Myct1, Nbea, P2ry14, Smarca2, Sox4, Stat4, and Zfp251. Knockdown of each of these genes yielded a loss of function, except in the cases of Armcx1 and Gprasp2, whose loss enhanced hematopoietic stem cell (HSC) repopulation. The discovery of multiple genes regulating vesicular trafficking, cell surface receptor turnover, and secretion of extracellular matrix components suggests active cross talk between HSCs and the niche and that HSCs may actively condition the niche to promote engraftment. We validated that Foxa3 is required for HSC repopulating activity, as Foxa3(-/-) HSC fails to repopulate ablated hosts efficiently, implicating for the first time Foxa genes as regulators of HSPCs. We further show that Foxa3 likely regulates the HSC response to hematologic stress. Each gene discovered here offers a window into the novel processes that regulate stable HSPC engraftment into an ablated host. PMID:26880577

  6. Effects of screen size on smartphone functionality and usability for stroke patients with hemiparalysis

    PubMed Central

    Jung, Nam-hae; Chang, Moonyoung

    2016-01-01

    [Purpose] The effect of screen size on smartphone functionality and usability for patients with stroke, considering both the non-dominant and dominant hand smartphone usage, was investigated in this study. [Subjects and Methods] Thirteen patients with stroke participated in this study—five pre-non-dominant hand users and eight pre-dominant hand users. The smartphone screen sizes used were 4.2, 4.5, and 5.6 inches. Usability was assessed in terms of discomfort experienced during dragging operations, which was self-reported using a four-point Likert scale. Functionality was assessed in terms of completion time and the frequency of errors in the task requiring users to quickly touch numbers 0 through 9 in order on the keypad. [Results] For all three screen sizes, a significant difference between the dominant and non-dominant hands was found in usability, completion time, and frequency of errors. For dominant hand users, differences in usability and completion time were found among the three screen sizes. Among the three screen sizes, no difference in the frequency of errors was found in either of the groups. [Conclusion] This study will be useful as basic research on usability and functionality with stroke patients using only pre-non-dominant or pre-dominant hand. PMID:27190477

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

    PubMed Central

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

    2015-01-01

    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. PMID:25944046

  8. An analysis of highly viscous flow using Imai's complex function method with application to screen printing

    NASA Astrophysics Data System (ADS)

    Kawabata, Jun-ichi; Shi-igai, Hiroyoshi; Yabuno, Kohei; Saito, Tadayuki

    1992-06-01

    The complex function method is applied to screen printing flow with low Reynolds number assuming that the printing ink is Newtonian. The screen printing flow is expressed through a Taylor flow in the corner. The pressure distribution, vorticity distribution and stream lines of the Taylor flow can easily be obtained by using the complex function method. A new model of the flow which may better express the screen printing is obtained through this method by placing a sink at the origin of the Taylor flow. The appropriate corner angle for printing, theoretically derived from this method as 65.4°, corresponds to the empirical angle in industrial use. The pressure distribution of this model shows good agreement with the experimental results obtained in the present work.

  9. Sudomotor function assessment as a screening tool for microvascular complications in type 2 diabetes.

    PubMed

    Eranki, V G; Santosh, R; Rajitha, K; Pillai, A; Sowmya, P; Dupin, J; Calvet, J H

    2013-09-01

    Sudoscan, a non invasive, quick, and simple method to measure sweat function, was evaluated as a screening tool for microvascular complications in type 2 diabetes. AUC of the ROC curve for detection of microvascular complication was 0.75 for an autonomic risk score, with a sensitivity of 82% and a specificity of 61%. PMID:23880037

  10. Functional genomics platform for pooled screening and mammalian genetic interaction maps

    PubMed Central

    Kampmann, Martin; Bassik, Michael C.; Weissman, Jonathan S.

    2014-01-01

    Systematic genetic interaction maps in microorganisms are powerful tools for identifying functional relationships between genes and defining the function of uncharacterized genes. We have recently implemented this strategy in mammalian cells as a two-stage approach. First, genes of interest are robustly identified in a pooled genome-wide screen using complex shRNA libraries. Second, phenotypes for all pairwise combinations of hit genes are measured in a double-shRNA screen and used to construct a genetic interaction map. Our protocol allows for rapid pooled screening under various conditions without a requirement for robotics, in contrast to arrayed approaches. Each stage of the protocol can be implemented in ~2 weeks, with additional time for analysis and generation of reagents. We discuss considerations for screen design, and present complete experimental procedures as well as a full computational analysis suite for identification of hits in pooled screens and generation of genetic interaction maps. While the protocols outlined here were developed for our original shRNA-based approach, they can be applied more generally, including to CRISPR-based approaches. PMID:24992097

  11. Development of a High-Throughput Functional Screen Using Nanowell-Assisted Cell Patterning.

    PubMed

    Ozkumur, Ayca Yalcin; Goods, Brittany A; Love, J Christopher

    2015-09-01

    Living-cell-based screens can facilitate lead discovery of functional therapeutics of interest. A versatile and scalable method is reported that uses dense arrays of nanowells for imparting defined patterns on monolayers of cells. It is shown that this approach can coordinate a multi-component biological assay by designing and implementing a high-throughput, functional nanoliter-scale neutralization assay to identify neutralizing antibodies against HIV. PMID:26121321

  12. Yeast functional screen to identify genetic determinants capable of conferring abiotic stress tolerance in Jatropha curcas

    PubMed Central

    2010-01-01

    Background Environmentally inflicted stresses such as salinity and drought limit the plant productivity both in natural and agricultural system. Increasing emphasis has been directed to molecular breeding strategies to enhance the intrinsic ability of plant to survive stress conditions. Functional screens in microorganisms with heterologous genes are a rapid, effective and powerful tool to identify stress tolerant genes in plants. Jatropha curcas (Physic nut) has been identified as a potential source of biodiesel plant. In order to improve its productivity under stress conditions to benefit commercial plantations, we initiated prospecting of novel genes expressed during stress in J. curcas that can be utilized to enhance stress tolerance ability of plant. Results To identify genes expressed during salt tolerance, cDNA expression libraries were constructed from salt-stressed roots of J. curcas, regulated under the control of the yeast GAL1 system. Using a replica based screening, twenty thousand yeast transformants were screened to identify transformants expressing heterologous gene sequences from J. curcas with enhanced ability to tolerate stress. From the screen we obtained 32 full length genes from J. curcas [GenBank accession numbers FJ489601-FJ489611, FJ619041-FJ619057 and FJ623457-FJ623460] that can confer abiotic stress tolerance. As a part of this screen, we optimized conditions for salt stress in J. curcas, defined parameters for salt stress in yeast, as well as isolated three salt hypersensitive yeast strains shs-2, shs-6 and shs-8 generated through a process of random mutagenesis, and exhibited growth retardation beyond 750 mM NaCl. Further, we demonstrated complementation of the salt sensitive phenotypes in the shs mutants, and analyzed the expression patterns for selected J. curcas genes obtained from the screen in both leaf and root tissues after salt stress treatments. Conclusions The approach described in this report provides a rapid and universal

  13. A genetic screen of the Drosophila X chromosome for mutations that modify Deformed function.

    PubMed Central

    Florence, B; McGinnis, W

    1998-01-01

    We have screened the Drosophila X chromosome for genes whose dosage affects the function of the homeotic gene Deformed. One of these genes, extradenticle, encodes a homeodomain transcription factor that heterodimerizes with Deformed and other homeotic Hox proteins. Mutations in the nejire gene, which encodes a transcriptional adaptor protein belonging to the CBP/p300 family, also interact with Deformed. The other previously characterized gene identified as a Deformed interactor is Notch, which encodes a transmembrane receptor. These three genes underscore the importance of transcriptional regulation and cell-cell signaling in Hox function. Four novel genes were also identified in the screen. One of these, rancor, is required for appropriate embryonic expression of Deformed and another homeotic gene, labial. Both Notch and nejire affect the function of another Hox gene, Ultrabithorax, indicating they may be required for homeotic activity in general. PMID:9832527

  14. Targeting multifunctional proteins by virtual screening: structurally diverse cytohesin inhibitors with differentiated biological functions.

    PubMed

    Stumpfe, Dagmar; Bill, Anke; Novak, Nina; Loch, Gerrit; Blockus, Heike; Geppert, Hanna; Becker, Thomas; Schmitz, Anton; Hoch, Michael; Kolanus, Waldemar; Famulok, Michael; Bajorath, Jürgen

    2010-09-17

    Virtual screening (VS) of chemical libraries formatted in silico provides an alternative to experimental high-throughput screening (HTS) for the identification of small molecule modulators of protein function. We have tailored a VS approach combining fingerprint similarity searching and support vector machine modeling toward the identification of small molecular probes for the study of cytohesins, a family of cytoplasmic regulator proteins with multiple cellular functions. A total of 40 new structurally diverse inhibitors were identified, and 26 of these compounds were more active than the primary VS template, a single known inhibitory chemotype, in at least one of three different assays (guanine nucleotide exchange, Drosophila insulin signaling, and human leukocyte cell adhesion). Moreover, these inhibitors displayed differential inhibitory profiles. Our findings demonstrate that, at least for the cytohesins, computational extrapolation from known active compounds was capable of identifying small molecular probes with highly diversified functional profiles. PMID:20614894

  15. Measuring error rates in genomic perturbation screens: gold standards for human functional genomics

    PubMed Central

    Hart, Traver; Brown, Kevin R; Sircoulomb, Fabrice; Rottapel, Robert; Moffat, Jason

    2014-01-01

    Technological advancement has opened the door to systematic genetics in mammalian cells. Genome-scale loss-of-function screens can assay fitness defects induced by partial gene knockdown, using RNA interference, or complete gene knockout, using new CRISPR techniques. These screens can reveal the basic blueprint required for cellular proliferation. Moreover, comparing healthy to cancerous tissue can uncover genes that are essential only in the tumor; these genes are targets for the development of specific anticancer therapies. Unfortunately, progress in this field has been hampered by off-target effects of perturbation reagents and poorly quantified error rates in large-scale screens. To improve the quality of information derived from these screens, and to provide a framework for understanding the capabilities and limitations of CRISPR technology, we derive gold-standard reference sets of essential and nonessential genes, and provide a Bayesian classifier of gene essentiality that outperforms current methods on both RNAi and CRISPR screens. Our results indicate that CRISPR technology is more sensitive than RNAi and that both techniques have nontrivial false discovery rates that can be mitigated by rigorous analytical methods. PMID:24987113

  16. The iBeetle large-scale RNAi screen reveals gene functions for insect development and physiology.

    PubMed

    Schmitt-Engel, Christian; Schultheis, Dorothea; Schwirz, Jonas; Ströhlein, Nadi; Troelenberg, Nicole; Majumdar, Upalparna; Dao, Van Anh; Grossmann, Daniela; Richter, Tobias; Tech, Maike; Dönitz, Jürgen; Gerischer, Lizzy; Theis, Mirko; Schild, Inga; Trauner, Jochen; Koniszewski, Nikolaus D B; Küster, Elke; Kittelmann, Sebastian; Hu, Yonggang; Lehmann, Sabrina; Siemanowski, Janna; Ulrich, Julia; Panfilio, Kristen A; Schröder, Reinhard; Morgenstern, Burkhard; Stanke, Mario; Buchhholz, Frank; Frasch, Manfred; Roth, Siegfried; Wimmer, Ernst A; Schoppmeier, Michael; Klingler, Martin; Bucher, Gregor

    2015-01-01

    Genetic screens are powerful tools to identify the genes required for a given biological process. However, for technical reasons, comprehensive screens have been restricted to very few model organisms. Therefore, although deep sequencing is revealing the genes of ever more insect species, the functional studies predominantly focus on candidate genes previously identified in Drosophila, which is biasing research towards conserved gene functions. RNAi screens in other organisms promise to reduce this bias. Here we present the results of the iBeetle screen, a large-scale, unbiased RNAi screen in the red flour beetle, Tribolium castaneum, which identifies gene functions in embryonic and postembryonic development, physiology and cell biology. The utility of Tribolium as a screening platform is demonstrated by the identification of genes involved in insect epithelial adhesion. This work transcends the restrictions of the candidate gene approach and opens fields of research not accessible in Drosophila. PMID:26215380

  17. The iBeetle large-scale RNAi screen reveals gene functions for insect development and physiology

    PubMed Central

    Schmitt-Engel, Christian; Schultheis, Dorothea; Schwirz, Jonas; Ströhlein, Nadi; Troelenberg, Nicole; Majumdar, Upalparna; Dao, Van Anh; Grossmann, Daniela; Richter, Tobias; Tech, Maike; Dönitz, Jürgen; Gerischer, Lizzy; Theis, Mirko; Schild, Inga; Trauner, Jochen; Koniszewski, Nikolaus D. B.; Küster, Elke; Kittelmann, Sebastian; Hu, Yonggang; Lehmann, Sabrina; Siemanowski, Janna; Ulrich, Julia; Panfilio, Kristen A.; Schröder, Reinhard; Morgenstern, Burkhard; Stanke, Mario; Buchhholz, Frank; Frasch, Manfred; Roth, Siegfried; Wimmer, Ernst A.; Schoppmeier, Michael; Klingler, Martin; Bucher, Gregor

    2015-01-01

    Genetic screens are powerful tools to identify the genes required for a given biological process. However, for technical reasons, comprehensive screens have been restricted to very few model organisms. Therefore, although deep sequencing is revealing the genes of ever more insect species, the functional studies predominantly focus on candidate genes previously identified in Drosophila, which is biasing research towards conserved gene functions. RNAi screens in other organisms promise to reduce this bias. Here we present the results of the iBeetle screen, a large-scale, unbiased RNAi screen in the red flour beetle, Tribolium castaneum, which identifies gene functions in embryonic and postembryonic development, physiology and cell biology. The utility of Tribolium as a screening platform is demonstrated by the identification of genes involved in insect epithelial adhesion. This work transcends the restrictions of the candidate gene approach and opens fields of research not accessible in Drosophila. PMID:26215380

  18. Toxicology screen

    MedlinePlus

    Barbiturates - screen; Benzodiazepines - screen; Amphetamines - screen; Analgesics - screen; Antidepressants - screen; Narcotics - screen; Phenothiazines - screen; Drug abuse screen; Blood alcohol test

  19. A kinome wide screen identifies novel kinases involved in regulation of monoamine transporter function.

    PubMed

    Vuorenpää, Anne; Ammendrup-Johnsen, Ina; Jørgensen, Trine N; Gether, Ulrik

    2016-09-01

    The high affinity transporters for the monoamine neurotransmitters, dopamine, norepinephrine, and serotonin, play a key role in controlling monoaminergic neurotransmission. It is believed that the transporters (DAT, NET and SERT, respectively) are subject to tight regulation by the cellular signaling machinery to maintain monoaminergic homeostasis. Kinases constitute a pivotal role in cellular signaling, however, the regulation of monoamine transporters by the entire ensemble of kinases is unknown. Here, we perform a whole human kinome RNA interference screen to identify novel kinases involved in regulation of monoamine transporter function and surface expression. A primary screen in HEK 293 cells stably expressing DAT or SERT with siRNAs against 573 human kinases revealed 93 kinases putatively regulating transporter function. All 93 hits, which also included kinases previously implicated in monoamine transporter regulation, such as Protein kinase B (Akt) and mitogen-activated protein kinases (MAPK), were validated with a new set of siRNAs in a secondary screen. In this screen we assessed both changes in uptake and surface expression leading to selection of 11 kinases for further evaluation in HEK 293 cells transiently expressing DAT, SERT or NET. Subsequently, three kinases; salt inducible kinase 3 (SIK3), cAMP-dependent protein kinase catalytic subunit alpha (PKA C-α) and protein kinase X-linked (PrKX); were selected for additional exploration in catecholaminergic CATH.a differentiated cells (CAD) and rat chromocytoma (PC12) cells. Whereas SIK3 likely transcriptionally regulated expression of the three transfected transporters, depletion of PKA C-α was shown to decrease SERT function. Depletion of PrKX caused decreased surface expression and function of DAT without changing protein levels, suggesting that PrKX stabilizes the transporter at the cell surface. Summarized, our data provide novel insight into kinome regulation of the monoamine transporters and

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

    Cancer.gov

    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.

  1. [Long-term functional effect of the photo screening in ocular diseases causing amblyopia].

    PubMed

    Dostálek, M; Belácek, J; Zárubová, A; Dusek, J

    2010-02-01

    Amblyopia represents the most common cause of insufficient monocular visual acuity in productive age.To begin the treatment in the early childhood is understood as the fundamental anticipation to achieve the optimal result. The research work concerns about the development of screening methods. Our retrospective study was not focused toward the efficacy of the screening itself, but it had to establish, how the participation of the suckling babies in the screening program influences the chance of good vision. The data analyzed in this study were obtained from retrospective review of medical records of two groups of patients of the Center for functional visual disorders. Four hundred and nineteen (419) patients with positive finding in the photo screening, selected by chance, were included into the study (SC group). In the second group, there were included 263 randomly selected patients who did not pass the photo screening procedure and to the first examination were referred by the pediatrician (PLDD group).The average age at the time of the first examination was 13 months in children from the SC group and 23 months in children from the PLDD group respectively.The difference was statistically highly significant. In both groups, the severity of the amblyopia related to the comparable degree of anisometropia and the degree of the involvement of binocular functions in strabismus related to the comparable degree of hypemetropia were compared. The data were evaluated according to the age of the child patients at the time of the examination used for the purpose of this study as well. The obtained data show, that the decrease of the visual acuity of the amblyopic eye in case of equal anisometropia is significantly higher in two to three years old children who were not screened. In older children, the results in the in the SC and PLDD groups did not significantly differ. The results of our study indicate that later (before the age of three years) beginning of systematic

  2. Certain Actions from the Functional Movement Screen Do Not Provide an Indication of Dynamic Stability.

    PubMed

    Lockie, Robert G; Callaghan, Samuel J; Jordan, Corrin A; Luczo, Tawni M; Jeffriess, Matthew D; Jalilvand, Farzad; Schultz, Adrian B

    2015-09-29

    Dynamic stability is an essential physical component for team sport athletes. Certain Functional Movement Screen (FMS) exercises (deep squat; left- and right-leg hurdle step; left- and right-leg in-line lunge [ILL]; left- and right-leg active straight-leg raise; and trunk stability push-up [TSPU]) have been suggested as providing an indication of dynamic stability. No research has investigated relationships between these screens and an established test of dynamic stability such as the modified Star Excursion Balance Test (mSEBT), which measures lower-limb reach distance in posteromedial, medial, and anteromedial directions, in team sport athletes. Forty-one male and female team sport athletes completed the screens and the mSEBT. Participants were split into high-, intermediate-, and low-performing groups according to the mean of the excursions when both the left and right legs were used for the mSEBT stance. Any between-group differences in the screens and mSEBT were determined via a one-way analysis of variance with Bonferroni post hoc adjustment (p < 0.05). Data was pooled for a correlation analysis (p < 0.05). There were no between-group differences in any of the screens, and only two positive correlations between the screens and the mSEBT (TSPU and right stance leg posteromedial excursion, r = 0.37; left-leg ILL and left stance leg posteromedial excursion, r = 0.46). The mSEBT clearly indicated participants with different dynamic stability capabilities. In contrast to the mSEBT, the selected FMS exercises investigated in this study have a limited capacity to identify dynamic stability in team sport athletes. PMID:26557187

  3. Certain Actions from the Functional Movement Screen Do Not Provide an Indication of Dynamic Stability

    PubMed Central

    Lockie, Robert G.; Callaghan, Samuel J.; Jordan, Corrin A.; Luczo, Tawni M.; Jeffriess, Matthew D.; Jalilvand, Farzad; Schultz, Adrian B.

    2015-01-01

    Dynamic stability is an essential physical component for team sport athletes. Certain Functional Movement Screen (FMS) exercises (deep squat; left- and right-leg hurdle step; left- and right-leg in-line lunge [ILL]; left- and right-leg active straight-leg raise; and trunk stability push-up [TSPU]) have been suggested as providing an indication of dynamic stability. No research has investigated relationships between these screens and an established test of dynamic stability such as the modified Star Excursion Balance Test (mSEBT), which measures lower-limb reach distance in posteromedial, medial, and anteromedial directions, in team sport athletes. Forty-one male and female team sport athletes completed the screens and the mSEBT. Participants were split into high-, intermediate-, and low-performing groups according to the mean of the excursions when both the left and right legs were used for the mSEBT stance. Any between-group differences in the screens and mSEBT were determined via a one-way analysis of variance with Bonferroni post hoc adjustment (p < 0.05). Data was pooled for a correlation analysis (p < 0.05). There were no between-group differences in any of the screens, and only two positive correlations between the screens and the mSEBT (TSPU and right stance leg posteromedial excursion, r = 0.37; left-leg ILL and left stance leg posteromedial excursion, r = 0.46). The mSEBT clearly indicated participants with different dynamic stability capabilities. In contrast to the mSEBT, the selected FMS exercises investigated in this study have a limited capacity to identify dynamic stability in team sport athletes. PMID:26557187

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

    PubMed

    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. PMID:25728500

  5. Calculations of properties of screened He-like systems using correlated wave functions.

    PubMed

    Dai, S T; Solovyova, A; Winkler, P

    2001-07-01

    The purpose of the present study is twofold. First, the techniques of correlated wave functions for two-electron systems have been extended to obtain results for P and D states in a screening environment, and in particular for Debye screening. In these calculations, the satisfaction of both the quantum virial theorem and a related sum rule has been enforced and found to provide a high degree of stability of the solutions. Second, in order to facilitate the general use of correlated wave functions in combination with sum rule stability criteria, a rather systematic computational approach to this notoriously cumbersome method has been developed and thoroughly discussed here. Accurate calculations for few-electron systems are of interest to plasma diagnostics; in particular, when inaccuracies in binding energies are drastically magnified as they occur in exponents of Boltzmann factors. PMID:11461411

  6. Function-specific virtual screening for GPCR ligands using a combined scoring method

    PubMed Central

    Kooistra, Albert J.; Vischer, Henry F.; McNaught-Flores, Daniel; Leurs, Rob; de Esch, Iwan J. P.; de Graaf, Chris

    2016-01-01

    The ability of scoring functions to correctly select and rank docking poses of small molecules in protein binding sites is highly target dependent, which presents a challenge for structure-based drug discovery. Here we describe a virtual screening method that combines an energy-based docking scoring function with a molecular interaction fingerprint (IFP) to identify new ligands based on G protein-coupled receptor (GPCR) crystal structures. The consensus scoring method is prospectively evaluated by: 1) the discovery of chemically novel, fragment-like, high affinity histamine H1 receptor (H1R) antagonists/inverse agonists, 2) the selective structure-based identification of ß2-adrenoceptor (ß2R) agonists, and 3) the experimental validation and comparison of the combined and individual scoring approaches. Systematic retrospective virtual screening simulations allowed the definition of scoring cut-offs for the identification of H1R and ß2R ligands and the selection of an optimal ß-adrenoceptor crystal structure for the discrimination between ß2R agonists and antagonists. The consensus approach resulted in the experimental validation of 53% of the ß2R and 73% of the H1R virtual screening hits with up to nanomolar affinities and potencies. The selective identification of ß2R agonists shows the possibilities of structure-based prediction of GPCR ligand function by integrating protein-ligand binding mode information. PMID:27339552

  7. Effects of d-electrons in pseudopotential screened-exchange density functional calculations

    NASA Astrophysics Data System (ADS)

    Lee, Byounghak; Wang, Lin-Wang; Canning, Andrew

    2008-06-01

    We report a theoretical study on the role of shallow d states in the screened-exchange local density approximation (sX-LDA) band structure of binary semiconductor systems. We found that inaccurate pseudo-wave functions can lead to (1) an overestimation of the screened-exchange interaction between the localized d states and the delocalized higher energy s and p states, and (2) an underestimation of the screened-exchange interaction between the d states. The resulting sX-LDA band structures have substantially smaller band gaps compared with experiments. We correct the pseudo-wave functions of d states by including the semicore s and p states of the same shell in the valence states. The correction of pseudo-wave functions yields band gaps and d-state binding energies in good agreement with experiments and the full potential linearized augmented plane wave sX-LDA calculations. Compared with the quasiparticle GW method, our sX-LDA results shows not only similar quality on the band gaps but also much better d-state binding energies. Combined with its capability of ground-state structure calculation, the sX-LDA is expected to be a valuable theoretical tool for the II-VI and III-V (especially the III-N) bulk semiconductors and nanostructure studies.

  8. MiRNA mimic screen for improved functional expression of neurotensin receptor

    PubMed Central

    Xiao, Su; Chen, Yu-Chi; Betenbaugh, Michael J.; Martin, Scott E.; Shiloach, Joseph

    2015-01-01

    Obtaining adequate quantities of functional mammalian membrane proteins has been a bottleneck in their structural and functional studies because the expression of these proteins from mammalian cells is relatively low. To explore the possibility of enhancing expression of these proteins using miRNA, a stable T-REx-293 cell line expressing the neurotensin receptor type 1 (NTSR1), a hard-to-express G protein-coupled receptor (GPCR), was constructed. The cell line was then subjected to human miRNA mimic library screening. In parallel, an HEK293 cell line expressing luciferase was also screened with the same human miRNA mimic library. Five microRNA mimics: hsa-miR-22-5p, hsa-miR-18a-5p, hsa-miR-22-3p, hsa-miR-429 and hsa-miR-2110 were identified from both screens. They led to 48% increase in the expression of functional NTSR1 and to 239% increase of luciferase expression. These miRNAs were also effective in enhancing the expression of secreted glypican-3 hFc-fusion protein from HEK293 cells. The results indicate that these molecules may have a wide role in enhancing the production of proteins with biomedical interest. PMID:25676429

  9. Function-specific virtual screening for GPCR ligands using a combined scoring method.

    PubMed

    Kooistra, Albert J; Vischer, Henry F; McNaught-Flores, Daniel; Leurs, Rob; de Esch, Iwan J P; de Graaf, Chris

    2016-01-01

    The ability of scoring functions to correctly select and rank docking poses of small molecules in protein binding sites is highly target dependent, which presents a challenge for structure-based drug discovery. Here we describe a virtual screening method that combines an energy-based docking scoring function with a molecular interaction fingerprint (IFP) to identify new ligands based on G protein-coupled receptor (GPCR) crystal structures. The consensus scoring method is prospectively evaluated by: 1) the discovery of chemically novel, fragment-like, high affinity histamine H1 receptor (H1R) antagonists/inverse agonists, 2) the selective structure-based identification of ß2-adrenoceptor (ß2R) agonists, and 3) the experimental validation and comparison of the combined and individual scoring approaches. Systematic retrospective virtual screening simulations allowed the definition of scoring cut-offs for the identification of H1R and ß2R ligands and the selection of an optimal ß-adrenoceptor crystal structure for the discrimination between ß2R agonists and antagonists. The consensus approach resulted in the experimental validation of 53% of the ß2R and 73% of the H1R virtual screening hits with up to nanomolar affinities and potencies. The selective identification of ß2R agonists shows the possibilities of structure-based prediction of GPCR ligand function by integrating protein-ligand binding mode information. PMID:27339552

  10. A TR-FRET-based functional assay for screening activators of CARM1.

    PubMed

    Zeng, Hao; Wu, Jiacai; Bedford, Mark T; Sbardella, Gianluca; Hoffmann, F Michael; Bi, Kun; Xu, Wei

    2013-05-10

    Epigenetics is an emerging field that demands selective cell-permeable chemical probes to perturb, especially in vivo, the activity of specific enzymes involved in modulating the epigenetic codes. Coactivator-associated arginine methyltransferase 1 (CARM1) is a coactivator of estrogen receptor α (ERα), the main target in human breast cancer. We previously showed that twofold overexpression of CARM1 in MCF7 breast cancer cells increased the expression of ERα-target genes involved in differentiation and reduced cell proliferation, thus leading to the hypothesis that activating CARM1 by chemical activators might be therapeutically effective in breast cancer. Selective, potent, cell-permeable CARM1 activators will be essential to test this hypothesis. Here we report the development of a cell-based, time-resolved (TR) FRET assay that uses poly(A) binding protein 1 (PABP1) methylation to monitor cellular activity of CARM1. The LanthaScreen TR-FRET assay uses MCF7 cells expressing GFP-PABP1 fusion protein through BacMam gene delivery system, methyl-PABP1 specific antibody, and terbium-labeled secondary antibody. This assay has been validated as reflecting the expression and/or activity of CARM1 and optimized for high throughput screening to identify CARM1 allosteric activators. This TR-FRET platform serves as a generic tool for functional screening of cell-permeable, chemical modulators of CARM1 for elucidation of its in vivo functions. PMID:23585185

  11. Yeast functional screen to identify genes conferring salt stress tolerance in Salicornia europaea

    PubMed Central

    Nakahara, Yoshiki; Sawabe, Shogo; Kainuma, Kenta; Katsuhara, Maki; Shibasaka, Mineo; Suzuki, Masanori; Yamamoto, Kosuke; Oguri, Suguru; Sakamoto, Hikaru

    2015-01-01

    Salinity is a critical environmental factor that adversely affects crop productivity. Halophytes have evolved various mechanisms to adapt to saline environments. Salicornia europaea L. is one of the most salt-tolerant plant species. It does not have special salt-secreting structures like a salt gland or salt bladder, and is therefore a good model for studying the common mechanisms underlying plant salt tolerance. To identify candidate genes encoding key proteins in the mediation of salt tolerance in S. europaea, we performed a functional screen of a cDNA library in yeast. The library was screened for genes that allowed the yeast to grow in the presence of 1.3 M NaCl. We obtained three full-length S. europaea genes that confer salt tolerance. The genes are predicted to encode (1) a novel protein highly homologous to thaumatin-like proteins, (2) a novel coiled-coil protein of unknown function, and (3) a novel short peptide of 32 residues. Exogenous application of a synthetic peptide corresponding to the 32 residues improved salt tolerance of Arabidopsis. The approach described in this report provides a rapid assay system for large-scale screening of S. europaea genes involved in salt stress tolerance and supports the identification of genes responsible for such mechanisms. These genes may be useful candidates for improving crop salt tolerance by genetic transformation. PMID:26579166

  12. UCLA's Molecular Screening Shared Resource: enhancing small molecule discovery with functional genomics and new technology.

    PubMed

    Damoiseaux, Robert

    2014-05-01

    The Molecular Screening Shared Resource (MSSR) offers a comprehensive range of leading-edge high throughput screening (HTS) services including drug discovery, chemical and functional genomics, and novel methods for nano and environmental toxicology. The MSSR is an open access environment with investigators from UCLA as well as from the entire globe. Industrial clients are equally welcome as are non-profit entities. The MSSR is a fee-for-service entity and does not retain intellectual property. In conjunction with the Center for Environmental Implications of Nanotechnology, the MSSR is unique in its dedicated and ongoing efforts towards high throughput toxicity testing of nanomaterials. In addition, the MSSR engages in technology development eliminating bottlenecks from the HTS workflow and enabling novel assays and readouts currently not available. PMID:24661210

  13. Reliability of clinician scoring of the functional movement screen to assess movement patterns.

    PubMed

    Stobierski, Lisa M; Fayson, Shirleeah D; Minthorn, Lindsay M; Valovich McLeod, Tamara C; Welch, Cailee E

    2015-05-01

    Clinical Scenario: Injuries are inevitable in the physically active population. As a part of preventive medicine, health care professionals often seek clinical tools that can be used in real time to identify factors that may predispose individuals to these injuries. The Functional Movement Screen (FMS), a clinical tool consisting of 7 individual tasks, has been reported as useful in identifying individuals in various populations that may be susceptible to musculoskeletal injuries. If factors that may predispose physically active individuals to injury could be identified before participation, clinicians may be able to develop a training plan based on FMS scores, which could potentially decrease the likelihood of injury and overall time missed from physical activities. However, in order for a screening tool to be used clinically, it must demonstrate acceptable reliability. Focused Clinical Question: Are clinicians reliable at scoring the FMS, in real time, to assess movement patterns of physically active individuals? PMID:25054658

  14. A human genome-wide loss-of-function screen identifies effective chikungunya antiviral drugs

    PubMed Central

    Karlas, Alexander; Berre, Stefano; Couderc, Thérèse; Varjak, Margus; Braun, Peter; Meyer, Michael; Gangneux, Nicolas; Karo-Astover, Liis; Weege, Friderike; Raftery, Martin; Schönrich, Günther; Klemm, Uwe; Wurzlbauer, Anne; Bracher, Franz; Merits, Andres; Meyer, Thomas F.; Lecuit, Marc

    2016-01-01

    Chikungunya virus (CHIKV) is a globally spreading alphavirus against which there is no commercially available vaccine or therapy. Here we use a genome-wide siRNA screen to identify 156 proviral and 41 antiviral host factors affecting CHIKV replication. We analyse the cellular pathways in which human proviral genes are involved and identify druggable targets. Twenty-one small-molecule inhibitors, some of which are FDA approved, targeting six proviral factors or pathways, have high antiviral activity in vitro, with low toxicity. Three identified inhibitors have prophylactic antiviral effects in mouse models of chikungunya infection. Two of them, the calmodulin inhibitor pimozide and the fatty acid synthesis inhibitor TOFA, have a therapeutic effect in vivo when combined. These results demonstrate the value of loss-of-function screening and pathway analysis for the rational identification of small molecules with therapeutic potential and pave the way for the development of new, host-directed, antiviral agents. PMID:27177310

  15. A small-molecule screen identifies new functions for the plant hormone strigolactone.

    PubMed

    Tsuchiya, Yuichiro; Vidaurre, Danielle; Toh, Shigeo; Hanada, Atsushi; Nambara, Eiji; Kamiya, Yuji; Yamaguchi, Shinjiro; McCourt, Peter

    2010-10-01

    Parasitic weeds of the genera Striga and Orobanche are considered the most damaging agricultural agents in the developing world. An essential step in parasitic seed germination is sensing a group of structurally related compounds called strigolactones that are released by host plants. Although this makes strigolactone synthesis and action a major target of biotechnology, little fundamental information is known about this hormone. Chemical genetic screening using Arabidopsis thaliana as a platform identified a collection of related small molecules, cotylimides, which perturb strigolactone accumulation. Suppressor screens against select cotylimides identified light-signaling genes as positive regulators of strigolactone levels. Molecular analysis showed strigolactones regulate the nuclear localization of the COP1 ubiquitin ligase, which in part determines the levels of light regulators such as HY5. This information not only uncovers new functions for strigolactones but was also used to identify rice cultivars with reduced capacity to germinate parasitic seed. PMID:20818397

  16. Differences in performance on the functional movement screen between chronic low back pain patients and healthy control subjects.

    PubMed

    Ko, Min-Joo; Noh, Kyung-Hee; Kang, Min-Hyeok; Oh, Jae-Seop

    2016-07-01

    [Purpose] Differences in scores on the Functional Movement Screen between patients with chronic lower back pain and healthy control subjects were investigated. [Subjects and Methods] In all, 20 chronic lower back pain patients and 20 healthy control subjects were recruited. Chronic lower back pain patients and healthy controls performed the Functional Movement Screen (deep squat, hurdle step, inline lunge, shoulder mobility, active straight leg raise, trunk stability pushup, and rotary stability). The Mann-Whitney test was used to analyze differences in Functional Movement Screen scores between the two groups. [Results] Chronic lower back pain patients scored lower on the Functional Movement Screen total composite compared with healthy control subjects. Chronic lower back pain patients scored lower on Functional Movement Screen subtests including the deep squat, hurdle step, active straight leg raise, and rotary stability tests. [Conclusion] The deep squat, hurdle step, active straight leg raise, and rotary stability tasks of the Functional Movement Screen can be recommended as a functional assessment tools to identify functional deficits in chronic lower back pain patients. PMID:27512272

  17. Differences in performance on the functional movement screen between chronic low back pain patients and healthy control subjects

    PubMed Central

    Ko, Min-Joo; Noh, Kyung-Hee; Kang, Min-Hyeok; Oh, Jae-Seop

    2016-01-01

    [Purpose] Differences in scores on the Functional Movement Screen between patients with chronic lower back pain and healthy control subjects were investigated. [Subjects and Methods] In all, 20 chronic lower back pain patients and 20 healthy control subjects were recruited. Chronic lower back pain patients and healthy controls performed the Functional Movement Screen (deep squat, hurdle step, inline lunge, shoulder mobility, active straight leg raise, trunk stability pushup, and rotary stability). The Mann-Whitney test was used to analyze differences in Functional Movement Screen scores between the two groups. [Results] Chronic lower back pain patients scored lower on the Functional Movement Screen total composite compared with healthy control subjects. Chronic lower back pain patients scored lower on Functional Movement Screen subtests including the deep squat, hurdle step, active straight leg raise, and rotary stability tests. [Conclusion] The deep squat, hurdle step, active straight leg raise, and rotary stability tasks of the Functional Movement Screen can be recommended as a functional assessment tools to identify functional deficits in chronic lower back pain patients. PMID:27512272

  18. Functional Screening Identifies miRNAs Influencing Apoptosis and Proliferation in Colorectal Cancer

    PubMed Central

    Rantala, Juha; Kallioniemi, Olli; Rasmussen, Mads H.; Ostenfeld, Marie S.; Dagnaes-Hansen, Frederik; Øster, Bodil; Schepeler, Troels; Tobiasen, Heidi; Thorsen, Kasper; Sieber, Oliver M.; Gibbs, Peter; Lamy, Philippe; Hansen, Torben F.; Jakobsen, Anders; Riising, Eva M.; Helin, Kristian; Lubinski, Jan; Hagemann-Madsen, Rikke; Laurberg, Søren; Ørntoft, Torben F.; Andersen, Claus L.

    2014-01-01

    MicroRNAs (miRNAs) play a critical role in many biological processes and are aberrantly expressed in human cancers. Particular miRNAs function either as tumor suppressors or oncogenes and appear to have diagnostic and prognostic significance. Although numerous miRNAs are dys-regulated in colorectal cancer (CRC) only a small fraction has been characterized functionally. Using high-throughput functional screening and miRNA profiling of clinical samples the present study aims at identifying miRNAs important for the control of cellular growth and/or apoptosis in CRC. The high-throughput functional screening was carried out in six CRC cell lines transfected with a pre-miR library including 319 synthetic human pre-miRs. Phenotypic alterations were evaluated by immunostaining of cleaved cPARP (apoptosis) or MKI67 (proliferation). Additionally, TaqMan Human MicroRNA Array Set v2.0 was used to profile the expression of 667 miRNAs in 14 normal colon mucosa and 46 microsatellite stable stage II CRC patients. Among the miRNAs that induced growth arrest and apoptosis in the CRC cell lines, and at same time were dys-regulated in the clinical samples, miR-375 was selected for further analysis. Independent in vitro analysis of transient and stable transfected CRC cell lines confirmed that miR-375 reduces cell viability through the induction of apoptotic death. We identified YAP1 as a direct miR-375 target in CRC and show that HELLS and NOLC1 are down-stream targets. Knock-down of YAP1 mimicked the phenotype induced by miR-375 over-expression indicating that miR-375 most likely exerts its pro-apoptotic role through YAP1 and its anti-apoptotic down-stream targets BIRC5 and BCL2L1. Finally, in vivo analysis of mouse xenograft tumors showed that miR-375 expression significantly reduced tumor growth. We conclude that the high-throughput screening successfully identified miRNAs that induce apoptosis and/or inhibit proliferation in CRC cells. Finally, combining the functional screening

  19. Vinardo: A Scoring Function Based on Autodock Vina Improves Scoring, Docking, and Virtual Screening

    PubMed Central

    Villarreal, Marcos A.

    2016-01-01

    Autodock Vina is a very popular, and highly cited, open source docking program. Here we present a scoring function which we call Vinardo (Vina RaDii Optimized). Vinardo is based on Vina, and was trained through a novel approach, on state of the art datasets. We show that the traditional approach to train empirical scoring functions, using linear regression to optimize the correlation of predicted and experimental binding affinities, does not result in a function with optimal docking capabilities. On the other hand, a combination of scoring, minimization, and re-docking on carefully curated training datasets allowed us to develop a simplified scoring function with optimum docking performance. This article provides an overview of the development of the Vinardo scoring function, highlights its differences with Vina, and compares the performance of the two scoring functions in scoring, docking and virtual screening applications. Vinardo outperforms Vina in all tests performed, for all datasets analyzed. The Vinardo scoring function is available as an option within Smina, a fork of Vina, which is freely available under the GNU Public License v2.0 from http://smina.sf.net. Precompiled binaries, source code, documentation and a tutorial for using Smina to run the Vinardo scoring function are available at the same address. PMID:27171006

  20. Identification of APC mutations and evaluation of their expression level using a functional screening assay

    SciTech Connect

    Varesco, L.; Gismondi, V.; Bafico, A.

    1994-09-01

    A functional screen for chain-terminating mutations in the APC gene recently has been developed. It is based on the PCR and cloning of a segment of the gene in-frame with a colorimetric marker gene (lacz) followed by screening for the level of activity of the marker polypeptide (beta-galactosidase). This method scores colony number with different blue colors that are produced by bacteria containing normal and mutant APC segments. In the present work this method was used to screen the entire APC coding region by using eight primer pairs. DNA segments with known APC mutations at different positions in the gene were used as controls and were clearly identifiable with this assay. In addition, the entire APC coding region has been examined in 21 APC patients in whom PCR-SSCP did not identify an APC mutation. Novel mutations (n=14) were identified by the blue/white assay and were all confirmed by sequence analysis. This method also was used to quantitate the expression of paternal and maternal APC alleles taking advantage of an RsaI site polymorphism at position 1458 in a small number of informative individuals. Differential expression of some known mutant APC mRNAs was observed.

  1. COLT-Cancer: functional genetic screening resource for essential genes in human cancer cell lines

    PubMed Central

    Koh, Judice L. Y.; Brown, Kevin R.; Sayad, Azin; Kasimer, Dahlia; Ketela, Troy; Moffat, Jason

    2012-01-01

    Genome-wide pooled shRNA screens enable global identification of the genes essential for cancer cell survival and proliferation and provide a ‘functional genetic’ map of human cancer to complement genomic studies. Using a lentiviral shRNA library targeting approximately 16 000 human genes and a newly developed scoring approach, we identified essential gene profiles in more than 70 breast, pancreatic and ovarian cancer cell lines. We developed a web-accessible database system for capturing information from each step in our standardized screening pipeline and a gene-centric search tool for exploring shRNA activities within a given cell line or across multiple cell lines. The database consists of a laboratory information and management system for tracking each step of a pooled shRNA screen as well as a web interface for querying and visualization of shRNA and gene-level performance across multiple cancer cell lines. COLT-Cancer Version 1.0 is currently accessible at http://colt.ccbr.utoronto.ca/cancer. PMID:22102578

  2. High-throughput functional screening using a homemade dual-glow luciferase assay.

    PubMed

    Baker, Jessica M; Boyce, Frederick M

    2014-01-01

    We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with Parkinson's disease. 293T cells are transiently transfected with plasmids from an arrayed ORF expression library, together with luciferase reporter plasmids, in a one-gene-per-well microplate format. Firefly luciferase activity is assayed after 48 hr to determine the effects of each library gene upon alpha-synuclein transcription, normalized to expression from an internal control construct (a hCMV promoter directing Renilla luciferase). This protocol is facilitated by a bench-top robot enclosed in a biosafety cabinet, which performs aseptic liquid handling in 96-well format. Our automated transfection protocol is readily adaptable to high-throughput lentiviral library production or other functional screening protocols requiring triple-transfections of large numbers of unique library plasmids in conjunction with a common set of helper plasmids. We also present an inexpensive and validated alternative to commercially-available, dual luciferase reagents which employs PTC124, EDTA, and pyrophosphate to suppress firefly luciferase activity prior to measurement of Renilla luciferase. Using these methods, we screened 7,670 human genes and identified 68 regulators of alpha-synuclein. This protocol is easily modifiable to target other genes of interest. PMID:24962249

  3. High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay

    PubMed Central

    Baker, Jessica M.; Boyce, Frederick M.

    2014-01-01

    We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with Parkinson's disease. 293T cells are transiently transfected with plasmids from an arrayed ORF expression library, together with luciferase reporter plasmids, in a one-gene-per-well microplate format. Firefly luciferase activity is assayed after 48 hr to determine the effects of each library gene upon alpha-synuclein transcription, normalized to expression from an internal control construct (a hCMV promoter directing Renilla luciferase). This protocol is facilitated by a bench-top robot enclosed in a biosafety cabinet, which performs aseptic liquid handling in 96-well format. Our automated transfection protocol is readily adaptable to high-throughput lentiviral library production or other functional screening protocols requiring triple-transfections of large numbers of unique library plasmids in conjunction with a common set of helper plasmids. We also present an inexpensive and validated alternative to commercially-available, dual luciferase reagents which employs PTC124, EDTA, and pyrophosphate to suppress firefly luciferase activity prior to measurement of Renilla luciferase. Using these methods, we screened 7,670 human genes and identified 68 regulators of alpha-synuclein. This protocol is easily modifiable to target other genes of interest. PMID:24962249

  4. Efficacy of functional movement screening for predicting injuries in coast guard cadets.

    PubMed

    Knapik, Joseph J; Cosio-Lima, Ludimila M; Reynolds, Katy L; Shumway, Richard S

    2015-05-01

    Functional movement screening (FMS) examines the ability of individuals to perform highly specific movements with the aim of identifying individuals who have functional limitations or asymmetries. It is assumed that individuals who can more effectively accomplish the required movements have a lower injury risk. This study determined the ability of FMS to predict injuries in the United States Coast Guard (USCG) cadets. Seven hundred seventy male and 275 female USCG freshman cadets were administered the 7 FMS tests before the physically intense 8-week Summer Warfare Annual Basic (SWAB) training. Physical training-related injuries were recorded during SWAB training. Cumulative injury incidence was calculated at various FMS cutpoint scores. The ability of the FMS total score to predict injuries was examined by calculating sensitivity and specificity. Determination of the FMS cutpoint that maximized specificity and sensitivity was determined from the Youden's index (sensitivity + specificity - 1). For men, FMS scores ≤ 12 were associated with higher injury risk than scores >12; for women, FMS scores ≤ 15 were associated with higher injury risk than scores >15. The Youden's Index indicated that the optimal FMS cutpoint was ≤ 11 for men (22% sensitivity, 87% specificity) and ≤ 14 for women (60% sensitivity, 61% specificity). Functional movement screening demonstrated moderate prognostic accuracy for determining injury risk among female Coast Guard cadets but relatively low accuracy among male cadets. Attempting to predict injury risk based on the FMS test seems to have some limited promise based on the present and past investigations. PMID:25264669

  5. A functional siRNA screen identifies genes modulating angiotensin II-mediated EGFR transactivation

    PubMed Central

    George, Amee J.; Purdue, Brooke W.; Gould, Cathryn M.; Thomas, Daniel W.; Handoko, Yanny; Qian, Hongwei; Quaife-Ryan, Gregory A.; Morgan, Kylie A.; Simpson, Kaylene J.; Thomas, Walter G.; Hannan, Ross D.

    2013-01-01

    Summary The angiotensin type 1 receptor (AT1R) transactivates the epidermal growth factor receptor (EGFR) to mediate cellular growth, however, the molecular mechanisms involved have not yet been resolved. To address this, we performed a functional siRNA screen of the human kinome in human mammary epithelial cells that demonstrate a robust AT1R–EGFR transactivation. We identified a suite of genes encoding proteins that both positively and negatively regulate AT1R–EGFR transactivation. Many candidates are components of EGFR signalling networks, whereas others, including TRIO, BMX and CHKA, have not been previously linked to EGFR transactivation. Individual knockdown of TRIO, BMX or CHKA attenuated tyrosine phosphorylation of the EGFR by angiotensin II stimulation, but this did not occur following direct stimulation of the EGFR with EGF, indicating that these proteins function between the activated AT1R and the EGFR. Further investigation of TRIO and CHKA revealed that their activity is likely to be required for AT1R–EGFR transactivation. CHKA also mediated EGFR transactivation in response to another G protein-coupled receptor (GPCR) ligand, thrombin, indicating a pervasive role for CHKA in GPCR–EGFR crosstalk. Our study reveals the power of unbiased, functional genomic screens to identify new signalling mediators important for tissue remodelling in cardiovascular disease and cancer. PMID:24046455

  6. A functional siRNA screen identifies genes modulating angiotensin II-mediated EGFR transactivation.

    PubMed

    George, Amee J; Purdue, Brooke W; Gould, Cathryn M; Thomas, Daniel W; Handoko, Yanny; Qian, Hongwei; Quaife-Ryan, Gregory A; Morgan, Kylie A; Simpson, Kaylene J; Thomas, Walter G; Hannan, Ross D

    2013-12-01

    The angiotensin type 1 receptor (AT1R) transactivates the epidermal growth factor receptor (EGFR) to mediate cellular growth, however, the molecular mechanisms involved have not yet been resolved. To address this, we performed a functional siRNA screen of the human kinome in human mammary epithelial cells that demonstrate a robust AT1R-EGFR transactivation. We identified a suite of genes encoding proteins that both positively and negatively regulate AT1R-EGFR transactivation. Many candidates are components of EGFR signalling networks, whereas others, including TRIO, BMX and CHKA, have not been previously linked to EGFR transactivation. Individual knockdown of TRIO, BMX or CHKA attenuated tyrosine phosphorylation of the EGFR by angiotensin II stimulation, but this did not occur following direct stimulation of the EGFR with EGF, indicating that these proteins function between the activated AT1R and the EGFR. Further investigation of TRIO and CHKA revealed that their activity is likely to be required for AT1R-EGFR transactivation. CHKA also mediated EGFR transactivation in response to another G protein-coupled receptor (GPCR) ligand, thrombin, indicating a pervasive role for CHKA in GPCR-EGFR crosstalk. Our study reveals the power of unbiased, functional genomic screens to identify new signalling mediators important for tissue remodelling in cardiovascular disease and cancer. PMID:24046455

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

    PubMed Central

    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. PMID:26442115

  8. Functional Screening of Metagenome and Genome Libraries for Detection of Novel Flavonoid-Modifying Enzymes

    PubMed Central

    Rabausch, U.; Juergensen, J.; Ilmberger, N.; Böhnke, S.; Fischer, S.; Schubach, B.; Schulte, M.

    2013-01-01

    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. PMID:23686272

  9. Discovering novel enzymes by functional screening of plurigenomic libraries from alga-associated Flavobacteriia and Gammaproteobacteria.

    PubMed

    Martin, Marjolaine; Vandermies, Marie; Joyeux, Coline; Martin, Renée; Barbeyron, Tristan; Michel, Gurvan; Vandenbol, Micheline

    2016-01-01

    Alga-associated microorganisms, in the context of their numerous interactions with the host and the complexity of the marine environment, are known to produce diverse hydrolytic enzymes with original biochemistry. We recently isolated several macroalgal-polysaccharide-degrading bacteria from the surface of the brown alga Ascophyllum nodosum. These active isolates belong to two classes: the Flavobacteriia and the Gammaproteobacteria. In the present study, we constructed two "plurigenomic" (with multiple bacterial genomes) libraries with the 5 most interesting isolates (regarding their phylogeny and their enzymatic activities) of each class (Fv and Gm libraries). Both libraries were screened for diverse hydrolytic activities. Five activities, out of the 48 previously identified in the natural polysaccharolytic isolates, were recovered by functional screening: a xylanase (GmXyl7), a beta-glucosidase (GmBg1), an esterase (GmEst7) and two iota-carrageenases (Fvi2.5 and Gmi1.3). We discuss here the potential role of the used host-cell, the average DNA insert-sizes and the used restriction enzymes on the divergent screening yields obtained for both libraries and get deeper inside the "great screen anomaly". Interestingly, the discovered esterase probably stands for a novel family of homoserine o-acetyltransferase-like-esterases, while the two iota-carrageenases represent new members of the poorly known GH82 family (containing only 19 proteins since its description in 2000). These original results demonstrate the efficiency of our uncommon "plurigenomic" library approach and the underexplored potential of alga-associated cultivable microbiota for the identification of novel and algal-specific enzymes. PMID:27242143

  10. Functional genomic screen and network analysis reveal novel modifiers of tauopathy dissociated from tau phosphorylation

    PubMed Central

    Ambegaokar, Surendra S.; Jackson, George R.

    2011-01-01

    A functional genetic screen using loss-of-function and gain-of-function alleles was performed to identify modifiers of tau-induced neurotoxicity using the 2N/4R (full-length) isoform of wild-type human tau expressed in the fly retina. We previously reported eye pigment mutations, which create dysfunctional lysosomes, as potent modifiers; here, we report 37 additional genes identified from ∼1900 genes screened, including the kinases shaggy/GSK-3beta, par-1/MARK, CamKI and Mekk1. Tau acts synergistically with Mekk1 and p38 to down-regulate extracellular regulated kinase activity, with a corresponding decrease in AT8 immunoreactivity (pS202/T205), suggesting that tau can participate in signaling pathways to regulate its own kinases. Modifiers showed poor correlation with tau phosphorylation (using the AT8, 12E8 and AT270 epitopes); moreover, tested suppressors of wild-type tau were equally effective in suppressing toxicity of a phosphorylation-resistant S11A tau construct, demonstrating that changes in tau phosphorylation state are not required to suppress or enhance its toxicity. Genes related to autophagy, the cell cycle, RNA-associated proteins and chromatin-binding proteins constitute a large percentage of identified modifiers. Other functional categories identified include mitochondrial proteins, lipid trafficking, Golgi proteins, kinesins and dynein and the Hsp70/Hsp90-organizing protein (Hop). Network analysis uncovered several other genes highly associated with the functional modifiers, including genes related to the PI3K, Notch, BMP/TGF-β and Hedgehog pathways, and nuclear trafficking. Activity of GSK-3β is strongly upregulated due to TDP-43 expression, and reduced GSK-3β dosage is also a common suppressor of Aβ42 and TDP-43 toxicity. These findings suggest therapeutic targets other than mitigation of tau phosphorylation. PMID:21949350

  11. A large-scale functional screen identifies Nova1 and Ncoa3 as regulators of neuronal miRNA function

    PubMed Central

    Störchel, Peter H; Thümmler, Juliane; Siegel, Gabriele; Aksoy-Aksel, Ayla; Zampa, Federico; Sumer, Simon; Schratt, Gerhard

    2015-01-01

    MicroRNAs (miRNAs) are important regulators of neuronal development, network connectivity, and synaptic plasticity. While many neuronal miRNAs were previously shown to modulate neuronal morphogenesis, little is known regarding the regulation of miRNA function. In a large-scale functional screen, we identified two novel regulators of neuronal miRNA function, Nova1 and Ncoa3. Both proteins are expressed in the nucleus and the cytoplasm of developing hippocampal neurons. We found that Nova1 and Ncoa3 stimulate miRNA function by different mechanisms that converge on Argonaute (Ago) proteins, core components of the miRNA-induced silencing complex (miRISC). While Nova1 physically interacts with Ago proteins, Ncoa3 selectively promotes the expression of Ago2 at the transcriptional level. We further show that Ncoa3 regulates dendritic complexity and dendritic spine maturation of hippocampal neurons in a miRNA-dependent fashion. Importantly, both the loss of miRNA activity and increased dendrite complexity upon Ncoa3 knockdown were rescued by Ago2 overexpression. Together, we uncovered two novel factors that control neuronal miRISC function at the level of Ago proteins, with possible implications for the regulation of synapse development and plasticity. PMID:26105073

  12. A large-scale functional screen identifies Nova1 and Ncoa3 as regulators of neuronal miRNA function.

    PubMed

    Störchel, Peter H; Thümmler, Juliane; Siegel, Gabriele; Aksoy-Aksel, Ayla; Zampa, Federico; Sumer, Simon; Schratt, Gerhard

    2015-09-01

    MicroRNAs (miRNAs) are important regulators of neuronal development, network connectivity, and synaptic plasticity. While many neuronal miRNAs were previously shown to modulate neuronal morphogenesis, little is known regarding the regulation of miRNA function. In a large-scale functional screen, we identified two novel regulators of neuronal miRNA function, Nova1 and Ncoa3. Both proteins are expressed in the nucleus and the cytoplasm of developing hippocampal neurons. We found that Nova1 and Ncoa3 stimulate miRNA function by different mechanisms that converge on Argonaute (Ago) proteins, core components of the miRNA-induced silencing complex (miRISC). While Nova1 physically interacts with Ago proteins, Ncoa3 selectively promotes the expression of Ago2 at the transcriptional level. We further show that Ncoa3 regulates dendritic complexity and dendritic spine maturation of hippocampal neurons in a miRNA-dependent fashion. Importantly, both the loss of miRNA activity and increased dendrite complexity upon Ncoa3 knockdown were rescued by Ago2 overexpression. Together, we uncovered two novel factors that control neuronal miRISC function at the level of Ago proteins, with possible implications for the regulation of synapse development and plasticity. PMID:26105073

  13. Functional Genomic Screening Approaches in Mechanistic Toxicology and Potential Future Applications of CRISPR-Cas9

    PubMed Central

    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

  14. Fluorescence Polarization Screening Assays for Small Molecule Allosteric Modulators of ABL Kinase Function

    PubMed Central

    Grover, Prerna; Shi, Haibin; Baumgartner, Matthew; Camacho, Carlos J.; Smithgall, Thomas E.

    2015-01-01

    The ABL protein-tyrosine kinase regulates intracellular signaling pathways controlling diverse cellular processes and contributes to several forms of cancer. The kinase activity of ABL is repressed by intramolecular interactions involving its regulatory Ncap, SH3 and SH2 domains. Small molecules that allosterically regulate ABL kinase activity through its non-catalytic domains may represent selective probes of ABL function. Here we report a screening assay for chemical modulators of ABL kinase activity that target the regulatory interaction of the SH3 domain with the SH2-kinase linker. This fluorescence polarization (FP) assay is based on a purified recombinant ABL protein consisting of the N-cap, SH3 and SH2 domains plus the SH2-kinase linker (N32L protein) and a short fluorescein-labeled probe peptide that binds to the SH3 domain. In assay development experiments, we found that the probe peptide binds to the recombinant ABL N32L protein in vitro, producing a robust FP signal that can be competed with an excess of unlabeled peptide. The FP signal is not observed with control N32L proteins bearing either an inactivating mutation in the SH3 domain or enhanced SH3:linker interaction. A pilot screen of 1200 FDA-approved drugs identified four compounds that specifically reduced the FP signal by at least three standard deviations from the untreated controls. Secondary assays showed that one of these hit compounds, the antithrombotic drug dipyridamole, enhances ABL kinase activity in vitro to a greater extent than the previously described ABL agonist, DPH. Docking studies predicted that this compound binds to a pocket formed at the interface of the SH3 domain and the linker, suggesting that it activates ABL by disrupting this regulatory interaction. These results show that screening assays based on the non-catalytic domains of ABL can identify allosteric small molecule regulators of kinase function, providing a new approach to selective drug discovery for this important

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

    PubMed

    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

  16. Functional screen identifies kinases driving prostate cancer visceral and bone metastasis

    PubMed Central

    Faltermeier, Claire M.; Drake, Justin M.; Clark, Peter M.; Smith, Bryan A.; Zong, Yang; Volpe, Carmen; Mathis, Colleen; Morrissey, Colm; Castor, Brandon; Huang, Jiaoti; Witte, Owen N.

    2016-01-01

    Mutationally activated kinases play an important role in the progression and metastasis of many cancers. Despite numerous oncogenic alterations implicated in metastatic prostate cancer, mutations of kinases are rare. Several lines of evidence suggest that nonmutated kinases and their pathways are involved in prostate cancer progression, but few kinases have been mechanistically linked to metastasis. Using a mass spectrometry-based phosphoproteomics dataset in concert with gene expression analysis, we selected over 100 kinases potentially implicated in human metastatic prostate cancer for functional evaluation. A primary in vivo screen based on overexpression of candidate kinases in murine prostate cells identified 20 wild-type kinases that promote metastasis. We queried these 20 kinases in a secondary in vivo screen using human prostate cells. Strikingly, all three RAF family members, MERTK, and NTRK2 drove the formation of bone and visceral metastasis confirmed by positron-emission tomography combined with computed tomography imaging and histology. Immunohistochemistry of tissue microarrays indicated that these kinases are highly expressed in human metastatic castration-resistant prostate cancer tissues. Our functional studies reveal the strong capability of select wild-type protein kinases to drive critical steps of the metastatic cascade, and implicate these kinases in possible therapeutic intervention. PMID:26621741

  17. Functional screen identifies kinases driving prostate cancer visceral and bone metastasis.

    PubMed

    Faltermeier, Claire M; Drake, Justin M; Clark, Peter M; Smith, Bryan A; Zong, Yang; Volpe, Carmen; Mathis, Colleen; Morrissey, Colm; Castor, Brandon; Huang, Jiaoti; Witte, Owen N

    2016-01-12

    Mutationally activated kinases play an important role in the progression and metastasis of many cancers. Despite numerous oncogenic alterations implicated in metastatic prostate cancer, mutations of kinases are rare. Several lines of evidence suggest that nonmutated kinases and their pathways are involved in prostate cancer progression, but few kinases have been mechanistically linked to metastasis. Using a mass spectrometry-based phosphoproteomics dataset in concert with gene expression analysis, we selected over 100 kinases potentially implicated in human metastatic prostate cancer for functional evaluation. A primary in vivo screen based on overexpression of candidate kinases in murine prostate cells identified 20 wild-type kinases that promote metastasis. We queried these 20 kinases in a secondary in vivo screen using human prostate cells. Strikingly, all three RAF family members, MERTK, and NTRK2 drove the formation of bone and visceral metastasis confirmed by positron-emission tomography combined with computed tomography imaging and histology. Immunohistochemistry of tissue microarrays indicated that these kinases are highly expressed in human metastatic castration-resistant prostate cancer tissues. Our functional studies reveal the strong capability of select wild-type protein kinases to drive critical steps of the metastatic cascade, and implicate these kinases in possible therapeutic intervention. PMID:26621741

  18. Validity of the lower extremity functional movement screen in patients with chronic ankle instability

    PubMed Central

    Choi, Ho-Suk; Shin, Won-Seob

    2015-01-01

    [Purpose] The purpose of this study was to provide evidence of construct validity for the lower extremity functional movement screen (LE-FMS) based on hypothesis testing in patients with chronic ankle instability (CAI). [Subjects] The subjects were 20 healthy subjects and 20 patients with CAI who had a history of ankle sprain with pain for more than 1 day. [Methods] All participants were measured using the Foot and Ankle Disability Index (FADI) and evaluated with the LE-FMS. The screen included the deep squat, the hurdle step (HS) and the in-line lunge (ILL). The symmetry ratios (RS) were accurately measured during the deep squat trial. [Results] Between the two groups, there were significant differences in scores on the LE-FMS, HS, ILL, RS, FADI, and FADI-sport. The FADI was strongly correlated with both LE-FMS score (r=0.807) and ILL score (r=0.896). There was a strong relationship (r=0.818) between LE-FMS score and FADI-sport. [Conclusion] These results suggest that the LE-FMS may be used to detect deficits related to CAI. Additionally, this instrument is reliable in detecting functional limitations in patients with CAI. PMID:26180349

  19. Nickel-Resistance Determinants in Acidiphilium sp. PM Identified by Genome-Wide Functional Screening

    PubMed Central

    San Martin-Uriz, Patxi; Mirete, Salvador; Alcolea, Pedro J.; Gomez, Manuel J.; Amils, Ricardo; Gonzalez-Pastor, Jose E.

    2014-01-01

    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. PMID:24740277

  20. A function-based screen for seeking RubisCO active clones from metagenomes: novel enzymes influencing RubisCO activity

    PubMed Central

    Böhnke, Stefanie; Perner, Mirjam

    2015-01-01

    Ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) is a key enzyme of the Calvin cycle, which is responsible for most of Earth's primary production. Although research on RubisCO genes and enzymes in plants, cyanobacteria and bacteria has been ongoing for years, still little is understood about its regulation and activation in bacteria. Even more so, hardly any information exists about the function of metagenomic RubisCOs and the role of the enzymes encoded on the flanking DNA owing to the lack of available function-based screens for seeking active RubisCOs from the environment. Here we present the first solely activity-based approach for identifying RubisCO active fosmid clones from a metagenomic library. We constructed a metagenomic library from hydrothermal vent fluids and screened 1056 fosmid clones. Twelve clones exhibited RubisCO activity and the metagenomic fragments resembled genes from Thiomicrospira crunogena. One of these clones was further analyzed. It contained a 35.2 kb metagenomic insert carrying the RubisCO gene cluster and flanking DNA regions. Knockouts of twelve genes and two intergenic regions on this metagenomic fragment demonstrated that the RubisCO activity was significantly impaired and was attributed to deletions in genes encoding putative transcriptional regulators and those believed to be vital for RubisCO activation. Our new technique revealed a novel link between a poorly characterized gene and RubisCO activity. This screen opens the door to directly investigating RubisCO genes and respective enzymes from environmental samples. PMID:25203835

  1. A genome-wide screen for genes affecting eisosomes reveals Nce102 function in sphingolipid signaling

    PubMed Central

    Fröhlich, Florian; Moreira, Karen; Aguilar, Pablo S.; Hubner, Nina C.; Mann, Matthias; Walter, Peter

    2009-01-01

    The protein and lipid composition of eukaryotic plasma membranes is highly dynamic and regulated according to need. The sphingolipid-responsive Pkh kinases are candidates for mediating parts of this regulation, as they affect a diverse set of plasma membrane functions, such as cortical actin patch organization, efficient endocytosis, and eisosome assembly. Eisosomes are large protein complexes underlying the plasma membrane and help to sort a group of membrane proteins into distinct domains. In this study, we identify Nce102 in a genome-wide screen for genes involved in eisosome organization and Pkh kinase signaling. Nce102 accumulates in membrane domains at eisosomes where Pkh kinases also localize. The relative abundance of Nce102 in these domains compared with the rest of the plasma membrane is dynamically regulated by sphingolipids. Furthermore, Nce102 inhibits Pkh kinase signaling and is required for plasma membrane organization. Therefore, Nce102 might act as a sensor of sphingolipids that regulates plasma membrane function. PMID:19564405

  2. Screened exchange hybrid density functional for accurate and efficient structures and interaction energies.

    PubMed

    Brandenburg, Jan Gerit; Caldeweyher, Eike; Grimme, Stefan

    2016-06-21

    We extend the recently introduced PBEh-3c global hybrid density functional [S. Grimme et al., J. Chem. Phys., 2015, 143, 054107] by a screened Fock exchange variant based on the Henderson-Janesko-Scuseria exchange hole model. While the excellent performance of the global hybrid is maintained for small covalently bound molecules, its performance for computed condensed phase mass densities is further improved. Most importantly, a speed up of 30 to 50% can be achieved and especially for small orbital energy gap cases, the method is numerically much more robust. The latter point is important for many applications, e.g., for metal-organic frameworks, organic semiconductors, or protein structures. This enables an accurate density functional based electronic structure calculation of a full DNA helix structure on a single core desktop computer which is presented as an example in addition to comprehensive benchmark results. PMID:27240749

  3. Tailor-Made Pore Surface Engineering in Covalent Organic Frameworks: Systematic Functionalization for Performance Screening.

    PubMed

    Huang, Ning; Krishna, Rajamani; Jiang, Donglin

    2015-06-10

    Imine-linked covalent organic frameworks (COFs) were synthesized to bear content-tunable, accessible, and reactive ethynyl groups on the walls of one-dimensional pores. These COFs offer an ideal platform for pore-wall surface engineering aimed at anchoring diverse functional groups ranging from hydrophobic to hydrophilic units and from basic to acidic moieties with controllable loading contents. This approach enables the development of various tailor-made COFs with systematically tuned porosities and functionalities while retaining the crystallinity. We demonstrate that this strategy can be used to efficiently screen for suitable pore structures for use as CO2 adsorbents. The pore-surface-engineered walls exhibit an enhanced affinity for CO2, resulting in COFs that can capture and separate CO2 with high performance. PMID:26028183

  4. Functional analysis of HBO1 in tumor development and inhibitor screening.

    PubMed

    Guo, Ling-Li; Yu, Su-Yang; Li, Meng

    2016-07-01

    The aim of the present study was to explore the functions of histone acetyltransferase binding to origin recog-nition complex (ORC) 1 (HBO1) during tumor development and to screen for HBO1 inhibitors. The chromatin immuno-precipitation sequencing (ChIP-seq) data of HBO1 in the RKO human colon cancer cell line (GSE33007) were downloaded from the Gene Expression Omnibus (GEO) database. The reads were then mapped back to a reference genome hg19. The PCR duplicate reads were removed by using SAMtools software and the shift was calculated using SPP and MaSC software. The peak calling was carried out using MACS 1.4.0 software. Furthermore, the inhibitors of HBO1 were screened out from the Specs database using Dock 6.6 software. The binding sites of HBO1 were mainly distributed in the intergenic, intronic and 3'-end regions. Further analysis revealed that a total of 9,467 target genes was identified around HBO1 binding sites in the RKO cell lines and those genes mainly participated in the cell cycle, biosynthetic process, as well as other processes. Finally, 5 inhibitors with best binding affinity in the positively charged cavity of HBO1 were screened out: i) 5-[(2-hydroxybenzylidene)amino] -2-(2‑{4‑[(2‑hydroxy-benzylidene)amino]-2-sulfonatophenyl}vinyl)benzenesulfonate, ii) 3-[4-(3-bromo-4-{2-[4-(ethoxycarbonyl)anilino]-2-oxo-ethoxy}-5-methoxybenzylidene)‑3‑methyl‑5‑oxo -4,5-dihydro-1H-pyrazol-1-yl]benzoic acid, iii) 4-(4-{3-iodo‑5‑ methoxy‑4-[2-(2-methoxyanilino)-2-oxoethoxy]benzylidene}-3-methyl-5-oxo-4,5-dihydro-1H-pyrazol-1-yl)benzoic acid, iv) 5-chloro-1,3-bis{[3,5,6-trihydroxy-4-(octyloxy)tetrahydro-2H-pyran-2-yl]methyl}-1,3-dihydro-2H-benzimidazol-2-one and v) 4-{[4-(tetradecylamino)-1-naphthyl]diazenyl}benzoic acid. As a whole, in this study, we identified the possible binding sites and biological functions of HBO1. The potential inhibitors of HBO1 were also screened, which prove to be helpful for the inhibition of HBO1 during

  5. Screening and functional pathway analysis of genes associated with pediatric allergic asthma using a DNA microarray

    PubMed Central

    LU, LI-QUN; LIAO, WEI

    2015-01-01

    The present study aimed to identify differentially expressed genes (DEGs) associated with pediatric allergic asthma, and to analyze the functional pathways of the selected target genes, in order to explore the pathogenesis of the disease. The GSE18965 gene expression profile was downloaded from the Gene Expression Omnibus database and was preprocessed. This gene expression profile consisted of seven normal samples and nine samples from patients with pediatric allergic asthma. The DEGs between the normal and pediatric allergic asthma samples were screened using limma package in R, and the cut-off value was set at false discovery rate <0.05 and log fold change >1. Following hierarchical clustering of the DEGs based on the expression profiles, the up- and downregulated genes underwent a functional enrichment analysis by topological approach (P<0.05), using the Database for Annotation, Visualization and Integrated Discovery. A total of 127 DEGs were identified between the normal and pediatric allergic asthma samples. The up- and downregulated genes were significantly enriched in the actin filament-based process and the monosaccharide metabolic process, respectively. Seven downregulated DEGs (M6PR, TPP1, GLB1, NEU1, ACP2, LAMP1 and HGSNAT) were identified in the lysosomal pathway, with P=6.4×10−9. These results suggested that variation in lysosomal function, triggered by the seven downregulated genes, may lead to aberrant functioning of the T lymphocytes, resulting in asthma. Further research regarding the treatment of pediatric allergic asthma through targeting lysosomal function is required. PMID:25633562

  6. Facilitating Structure-Function Studies of CFTR Modulator Sites with Efficiencies in Mutagenesis and Functional Screening.

    PubMed

    Molinski, Steven V; Ahmadi, Saumel; Hung, Maurita; Bear, Christine E

    2015-12-01

    There are nearly 2000 mutations in the CFTR gene associated with cystic fibrosis disease, and to date, the only approved drug, Kalydeco, has been effective in rescuing the functional expression of a small subset of these mutant proteins with defects in channel activation. However, there is currently an urgent need to assess other mutations for possible rescue by Kalydeco, and further, definition of the binding site of such modulators on CFTR would enhance our understanding of the mechanism of action of such therapeutics. Here, we describe a simple and rapid one-step PCR-based site-directed mutagenesis method to generate mutations in the CFTR gene. This method was used to generate CFTR mutants bearing deletions (p.Gln2_Trp846del, p.Ser700_Asp835del, p.Ile1234_Arg1239del) and truncation with polyhistidine tag insertion (p.Glu1172-3Gly-6-His*), which either recapitulate a disease phenotype or render tools for modulator binding site identification, with subsequent evaluation of drug responses using a high-throughput (384-well) membrane potential-sensitive fluorescence assay of CFTR channel activity within a 1 wk time frame. This proof-of-concept study shows that these methods enable rapid and quantitative comparison of multiple CFTR mutants to emerging drugs, facilitating future large-scale efforts to stratify mutants according to their "theratype" or most promising targeted therapy. PMID:26385858

  7. Functional movement screen differences between male and female secondary school athletes.

    PubMed

    Anderson, Barton E; Neumann, Matthew L; Huxel Bliven, Kellie C

    2015-04-01

    The functional movement screen (FMS) is commonly used to assess movement capacity and determine injury risk. Evidence suggests that athletes who score 14 points or less on the FMS are at increased risk for injury, but differences between males and females have been minimally studied. The purpose of this study was to investigate sex differences in FMS scores of secondary school athletes. Using a cross-sectional study design, 60 healthy secondary school athletes performed the FMS, which is composed of 7 functional movement tasks (deep squat, hurdle step, inline lunge, shoulder mobility, active straight-leg raise, trunk stability push-up, and rotary stability) and 3 clearance screens. Dependent variables were FMS total composite score and individual task scores; secondary analyses were performed using total research score and individual task research scores when indicated. Lower scores indicated functional movement deficits and increased injury risk. Healthy secondary school female athletes scored lower on the total composite (p = 0.004) than healthy secondary school male athletes. Females also scored lower on the following individual FMS tasks: inline lunge (p < 0.04) and trunk stability push-up (p = 0.001). Healthy secondary school female athletes scored 14 or less on the FMS total composite score and significantly lower in general compared with healthy secondary school male athletes, which suggests these female athletes may be at higher risk for injury. Factors that may contribute to increased injury risk include deficits in mobility, core stabilization, and coordinated movement patterns. Clinicians should be aware of possible sex differences when using the FMS and developing injury prevention programs. PMID:25330082

  8. Use of large-scale expression cloning screens in the Xenopus laevis tadpole to identify gene function.

    PubMed

    Grammer, T C; Liu, K J; Mariani, F V; Harland, R M

    2000-12-15

    We have conducted an expression cloning screen of approximately 50, 000 cDNAs from a tadpole stage Xenopus laevis cDNA library to functionally identify genes affecting a wide range of cellular and developmental processes. Fifty-seven cDNAs were isolated for their ability to alter gross tadpole morphology or the expression patterns of tissue-specific markers. Thirty-seven of the cDNAs have not been previously described for Xenopus, and 15 of these show little or no similarity to sequences in the NCBI database. The screen and the identified genes are presented in this paper to demonstrate the power, ease, speed, and flexibility of expression cloning in the X. laevis embryo. Future screens such as this one can be done on a larger scale and will complement the sequence-based screens and genome-sequencing projects which are producing a large body of novel genes without ascribed functions. PMID:11112324

  9. A screen for genes that function in abscisic acid signaling in Arabidopsis thaliana.

    PubMed Central

    Nambara, Eiji; Suzuki, Masaharu; Abrams, Suzanne; McCarty, Donald R; Kamiya, Yuji; McCourt, Peter

    2002-01-01

    The plant hormone abscisic acid (ABA) controls many aspects of plant growth and development under a diverse range of environmental conditions. To identify genes functioning in ABA signaling, we have carried out a screen for mutants that takes advantage of the ability of wild-type Arabidopsis seeds to respond to (-)-(R)-ABA, an enantiomer of the natural (+)-(S)-ABA. The premise of the screen was to identify mutations that preferentially alter their germination response in the presence of one stereoisomer vs. the other. Twenty-six mutants were identified and genetic analysis on 23 lines defines two new loci, designated CHOTTO1 and CHOTTO2, and a collection of new mutant alleles of the ABA-insensitive genes, ABI3, ABI4, and ABI5. The abi5 alleles are less sensitive to (+)-ABA than to (-)-ABA. In contrast, the abi3 alleles exhibit a variety of differences in response to the ABA isomers. Genetic and molecular analysis of these alleles suggests that the ABI3 transcription factor may perceive multiple ABA signals. PMID:12136027

  10. Value of phagocyte function screening for immunotoxicity of nanoparticles in vivo.

    PubMed

    Fröhlich, Eleonore

    2015-01-01

    Nanoparticles (NPs) present in the environment and in consumer products can cause immunotoxic effects. The immune system is very complex, and in vivo studies are the gold standard for evaluation. Due to the increased amount of NPs that are being developed, cellular screening assays to decrease the amount of NPs that have to be tested in vivo are highly needed. Effects on the unspecific immune system, such as effects on phagocytes, might be suitable for screening for immunotoxicity because these cells mediate unspecific and specific immune responses. They are present at epithelial barriers, in the blood, and in almost all organs. This review summarizes the effects of carbon, metal, and metal oxide NPs used in consumer and medical applications (gold, silver, titanium dioxide, silica dioxide, zinc oxide, and carbon nanotubes) and polystyrene NPs on the immune system. Effects in animal exposures through different routes are compared to the effects on isolated phagocytes. In addition, general problems in the testing of NPs, such as unknown exposure doses, as well as interference with assays are mentioned. NPs appear to induce a specific immunotoxic pattern consisting of the induction of inflammation in normal animals and aggravation of pathologies in disease models. The evaluation of particle action on several phagocyte functions in vitro may provide an indication on the potency of the particles to induce immunotoxicity in vivo. In combination with information on realistic exposure levels, in vitro studies on phagocytes may provide useful information on the health risks of NPs. PMID:26060398

  11. Complex-scaling of screened Coulomb potentials for resonance calculations utilizing the modified Bessel functions

    NASA Astrophysics Data System (ADS)

    Jiao, Li-Guang; Ho, Yew Kam

    2014-05-01

    The screened Coulomb potential (SCP) has been extensively used in atomic physics, nuclear physics, quantum chemistry and plasma physics. However, an accurate calculation for atomic resonances under SCP is still a challenging task for various methods. Within the complex-scaling computational scheme, we have developed a method utilizing the modified Bessel functions to calculate doubly-excited resonances in two-electron atomic systems with configuration interaction-type basis. To test the validity of our method, we have calculated S- and P-wave resonance states of the helium atom with various screening strengths, and have found good agreement with earlier calculations using different methods. Our present method can be applied to calculate high-lying resonances associated with high excitation thresholds of the He+ ion, and with high-angular-momentum states. The derivation and calculation details of our present investigation together with new results of high-angular-momentum states will be presented at the meeting. Supported by NSC of Taiwan.

  12. Characterization of MODIS SD screen vignetting function using observations from spacecraft yaw maneuvers

    NASA Astrophysics Data System (ADS)

    Wang, Zhipeng; Xiong, Xiaoxiong

    2009-08-01

    The MODIS reflective solar bands (RSB) include both the low-gain and high-gain spectral bands depending on their specific applications. MODIS RSBs are calibrated on-orbit by an on-board solar diffuser. In order to avoid detector response saturation when calibrating the high-gain bands, an optional attenuation screen, made of a metal plate with pinhole arrays, is placed in front of the SD panel. Since no pre-launch system-level characterization was made for the SD screen (SDS) vignetting function (VF), a series of spacecraft (Terra and Aqua) yaw maneuvers were carried out to perform on-orbit characterization of the VF. Assuming that the low-gain bands and the high-gain bands have the same VF, the current VF was derived from yaw observations using the MODIS low-gain bands through taking the ratio of their SD responses with and without the SDS in place. In this study, we attempt to characterize the SDS VF directly using detector responses of individual high-gain bands with the SDS in place only. The corresponding SD responses without the SDS, not available from measurements due to saturation, are calculated using detector gains, the SD bi-directional reflectance factor (BRF), and the view geometry that matches the yaw observations with the SDS in place. Results and discussions are focused on the band dependent and detector dependent features of the SDS VF, and their potential impact on the RSB calibration.

  13. Functional movement ScreenTM--normative values in healthy distance runners.

    PubMed

    Agresta, C; Slobodinsky, M; Tucker, C

    2014-12-01

    Recreational runners have an estimated overuse injury incidence rate of up to 79% and 90% for marathoners. A pre-participation screening tool that can identify risk for injury may help reduce overuse injury in runners. The Functional Movement Screen (FMS(TM)) is a reliable clinical tool used with athletes to help predict injury. To date, the FMS(TM) has not been used with endurance athletes. The purpose of this article is to establish normative FMS(TM) values for distance runners. 45 healthy runners performed the FMS(TM). Descriptive statistics were calculated; independent t-tests were performed to examine the effect of gender, experience and injury on scores. A Chi-square test was used to evaluate whether significant differences in scores exist for any component of the FMS(TM). The mean FMS(TM) score was 13.13±1.8. No significant differences in FMS(TM) scores were found between novice and experienced runners (p=0.71) or runners with a history of injury and those without (p=0.20). While male and female runners did not differ significantly in their total FMS(TM) score (p=0.65), significant differences were found in the deep squat (p<0.05), trunk stability push-up (p<0.001) and active straight leg raise components (p=0.002). This study provides normative values for FMS(TM) scores when testing uninjured distance runners. PMID:25144432

  14. Value of phagocyte function screening for immunotoxicity of nanoparticles in vivo

    PubMed Central

    Fröhlich, Eleonore

    2015-01-01

    Nanoparticles (NPs) present in the environment and in consumer products can cause immunotoxic effects. The immune system is very complex, and in vivo studies are the gold standard for evaluation. Due to the increased amount of NPs that are being developed, cellular screening assays to decrease the amount of NPs that have to be tested in vivo are highly needed. Effects on the unspecific immune system, such as effects on phagocytes, might be suitable for screening for immunotoxicity because these cells mediate unspecific and specific immune responses. They are present at epithelial barriers, in the blood, and in almost all organs. This review summarizes the effects of carbon, metal, and metal oxide NPs used in consumer and medical applications (gold, silver, titanium dioxide, silica dioxide, zinc oxide, and carbon nanotubes) and polystyrene NPs on the immune system. Effects in animal exposures through different routes are compared to the effects on isolated phagocytes. In addition, general problems in the testing of NPs, such as unknown exposure doses, as well as interference with assays are mentioned. NPs appear to induce a specific immunotoxic pattern consisting of the induction of inflammation in normal animals and aggravation of pathologies in disease models. The evaluation of particle action on several phagocyte functions in vitro may provide an indication on the potency of the particles to induce immunotoxicity in vivo. In combination with information on realistic exposure levels, in vitro studies on phagocytes may provide useful information on the health risks of NPs. PMID:26060398

  15. Tissue matrix arrays for high throughput screening and systems analysis of cell function

    PubMed Central

    Beachley, Vince Z.; Wolf, Matthew T.; Sadtler, Kaitlyn; Manda, Srikanth S.; Jacobs, Heather; Blatchley, Michael; Bader, Joel S.; Pandey, Akhilesh; Pardoll, Drew; Elisseeff, Jennifer H.

    2015-01-01

    Cell and protein arrays have demonstrated remarkable utility in the high-throughput evaluation of biological responses; however, they lack the complexity of native tissue and organs. Here, we describe tissue extracellular matrix (ECM) arrays for screening biological outputs and systems analysis. We spotted processed tissue ECM particles as two-dimensional arrays or incorporated them with cells to generate three-dimensional cell-matrix microtissue arrays. We then investigated the response of human stem, cancer, and immune cells to tissue ECM arrays originating from 11 different tissues, and validated the 2D and 3D arrays as representative of the in vivo microenvironment through quantitative analysis of tissue-specific cellular responses, including matrix production, adhesion and proliferation, and morphological changes following culture. The biological outputs correlated with tissue proteomics, and network analysis identified several proteins linked to cell function. Our methodology enables broad screening of ECMs to connect tissue-specific composition with biological activity, providing a new resource for biomaterials research and translation. PMID:26480475

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

    PubMed Central

    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

    2012-01-01

    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. PMID:21482774

  17. G-centers in irradiated silicon revisited: A screened hybrid density functional theory approach

    SciTech Connect

    Wang, H.; Schwingenschlögl, U.; Chroneos, A.; Londos, C. A.; Sgourou, E. N.

    2014-05-14

    Electronic structure calculations employing screened hybrid density functional theory are used to gain fundamental insight into the interaction of carbon interstitial (C{sub i}) and substitutional (C{sub s}) atoms forming the C{sub i}C{sub s} defect known as G-center in silicon (Si). The G-center is one of the most important radiation related defects in Czochralski grown Si. We systematically investigate the density of states and formation energy for different types of C{sub i}C{sub s} defects with respect to the Fermi energy for all possible charge states. Prevalence of the neutral state for the C-type defect is established.

  18. The nylon column dye test: a possible screening test of phagocyte function.

    PubMed

    Segal, A W; Peters, T J

    1975-12-01

    1. A simple quantitative test has been developed to investigate phagocyte function. 2. This test is performed by the addition of Nitroblue Tetrazolium to whole blood, followed by the isolation of leucocytes on a column of nylon wool. Dye reduction by phagocytes is apparent as a blue coloration of the column due to the formation of formazan. The formazan can be extracted from the column and measured spectrophotometrically. 3. The formation of formazan was found to be directly related to the number of phagocytes in blood. 4. Two patients with chronic granulomatous disease gave abnormal results, suggesting that the test procedure may be of value as a screening procedure for this disease. PMID:1106938

  19. Impact ionization in GaAs: A screened exchange density-functional approach

    SciTech Connect

    Picozzi, S.; Asahi, R.; Geller, C. B.; Continenza, A.; Freeman, A. J.

    2001-08-13

    Results are presented of a fully ab initio calculation of impact ionization rates in GaAs within the density functional theory framework, using a screened-exchange formalism and the highly precise all-electron full-potential linearized augmented plane wave method. The calculated impact ionization rates show a marked orientation dependence in k space, indicating the strong restrictions imposed by the conservation of energy and momentum. This anisotropy diminishes as the impacting electron energy increases. A Keldysh type fit performed on the energy-dependent rate shows a rather soft edge and a threshold energy greater than the direct band gap. The consistency with available Monte Carlo and empirical pseudopotential calculations shows the reliability of our approach and paves the way to ab initio calculations of pair production rates in new and more complex materials.

  20. A functional genomic screen in planarians identifies novel regulators of germ cell development

    PubMed Central

    Wang, Yuying; Stary, Joel M.; Wilhelm, James E.; Newmark, Phillip A.

    2010-01-01

    Germ cells serve as intriguing examples of differentiated cells that retain the capacity to generate all cell types of an organism. Here we used functional genomic approaches in planarians to identify genes required for proper germ cell development. We conducted microarray analyses and in situ hybridization to discover and validate germ cell-enriched transcripts, and then used RNAi to screen for genes required for discrete stages of germ cell development. The majority of genes we identified encode conserved RNA-binding proteins, several of which have not been implicated previously in germ cell development. We also show that a germ cell-specific subunit of the conserved transcription factor CCAAT-binding protein/nuclear factor-Y is required for maintaining spermatogonial stem cells. Our results demonstrate that conserved transcriptional and post-transcriptional mechanisms regulate germ cell development in planarians. These findings suggest that studies of planarians will inform our understanding of germ cell biology in higher organisms. PMID:20844018

  1. The Student Risk Screening Scale: Exploring Dimensionality and Differential Item Functioning

    ERIC Educational Resources Information Center

    Schatschneider, Christopher; Lane, Kathleen Lynne; Oakes, Wendy Peia; Kalberg, Jemma Robertson

    2014-01-01

    Screening of students at risk for antisocial behaviors in school is an essential step in the implementation of evidence-based supports for academic, behavioral, and social domains at the first sign of concern. This study examined the measurement properties of a free-access systematic behavior screening tool: the Student Risk Screening Scale…

  2. Effects of interlayer screening and temperature on dielectric functions of graphene by first-principles

    NASA Astrophysics Data System (ADS)

    Yang, J. Y.; Liu, L. H.

    2016-07-01

    The dielectric functions of few-layer graphene and the related temperature dependence are investigated from the atomic scale using first-principles calculations. Compared with ellipsometry experiments in the spectral range of 190-2500 nm, the normalized optical constants of mono-layer graphene demonstrate good agreement and further validate first-principles calculations. To interpret dielectric function of mono-layer graphene, the electronic band structure and density of states are analyzed. By comparing dielectric functions of mono-, bi-, and tri-layer graphene, it shows that interlayer screening strengthens intraband transition and greatly enhances the absorption peak located around 1 eV. The strengthened optical absorption is intrinsically caused by the increasing electron states near the Fermi level. To investigate temperature effect, the first-principles calculations and lattice dynamics are combined. The lattice vibration enhances parallel optical absorption peak around 1 eV and induces redshift. Moreover, it is observed that the van der Waals force plays a key role in keeping the interlayer distance stable during dynamics simulations.

  3. Urodynamics: a noninvasive screening of lower urinary tract function in children with radioisotopes.

    PubMed

    van der Vis-Melsen, M J; Baert, R J; Rajnherc, J R; Groen, J M; Benelmans, L M; De Nef, J J

    1988-01-01

    A noninvasive intravenous assessment of lower urinary tract function with 123I-hippurate was carried out in 129 children without suspicion of lower urinary tract outflow pathology. Without increasing the radiation burden standard renography was extended by lower urinary tract function analysis in the same session. The maximum bladder capacity, voiding and residual bladder volumes, average and maximum urine flow rates were calculated and the relation between the urine flow rate and bladder volume expressed as the index of urine transport (IUT). This index seems to be a much more reliable standard than isolated measurements of urine flow rates and bladder volumes in screening lower urinary tract function. Three different urine flow patterns were recognized: a single sharp peak (in 70%) and a biphasic curve (in 20%) were considered to be normal. A sawtooth-shaped pattern, observed in 10% of the children, may be caused by detrusor malfunction. The prolonged time necessary for this more extensive analysis of the lower urinary tract is well compensated by the additional information gained. PMID:3386971

  4. An Evolution-Based Screen for Genetic Differentiation between Anopheles Sister Taxa Enriches for Detection of Functional Immune Factors

    PubMed Central

    Takashima, Eizo; Williams, Marni; Eiglmeier, Karin; Pain, Adrien; Guelbeogo, Wamdaogo M.; Gneme, Awa; Brito-Fravallo, Emma; Holm, Inge; Lavazec, Catherine; Sagnon, N’Fale; Baxter, Richard H.; Riehle, Michelle M.; Vernick, Kenneth D.

    2015-01-01

    Nucleotide variation patterns across species are shaped by the processes of natural selection, including exposure to environmental pathogens. We examined patterns of genetic variation in two sister species, Anopheles gambiae and Anopheles coluzzii, both efficient natural vectors of human malaria in West Africa. We used the differentiation signature displayed by a known coordinate selective sweep of immune genes APL1 and TEP1 in A. coluzzii to design a population genetic screen trained on the sweep, classified a panel of 26 potential immune genes for concordance with the signature, and functionally tested their immune phenotypes. The screen results were strongly predictive for genes with protective immune phenotypes: genes meeting the screen criteria were significantly more likely to display a functional phenotype against malaria infection than genes not meeting the criteria (p = 0.0005). Thus, an evolution-based screen can efficiently prioritize candidate genes for labor-intensive downstream functional testing, and safely allow the elimination of genes not meeting the screen criteria. The suite of immune genes with characteristics similar to the APL1-TEP1 selective sweep appears to be more widespread in the A. coluzzii genome than previously recognized. The immune gene differentiation may be a consequence of adaptation of A. coluzzii to new pathogens encountered in its niche expansion during the separation from A. gambiae, although the role, if any of natural selection by Plasmodium is unknown. Application of the screen allowed identification of new functional immune factors, and assignment of new functions to known factors. We describe biochemical binding interactions between immune proteins that underlie functional activity for malaria infection, which highlights the interplay between pathogen specificity and the structure of immune complexes. We also find that most malaria-protective immune factors display phenotypes for either human or rodent malaria, with

  5. An Evolution-Based Screen for Genetic Differentiation between Anopheles Sister Taxa Enriches for Detection of Functional Immune Factors.

    PubMed

    Mitri, Christian; Bischoff, Emmanuel; Takashima, Eizo; Williams, Marni; Eiglmeier, Karin; Pain, Adrien; Guelbeogo, Wamdaogo M; Gneme, Awa; Brito-Fravallo, Emma; Holm, Inge; Lavazec, Catherine; Sagnon, N'Fale; Baxter, Richard H; Riehle, Michelle M; Vernick, Kenneth D

    2015-12-01

    Nucleotide variation patterns across species are shaped by the processes of natural selection, including exposure to environmental pathogens. We examined patterns of genetic variation in two sister species, Anopheles gambiae and Anopheles coluzzii, both efficient natural vectors of human malaria in West Africa. We used the differentiation signature displayed by a known coordinate selective sweep of immune genes APL1 and TEP1 in A. coluzzii to design a population genetic screen trained on the sweep, classified a panel of 26 potential immune genes for concordance with the signature, and functionally tested their immune phenotypes. The screen results were strongly predictive for genes with protective immune phenotypes: genes meeting the screen criteria were significantly more likely to display a functional phenotype against malaria infection than genes not meeting the criteria (p = 0.0005). Thus, an evolution-based screen can efficiently prioritize candidate genes for labor-intensive downstream functional testing, and safely allow the elimination of genes not meeting the screen criteria. The suite of immune genes with characteristics similar to the APL1-TEP1 selective sweep appears to be more widespread in the A. coluzzii genome than previously recognized. The immune gene differentiation may be a consequence of adaptation of A. coluzzii to new pathogens encountered in its niche expansion during the separation from A. gambiae, although the role, if any of natural selection by Plasmodium is unknown. Application of the screen allowed identification of new functional immune factors, and assignment of new functions to known factors. We describe biochemical binding interactions between immune proteins that underlie functional activity for malaria infection, which highlights the interplay between pathogen specificity and the structure of immune complexes. We also find that most malaria-protective immune factors display phenotypes for either human or rodent malaria, with

  6. Construction and functional screening of a metagenomic library using a T7 RNA polymerase-based expression cosmid vector.

    PubMed

    Lussier, François-Xavier; Chambenoit, Olivier; Côté, Amélie; Hupé, Jean-François; Denis, François; Juteau, Pierre; Beaudet, Réjean; Shareck, François

    2011-09-01

    The metagenomic approach has greatly accelerated the discovery of new enzymes by giving access to the genetic potential of microorganisms from various environments. Function-based screening depends on adequate expression of the foreign genes in the heterologous host, which can be challenging in large-insert libraries. In this study, the shuttle cosmid vector pFX583 was used for the construction and screening of a metagenomic library. This vector allows T7 RNA polymerase-directed transcription of the cloned DNA and can be used in Escherichia coli and Streptomyces lividans. The DNA used for the library construction was obtained from an enriched biomass. The library was screened for lipolytic and proteolytic activities using E. coli and S. lividans as hosts. Numerous E. coli clones with lipolytic activity were detected. Unfortunately, proteases could not be detected in both hosts. From the lipolytic activity screen, a gene coding for a new lipase was isolated, and partial characterization was conducted. PMID:21108039

  7. Risk miRNA screening of ovarian cancer based on miRNA functional synergistic network

    PubMed Central

    2014-01-01

    Background miRNAs are proved to have causal roles in tumorgenesis involving various types of human cancers, but the mechanism is not clear. We aimed to explore the effect of miRNAs on the development of ovarian cancer and the underlying mechanism. Methods The miRNA expression profile GSE31801 was downloaded from GEO (Gene Expression Omnibus) database. Firstly, the differentially expressed miRNAs were screened. Target genes of the miRNAs were collected from TargetScan, PicTar, miRanda, and DIANA-microT database, then the miRNA-miRNA co-regulating network was constructed using miRNA pairs with common regulated target genes. Next, the functional modules in the network were studied, the miRNA pairs regulated at least one modules were enriched to form the miRNA functional synergistic network (MFSN). Results Risk miRNA were selected in MFSN according to the topological structure. Transcript factors (TFs) in MFSN were identified, followed by the miRNA-transcript factor networks construction. Totally, 42 up- and 61 down-regulated differentially expressed miRNAs were identified, of which 68 formed 2292 miRNA pairs in the miRNA-miRNA co-regulating network. GO: 0007268 (synaptic transmission) and GO: 0019226 (transmission of nerve impulse) were the two common functions of miRNAs in MFSN, and hsa-miR-579 (36), hsa-miR-942 (31), hsa-miR-105 (31), hsa-miR-150 (34), and hsa-miR-27a* (32) were selected as the hub nodes in MFSN. Conclusions In all, 17 TFs, including CREM, ERG, and CREB1 were screened as the cancer related TFs in MFSN. Other TFs, such as BIN1, FOXN3, FOXK1, FOXP2, and ESRRG with high degrees may be inhibited in ovarian cancer. MFSN gave us a new shed light on the mechanism studies in ovarian cancer. PMID:24444095

  8. Design and Synthesis of Activity-Based Probes and Inhibitors for Bleomycin Hydrolase.

    PubMed

    van der Linden, Wouter A; Segal, Ehud; Child, Matthew A; Byzia, Anna; Drąg, Marcin; Bogyo, Matthew

    2015-08-20

    Bleomycin hydrolase (BLMH) is a neutral cysteine aminopeptidase that has been ascribed roles in many physiological and pathological processes, yet its primary biological function remains enigmatic. In this work, we describe the results of screening of a library of fluorogenic substrates to identify non-natural amino acids that are optimally recognized by BLMH. This screen identified several substrates with kcat/KM values that are substantially improved over the previously reported fluorogenic substrates for this enzyme. The substrate sequences were used to design activity-based probes that showed potent labeling of recombinant BLMH as well as endogenously expressed BLMH in cell extracts, and in intact cells. Importantly, we identify potent BLMH inhibitors that are able to fully inhibit endogenous BLMH activity in intact cells. These probes and inhibitors will be valuable new reagents to study BLMH function in cellular and animal models of human diseases where BLMH is likely to be involved. PMID:26256478

  9. A screen for hydroxymethylcytosine and formylcytosine binding proteins suggests functions in transcription and chromatin regulation

    PubMed Central

    2013-01-01

    Background DNA methylation (5mC) plays important roles in epigenetic regulation of genome function. Recently, TET hydroxylases have been found to oxidise 5mC to hydroxymethylcytosine (5hmC), formylcytosine (5fC) and carboxylcytosine (5caC) in DNA. These derivatives have a role in demethylation of DNA but in addition may have epigenetic signaling functions in their own right. A recent study identified proteins which showed preferential binding to 5-methylcytosine (5mC) and its oxidised forms, where readers for 5mC and 5hmC showed little overlap, and proteins bound to further oxidation forms were enriched for repair proteins and transcription regulators. We extend this study by using promoter sequences as baits and compare protein binding patterns to unmodified or modified cytosine using DNA from mouse embryonic stem cell extracts. Results We compared protein enrichments from two DNA probes with different CpG composition and show that, whereas some of the enriched proteins show specificity to cytosine modifications, others are selective for both modification and target sequences. Only a few proteins were identified with a preference for 5hmC (such as RPL26, PRP8 and the DNA mismatch repair protein MHS6), but proteins with a strong preference for 5fC were more numerous, including transcriptional regulators (FOXK1, FOXK2, FOXP1, FOXP4 and FOXI3), DNA repair factors (TDG and MPG) and chromatin regulators (EHMT1, L3MBTL2 and all components of the NuRD complex). Conclusions 0ur screen has identified novel proteins that bind to 5fC in genomic sequences with different CpG composition and suggests they regulate transcription and chromatin, hence opening up functional investigations of 5fC readers. PMID:24156278

  10. A functional screen for copper homeostasis genes identifies a pharmacologically tractable cellular system

    PubMed Central

    2014-01-01

    Background Copper is essential for the survival of aerobic organisms. If copper is not properly regulated in the body however, it can be extremely cytotoxic and genetic mutations that compromise copper homeostasis result in severe clinical phenotypes. Understanding how cells maintain optimal copper levels is therefore highly relevant to human health. Results We found that addition of copper (Cu) to culture medium leads to increased respiratory growth of yeast, a phenotype which we then systematically and quantitatively measured in 5050 homozygous diploid deletion strains. Cu’s positive effect on respiratory growth was quantitatively reduced in deletion strains representing 73 different genes, the function of which identify increased iron uptake as a cause of the increase in growth rate. Conversely, these effects were enhanced in strains representing 93 genes. Many of these strains exhibited respiratory defects that were specifically rescued by supplementing the growth medium with Cu. Among the genes identified are known and direct regulators of copper homeostasis, genes required to maintain low vacuolar pH, and genes where evidence supporting a functional link with Cu has been heretofore lacking. Roughly half of the genes are conserved in man, and several of these are associated with Mendelian disorders, including the Cu-imbalance syndromes Menkes and Wilson’s disease. We additionally demonstrate that pharmacological agents, including the approved drug disulfiram, can rescue Cu-deficiencies of both environmental and genetic origin. Conclusions A functional screen in yeast has expanded the list of genes required for Cu-dependent fitness, revealing a complex cellular system with implications for human health. Respiratory fitness defects arising from perturbations in this system can be corrected with pharmacological agents that increase intracellular copper concentrations. PMID:24708151

  11. A Preliminary Screening Program to Identify Functioning Strengths and Weaknesses in Preschool Children.

    ERIC Educational Resources Information Center

    Amundson, Marian Stuehrenberg

    The purpose of this study was to compare two instruments for screening preschool children for potential learning problems. The two instruments used were the Metropolitan Readiness Tests (MRT) and the Wizard of Oz Preschool Preliminary Screening Program. The children tested on both measures were members of a self-contained kindergarten class. MRT…

  12. Associations Between Functional Movement Screening, the Y Balance Test, and Injuries in Coast Guard Training.

    PubMed

    Cosio-Lima, Ludmila; Knapik, Joseph J; Shumway, Richard; Reynolds, Katy; Lee, Youngil; Greska, Eric; Hampton, Michael

    2016-07-01

    Tests that have the ability to predict injuries in various military and athletic populations are important because of the role they could play in primary prevention. Functional Movement Screen (FMS) and Y Balance Tests (YBT) may provide this prognostic ability. This study examined the association between injuries and age, physical characteristics, FMS, and upper and lower body YBTs among Coast Guard Maritime Security Response Team (MSRT) candidates. Thirty-one male Coast Guard Maritime Security Response Team candidates were administered the 7 FMS tests and lower- and upper-body YBTs before their intense 2-month training course. Age, height, weight, and body mass index were also obtained. Physical training-related injuries were recorded during the course. Injury incidence was 41%. Older age and lower scores on either FMS or the upper-body YBT were associated with higher injury risk. Performance of the lower-body YBT was not associated with injury risk. This is the first investigation showing that lower scores on the upper-body YBT were associated with higher injury risk and is in consonance with previous investigations demonstrating associations between lower FMS scores and higher injury risk. Certain limitations need to be addressed. Future studies should determine if FMS and the YBTs have prognostic ability in other populations. PMID:27391617

  13. Functional genomic screening reveals asparagine dependence as a metabolic vulnerability in sarcoma

    PubMed Central

    Hettmer, Simone; Schinzel, Anna C; Tchessalova, Daria; Schneider, Michaela; Parker, Christina L; Bronson, Roderick T; Richards, Nigel GJ; Hahn, William C; Wagers, Amy J

    2015-01-01

    Current therapies for sarcomas are often inadequate. This study sought to identify actionable gene targets by selective targeting of the molecular networks that support sarcoma cell proliferation. Silencing of asparagine synthetase (ASNS), an amidotransferase that converts aspartate into asparagine, produced the strongest inhibitory effect on sarcoma growth in a functional genomic screen of mouse sarcomas generated by oncogenic Kras and disruption of Cdkn2a. ASNS silencing in mouse and human sarcoma cell lines reduced the percentage of S phase cells and impeded new polypeptide synthesis. These effects of ASNS silencing were reversed by exogenous supplementation with asparagine. Also, asparagine depletion via the ASNS inhibitor amino sulfoximine 5 (AS5) or asparaginase inhibited mouse and human sarcoma growth in vitro, and genetic silencing of ASNS in mouse sarcoma cells combined with depletion of plasma asparagine inhibited tumor growth in vivo. Asparagine reliance of sarcoma cells may represent a metabolic vulnerability with potential anti-sarcoma therapeutic value. DOI: http://dx.doi.org/10.7554/eLife.09436.001 PMID:26499495

  14. Functional Screening of Antibiotic Resistance Genes from a Representative Metagenomic Library of Food Fermenting Microbiota

    PubMed Central

    Devirgiliis, Chiara; Barile, Simona; Perozzi, Giuditta

    2014-01-01

    Lactic acid bacteria (LAB) represent the predominant microbiota in fermented foods. Foodborne LAB have received increasing attention as potential reservoir of antibiotic resistance (AR) determinants, which may be horizontally transferred to opportunistic pathogens. We have previously reported isolation of AR LAB from the raw ingredients of a fermented cheese, while AR genes could be detected in the final, marketed product only by PCR amplification, thus pointing at the need for more sensitive microbial isolation techniques. We turned therefore to construction of a metagenomic library containing microbial DNA extracted directly from the food matrix. To maximize yield and purity and to ensure that genomic complexity of the library was representative of the original bacterial population, we defined a suitable protocol for total DNA extraction from cheese which can also be applied to other lipid-rich foods. Functional library screening on different antibiotics allowed recovery of ampicillin and kanamycin resistant clones originating from Streptococcus salivarius subsp. thermophilus and Lactobacillus helveticus genomes. We report molecular characterization of the cloned inserts, which were fully sequenced and shown to confer AR phenotype to recipient bacteria. We also show that metagenomics can be applied to food microbiota to identify underrepresented species carrying specific genes of interest. PMID:25243126

  15. Integration of the functional movement screen into the National Hockey League Combine.

    PubMed

    Rowan, Chip P; Kuropkat, Christiane; Gumieniak, Robert J; Gledhill, Norman; Jamnik, Veronica K

    2015-05-01

    The sport of ice hockey requires coordination of complex skills involving musculoskeletal and physiological abilities while simultaneously exposing players to a high risk for injury. The Functional Movement Screen (FMS) was developed to assess fundamental movement patterns that underlie both sport performance and injury risk. The top 111 elite junior hockey players from around the world took part in the 2013 National Hockey League Entry Draft Combine (NHL Combine). The FMS was integrated into the comprehensive medical and physiological fitness evaluations at the request of strength and conditioning coaches with affiliations to NHL teams. The inclusion of the FMS aimed to help develop strategies that could maximize its utility among elite hockey players and to encourage or inform further research in this field. This study evaluated the outcomes of integrating the FMS into the NHL Combine and identified any links to other medical plus physical and physiological fitness assessment outcomes. These potential associations may provide valuable information to identify elements of future training programs that are individualized to athletes' specific needs. The results of the FMS (total score and number of asymmetries identified) were significantly correlated to various body composition measures, aerobic and anaerobic fitness, leg power, timing of recent workouts, and the presence of lingering injury at the time of the NHL Combine. Although statistically significant correlations were observed, the implications of the FMS assessment outcomes remain difficult to quantify until ongoing assessment of FMS patterns, tracking of injuries, and hockey performance are available. PMID:25719918

  16. Screening SIRT1 Activators from Medicinal Plants as Bioactive Compounds against Oxidative Damage in Mitochondrial Function

    PubMed Central

    Wang, Yi; Liang, Xinying; Chen, Yaqi; Zhao, Xiaoping

    2016-01-01

    Sirtuin type 1 (SIRT1) belongs to the family of NAD+ dependent histone deacetylases and plays a critical role in cellular metabolism and response to oxidative stress. Traditional Chinese medicines (TCMs), as an important part of natural products, have been reported to exert protective effect against oxidative stress in mitochondria. In this study, we screened SIRT1 activators from TCMs and investigated their activities against mitochondrial damage. 19 activators were found in total by in vitro SIRT1 activity assay. Among those active compounds, four compounds, ginsenoside Rb2, ginsenoside F1, ginsenoside Rc, and schisandrin A, were further studied to validate the SIRT1-activation effects by liquid chromatography-mass spectrometry and confirm their activities against oxidative damage in H9c2 cardiomyocytes exposed to tert-butyl hydroperoxide (t-BHP). The results showed that those compounds enhanced the deacetylated activity of SIRT1, increased ATP content, and inhibited intracellular ROS formation as well as regulating the activity of Mn-SOD. These SIRT1 activators also showed moderate protective effects on mitochondrial function in t-BHP cells by recovering oxygen consumption and increasing mitochondrial DNA content. Our results suggested that those compounds from TCMs attenuated oxidative stress-induced mitochondrial damage in cardiomyocytes through activation of SIRT1. PMID:26981165

  17. Tissue matrix arrays for high-throughput screening and systems analysis of cell function.

    PubMed

    Beachley, Vince Z; Wolf, Matthew T; Sadtler, Kaitlyn; Manda, Srikanth S; Jacobs, Heather; Blatchley, Michael R; Bader, Joel S; Pandey, Akhilesh; Pardoll, Drew; Elisseeff, Jennifer H

    2015-12-01

    Cell and protein arrays have demonstrated remarkable utility in the high-throughput evaluation of biological responses; however, they lack the complexity of native tissue and organs. Here we spotted tissue extracellular matrix (ECM) particles as two-dimensional (2D) arrays or incorporated them with cells to generate three-dimensional (3D) cell-matrix microtissue arrays. We then investigated the responses of human stem, cancer and immune cells to tissue ECM arrays originating from 11 different tissues. We validated the 2D and 3D arrays as representative of the in vivo microenvironment by means of quantitative analysis of tissue-specific cellular responses, including matrix production, adhesion and proliferation, and morphological changes after culture. The biological outputs correlated with tissue proteomics, and network analysis identified several proteins linked to cell function. Our methodology enables broad screening of ECMs to connect tissue-specific composition with biological activity, providing a new resource for biomaterials research and further understanding of regeneration and disease mechanisms. PMID:26480475

  18. Identification of an abscisic acid transporter by functional screening using the receptor complex as a sensor

    PubMed Central

    Kanno, Yuri; Hanada, Atsushi; Chiba, Yasutaka; Ichikawa, Takanari; Nakazawa, Miki; Matsui, Minami; Koshiba, Tomokazu; Kamiya, Yuji; Seo, Mitsunori

    2012-01-01

    Movement of the plant hormone abscisic acid (ABA) within plants has been documented; however, the molecular mechanisms that regulate ABA transport are not fully understood. By using a modified yeast two-hybrid system, we screened Arabidopsis cDNAs capable of inducing interactions between the ABA receptor PYR/PYL/RCAR and PP2C protein phosphatase under low ABA concentrations. By using this approach, we identified four members of the NRT1/PTR family as candidates for ABA importers. Transport assays in yeast and insect cells demonstrated that at least one of the candidates ABA-IMPORTING TRANSPORTER (AIT) 1, which had been characterized as the low-affinity nitrate transporter NRT1.2, mediates cellular ABA uptake. Compared with WT, the ait1/nrt1.2 mutants were less sensitive to exogenously applied ABA during seed germination and/or postgermination growth, whereas overexpression of AIT1/NRT1.2 resulted in ABA hypersensitivity in the same conditions. Interestingly, the inflorescence stems of ait1/nrt1.2 had a lower surface temperature than those of the WT because of excess water loss from open stomata. We detected promoter activities of AIT1/NRT1.2 around vascular tissues in inflorescence stems, leaves, and roots. These data suggest that the function of AIT1/NRT1.2 as an ABA importer at the site of ABA biosynthesis is important for the regulation of stomatal aperture in inflorescence stems. PMID:22645333

  19. Acceleration of screened-exchange density-functional calculations with approximate differential overlap

    NASA Astrophysics Data System (ADS)

    Moussa, Jonathan; Schultz, Peter

    2014-03-01

    We implement the Heyd-Scuseria-Ernzerhof (HSE) screened-exchange density functional in the SEQQUEST electronic structure code. HSE calculations are accelerated by approximating differential overlap in the Fock exchange based on an atomic-orbital partitioning scheme. All one-center and two-center exchange integrals are calculated. A subset of three-center exchange integrals are calculated for one-center Fock exchange matrix elements and for exchange mediated by one-center density matrix elements. Four-center exchange integrals are not calculated. We test the validity of this approximation by examining the number and magnitude of these different classes of exchange integrals. Basis set and pseudopotential errors in HSE calculations are benchmarked on atoms. Differential overlap approximation errors are benchmarked on small molecules. Sandia National Labs is a multi-program laboratory managed and operated by Sandia Corp., a wholly owned subsidiary of Lockheed Martin Corp., for the U.S. Department of Energy's National Nuclear Security Administration under contract DE-AC04-94AL85000.

  20. In vitro functional screening as a means to identify new plasticizers devoid of reproductive toxicity.

    PubMed

    Boisvert, Annie; Jones, Steven; Issop, Leeyah; Erythropel, Hanno C; Papadopoulos, Vassilios; Culty, Martine

    2016-10-01

    Plasticizers are indispensable additives providing flexibility and malleability to plastics. Among them, several phthalates, including di (2-ethylhexyl) phthalate (DEHP), have emerged as endocrine disruptors, leading to their restriction in consumer products and creating a need for new, safer plasticizers. The goal of this project was to use in vitro functional screening tools to select novel non-toxic plasticizers suitable for further in vivo evaluation. A panel of novel compounds with satisfactory plasticizer properties and biodegradability were tested, along with several commercial plasticizers, such as diisononyl-cyclohexane-1,2-dicarboxylate (DINCH®). MEHP, the monoester metabolite of DEHP was also included as reference compound. Because phthalates target mainly testicular function, including androgen production and spermatogenesis, we used the mouse MA-10 Leydig and C18-4 spermatogonial cell lines as surrogates to examine cell survival, proliferation, steroidogenesis and mitochondrial integrity. The most promising compounds were further assessed on organ cultures of rat fetal and neonatal testes, corresponding to sensitive developmental windows. Dose-response studies revealed the toxicity of most maleates and fumarates, while identifying several dibenzoate and succinate plasticizers as innocuous on Leydig and germ cells. Interestingly, DINCH®, a plasticizer marketed as a safe alternative to phthalates, exerted a biphasic effect on steroid production in MA-10 and fetal Leydig cells. MEHP was the only plasticizer inducing the formation of multinucleated germ cells (MNG) in organ culture. Overall, organ cultures corroborated the cell line data, identifying one dibenzoate and one succinate as the most promising candidates. The adoption of such collaborative approaches for developing new chemicals should help prevent the development of compounds potentially harmful to human health. PMID:27423704

  1. Functional screening identifies CRLF2 in precursor B-cell acute lymphoblastic leukemia.

    PubMed

    Yoda, Akinori; Yoda, Yuka; Chiaretti, Sabina; Bar-Natan, Michal; Mani, Kartik; Rodig, Scott J; West, Nathan; Xiao, Yun; Brown, Jennifer R; Mitsiades, Constantine; Sattler, Martin; Kutok, Jeffrey L; DeAngelo, Daniel J; Wadleigh, Martha; Piciocchi, Alfonso; Dal Cin, Paola; Bradner, James E; Griffin, James D; Anderson, Kenneth C; Stone, Richard M; Ritz, Jerome; Foà, Robin; Aster, Jon C; Frank, David A; Weinstock, David M

    2010-01-01

    The prognosis for adults with precursor B-cell acute lymphoblastic leukemia (B-ALL) remains poor, in part from a lack of therapeutic targets. We identified the type I cytokine receptor subunit CRLF2 in a functional screen for B-ALL-derived mRNA transcripts that can substitute for IL3 signaling. We demonstrate that CRLF2 is overexpressed in approximately 15% of adult and high-risk pediatric B-ALL that lack MLL, TCF3, TEL, and BCR/ABL rearrangements, but not in B-ALL with these rearrangements or other lymphoid malignancies. CRLF2 overexpression can result from translocation with the IGH locus or intrachromosomal deletion and is associated with poor outcome. CRLF2 overexpressing B-ALLs share a transcriptional signature that significantly overlaps with a BCR/ABL signature, and is enriched for genes involved in cytokine receptor and JAK-STAT signaling. In a subset of cases, CRLF2 harbors a Phe232Cys gain-of-function mutation that promotes constitutive dimerization and cytokine independent growth. A mutually exclusive subset harbors activating mutations in JAK2. In fact, all 22 B-ALLs with mutant JAK2 that we analyzed overexpress CRLF2, distinguishing CRLF2 as the key scaffold for mutant JAK2 signaling in B-ALL. Expression of WT CRLF2 with mutant JAK2 also promotes cytokine independent growth that, unlike CRLF2 Phe232Cys or ligand-induced signaling by WT CRLF2, is accompanied by JAK2 phosphorylation. Finally, cells dependent on CRLF2 signaling are sensitive to small molecule inhibitors of either JAKs or protein kinase C family kinases. Together, these findings implicate CRLF2 as an important factor in B-ALL with diagnostic, prognostic, and therapeutic implications. PMID:20018760

  2. Preparation of Protein Samples for NMR Structure, Function, and Small Molecule Screening Studies

    PubMed Central

    Acton, Thomas B.; Xiao, Rong; Anderson, Stephen; Aramini, James; Buchwald, William A.; Ciccosanti, Colleen; Conover, Ken; Everett, John; Hamilton, Keith; Huang, Yuanpeng Janet; Janjua, Haleema; Kornhaber, Gregory; Lau, Jessica; Lee, Dong Yup; Liu, Gaohua; Maglaqui, Melissa; Ma, Lichung; Mao, Lei; Patel, Dayaban; Rossi, Paolo; Sahdev, Seema; Shastry, Ritu; Swapna, G.V.T.; Tang, Yeufeng; Tong, Saichiu; Wang, Dongyan; Wang, Huang; Zhao, Li; Montelione, Gaetano T.

    2014-01-01

    technologies are suitable for implementation in a large individual laboratory or by a small group of collaborating investigators for structural biology, functional proteomics, ligand screening and structural genomics research. PMID:21371586

  3. Comparative RNAi Screens in C. elegans and C. briggsae Reveal the Impact of Developmental System Drift on Gene Function

    PubMed Central

    Verster, Adrian J.; Ramani, Arun K.; McKay, Sheldon J.; Fraser, Andrew G.

    2014-01-01

    Although two related species may have extremely similar phenotypes, the genetic networks underpinning this conserved biology may have diverged substantially since they last shared a common ancestor. This is termed Developmental System Drift (DSD) and reflects the plasticity of genetic networks. One consequence of DSD is that some orthologous genes will have evolved different in vivo functions in two such phenotypically similar, related species and will therefore have different loss of function phenotypes. Here we report an RNAi screen in C. elegans and C. briggsae to identify such cases. We screened 1333 genes in both species and identified 91 orthologues that have different RNAi phenotypes. Intriguingly, we find that recently evolved genes of unknown function have the fastest evolving in vivo functions and, in several cases, we identify the molecular events driving these changes. We thus find that DSD has a major impact on the evolution of gene function and we anticipate that the C. briggsae RNAi library reported here will drive future studies on comparative functional genomics screens in these nematodes. PMID:24516395

  4. A gain-of-function screen for genes that affect the development of the Drosophila adult external sensory organ.

    PubMed Central

    Abdelilah-Seyfried, S; Chan, Y M; Zeng, C; Justice, N J; Younger-Shepherd, S; Sharp, L E; Barbel, S; Meadows, S A; Jan, L Y; Jan, Y N

    2000-01-01

    The Drosophila adult external sensory organ, comprising a neuron and its support cells, is derived from a single precursor cell via several asymmetric cell divisions. To identify molecules involved in sensory organ development, we conducted a tissue-specific gain-of-function screen. We screened 2293 independent P-element lines established by P. Rorth and identified 105 lines, carrying insertions at 78 distinct loci, that produced misexpression phenotypes with changes in number, fate, or morphology of cells of the adult external sensory organ. On the basis of the gain-of-function phenotypes of both internal and external support cells, we subdivided the candidate lines into three classes. The first class (52 lines, 40 loci) exhibits partial or complete loss of adult external sensory organs. The second class (38 lines, 28 loci) is associated with increased numbers of entire adult external sensory organs or subsets of sensory organ cells. The third class (15 lines, 10 loci) results in potential cell fate transformations. Genetic and molecular characterization of these candidate lines reveals that some loci identified in this screen correspond to genes known to function in the formation of the peripheral nervous system, such as big brain, extra macrochaetae, and numb. Also emerging from the screen are a large group of previously uncharacterized genes and several known genes that have not yet been implicated in the development of the peripheral nervous system. PMID:10835395

  5. Screening of central functions of amino acids and their metabolites for sedative and hypnotic effects using chick models.

    PubMed

    Furuse, Mitsuhiro

    2015-09-01

    The chick has a practical advantage in the screening process in that chicks require only small quantities of drugs. The chick separation stress paradigm has traditionally been recognized as a valid form of anxiolytic screening. Further, chick behavior involving standing motionless with eyes closed or sitting motionless with head drooped is nearly always associated with electrophysiological sleep. When centrally administered, some DNA-encoded L-α-amino acids, as well as some DNA-non-encoded amino acids, such as metabolites of L-α-amino acids, D-amino acid and β-amino acid, have shown sedative and/or hypnotic effects in chicks. The effects of some of these amino acids have subsequently been confirmed in humans. In conclusion, the chick model is convenient and useful for screening central functions of amino acids and their metabolites for hypnosis and sedation. PMID:26101060

  6. Identification of Genes That Modulate Susceptibility to Formaldehyde and Imatinib by Functional Genomic Screening in Human Haploid KBM7 Cells.

    PubMed

    Shen, Hua; McHale, Cliona M; Haider, Syed I; Jung, Cham; Zhang, Susie; Smith, Martyn T; Zhang, Luoping

    2016-05-01

    Though current functional genomic screening systems are useful for investigating human susceptibility to chemical toxicity, they have limitations. Well-established, high-throughput yeast mutant screens identify only evolutionarily conserved processes. RNA interference can be applied in human cells but is limited by incomplete gene knockout and off-target effects. Human haploid cell screening is advantageous as it requires knockdown of only a single copy of each gene. A human haploid cell mutant library (KBM7-Mu), derived from a chronic myeloid leukemia (CML) patient, was recently developed and has been used to identify genes that modulate sensitivity to infectious agents and pharmaceutical drugs. Here, we sought to improve the KBM7-Mu screening process to enable efficient screening of environmental chemicals. We developed a semi-solid medium based screening approach that cultures individual mutant colonies from chemically resistant cells, faster (by 2-3 weeks) and with less labor than the original liquid medium-based approach. As proof of principle, we identified genetic mutants that confer resistance to the carcinogen formaldehyde (FA, 12 genes, 18 hits) and the CML chemotherapeutic agent imatinib (6 genes, 13 hits). Validation experiments conducted on KBM7 mutants lacking each of the 18 genes confirmed resistance of 6 FA mutants (CTC1, FCRLA, GOT1, LPR5, M1AP, and MAP2K5) and 1 imatinib-resistant mutant (LYRM9). Despite the improvements to the method, it remains technically challenging to limit false positive findings. Nonetheless, our findings demonstrate the broad applicability of this optimized haploid approach to screen toxic chemicals to identify novel susceptibility genes and gain insight into potential mechanisms of toxicity. PMID:27008852

  7. Advantages of Crystallographic Fragment Screening: Functional and Mechanistic Insights from a Powerful Platform for Efficient Drug Discovery

    PubMed Central

    Patel, Disha; Bauman, Joseph D.; Arnold, Eddy

    2015-01-01

    X-ray crystallography has been an under-appreciated screening tool for fragment-based drug discovery due to the perception of low throughput and technical difficulty. Investigators in industry and academia have overcome these challenges by taking advantage of key factors that contribute to a successful crystallographic screening campaign. Efficient cocktail design and soaking methodologies have evolved to maximize throughput while minimizing false positives/negatives. In addition, technical improvements at synchrotron beamlines have dramatically increased data collection rates thus enabling screening on a timescale comparable to other techniques. The combination of available resources and efficient experimental design has resulted in many successful crystallographic screening campaigns. The three-dimensional crystal structure of the bound fragment complexed to its target, a direct result of the screening effort, enables structure-based drug design while revealing insights regarding protein dynamics and function not readily obtained through other experimental approaches. Furthermore, this “chemical interrogation” of the target protein crystals can lead to the identification of useful reagents for improving diffraction resolution or compound solubility. PMID:25117499

  8. Brief Functional Screening for Transition Difficulties Prior to Enrolment Predicts Socio-Emotional Competence and School Adjustment in Head Start Preschoolers

    ERIC Educational Resources Information Center

    Miller, Alison L.; Gouley, Kathleen Kiely; Shields, Ann; Dickstein, Susan; Seifer, Ronald; Magee, Karin Dodge; Fox, Christina

    2003-01-01

    Successful preschool transition is important for future educational success. We used brief functional screenings to identify low-income children at risk for difficulty transitioning into preschool. Functional screenings were conducted for 163 children prior to enrollment, in a naturalistic peer setting, and focused on multiple domains important…

  9. Genome-wide gain-of-function screen identifies novel regulators of pluripotency.

    PubMed

    Abujarour, Ramzey; Efe, Jem; Ding, Sheng

    2010-09-01

    Pluripotent stem cells are characterized by the capacity to self-renew and to differentiate into all the cell types of the body. To identify novel regulators of pluripotency, we screened cDNA libraries (>30,000 clones) in P19 embryonal carcinoma cells for factors that modulate the expression of a luciferase reporter driven by the promoter of the pluripotency master regulator Nanog. Ninety confirmed hits activated the reporter and 14 confirmed hits inhibited the reporter by more than two-fold. The identified hits were evaluated by gain- and loss-of-functions approaches. The reporter-activating hits Timp2, Hig2, and Mki67ip promoted embryonic stem (ES) cell self-renewal when episomally overexpressed in ES cells, whereas the reporter-inhibiting hits PU.1/Spi1, Prkaca, and Jun induced differentiation of ES cells. Conversely, the knockdown of the activating hits Timp2, Mki67ip, Esrrg, and Dusp7 in ES cells induced differentiation, whereas the knockdown of the reporter-inhibiting hit PU.1/Spi1 led to inhibition of differentiation. One of the novel hits, the RNA-binding protein Mki67ip was further characterized, and found to be overexpressed in ES cells and in early development and downregulated during differentiation. The knockdown of Mki67ip led to the differentiation of ES cells, decreased growth rate, reduction in pluripotency markers, and induction of lineage-specific markers. In addition, colocalization and coimmunoprecipitation experiments suggest that Mki67ip promotes ES cell self-renewal via a mechanism involving nucleophosmin, a multifunctional nucleolar protein upregulated in stem cells and cancer. PMID:20629179

  10. Screening of differentially expressed genes associated with human glioblastoma and functional analysis using a DNA microarray.

    PubMed

    Wang, Lina; Wei, Bo; Hu, Guozhang; Wang, Le; Bi, Miaomiao; Sun, Zhigang; Jin, Ying

    2015-08-01

    Glioblastoma multiforme (GBM) is the most malignant type of human glioma, and has a poor prognosis. Screening differentially expressed genes (DEGs) in brain tumor samples and normal brain samples is of importance for identifying GBM and to design specific-targeting drugs. The transcriptional profile of GSE30563, containing three genechips of brain tumor samples and three genechips of normal brain samples, was downloaded from Gene Expression Omnibus to identify the DEGs. The differences in the expression of the DEGs in the two different samples were compared through hierarchical biclustering. The co-expression coefficient of the DEGs was calculated using the information from COXPRESdb, the network of the DEGs was constructed and functional enrichment and pathway analysis were performed. Finally, the transcription factors of important DEGs were predicted. A total of 1,006 DEGs, including 368 upregulated and 638 downregulated DEGs, were identified. A close correlation was demonstrated between six important genes, associated with immune response, HLA-DQB1, HLA-DRB1, HLA-DPA1, HLA-B, HLA-DMA and HLA-DRA, and the immune response. Allograft rejection was selected as the most significant pathway. A total of 17 transcription factors, including nuclear factor (NF)-κB and NF-κB1, and their binding sites containing these six DEGs, were also identified. The DEGs, including major histocompatibility complex (MHC) class II, DQβ1, MHC class II, DRβ1, MHC class IB, MHC class II, DMα, MHC class II, DPα1, MHC class II, DRα, may provide novel targets for the diagnosis and treatment of GBM. The transcription factors of these six genes and their binding sites may also provide evidence and direction for identifying target-specific drugs. PMID:25901754

  11. Factor Structure and Internal Validity of the Functional Movement Screen in Adults.

    PubMed

    Koehle, Michael S; Saffer, Boaz Y; Sinnen, Nadine M; MacInnis, Martin J

    2016-02-01

    The factor structure and internal consistency of the Functional Movement Screen (FMS) have not been examined in a general healthcare population. Replicating the factor structure of the FMS is important because it illustrates the interdependence between each of the subtests, enabling the strength and conditioning professional to better interpret and act on an individual's FMS score. Anthropometric data and FMS scores were collected from 1,113 clients of a multidisciplinary healthcare clinic in Vancouver, BC The mean (SD) ages were 53.4 (11.1) for men (n = 656) and 49.3 (12.3) for women (n = 457). The mean FMS Summary Score was 13.7 (2.9) and was significantly negatively correlated with both age (r = -0.25; p < 0.001) and body mass index (r = -0.37; p < 0.001). The internal consistency of the FMS scale, which was assessed with both ordinal and Cronbach's alpha, was 0.73 and 0.64, respectively. Polychoric correlations between individual movements ranged from 0.03 to 0.59. Exploratory and confirmatory factor analyses (CFA) revealed that the FMS showed 2 main factors, a basic movement factor (shoulder mobility and active straight leg raise) and a complex movement factor (squat, hurdle step, inline lunge, and the trunk stability push-up). Rotary stability loaded onto both factors in the CFA, and its exclusion from the model had little effect. The findings of this study broadly replicated the intended factor structure of the FMS, as the individual movements aligned well with the intended factors. PMID:26200190

  12. A Drosophila Gain-of-Function Screen for Candidate Genes Involved in Steroid-Dependent Neuroendocrine Cell Remodeling

    PubMed Central

    Zhao, Tao; Gu, Tingting; Rice, Heather C.; McAdams, Kathleen L.; Roark, Kimberly M.; Lawson, Kaylan; Gauthier, Sebastien A.; Reagan, Kathleen L.; Hewes, Randall S.

    2008-01-01

    The normal functioning of neuroendocrine systems requires that many neuropeptidergic cells change, to alter transmitter identity and concentration, electrical properties, and cellular morphology in response to hormonal cues. During insect metamorphosis, a pulse of circulating steroids, ecdysteroids, governs the dramatic remodeling of larval neurons to serve adult-specific functions. To identify molecular mechanisms underlying metamorphic remodeling, we conducted a neuropeptidergic cell-targeted, gain-of-function genetic screen. We screened 6097 lines. Each line permitted Gal4-regulated transcription of flanking genes. A total of 58 lines, representing 51 loci, showed defects in neuropeptide-mediated developmental transitions (ecdysis or wing expansion) when crossed to the panneuropeptidergic Gal4 driver, 386Y-Gal4. In a secondary screen, we found 29 loci that produced wing expansion defects when crossed to a crustacean cardioactive peptide (CCAP)/bursicon neuron-specific Gal4 driver. At least 14 loci disrupted the formation or maintenance of adult-specific CCAP/bursicon cell projections during metamorphosis. These include components of the insulin and epidermal growth factor signaling pathways, an ecdysteroid-response gene, cabut, and an ubiquitin-specific protease gene, fat facets, with known functions in neuronal development. Several additional genes, including three micro-RNA loci and two factors related to signaling by Myb-like proto-oncogenes, have not previously been implicated in steroid signaling or neuronal remodeling. PMID:18245346

  13. A Novel Dual Expression Platform for High Throughput Functional Screening of Phage Libraries in Product like Format

    PubMed Central

    Mugabe, Sheila; Gao, Changshou; Tkaczyk, Christine; Mazor, Yariv; Pavlik, Peter; Wu, Herren; Dall’Acqua, William; Chowdhury, Partha Sarathi

    2015-01-01

    High throughput screenings of single chain Fv (scFv) antibody phage display libraries are currently done as soluble scFvs produced in E.coli. Due to endotoxin contaminations from bacterial cells these preparations cannot be reliably used in mammalian cell based assays. The monovalent nature and lack of Fc in soluble scFvs prevent functional assays that are dependent on target cross linking and/or Fc functions. A convenient approach is to convert scFvs into scFv.Fc fusion proteins and express them in mammalian cell lines for screening. This approach is low throughput and is only taken after primary screening of monovalent scFvs that are expressed in bacteria. There is no platform at present that combines the benefits of both bacterial and mammalian expression system for screening phage library output. We have, therefore, developed a novel dual expression vector, called pSplice, which can be used to express scFv.Fc fusion proteins both in E.coli and mammalian cell lines. The hallmark of the vector is an engineered intron which houses the bacterial promoter and signal peptide for expression and secretion of scFv.Fc in E.coli. When the vector is transfected into a mammalian cell line, the intron is efficiently spliced out resulting in a functional operon for expression and secretion of the scFv.Fc fusion protein into the culture medium. By applying basic knowledge of mammalian introns and splisosome, we designed this vector to enable screening of phage libraries in a product like format. Like IgG, the scFv.Fc fusion protein is bi-valent for the antigen and possesses Fc effector functions. Expression in E.coli maintains the speed of the bacterial expression platform and is used to triage clones based on binding and other assays that are not sensitive to endotoxin. Triaged clones are then expressed in a mammalian cell line without the need for any additional cloning steps. Conditioned media from the mammalian cell line containing the fusion proteins are then used for

  14. A Novel Dual Expression Platform for High Throughput Functional Screening of Phage Libraries in Product like Format.

    PubMed

    Xiao, Xiaodong; Chen, Yan; Mugabe, Sheila; Gao, Changshou; Tkaczyk, Christine; Mazor, Yariv; Pavlik, Peter; Wu, Herren; Dall'Acqua, William; Chowdhury, Partha Sarathi

    2015-01-01

    High throughput screenings of single chain Fv (scFv) antibody phage display libraries are currently done as soluble scFvs produced in E.coli. Due to endotoxin contaminations from bacterial cells these preparations cannot be reliably used in mammalian cell based assays. The monovalent nature and lack of Fc in soluble scFvs prevent functional assays that are dependent on target cross linking and/or Fc functions. A convenient approach is to convert scFvs into scFv.Fc fusion proteins and express them in mammalian cell lines for screening. This approach is low throughput and is only taken after primary screening of monovalent scFvs that are expressed in bacteria. There is no platform at present that combines the benefits of both bacterial and mammalian expression system for screening phage library output. We have, therefore, developed a novel dual expression vector, called pSplice, which can be used to express scFv.Fc fusion proteins both in E.coli and mammalian cell lines. The hallmark of the vector is an engineered intron which houses the bacterial promoter and signal peptide for expression and secretion of scFv.Fc in E.coli. When the vector is transfected into a mammalian cell line, the intron is efficiently spliced out resulting in a functional operon for expression and secretion of the scFv.Fc fusion protein into the culture medium. By applying basic knowledge of mammalian introns and splisosome, we designed this vector to enable screening of phage libraries in a product like format. Like IgG, the scFv.Fc fusion protein is bi-valent for the antigen and possesses Fc effector functions. Expression in E.coli maintains the speed of the bacterial expression platform and is used to triage clones based on binding and other assays that are not sensitive to endotoxin. Triaged clones are then expressed in a mammalian cell line without the need for any additional cloning steps. Conditioned media from the mammalian cell line containing the fusion proteins are then used for

  15. Injury History, Sex, and Performance on the Functional Movement Screen and Y Balance Test

    PubMed Central

    Chimera, Nicole J.; Smith, Craig A.; Warren, Meghan

    2015-01-01

    Context: Research is limited regarding the effects of injury or surgery history and sex on the Functional Movement Screen (FMS) and Y Balance Test (YBT). Objective: To determine if injury or surgery history or sex affected results on the FMS and YBT. Design: Cross-sectional study. Setting: Athletic training facilities. Patients or Other Participants: A total of 200 National Collegiate Athletic Association Division I female (n = 92; age = 20.0 ± 1.4 years, body mass index = 22.8 ± 3.1 kg/m2) and male (n = 108; age = 20.0 ± 1.5 years, body mass index = 27.0 ± 4.6 kg/m2) athletes were screened; 170 completed the FMS, and 190 completed the YBT. Intervention(s): A self-reported questionnaire identified injury or surgery history and sex. The FMS assessed movement during the patterns of deep squat, hurdle step, in-line lunge, shoulder mobility, impingement-clearing test, straight-leg raise, trunk stability push-up, press-up clearing test, rotary stability, and posterior-rocking clearing test. The YBT assessed balance while participants reached in anterior, posteromedial, and posterolateral directions. Main Outcome Measure(s): The FMS composite score (CS; range, 0–21) and movement pattern score (range, 0–3), the YBT CS (% lower extremity length), and YBT anterior, posteromedial, and posterolateral asymmetry (difference between limbs in centimeters). Independent-samples t tests established differences in mean FMS CS, YBT CS, and YBT asymmetry. The Mann-Whitney U test identified differences in FMS movement patterns. Results: We found lower overall FMS CSs for the following injuries or surgeries: hip (injured = 12.7 ± 3.1, uninjured = 14.4 ± 2.3; P = .005), elbow (injured = 12.1 ± 2.8, uninjured = 14.3 ± 2.4; P = .02), and hand (injured = 12.3 ± 2.9, uninjured = 14.3 ± 2.3; P = .006) injuries and shoulder surgery (surgery = 12.0 ± 1.0, no surgery = 14.3 ± 2.4; P < .001). We observed worse FMS movement pattern performance for knee surgery (rotary stability: P

  16. Coupled high-throughput functional screening and next generation sequencing for identification of plant polymer decomposing enzymes in metagenomic libraries

    PubMed Central

    Nyyssönen, Mari; Tran, Huu M.; Karaoz, Ulas; Weihe, Claudia; Hadi, Masood Z.; Martiny, Jennifer B. H.; Martiny, Adam C.; Brodie, Eoin L.

    2013-01-01

    Recent advances in sequencing technologies generate new predictions and hypotheses about the functional roles of environmental microorganisms. Yet, until we can test these predictions at a scale that matches our ability to generate them, most of them will remain as hypotheses. Function-based mining of metagenomic libraries can provide direct linkages between genes, metabolic traits and microbial taxa and thus bridge this gap between sequence data generation and functional predictions. Here we developed high-throughput screening assays for function-based characterization of activities involved in plant polymer decomposition from environmental metagenomic libraries. The multiplexed assays use fluorogenic and chromogenic substrates, combine automated liquid handling and use a genetically modified expression host to enable simultaneous screening of 12,160 clones for 14 activities in a total of 170,240 reactions. Using this platform we identified 374 (0.26%) cellulose, hemicellulose, chitin, starch, phosphate and protein hydrolyzing clones from fosmid libraries prepared from decomposing leaf litter. Sequencing on the Illumina MiSeq platform, followed by assembly and gene prediction of a subset of 95 fosmid clones, identified a broad range of bacterial phyla, including Actinobacteria, Bacteroidetes, multiple Proteobacteria sub-phyla in addition to some Fungi. Carbohydrate-active enzyme genes from 20 different glycoside hydrolase (GH) families were detected. Using tetranucleotide frequency (TNF) binning of fosmid sequences, multiple enzyme activities from distinct fosmids were linked, demonstrating how biochemically-confirmed functional traits in environmental metagenomes may be attributed to groups of specific organisms. Overall, our results demonstrate how functional screening of metagenomic libraries can be used to connect microbial functionality to community composition and, as a result, complement large-scale metagenomic sequencing efforts. PMID:24069019

  17. A Functional Genome-Wide In Vivo Screen Identifies New Regulators of Signalling Pathways during Early Xenopus Embryogenesis

    PubMed Central

    Zhang, Siwei; Li, Jingjing; Lea, Robert; Amaya, Enrique; Dorey, Karel

    2013-01-01

    Embryonic development requires exquisite regulation of several essential processes, such as patterning of tissues and organs, cell fate decisions, and morphogenesis. Intriguingly, these diverse processes are controlled by only a handful of signalling pathways, and mis-regulation in one or more of these pathways may result in a variety of congenital defects and diseases. Consequently, investigating how these signalling pathways are regulated at the molecular level is essential to understanding the mechanisms underlying vertebrate embryogenesis, as well as developing treatments for human diseases. Here, we designed and performed a large-scale gain-of-function screen in Xenopus embryos aimed at identifying new regulators of MAPK/Erk, PI3K/Akt, BMP, and TGF-β/Nodal signalling pathways. Our gain-of-function screen is based on the identification of gene products that alter the phosphorylation state of key signalling molecules, which report the activation state of the pathways. In total, we have identified 20 new molecules that regulate the activity of one or more signalling pathways during early Xenopus development. This is the first time that such a functional screen has been performed, and the findings pave the way toward a more comprehensive understanding of the molecular mechanisms regulating the activity of important signalling pathways under normal and pathological conditions. PMID:24244509

  18. Identification of Genes Important for Cutaneous Function Revealed by a Large Scale Reverse Genetic Screen in the Mouse

    PubMed Central

    DiTommaso, Tia; Jones, Lynelle K.; Cottle, Denny L.; Gerdin, Anna-Karin; Vancollie, Valerie E.; Watt, Fiona M.; Ramirez-Solis, Ramiro; Bradley, Allan; Steel, Karen P.; Sundberg, John P.; White, Jacqueline K.; Smyth, Ian M.

    2014-01-01

    The skin is a highly regenerative organ which plays critical roles in protecting the body and sensing its environment. Consequently, morbidity and mortality associated with skin defects represent a significant health issue. To identify genes important in skin development and homeostasis, we have applied a high throughput, multi-parameter phenotype screen to the conditional targeted mutant mice generated by the Wellcome Trust Sanger Institute's Mouse Genetics Project (Sanger-MGP). A total of 562 different mouse lines were subjected to a variety of tests assessing cutaneous expression, macroscopic clinical disease, histological change, hair follicle cycling, and aberrant marker expression. Cutaneous lesions were associated with mutations in 23 different genes. Many of these were not previously associated with skin disease in the organ (Mysm1, Vangl1, Trpc4ap, Nom1, Sparc, Farp2, and Prkab1), while others were ascribed new cutaneous functions on the basis of the screening approach (Krt76, Lrig1, Myo5a, Nsun2, and Nf1). The integration of these skin specific screening protocols into the Sanger-MGP primary phenotyping pipelines marks the largest reported reverse genetic screen undertaken in any organ and defines approaches to maximise the productivity of future projects of this nature, while flagging genes for further characterisation. PMID:25340873

  19. The Inter-rater Reliability of the Functional Movement Screen within an athletic population using Untrained Raters.

    PubMed

    Elias, Jade E

    2013-07-01

    The Functional Movement Screen (FMS) is a commonly used screening tool designed to identify restrictions to movement patterns and increased injury risk using 7 predesigned tests. The purpose of this study was to analyse the inter- rater reliability of scoring of the FMS using a group of 'untrained' subjects. Additionally, the study also examined if clinical experience level had any effect on reliability. Twenty fully qualified Physiotherapists working at the English Institute of Sport, with elite athletes, volunteered to participate in the study. The group comprised of both Level 2 and Level 3 physiotherapists, based on clinical experience levels. Five elite athletes, free from injury, were recruited and videoed completing 6 of the 7 FMS tests, using a 3 camera system. The videos were scored by each Physiotherapist using the standardised scoring sheet, as developed by Cook et al. (2006) (4,5). Each practitioner marked each athlete completing the 6 tests. The total scores were calculated for each athlete (maximum score of 18). The inter- rater reliability of the test was shown to be high, ICC 0.906. An independent t-test showed no significant differences between the Level 2 and Level 3 practitioners in the total scores (p = 0.502). The results of the test indicate that the FMS is a reliable screening tool when used by 'untrained' practitioners in determining faulty movement patterns and that clinical experience level does not affect the reliability, therefore it may be a useful tool in the screening of athletic populations. PMID:23838983

  20. 49 CFR 1544.407 - Training, testing, and knowledge of individuals who perform screening functions.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 49 Transportation 9 2011-10-01 2011-10-01 false Training, testing, and knowledge of individuals... Qualifications When the Aircraft Operator Performs Screening § 1544.407 Training, testing, and knowledge of... on-the-job training test prescribed by TSA. (f) Knowledge requirements. Each aircraft operator...

  1. Functional Assays and Alternative Species: Using Larval Zebrafish in Developmental Neurotoxicity Screening

    EPA Science Inventory

    The U.S. Environmental Protection Agency is developing and evaluating methods to screen and prioritize large numbers of chemicals for developmental toxicity. Towards this goal, we are exploring methods to detect developmental neurotoxicants in very young larval zebrafish. We have...

  2. Functional Assays and Alternative Species: Using Larval Zebrafish in Developmental Neurotoxicity Screening**

    EPA Science Inventory

    The U.S. Environmental Protection Agency is evaluating methods to screen and prioritize large numbers of chemicals for developmental toxicity. As such, we are exploring a behavioral testing paradigm, which can assess the effect of sublethal and subteratogenic concentrations of de...

  3. Functional Genetic Screen to Identify Interneurons Governing Behaviorally Distinct Aspects of Drosophila Larval Motor Programs.

    PubMed

    Clark, Matt Q; McCumsey, Stephanie J; Lopez-Darwin, Sereno; Heckscher, Ellie S; Doe, Chris Q

    2016-01-01

    Drosophila larval crawling is an attractive system to study rhythmic motor output at the level of animal behavior. Larval crawling consists of waves of muscle contractions generating forward or reverse locomotion. In addition, larvae undergo additional behaviors, including head casts, turning, and feeding. It is likely that some neurons (e.g., motor neurons) are used in all these behaviors, but the identity (or even existence) of neurons dedicated to specific aspects of behavior is unclear. To identify neurons that regulate specific aspects of larval locomotion, we performed a genetic screen to identify neurons that, when activated, could elicit distinct motor programs. We used 165 Janelia CRM-Gal4 lines-chosen for sparse neuronal expression-to ectopically express the warmth-inducible neuronal activator TrpA1, and screened for locomotor defects. The primary screen measured forward locomotion velocity, and we identified 63 lines that had locomotion velocities significantly slower than controls following TrpA1 activation (28°). A secondary screen was performed on these lines, revealing multiple discrete behavioral phenotypes, including slow forward locomotion, excessive reverse locomotion, excessive turning, excessive feeding, immobile, rigid paralysis, and delayed paralysis. While many of the Gal4 lines had motor, sensory, or muscle expression that may account for some or all of the phenotype, some lines showed specific expression in a sparse pattern of interneurons. Our results show that distinct motor programs utilize distinct subsets of interneurons, and provide an entry point for characterizing interneurons governing different elements of the larval motor program. PMID:27172197

  4. Functional Genetic Screen to Identify Interneurons Governing Behaviorally Distinct Aspects of Drosophila Larval Motor Programs

    PubMed Central

    Clark, Matt Q.; McCumsey, Stephanie J.; Lopez-Darwin, Sereno; Heckscher, Ellie S.; Doe, Chris Q.

    2016-01-01

    Drosophila larval crawling is an attractive system to study rhythmic motor output at the level of animal behavior. Larval crawling consists of waves of muscle contractions generating forward or reverse locomotion. In addition, larvae undergo additional behaviors, including head casts, turning, and feeding. It is likely that some neurons (e.g., motor neurons) are used in all these behaviors, but the identity (or even existence) of neurons dedicated to specific aspects of behavior is unclear. To identify neurons that regulate specific aspects of larval locomotion, we performed a genetic screen to identify neurons that, when activated, could elicit distinct motor programs. We used 165 Janelia CRM-Gal4 lines—chosen for sparse neuronal expression—to ectopically express the warmth-inducible neuronal activator TrpA1, and screened for locomotor defects. The primary screen measured forward locomotion velocity, and we identified 63 lines that had locomotion velocities significantly slower than controls following TrpA1 activation (28°). A secondary screen was performed on these lines, revealing multiple discrete behavioral phenotypes, including slow forward locomotion, excessive reverse locomotion, excessive turning, excessive feeding, immobile, rigid paralysis, and delayed paralysis. While many of the Gal4 lines had motor, sensory, or muscle expression that may account for some or all of the phenotype, some lines showed specific expression in a sparse pattern of interneurons. Our results show that distinct motor programs utilize distinct subsets of interneurons, and provide an entry point for characterizing interneurons governing different elements of the larval motor program. PMID:27172197

  5. Effects of semicore d-electrons in screened-exchange density functional methods

    NASA Astrophysics Data System (ADS)

    Lee, Byounghak; Wang, Lin-Wang

    2007-03-01

    We report a theoretical study on the role of shallow d states in the screened-exchange local density approximation (sX-LDA) band structure of binary semiconductor systems. We found that the inaccurate pseudo-wavefunctions can lead to 1) an overestimation of the screened-exchange interaction between the localized d states and the delocalized higher energy s and p states and 2) an underestimation of the screened-exchange interaction between the d states. The resulting sX-LDA band structures have substantially smaller band gaps compared with experiments. We correct the pseudo-wavefunctions of d states by including the s and p states of the same shell in the valence states. The correction of pseudo-wavefunctions yields band gaps and the d state binding energy in good agreement with experiments. Compared with the quasi-particle GW method, our sX-LDA results shows not only similar quality band gaps but also much better d state binding energy. As an example, we present sX-LDA results of s-d coupling in zinc-blende semiconductors and compare them with LDA+U results. We also present an efficient method to correct the pseudo-wavefunction exchange-integral error by using projection of wavefunctions onto atomic orbitals.

  6. The m/r SEBT: development of a functional screening tool for dance educators.

    PubMed

    Wilson, Margaret; Batson, Glenna

    2014-12-01

    Dance screenings provide direct and indirect data bearing on a dancer's readiness to undertake rigorous physical training. Rarely, however, are dance teachers able to translate results from these screenings into practical technical knowledge. In this article, an example of a preseason assessment tool is presented that translates scientific findings into useful information for dance teachers conducting auditions. Designed as a baseline assessment of the dancer during auditioning, the m/r SEBT tool helps teachers stratify technical levels, identify injury risk, and consequently assist with immediate and appropriate recommendations for supplemental training and//or follow-up with a medical professional. The tool evolved out of more than 3 years of collaborative, multisite research utilizing the Star Excursion Balance Test (SEBT) as a dynamic test of balance. Modifications were made to render the test more dance-specific and to increase balance challenges. Within the 3-year period, more than 100 dancers were tested in four sites, two in the United States and two in the United Kingdom. Despite the relatively large collective sample size, neither the original SEBT nor its modifications (m/r SEBT) held robust face or content validity as balance screens. What did emerge, however, were qualitative criteria that the authors organized into a feasible assessment tool for preseason auditions. While this tool awaits further validation, its current evolution helps serve as a bridge between dance teachers' clinical and practical knowledge. PMID:25433257

  7. Functional CRISPR screening identifies the ufmylation pathway as a regulator of SQSTM1/p62.

    PubMed

    DeJesus, Rowena; Moretti, Francesca; McAllister, Gregory; Wang, Zuncai; Bergman, Phil; Liu, Shanming; Frias, Elizabeth; Alford, John; Reece-Hoyes, John S; Lindeman, Alicia; Kelliher, Jennifer; Russ, Carsten; Knehr, Judith; Carbone, Walter; Beibel, Martin; Roma, Guglielmo; Ng, Aylwin; Tallarico, John A; Porter, Jeffery A; Xavier, Ramnik J; Mickanin, Craig; Murphy, Leon O; Hoffman, Gregory R; Nyfeler, Beat

    2016-01-01

    SQSTM1 is an adaptor protein that integrates multiple cellular signaling pathways and whose expression is tightly regulated at the transcriptional and post-translational level. Here, we describe a forward genetic screening paradigm exploiting CRISPR-mediated genome editing coupled to a cell selection step by FACS to identify regulators of SQSTM1. Through systematic comparison of pooled libraries, we show that CRISPR is superior to RNAi in identifying known SQSTM1 modulators. A genome-wide CRISPR screen exposed MTOR signalling and the entire macroautophagy machinery as key regulators of SQSTM1 and identified several novel modulators including HNRNPM, SLC39A14, SRRD, PGK1 and the ufmylation cascade. We show that ufmylation regulates SQSTM1 by eliciting a cell type-specific ER stress response which induces SQSTM1 expression and results in its accumulation in the cytosol. This study validates pooled CRISPR screening as a powerful method to map the repertoire of cellular pathways that regulate the fate of an individual target protein. PMID:27351204

  8. Functional CRISPR screening identifies the ufmylation pathway as a regulator of SQSTM1/p62

    PubMed Central

    DeJesus, Rowena; Moretti, Francesca; McAllister, Gregory; Wang, Zuncai; Bergman, Phil; Liu, Shanming; Frias, Elizabeth; Alford, John; Reece-Hoyes, John S; Lindeman, Alicia; Kelliher, Jennifer; Russ, Carsten; Knehr, Judith; Carbone, Walter; Beibel, Martin; Roma, Guglielmo; Ng, Aylwin; Tallarico, John A; Porter, Jeffery A; Xavier, Ramnik J; Mickanin, Craig; Murphy, Leon O; Hoffman, Gregory R; Nyfeler, Beat

    2016-01-01

    SQSTM1 is an adaptor protein that integrates multiple cellular signaling pathways and whose expression is tightly regulated at the transcriptional and post-translational level. Here, we describe a forward genetic screening paradigm exploiting CRISPR-mediated genome editing coupled to a cell selection step by FACS to identify regulators of SQSTM1. Through systematic comparison of pooled libraries, we show that CRISPR is superior to RNAi in identifying known SQSTM1 modulators. A genome-wide CRISPR screen exposed MTOR signalling and the entire macroautophagy machinery as key regulators of SQSTM1 and identified several novel modulators including HNRNPM, SLC39A14, SRRD, PGK1 and the ufmylation cascade. We show that ufmylation regulates SQSTM1 by eliciting a cell type-specific ER stress response which induces SQSTM1 expression and results in its accumulation in the cytosol. This study validates pooled CRISPR screening as a powerful method to map the repertoire of cellular pathways that regulate the fate of an individual target protein. DOI: http://dx.doi.org/10.7554/eLife.17290.001 PMID:27351204

  9. Effects of d-electrons in pseudopotential screened-exchange density functional calculations

    SciTech Connect

    Lee, Byounghak; Canning, Andrew; Wang, Lin-Wang

    2007-09-12

    We report a theoretical study on the role of shallow d states in the screened-exchange local density approximation (sX-LDA) band structure of binary semiconductor systems.We found that inaccurate pseudo-wavefunctions can lead to 1) an overestimation of the screened-exchange interaction betweenthe localized d states and the delocalized higher energy s and p states and 2) an underestimation of the screened-exchange interaction between the d states. The resulting sX-LDA band structures have substantially smaller band gaps compared with experiments. We correct the pseudo-wavefunctions of d states by including the semicore s and p states of the same shell in the valence states. The correction of pseudo-wavefunctions yields band gaps and d state binding energies in good agreement with experiments and the full potential linearized augmented plane wave sX-LDA calculations. Compared with the quasi-particle GW method, our sX-LDA results shows not only similar quality on the band gaps but also much better d state binding energies. Combined with its capability of ground state structure calculation, the sX-LDA is expected to be a valuable theoretical tool for the II-VI and III-V (especially the III-N) bulk semiconductors and nanostructure studies.

  10. Effects of d-electrons in pseudopotential screened-exchange density functional calculations

    SciTech Connect

    Wang, Lin-Wang; Lee, Byounghak; Canning, Andrew; Wang, Lin-Wang

    2008-08-11

    We report a theoretical study on the role of shallow d states in the screened-exchange local density approximation (sX-LDA) band structure of binary semiconductor systems. We found that the inaccurate pseudo-wavefunctions can lead to (1) an overestimation of the screened-exchange interaction between the localized d states and the delocalized higher energy s and p states and (2) an underestimation of the screened-exchange interaction between the d states. The resulting sX-LDA band structures have substantially smaller band gaps compared with experiments. We correct the pseudo-wavefunctions of d states by including the semicore s and p states of the same shell in the valence states. The correction of pseudo-wavefunctions yields band gaps and the d state binding energy with good agreements with experiments and the full potential linearized augmented planewave (FLAPW) calculations. Compared with the quasi-particle GW method, our sX-LDA results shows not only similar quality on the band gaps but also much better d state binding energy. Combined with its capability of ground state structure calculation, the sX-LDA is expected to be a valuable theoretical tool for the II-VI and III-V (especially the III-N) bulk semiconductors and nanostructure studies.

  11. Functional Genomics Screening Utilizing Mutant Mouse Embryonic Stem Cells Identifies Novel Radiation-Response Genes

    PubMed Central

    Loesch, Kimberly; Galaviz, Stacy; Hamoui, Zaher; Clanton, Ryan; Akabani, Gamal; Deveau, Michael; DeJesus, Michael; Ioerger, Thomas; Sacchettini, James C.; Wallis, Deeann

    2015-01-01

    Elucidating the genetic determinants of radiation response is crucial to optimizing and individualizing radiotherapy for cancer patients. In order to identify genes that are involved in enhanced sensitivity or resistance to radiation, a library of stable mutant murine embryonic stem cells (ESCs), each with a defined mutation, was screened for cell viability and gene expression in response to radiation exposure. We focused on a cancer-relevant subset of over 500 mutant ESC lines. We identified 13 genes; 7 genes that have been previously implicated in radiation response and 6 other genes that have never been implicated in radiation response. After screening, proteomic analysis showed enrichment for genes involved in cellular component disassembly (e.g. Dstn and Pex14) and regulation of growth (e.g. Adnp2, Epc1, and Ing4). Overall, the best targets with the highest potential for sensitizing cancer cells to radiation were Dstn and Map2k6, and the best targets for enhancing resistance to radiation were Iqgap and Vcan. Hence, we provide compelling evidence that screening mutant ESCs is a powerful approach to identify genes that alter radiation response. Ultimately, this knowledge can be used to define genetic variants or therapeutic targets that will enhance clinical therapy. PMID:25853515

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

    PubMed Central

    2012-01-01

    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 of B2 may provide the

  13. Experimental Strategies for Functional Annotation and Metabolism Discovery: Targeted Screening of Solute Binding Proteins and Unbiased Panning of Metabolomes

    PubMed Central

    2015-01-01

    The rate at which genome sequencing data is accruing demands enhanced methods for functional annotation and metabolism discovery. Solute binding proteins (SBPs) facilitate the transport of the first reactant in a metabolic pathway, thereby constraining the regions of chemical space and the chemistries that must be considered for pathway reconstruction. We describe high-throughput protein production and differential scanning fluorimetry platforms, which enabled the screening of 158 SBPs against a 189 component library specifically tailored for this class of proteins. Like all screening efforts, this approach is limited by the practical constraints imposed by construction of the library, i.e., we can study only those metabolites that are known to exist and which can be made in sufficient quantities for experimentation. To move beyond these inherent limitations, we illustrate the promise of crystallographic- and mass spectrometric-based approaches for the unbiased use of entire metabolomes as screening libraries. Together, our approaches identified 40 new SBP ligands, generated experiment-based annotations for 2084 SBPs in 71 isofunctional clusters, and defined numerous metabolic pathways, including novel catabolic pathways for the utilization of ethanolamine as sole nitrogen source and the use of d-Ala-d-Ala as sole carbon source. These efforts begin to define an integrated strategy for realizing the full value of amassing genome sequence data. PMID:25540822

  14. Zebrafish eleutheroembryos as an alternative system for screening chemicals disrupting the mammalian thyroid gland morphogenesis and function.

    PubMed

    Raldúa, Demetrio; Thienpont, Benedicte; Babin, Patrick J

    2012-04-01

    The importance and irreversibility of the effects of thyroid hormone deficiency on human brain development highlight the importance of identifying environmental agents that interfere with thyroid gland morphogenesis and function. Zebrafish eleutheroembryos are currently used by many pharmaceutical companies in drug discovery as a vertebrate model, not subjected to regulations for animal experiments, that provides an intermediate step between in vitro and rodent assay. The mechanisms of zebrafish thyroid development are generally comparable to those in humans, and moreover, molecular and functional studies of zebrafish thyroid follicles have demonstrated a high degree of conservation with upper vertebrates, opening up the possibility of designing alternative methods for screening individual chemicals and mixtures that impairing thyroid gland morphogenesis and/or function. Analysis of the intrafollicular thyroxine-content of zebrafish larvae exposed to potential disruptors has proved to be a reliable, physiologically relevant endpoint to estimate effects of chemicals on the mammalian thyroid gland. PMID:21978863

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

    PubMed Central

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

    2014-01-01

    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. PMID:25405319

  16. A Gain-of-Function Screen Identifying Genes Required for Growth and Pattern Formation of the Drosophila melanogaster Wing

    PubMed Central

    Cruz, Cristina; Glavic, Alvaro; Casado, Mar; de Celis, Jose F.

    2009-01-01

    The Drosophila melanogaster wing is a model system for analyzing the genetic control of organ size, shape, and pattern formation. The formation of the wing involves a variety of processes, such as cell growth, proliferation, pattern formation, and differentiation. These developmental processes are under genetic control, and many genes participating in specific aspects of wing development have already being characterized. In this work, we aim to identify novel genes regulating wing growth and patterning. To this end, we have carried out a gain-of-function screen generating novel P-UAS (upstream activating sequences) insertions allowing forced gene expression. We produced 3340 novel P-UAS insertions and isolated 300 that cause a variety of wing phenotypes in combination with a Gal4 driver expressed exclusively in the central domain of the presumptive wing blade. The mapping of these P-UAS insertion sites allowed us to identify the gene that causes the gain-of-function phenotypes. We show that a fraction of these phenotypes are related to the induction of cell death in the domain of ectopic gene expression. Finally, we present a preliminary characterization of a gene identified in the screen, the function of which is required for the development of the L5 longitudinal vein. PMID:19737745

  17. The gated integration technique for the accurate measurement of the autocorrelation function of speckle intensities scattered from random phase screens

    NASA Astrophysics Data System (ADS)

    Zhang, Ningyu; Cheng, Chuanfu; Teng, Shuyun; Chen, Xiaoyi; Xu, Zhizhan

    2007-09-01

    A new approach based on the gated integration technique is proposed for the accurate measurement of the autocorrelation function of speckle intensities scattered from a random phase screen. The Boxcar used for this technique in the acquisition of the speckle intensity data integrates the photoelectric signal during its sampling gate open, and it repeats the sampling by a preset number, m. The average analog of the m samplings output by the Boxcar enhances the signal-to-noise ratio by √{m}, because the repeated sampling and the average make the useful speckle signals stable, while the randomly varied photoelectric noise is suppressed by 1/√{m}. In the experiment, we use an analog-to-digital converter module to synchronize all the actions such as the stepped movement of the phase screen, the repeated sampling, the readout of the averaged output of the Boxcar, etc. The experimental results show that speckle signals are better recovered from contaminated signals, and the autocorrelation function with the secondary maximum is obtained, indicating that the accuracy of the measurement of the autocorrelation function is greatly improved by the gated integration technique.

  18. Ag nanocluster functionalized glasses for efficient photonic conversion in light sources, solar cells and flexible screen monitors.

    PubMed

    Kuznetsov, A S; Tikhomirov, V K; Shestakov, M V; Moshchalkov, V V

    2013-11-01

    An ever growing demand for efficient energy conversion, for instance in luminescent lamps, flexible screens and solar cells, results in the current significant growth of research on functionalized nanomaterials for these applications. This paper reviews recent developments of a new class of optically active nanostructured materials based on glasses doped with luminescent Ag nanoclusters consisting of only a few Ag atoms, suitable for mercury-free white light generation and solar down-shifting. This new approach, based solely on Ag nanocluster doped glasses, is compared to other alternatives in the field of Ag and rare-earth ion co-doped materials. PMID:23948871

  19. Ag nanocluster functionalized glasses for efficient photonic conversion in light sources, solar cells and flexible screen monitors

    NASA Astrophysics Data System (ADS)

    Kuznetsov, A. S.; Tikhomirov, V. K.; Shestakov, M. V.; Moshchalkov, V. V.

    2013-10-01

    An ever growing demand for efficient energy conversion, for instance in luminescent lamps, flexible screens and solar cells, results in the current significant growth of research on functionalized nanomaterials for these applications. This paper reviews recent developments of a new class of optically active nanostructured materials based on glasses doped with luminescent Ag nanoclusters consisting of only a few Ag atoms, suitable for mercury-free white light generation and solar down-shifting. This new approach, based solely on Ag nanocluster doped glasses, is compared to other alternatives in the field of Ag and rare-earth ion co-doped materials.

  20. Determination of liquid's molecular interference function based on X-ray diffraction and dual-energy CT in security screening.

    PubMed

    Zhang, Li; YangDai, Tianyi

    2016-08-01

    A method for deriving the molecular interference function (MIF) of an unknown liquid for security screening is presented. Based on the effective atomic number reconstructed from dual-energy computed tomography (CT), equivalent molecular formula of the liquid is estimated. After a series of optimizations, the MIF and a new effective atomic number are finally obtained from the X-ray diffraction (XRD) profile. The proposed method generates more accurate results with less sensitivity to the noise and data deficiency of the XRD profile. PMID:27239986

  1. A discriminant function model as an alternative method to spirometry for COPD screening in primary care settings in China

    PubMed Central

    Cui, Jiangyu; Zhou, Yumin; Tian, Jia; Wang, Xinwang; Zheng, Jingping; Zhong, Nanshan

    2012-01-01

    Objective COPD is often underdiagnosed in a primary care setting where the spirometry is unavailable. This study was aimed to develop a simple, economical and applicable model for COPD screening in those settings. Methods First we established a discriminant function model based on Bayes’ Rule by stepwise discriminant analysis, using the data from 243 COPD patients and 112 non-COPD subjects from our COPD survey in urban and rural communities and local primary care settings in Guangdong Province, China. We then used this model to discriminate COPD in additional 150 subjects (50 non-COPD and 100 COPD ones) who had been recruited by the same methods as used to have established the model. All participants completed pre- and post-bronchodilator spirometry and questionnaires. COPD was diagnosed according to the Global Initiative for Chronic Obstructive Lung Disease criteria. The sensitivity and specificity of the discriminant function model was assessed. Results The established discriminant function model included nine variables: age, gender, smoking index, body mass index, occupational exposure, living environment, wheezing, cough and dyspnoea. The sensitivity, specificity, positive likelihood ratio, negative likelihood ratio, accuracy and error rate of the function model to discriminate COPD were 89.00%, 82.00%, 4.94, 0.13, 86.66% and 13.34%, respectively. The accuracy and Kappa value of the function model to predict COPD stages were 70% and 0.61 (95% CI, 0.50 to 0.71). Conclusions This discriminant function model may be used for COPD screening in primary care settings in China as an alternative option instead of spirometry. PMID:23205284

  2. The evolving role of physiotherapists in pre-employment screening for workplace injury prevention: are functional capacity evaluations the answer?

    PubMed Central

    Legge, Jennifer

    2013-01-01

    Background Musculoskeletal injuries account for the largest proportion of workplace injuries. In an attempt to predict, and subsequently manage, the risk of sprains and strains in the workplace, employers are turning to pre-employment screening. Functional capacity evaluations (FCEs) are increasing in popularity as a tool for pre-employment screening despite limited published evidence for their validity in healthy working populations. Objectives This narrative review will present an overview of the state of the evidence for pre-employment functional testing, propose a framework for decision-making to determine the suitability of assessment tools, and discuss the role and potential ethical challenges for physiotherapists conducting pre-employment functional testing. Major Findings Much of the evidence surrounding the validity of functional testing is in the context of the injured worker and prediction of return to work. In healthy populations, FCE components, such as aerobic fitness and manual handling activities, have demonstrated predictability of workplace injury in a small number of studies. This predictability improves when workers' performance is compared with the job demands. This job-specific approach is also required to meet anti-discrimination requirements. There are a number of practical limitations to functional testing, although these are not limited to the pre-employment domain. Physiotherapists need to have a clear understanding of the legal requirements and potential ethical challenges that they may face when conducting pre-employment functional assessments (PEFAs). Conclusions Further research is needed into the efficacy of pre-employment testing for workplace injury prevention. Physiotherapists and PEFAs are just one part of a holistic approach to workplace injury prevention. PMID:24124346

  3. Can selected functional movement screen assessments be used to identify movement deficiencies that could affect multidirectional speed and jump performance?

    PubMed

    Lockie, Robert G; Schultz, Adrian B; Jordan, Corrin A; Callaghan, Samuel J; Jeffriess, Matthew D; Luczo, Tawni M

    2015-01-01

    The Functional Movement Screen (FMS) includes lower-body focused tests (deep squat [DS], hurdle step, in-line lunge) that could assist in identifying movement deficiencies affecting multidirectional sprinting and jumping, which are important qualities for team sports. However, the hypothesized relationship with athletic performance lacks supportive research. This study investigated relationships between the lower-body focused screens and overall FMS performance and multidirectional speed and jumping capabilities in team sport athletes. Twenty-two healthy men were assessed in the FMS, and multidirectional speed (0- to 5-m, 0- to 10-m, 0- to 20-m sprint intervals; 505 and between-leg turn differences, modified T-test and differences between initial movement to the left or right); and bilateral and unilateral multidirectional jumping (vertical [VJ], standing long [SLJ], and lateral jump) tests. Pearson's correlations (r) were used to calculate relationships between screening scores and performance tests (p ≤ 0.05). After the determination of any screens relating to athletic performance, subjects were stratified into groups (3 = high-performing group; 2 = intermediate-performing group; 1 = low-performing group) to investigate movement compensations. A 1-way analysis of variance (p ≤ 0.05) determined any between-group differences. There were few significant correlations. The DS did moderately correlate with between-leg 505 difference (r = -0.423), and bilateral VJ (r = -0.428) and SLJ (r = -0.457). When stratified into groups according to DS score, high performers had a 13% greater SLJ when compared with intermediate performers, which was the only significant result. The FMS seems to have minimal capabilities for identifying movement deficiencies that could affect multidirectional speed and jumping in male team sport athletes. PMID:25028993

  4. Integration of high-content screening and untargeted metabolomics for comprehensive functional annotation of natural product libraries

    PubMed Central

    Kurita, Kenji L.; Glassey, Emerson; Linington, Roger G.

    2015-01-01

    Traditional natural products discovery using a combination of live/dead screening followed by iterative bioassay-guided fractionation affords no information about compound structure or mode of action until late in the discovery process. This leads to high rates of rediscovery and low probabilities of finding compounds with unique biological and/or chemical properties. By integrating image-based phenotypic screening in HeLa cells with high-resolution untargeted metabolomics analysis, we have developed a new platform, termed Compound Activity Mapping, that is capable of directly predicting the identities and modes of action of bioactive constituents for any complex natural product extract library. This new tool can be used to rapidly identify novel bioactive constituents and provide predictions of compound modes of action directly from primary screening data. This approach inverts the natural products discovery process from the existing ‟grind and find” model to a targeted, hypothesis-driven discovery model where the chemical features and biological function of bioactive metabolites are known early in the screening workflow, and lead compounds can be rationally selected based on biological and/or chemical novelty. We demonstrate the utility of the Compound Activity Mapping platform by combining 10,977 mass spectral features and 58,032 biological measurements from a library of 234 natural products extracts and integrating these two datasets to identify 13 clusters of fractions containing 11 known compound families and four new compounds. Using Compound Activity Mapping we discovered the quinocinnolinomycins, a new family of natural products with a unique carbon skeleton that cause endoplasmic reticulum stress. PMID:26371303

  5. Novel gene function revealed by mouse mutagenesis screens for models of age-related disease

    PubMed Central

    Potter, Paul K.; Bowl, Michael R.; Jeyarajan, Prashanthini; Wisby, Laura; Blease, Andrew; Goldsworthy, Michelle E.; Simon, Michelle M.; Greenaway, Simon; Michel, Vincent; Barnard, Alun; Aguilar, Carlos; Agnew, Thomas; Banks, Gareth; Blake, Andrew; Chessum, Lauren; Dorning, Joanne; Falcone, Sara; Goosey, Laurence; Harris, Shelley; Haynes, Andy; Heise, Ines; Hillier, Rosie; Hough, Tertius; Hoslin, Angela; Hutchison, Marie; King, Ruairidh; Kumar, Saumya; Lad, Heena V.; Law, Gemma; MacLaren, Robert E.; Morse, Susan; Nicol, Thomas; Parker, Andrew; Pickford, Karen; Sethi, Siddharth; Starbuck, Becky; Stelma, Femke; Cheeseman, Michael; Cross, Sally H.; Foster, Russell G.; Jackson, Ian J.; Peirson, Stuart N.; Thakker, Rajesh V.; Vincent, Tonia; Scudamore, Cheryl; Wells, Sara; El-Amraoui, Aziz; Petit, Christine; Acevedo-Arozena, Abraham; Nolan, Patrick M.; Cox, Roger; Mallon, Anne-Marie; Brown, Steve D. M.

    2016-01-01

    Determining the genetic bases of age-related disease remains a major challenge requiring a spectrum of approaches from human and clinical genetics to the utilization of model organism studies. Here we report a large-scale genetic screen in mice employing a phenotype-driven discovery platform to identify mutations resulting in age-related disease, both late-onset and progressive. We have utilized N-ethyl-N-nitrosourea mutagenesis to generate pedigrees of mutagenized mice that were subject to recurrent screens for mutant phenotypes as the mice aged. In total, we identify 105 distinct mutant lines from 157 pedigrees analysed, out of which 27 are late-onset phenotypes across a range of physiological systems. Using whole-genome sequencing we uncover the underlying genes for 44 of these mutant phenotypes, including 12 late-onset phenotypes. These genes reveal a number of novel pathways involved with age-related disease. We illustrate our findings by the recovery and characterization of a novel mouse model of age-related hearing loss. PMID:27534441

  6. Novel gene function revealed by mouse mutagenesis screens for models of age-related disease.

    PubMed

    Potter, Paul K; Bowl, Michael R; Jeyarajan, Prashanthini; Wisby, Laura; Blease, Andrew; Goldsworthy, Michelle E; Simon, Michelle M; Greenaway, Simon; Michel, Vincent; Barnard, Alun; Aguilar, Carlos; Agnew, Thomas; Banks, Gareth; Blake, Andrew; Chessum, Lauren; Dorning, Joanne; Falcone, Sara; Goosey, Laurence; Harris, Shelley; Haynes, Andy; Heise, Ines; Hillier, Rosie; Hough, Tertius; Hoslin, Angela; Hutchison, Marie; King, Ruairidh; Kumar, Saumya; Lad, Heena V; Law, Gemma; MacLaren, Robert E; Morse, Susan; Nicol, Thomas; Parker, Andrew; Pickford, Karen; Sethi, Siddharth; Starbuck, Becky; Stelma, Femke; Cheeseman, Michael; Cross, Sally H; Foster, Russell G; Jackson, Ian J; Peirson, Stuart N; Thakker, Rajesh V; Vincent, Tonia; Scudamore, Cheryl; Wells, Sara; El-Amraoui, Aziz; Petit, Christine; Acevedo-Arozena, Abraham; Nolan, Patrick M; Cox, Roger; Mallon, Anne-Marie; Brown, Steve D M

    2016-01-01

    Determining the genetic bases of age-related disease remains a major challenge requiring a spectrum of approaches from human and clinical genetics to the utilization of model organism studies. Here we report a large-scale genetic screen in mice employing a phenotype-driven discovery platform to identify mutations resulting in age-related disease, both late-onset and progressive. We have utilized N-ethyl-N-nitrosourea mutagenesis to generate pedigrees of mutagenized mice that were subject to recurrent screens for mutant phenotypes as the mice aged. In total, we identify 105 distinct mutant lines from 157 pedigrees analysed, out of which 27 are late-onset phenotypes across a range of physiological systems. Using whole-genome sequencing we uncover the underlying genes for 44 of these mutant phenotypes, including 12 late-onset phenotypes. These genes reveal a number of novel pathways involved with age-related disease. We illustrate our findings by the recovery and characterization of a novel mouse model of age-related hearing loss. PMID:27534441

  7. Functional genome-wide siRNA screen identifies KIAA0586 as mutated in Joubert syndrome.

    PubMed

    Roosing, Susanne; Hofree, Matan; Kim, Sehyun; Scott, Eric; Copeland, Brett; Romani, Marta; Silhavy, Jennifer L; Rosti, Rasim O; Schroth, Jana; Mazza, Tommaso; Miccinilli, Elide; Zaki, Maha S; Swoboda, Kathryn J; Milisa-Drautz, Joanne; Dobyns, William B; Mikati, Mohamed A; İncecik, Faruk; Azam, Matloob; Borgatti, Renato; Romaniello, Romina; Boustany, Rose-Mary; Clericuzio, Carol L; D'Arrigo, Stefano; Strømme, Petter; Boltshauser, Eugen; Stanzial, Franco; Mirabelli-Badenier, Marisol; Moroni, Isabella; Bertini, Enrico; Emma, Francesco; Steinlin, Maja; Hildebrandt, Friedhelm; Johnson, Colin A; Freilinger, Michael; Vaux, Keith K; Gabriel, Stacey B; Aza-Blanc, Pedro; Heynen-Genel, Susanne; Ideker, Trey; Dynlacht, Brian D; Lee, Ji Eun; Valente, Enza Maria; Kim, Joon; Gleeson, Joseph G

    2015-01-01

    Defective primary ciliogenesis or cilium stability forms the basis of human ciliopathies, including Joubert syndrome (JS), with defective cerebellar vermis development. We performed a high-content genome-wide small interfering RNA (siRNA) screen to identify genes regulating ciliogenesis as candidates for JS. We analyzed results with a supervised-learning approach, using SYSCILIA gold standard, Cildb3.0, a centriole siRNA screen and the GTex project, identifying 591 likely candidates. Intersection of this data with whole exome results from 145 individuals with unexplained JS identified six families with predominantly compound heterozygous mutations in KIAA0586. A c.428del base deletion in 0.1% of the general population was found in trans with a second mutation in an additional set of 9 of 163 unexplained JS patients. KIAA0586 is an orthologue of chick Talpid3, required for ciliogenesis and Sonic hedgehog signaling. Our results uncover a relatively high frequency cause for JS and contribute a list of candidates for future gene discoveries in ciliopathies. PMID:26026149

  8. Toward the discovery of functional transthyretin amyloid inhibitors: application of virtual screening methods.

    PubMed

    Simões, Carlos J V; Mukherjee, Trishna; Brito, Rui M M; Jackson, Richard M

    2010-10-25

    Inhibition of amyloid fibril formation by stabilization of the native form of the protein transthyretin (TTR) is a viable approach for the treatment of familial amyloid polyneuropathy that has been gaining momentum in the field of amyloid research. The TTR stabilizer molecules discovered to date have shown efficacy at inhibiting fibrilization in vitro but display impairing issues of solubility, affinity for TTR in the blood plasma and/or adverse effects. In this study we present a benchmark of four protein- and ligand-based virtual screening (VS) methods for identifying novel TTR stabilizers: (i) two-dimensional (2D) similarity searches with chemical hashed, pharmacophore, and UNITY fingerprints, (ii) 3D searches based on shape, chemical, and electrostatic similarity, (iii) LigMatch, a new ligand-based method which uses multiple templates and combines 3D geometric hashing with a 2D preselection process, and (iv) molecular docking to consensus X-ray crystal structures of TTR. We illustrate the potential of the best-performing VS protocols to retrieve promising new leads by ranking a tailored library of 2.3 million commercially available compounds. Our predictions show that the top-scoring molecules possess distinctive features from the known TTR binders, holding better solubility, fraction of halogen atoms, and binding affinity profiles. To the best of our knowledge, this is the first attempt to rationalize the utilization of a large battery of in silico screening techniques toward the identification of a new generation of TTR amyloid inhibitors. PMID:20883031

  9. High potency olfactory receptor agonists discovered by virtual high-throughput screening: molecular probes for receptor structure and olfactory function

    PubMed Central

    Triballeau, Nicolas; Van Name, Eric; Laslier, Guillaume; Cai, Diana; Paillard, Guillaume; Sorensen, Peter W.; Hoffmann, Rémy; Bertrand, Hugues-Olivier; Ngai, John; Acher, Francine C.

    2008-01-01

    The detection and discrimination of diverse chemical structures by the vertebrate olfactory system is accomplished by the recognition of odorous ligands by their cognate receptors. In the present study we used a computational high-throughput screening strategy to discover novel high affinity agonists of an olfactory G protein-coupled receptor tuned to recognize amino acid ligands. Functional testing of the top candidates validated several agonists with potencies higher than any of the receptor’s known natural ligands. Computational modeling revealed molecular interactions involved in ligand recognition by this receptor, and further highlighted interactions that have been conserved in evolutionarily divergent amino acid receptors. Significantly, the top compounds display robust activities as odorants in vivo, and include a natural product that may be used to signal the presence of bacteria in the aquatic environment. Our virtual screening approach should be applicable to the identification of new bioactive molecules for probing the structure of chemosensory receptors and the function of chemosensory systems in vivo. PMID:19081373

  10. Activity-Based Protein Profiling of Microbes

    SciTech Connect

    Sadler, Natalie C.; Wright, Aaron T.

    2015-02-01

    Activity-Based Protein Profiling (ABPP) in conjunction with multimodal characterization techniques has yielded impactful findings in microbiology, particularly in pathogen, bioenergy, drug discovery, and environmental research. Using small molecule chemical probes that react irreversibly with specific proteins or protein families in complex systems has provided insights in enzyme functions in central metabolic pathways, drug-protein interactions, and regulatory protein redox, for systems ranging from photoautotrophic cyanobacteria to mycobacteria, and combining live cell or cell extract ABPP with proteomics, molecular biology, modeling, and other techniques has greatly expanded our understanding of these systems. New opportunities for application of ABPP to microbial systems include: enhancing protein annotation, characterizing protein activities in myriad environments, and reveal signal transduction and regulatory mechanisms in microbial systems.

  11. Electronic and optical properties of (U,Th)O2 compound from screened hybrid density functional studies

    NASA Astrophysics Data System (ADS)

    Mo, Chongjie; Yang, Yu; Kang, Wei; Zhang, Ping

    2016-04-01

    The electronic structure and optical properties for the (U,Th)O2 compound are systematically studied by employing the Heyd-Scuseria-Ernzerh method (HSE) of screened hybrid density functional. The electronic band gap of (U,Th)O2 is predicted to be 3.06 eV, in the middle of the values of UO2 and ThO2. Based on wavefunction analysis, we conclude (U,Th)O2 to be a Mott insulator in its ground state. The frequency dependent dielectric functions and optical properties are then calculated and compared with those of ThO2 and UO2. At the visible light frequency range, the adsorption coefficients for ThO2, UO2 and (U,Th)O2 are totally different, which gives an accessible method to predict the proportion of U atoms in an arbitrary unknown (U,Th)O2 compounds from the adsorption spectrum of visible lights.

  12. Density-functional theory with screened van der Waals interactions for the modeling of hybrid inorganic-organic systems.

    PubMed

    Ruiz, Victor G; Liu, Wei; Zojer, Egbert; Scheffler, Matthias; Tkatchenko, Alexandre

    2012-04-01

    The electronic properties and the function of hybrid inorganic-organic systems (HIOS) are intimately linked to their interface geometry. Here we show that the inclusion of the many-body collective response of the substrate electrons inside the inorganic bulk enables us to reliably predict the HIOS geometries and energies. This is achieved by the combination of dispersion-corrected density-functional theory (the DFT+ van der Waals approach) [Phys. Rev. Lett. 102, 073005 (2009)], with the Lifshitz-Zaremba-Kohn theory for the nonlocal Coulomb screening within the bulk. Our method yields geometries in remarkable agreement (≈0.1 Å) with normal incidence x-ray standing wave measurements for the 3, 4, 9, 10-perylene-tetracarboxylic acid dianhydride (C(24)O(6)H(8), PTCDA) molecule on Cu(111), Ag(111), and Au(111) surfaces. Similarly accurate results are obtained for xenon and benzene adsorbed on metal surfaces. PMID:22540809

  13. Functionalized Carbon Quantum Dots with Dopamine for Tyrosinase Activity Monitoring and Inhibitor Screening: In Vitro and Intracellular Investigation.

    PubMed

    Chai, Lujing; Zhou, Jin; Feng, Hui; Tang, Cong; Huang, Yuanyuan; Qian, Zhaosheng

    2015-10-28

    Sensitive assay of tyrosinase (TYR) activity is in urgent demand for both fundamental research and practical application, but the exploration of functional materials with good biocompatibility for its activity evaluation at the intracellular level is still challenging until now. In this work, we develop a convenient and real-time assay with high sensitivity for TYR activity/level monitoring and its inhibitor screening based on biocompatible dopamine functionalized carbon quantum dots (Dopa-CQDs). Dopamine with redox property was functionalized on the surface of carbon quantum dots to construct a Dopa-CQDs conjugate with strong bluish green fluorescence. When the dopamine moiety in Dopa-CQDs conjugate was oxidized to a dopaquinone derivative under specific catalysis of TYR, an intraparticle photoinduced electron transfer (PET) process between CQDs and dopaquinone moiety took place, and then the fluorescence of the conjugate could be quenched simultaneously. Quantitative evaluation of TYR activity was established in terms of the relationship between fluorescence quenching efficiency and TYR activity. The assay covered a broad linear range of up to 800 U/L with a low detection limit of 7.0 U/L. Arbutin, a typical inhibitor of TYR, was chosen as an example to assess its function of inhibitor screening, and positive results were observed that fluorescence quenching extent of the probe was reduced in the presence of arbutin. It is also demonstrated that Dopa-CQD conjugate possesses excellent biocompatibility, and can sensitively monitor intracellular tyrosinase level in melanoma cells and intracellular pH changes in living cells, which provides great potential in application of TYR/pH-associated disease monitoring and medical diagnostics. PMID:26440479

  14. A Multistep High-Content Screening Approach to Identify Novel Functionally Relevant Target Genes in Pancreatic Cancer

    PubMed Central

    Buchholz, Malte; Honstein, Tatjana; Kirchhoff, Sandra; Kreider, Ramona; Schmidt, Harald; Sipos, Bence; Gress, Thomas M.

    2015-01-01

    In order to foster the systematic identification of novel genes with important functional roles in pancreatic cancer, we have devised a multi-stage screening strategy to provide a rational basis for the selection of highly relevant novel candidate genes based on the results of functional high-content analyses. The workflow comprised three consecutive stages: 1) serial gene expression profiling analyses of primary human pancreatic tissues as well as a number of in vivo and in vitro models of tumor-relevant characteristics in order to identify genes with conspicuous expression patterns; 2) use of ‘reverse transfection array’ technology for large-scale parallelized functional analyses of potential candidate genes in cell-based assays; and 3) selection of individual candidate genes for further in-depth examination of their cellular roles. A total of 14 genes, among them 8 from “druggable” gene families, were classified as high priority candidates for individual functional characterization. As an example to demonstrate the validity of the approach, comprehensive functional data on candidate gene ADRBK1/GRK2, which has previously not been implicated in pancreatic cancer, is presented. PMID:25849100

  15. Rapid Discovery of Functional Small Molecule Ligands against Proteomic Targets through Library-Against-Library Screening.

    PubMed

    Wu, Chun-Yi; Wang, Don-Hong; Wang, Xiaobing; Dixon, Seth M; Meng, Liping; Ahadi, Sara; Enter, Daniel H; Chen, Chao-Yu; Kato, Jason; Leon, Leonardo J; Ramirez, Laura M; Maeda, Yoshiko; Reis, Carolina F; Ribeiro, Brianna; Weems, Brittany; Kung, Hsing-Jien; Lam, Kit S

    2016-06-13

    Identifying "druggable" targets and their corresponding therapeutic agents are two fundamental challenges in drug discovery research. The one-bead-one-compound (OBOC) combinatorial library method has been developed to discover peptides or small molecules that bind to a specific target protein or elicit a specific cellular response. The phage display cDNA expression proteome library method has been employed to identify target proteins that interact with specific compounds. Here, we combined these two high-throughput approaches, efficiently interrogated approximately 10(13) possible molecular interactions, and identified 91 small molecule compound beads that interacted strongly with the phage library. Of 19 compounds resynthesized, 4 were cytotoxic against cancer cells; one of these compounds was found to interact with EIF5B and inhibit protein translation. As more binding pairs are confirmed and evaluated, the "library-against-library" screening approach and the resulting small molecule-protein domain interaction database may serve as a valuable tool for basic research and drug development. PMID:27053324

  16. Organotypic Spinal Cord Culture: a Proper Platform for the Functional Screening.

    PubMed

    Pandamooz, Sareh; Nabiuni, Mohammad; Miyan, Jaleel; Ahmadiani, Abolhassan; Dargahi, Leila

    2016-09-01

    Recent improvements in organotypic slice culturing and its accompanying technological innovations have made this biological preparation increasingly useful ex vivo experimental model. Among organotypic slice cultures obtained from various central nervous regions, spinal cord slice culture is an absorbing model that represents several unique advantages over other current in vitro and in vivo models. The culture of developing spinal cord slices, as allows real-time observation of embryonic cells behaviors, is an instrumental platform for developmental investigation. Importantly, due to the ability of ex vivo models to recapitulate different aspects of corresponding in vivo conditions, these models have been subject of various manipulations to derive disease-relevant slice models. Moreover spinal cord slice cultures represent a potential platform for screening of different pharmacological agents and evaluation of cell transplantation and neuroregenerative materials. In this review, we will focus on studies carried out using the ex vivo model of spinal cord slice cultures and main advantages linked to practicality of these slices in both normal and neuropathological diseases and summarize them in different categories based on application. PMID:26310972

  17. Identification of an antibacterial protein by functional screening of a human oral metagenomic library.

    PubMed

    Arivaradarajan, Preeti; Warburton, Philip J; Paramasamy, Gunasekaran; Nair, Sean P; Allan, Elaine; Mullany, Peter

    2015-09-01

    Screening of a bacterial artificial chromosome (BAC) library containing metagenomic DNA from human plaque and saliva allowed the isolation of four clones producing antimicrobial activity. Three of these were pigmented and encoded homologues of glutamyl-tRNA reductase (GluTR), an enzyme involved in the C5 pathway leading to tetrapyrole synthesis, and one clone had antibacterial activity with no pigmentation. The latter contained a BAC with an insert of 15.6 kb. Initial attempts to localize the gene(s) responsible for antimicrobial activity by subcloning into pUC-based vectors failed. A new plasmid for toxic gene expression (pTGEX) was designed enabling localization of the antibacterial activity to a 4.7-kb HindIII fragment. Transposon mutagenesis localized the gene to an open reading frame of 483 bp designated antibacterial protein1 (abp1). Abp1 was 94% identical to a hypothetical protein of Neisseria subflava (accession number WP_004519448.1). An Escherichia coli clone expressing Abp1 exhibited antibacterial activity against Bacillus subtilis BS78H, Staphylococcus epidermidis NCTC 11964 and B4268, and S. aureus NCTC 12493,ATCC 35696 and NCTC 11561. However, no antibacterial activity was observed against Pseudomonas aeruginosa ATCC 9027, N. subflava ATCC A1078, E. coli K12 JM109 and BL21(DE3) Fusobacterium nucleatum ATCC 25586 and NCTC 11326, Prevotella intermedia ATCC 25611, Veillonella parvula ATCC 10790 or Lactobacillus casei NCTC 6375. PMID:26347298

  18. Synthetic viability genomic screening defines Sae2 function in DNA repair

    PubMed Central

    Puddu, Fabio; Oelschlaegel, Tobias; Guerini, Ilaria; Geisler, Nicola J; Niu, Hengyao; Herzog, Mareike; Salguero, Israel; Ochoa-Montaño, Bernardo; Viré, Emmanuelle; Sung, Patrick; Adams, David J; Keane, Thomas M; Jackson, Stephen P

    2015-01-01

    DNA double-strand break (DSB) repair by homologous recombination (HR) requires 3′ single-stranded DNA (ssDNA) generation by 5′ DNA-end resection. During meiosis, yeast Sae2 cooperates with the nuclease Mre11 to remove covalently bound Spo11 from DSB termini, allowing resection and HR to ensue. Mitotic roles of Sae2 and Mre11 nuclease have remained enigmatic, however, since cells lacking these display modest resection defects but marked DNA damage hypersensitivities. By combining classic genetic suppressor screening with high-throughput DNA sequencing, we identify Mre11 mutations that strongly suppress DNA damage sensitivities of sae2Δ cells. By assessing the impacts of these mutations at the cellular, biochemical and structural levels, we propose that, in addition to promoting resection, a crucial role for Sae2 and Mre11 nuclease activity in mitotic DSB repair is to facilitate the removal of Mre11 from ssDNA associated with DSB ends. Thus, without Sae2 or Mre11 nuclease activity, Mre11 bound to partly processed DSBs impairs strand invasion and HR. PMID:25899817

  19. A loss-of-function genetic screening identifies novel mediators of thyroid cancer cell viability.

    PubMed

    Cantisani, Maria Carmela; Parascandolo, Alessia; Perälä, Merja; Allocca, Chiara; Fey, Vidal; Sahlberg, Niko; Merolla, Francesco; Basolo, Fulvio; Laukkanen, Mikko O; Kallioniemi, Olli Pekka; Santoro, Massimo; Castellone, Maria Domenica

    2016-05-10

    RET, BRAF and other protein kinases have been identified as major molecular players in thyroid cancer. To identify novel kinases required for the viability of thyroid carcinoma cells, we performed a RNA interference screening in the RET/PTC1(CCDC6-RET)-positive papillary thyroid cancer cell line TPC1 using a library of synthetic small interfering RNAs (siRNAs) targeting the human kinome and related proteins. We identified 14 hits whose silencing was able to significantly reduce the viability and the proliferation of TPC1 cells; most of them were active also in BRAF-mutant BCPAP (papillary thyroid cancer) and 8505C (anaplastic thyroid cancer) and in RAS-mutant CAL62 (anaplastic thyroid cancer) cells. These included members of EPH receptor tyrosine kinase family as well as SRC and MAPK (mitogen activated protein kinases) families. Importantly, silencing of the identified hits did not affect significantly the viability of Nthy-ori 3-1 (hereafter referred to as NTHY) cells derived from normal thyroid tissue, suggesting cancer cell specificity. The identified proteins are worth exploring as potential novel druggable thyroid cancer targets. PMID:27058903

  20. Systematic screen reveals new functional dynamics of histones H3 and H4 during gametogenesis.

    PubMed

    Govin, Jérôme; Dorsey, Jean; Gaucher, Jonathan; Rousseaux, Sophie; Khochbin, Saadi; Berger, Shelley L

    2010-08-15

    Profound epigenetic differences exist between genomes derived from male and female gametes; however, the nature of these changes remains largely unknown. We undertook a systematic investigation of chromatin reorganization during gametogenesis, using the model eukaryote Saccharomyces cerevisiae to examine sporulation, which has strong similarities with higher eukaryotic spermatogenesis. We established a mutational screen of histones H3 and H4 to uncover substitutions that reduce sporulation efficiency. We discovered two patches of residues-one on H3 and a second on H4-that are crucial for sporulation but not critical for mitotic growth, and likely comprise interactive nucleosomal surfaces. Furthermore, we identified novel histone post-translational modifications that mark the chromatin reorganization process during sporulation. First, phosphorylation of H3T11 appears to be a key modification during meiosis, and requires the meiotic-specific kinase Mek1. Second, H4 undergoes amino tail acetylation at Lys 5, Lys 8, and Lys 12, and these are synergistically important for post-meiotic chromatin compaction, occurring subsequent to the post-meiotic transient peak in phosphorylation at H4S1, and crucial for recruitment of Bdf1, a bromodomain protein, to chromatin in mature spores. Strikingly, the presence and temporal succession of the new H3 and H4 modifications are detected during mouse spermatogenesis, indicating that they are conserved through evolution. Thus, our results show that investigation of gametogenesis in yeast provides novel insights into chromatin dynamics, which are potentially relevant to epigenetic modulation of the mammalian process. PMID:20713519

  1. A Functional Misexpression Screen Uncovers a Role for Enabled in Progressive Neurodegeneration

    PubMed Central

    Rezával, Carolina; Berni, Jimena; Gorostiza, Ezequiel Axel; Werbajh, Santiago; Fagilde, María Marta; Fernández, María Paz; Beckwith, Esteban J.; Aranovich, Ezequiel J.; Sabio y García, Carmen A.; Ceriani, María Fernanda

    2008-01-01

    Drosophila is a well-established model to study the molecular basis of neurodegenerative diseases. We carried out a misexpression screen to identify genes involved in neurodegeneration examining locomotor behavior in young and aged flies. We hypothesized that a progressive loss of rhythmic activity could reveal novel genes involved in neurodegenerative mechanisms. One of the interesting candidates showing progressive arrhythmicity has reduced enabled (ena) levels. ena down-regulation gave rise to progressive vacuolization in specific regions of the adult brain. Abnormal staining of pre-synaptic markers such as cystein string protein (CSP) suggest that axonal transport could underlie the neurodegeneration observed in the mutant. Reduced ena levels correlated with increased apoptosis, which could be rescued in the presence of p35, a general Caspase inhibitor. Thus, this mutant recapitulates two important features of human neurodegenerative diseases, i.e., vulnerability of certain neuronal populations and progressive degeneration, offering a unique scenario in which to unravel the specific mechanisms in an easily tractable organism. PMID:18841196

  2. Use of organic functional group concentrations as a means of screening for energetics

    SciTech Connect

    Burgeson, I.E.; Bryan, S.A.; Camaioni, D.M.; Hallen, R.T.; Lerner, B.D.; Scheele, R.D.

    1996-06-01

    One of the safety concerns associated with the waste tanks on the Hanford site is the presence of organics in a highly oxidizing environment that could potentially act as a fuel source to maintain a propagating reaction. To determine this risk, it is necessary to determine the amount of high enthalpy organics present in the tanks. Currently, the primary ways of obtaining this information are to either rely on tank-fill histories, which are often unreliable and do not account for waste-aging processes, or obtain samples from the tank and speciate the organics present through a series of analytical procedures. While organic speciation has been successful in providing very valuable information about organics present in the tanks and the waste aging processes that are occurring in general, it can be costly and time consuming analyzing a large number of waste tanks. Differential scanning calorimetry has previously been used to obtain heat of reaction measurements of Hanford tank waste samples. However, differential scanning calorimetry is shown here to inadequately measure calculated heats of reaction of simulant tank mixtures. Overall, the preliminary results presented here, suggest that indeed Fourier transform infrared and Raman spectroscopy would be useful screening tools for determination of C-H and COO- organic content in tank waste samples analyzed in a hot cell environment. These techniques however, are not truly quantitative for this application and would be primarily used for identifying tanks of potential safety concern that would require further, more detailed confirmatory analysis by organic speciation techniques.

  3. Identification of thyroid tumor cell vulnerabilities through a siRNA-based functional screening

    PubMed Central

    Cetti, Elena; Fraietta, Ivan; Todoerti, Katia; Miranda, Claudia; Mazzoni, Mara; Re, Claudia; Colombo, Riccardo; Ukmar, Giorgio; Camisasca, Stefano; Pagliardini, Sonia; Pierotti, Marco A.; Neri, Antonino; Galvani, Arturo; Greco, Angela

    2015-01-01

    The incidence of thyroid carcinoma is rapidly increasing. Although generally associated with good prognosis, a fraction of thyroid tumors are not cured by standard therapy and progress to aggressive forms for which no effective treatments are currently available. In order to identify novel therapeutic targets for thyroid carcinoma, we focused on the discovery of genes essential for sustaining the oncogenic phenotype of thyroid tumor cells, but not required to the same degree for the viability of normal cells (non-oncogene addiction paradigm). We screened a siRNA oligonucleotide library targeting the human druggable genome in thyroid cancer BCPAP cell line in comparison with immortalized normal human thyrocytes (Nthy-ori 3–1). We identified a panel of hit genes whose silencing interferes with the growth of tumor cells, while sparing that of normal ones. Further analysis of three selected hit genes, namely Cyclin D1, MASTL and COPZ1, showed that they represent common vulnerabilities for thyroid tumor cells, as their inhibition reduced the viability of several thyroid tumor cell lines, regardless the histotype or oncogenic lesion. This work identified non-oncogenes essential for sustaining the phenotype of thyroid tumor cells, but not of normal cells, thus suggesting that they might represent promising targets for new therapeutic strategies. PMID:26431489

  4. Identification of thyroid tumor cell vulnerabilities through a siRNA-based functional screening.

    PubMed

    Anania, Maria; Gasparri, Fabio; Cetti, Elena; Fraietta, Ivan; Todoerti, Katia; Miranda, Claudia; Mazzoni, Mara; Re, Claudia; Colombo, Riccardo; Ukmar, Giorgio; Camisasca, Stefano; Pagliardini, Sonia; Pierotti, Marco; Neri, Antonino; Galvani, Arturo; Greco, Angela

    2015-10-27

    The incidence of thyroid carcinoma is rapidly increasing. Although generally associated with good prognosis, a fraction of thyroid tumors are not cured by standard therapy and progress to aggressive forms for which no effective treatments are currently available. In order to identify novel therapeutic targets for thyroid carcinoma, we focused on the discovery of genes essential for sustaining the oncogenic phenotype of thyroid tumor cells, but not required to the same degree for the viability of normal cells (non-oncogene addiction paradigm). We screened a siRNA oligonucleotide library targeting the human druggable genome in thyroid cancer BCPAP cell line in comparison with immortalized normal human thyrocytes (Nthy-ori 3-1). We identified a panel of hit genes whose silencing interferes with the growth of tumor cells, while sparing that of normal ones. Further analysis of three selected hit genes, namely Cyclin D1, MASTL and COPZ1, showed that they represent common vulnerabilities for thyroid tumor cells, as their inhibition reduced the viability of several thyroid tumor cell lines, regardless the histotype or oncogenic lesion. This work identified non-oncogenes essential for sustaining the phenotype of thyroid tumor cells, but not of normal cells, thus suggesting that they might represent promising targets for new therapeutic strategies. PMID:26431489

  5. Rapid Discovery of Functional Small Molecule Ligands against Proteomic Targets through Library-Against-Library Screening

    PubMed Central

    2016-01-01

    Identifying “druggable” targets and their corresponding therapeutic agents are two fundamental challenges in drug discovery research. The one-bead-one-compound (OBOC) combinatorial library method has been developed to discover peptides or small molecules that bind to a specific target protein or elicit a specific cellular response. The phage display cDNA expression proteome library method has been employed to identify target proteins that interact with specific compounds. Here, we combined these two high-throughput approaches, efficiently interrogated approximately 1013 possible molecular interactions, and identified 91 small molecule compound beads that interacted strongly with the phage library. Of 19 compounds resynthesized, 4 were cytotoxic against cancer cells; one of these compounds was found to interact with EIF5B and inhibit protein translation. As more binding pairs are confirmed and evaluated, the “library-against-library” screening approach and the resulting small molecule–protein domain interaction database may serve as a valuable tool for basic research and drug development. PMID:27053324

  6. Functional Screening of Hydrolytic Activities Reveals an Extremely Thermostable Cellulase from a Deep-Sea Archaeon

    PubMed Central

    Leis, Benedikt; Heinze, Simon; Angelov, Angel; Pham, Vu Thuy Trang; Thürmer, Andrea; Jebbar, Mohamed; Golyshin, Peter N.; Streit, Wolfgang R.; Daniel, Rolf; Liebl, Wolfgang

    2015-01-01

    Extreme habitats serve as a source of enzymes that are active under extreme conditions and are candidates for industrial applications. In this work, six large-insert mixed genomic libraries were screened for hydrolase activities in a broad temperature range (8–70°C). Among a variety of hydrolytic activities, one fosmid clone, derived from a library of pooled isolates of hyperthermophilic archaea from deep sea vents, displayed hydrolytic activity on carboxymethyl cellulose substrate plates at 70°C but not at lower temperatures. Sequence analysis of the fosmid insert revealed a gene encoding a novel glycoside hydrolase family 12 (GHF12) endo-1,4-β-glucanase, termed Cel12E. The enzyme shares 45% sequence identity with a protein from the archaeon Thermococcus sp. AM4 and displays a unique multidomain architecture. Biochemical characterization of Cel12E revealed a remarkably thermostable protein, which appears to be of archaeal origin. The enzyme displayed maximum activity at 92°C and was active on a variety of linear 1,4-β-glucans like carboxymethyl cellulose, β-glucan, lichenan, and phosphoric acid swollen cellulose. The protein is able to bind to various insoluble β-glucans. Product pattern analysis indicated that Cel12E is an endo-cleaving β-glucanase. Cel12E expands the toolbox of hyperthermostable archaeal cellulases with biotechnological potential. PMID:26191525

  7. Integrated phenotypic and activity-based profiling links Ces3 to obesity and diabetes

    PubMed Central

    Dominguez, Eduardo; Galmozzi, Andrea; Chang, Jae Won; Hsu, Ku-Lung; Pawlak, Joanna; Li, Weiwei; Godio, Cristina; Thomas, Jason; Partida, David; Niessen, Sherry; O'Brien, Paul E.; Russell, Aaron P.; Watt, Matthew J.; Nomura, Daniel K.; Cravatt, Benjamin F.; Saez, Enrique

    2014-01-01

    Phenotypic screening is making a comeback in drug discovery as the maturation of chemical proteomics methods has facilitated target identification for bioactive small molecules. A limitation of these approaches is that time-consuming genetic methods or other means is often required to determine the biologically relevant target(s) from among multiple protein-compound interactions that are typically detected. Here, we have combined phenotypic screening of a directed small-molecule library with competitive activity-based protein profiling to map and functionally characterize the targets of screening hits. Using this approach, we identify carboxylesterase 3 (Ces3 or Ces1d) as a primary molecular target of bioactive compounds that promote lipid storage in adipocytes. We further show that Ces3 activity is dramatically elevated during adipocyte differentiation. Treatment of two mouse models of obesity-diabetes with a Ces3 inhibitor ameliorates multiple features of metabolic syndrome, illustrating the power of the described strategy to accelerate the identification and pharmacologic validation of new therapeutic targets. PMID:24362705

  8. Using activity-based costing in surgery.

    PubMed

    Grandlich, Cheryl

    2004-01-01

    ACTIVITY-BASED COSTING is an accounting technique that allows organizations to determine actual costs associated with their services based on the resources they consume. THIS TECHNIQUE can be used in a variety of ways, including targeting high-cost activities, forecasting financial baselines, and supporting resource allocation. FOUR STEPS should be followed when applying activity-based costing to surgical procedures. THIS ARTICLE explores how Froedtert Memorial Lutheran Hospital, Milwaukee, used activity-based costing. PMID:14763586

  9. In silico structure-based screening of versatile P-glycoprotein inhibitors using polynomial empirical scoring functions.

    PubMed

    Shityakov, Sergey; Förster, Carola

    2014-01-01

    P-glycoprotein (P-gp) is an ATP (adenosine triphosphate)-binding cassette transporter that causes multidrug resistance of various chemotherapeutic substances by active efflux from mammalian cells. P-gp plays a pivotal role in limiting drug absorption and distribution in different organs, including the intestines and brain. Thus, the prediction of P-gp-drug interactions is of vital importance in assessing drug pharmacokinetic and pharmacodynamic properties. To find the strongest P-gp blockers, we performed an in silico structure-based screening of P-gp inhibitor library (1,300 molecules) by the gradient optimization method, using polynomial empirical scoring (POLSCORE) functions. We report a strong correlation (r (2)=0.80, F=16.27, n=6, P<0.0157) of inhibition constants (Kiexp or pKiexp; experimental Ki or negative decimal logarithm of Kiexp) converted from experimental IC50 (half maximal inhibitory concentration) values with POLSCORE-predicted constants (KiPOLSCORE or pKiPOLSCORE), using a linear regression fitting technique. The hydrophobic interactions between P-gp and selected drug substances were detected as the main forces responsible for the inhibition effect. The results showed that this scoring technique might be useful in the virtual screening and filtering of databases of drug-like compounds at the early stage of drug development processes. PMID:24711707

  10. Screening of CACNA1A and ATP1A2 genes in hemiplegic migraine: clinical, genetic, and functional studies.

    PubMed

    Carreño, Oriel; Corominas, Roser; Serra, Selma Angèlica; Sintas, Cèlia; Fernández-Castillo, Noèlia; Vila-Pueyo, Marta; Toma, Claudio; Gené, Gemma G; Pons, Roser; Llaneza, Miguel; Sobrido, María-Jesús; Grinberg, Daniel; Valverde, Miguel Ángel; Fernández-Fernández, José Manuel; Macaya, Alfons; Cormand, Bru

    2013-11-01

    Hemiplegic migraine (HM) is a rare and severe subtype of autosomal dominant migraine, characterized by a complex aura including some degree of motor weakness. Mutations in four genes (CACNA1A, ATP1A2, SCN1A and PRRT2) have been detected in familial and in sporadic cases. This genetically and clinically heterogeneous disorder is often accompanied by permanent ataxia, epileptic seizures, mental retardation, and chronic progressive cerebellar atrophy. Here we report a mutation screening in the CACNA1A and ATP1A2 genes in 18 patients with HM. Furthermore, intragenic copy number variant (CNV) analysis was performed in CACNA1A using quantitative approaches. We identified four previously described missense CACNA1A mutations (p.Ser218Leu, p.Thr501Met, p.Arg583Gln, and p.Thr666Met) and two missense changes in the ATP1A2 gene, the previously described p.Ala606Thr and the novel variant p.Glu825Lys. No structural variants were found. This genetic screening allowed the identification of more than 30% of the disease alleles, all present in a heterozygous state. Functional consequences of the CACNA1A-p.Thr501Met mutation, previously described only in association with episodic ataxia, and ATP1A2-p.Glu825Lys, were investigated by means of electrophysiological studies, cell viability assays or Western blot analysis. Our data suggest that both these variants are disease-causing. PMID:24498617

  11. Pharmacophore modeling, virtual screening, molecular docking studies and density functional theory approaches to identify novel ketohexokinase (KHK) inhibitors.

    PubMed

    Kavitha, Rengarajan; Karunagaran, Subramanian; Chandrabose, Subramaniam Subhash; Lee, Keun Woo; Meganathan, Chandrasekaran

    2015-12-01

    Fructose catabolism starts with phosphorylation of d-fructose to fructose 1-phosphate, which is performed by ketohexokinase (KHK). Fructose metabolism may be the key to understand the long-term consumption of fructose in human's obesity, diabetes and metabolic states in western populations. The inhibition of KHK has medicinally potential roles in fructose metabolism and the metabolic syndrome. To identify the essential chemical features for KHK inhibition, a three-dimensional (3D) chemical-feature-based QSAR pharmacophore model was developed for the first time by using Discovery Studio v2.5 (DS). The best pharmacophore hypothesis (Hypo1) consisting two hydrogen bond donor, two hydrophobic features and has exhibited high correlation co-efficient (0.97), cost difference (76.1) and low RMS (0.66) value. The robustness and predictability of Hypo1 was validated by fisher's randomization method, test set, and the decoy set. Subsequently, chemical databases like NCI, Chembridge and Maybridge were screened for validated Hypo1. The screened compounds were further analyzed by applying drug-like filters such as Lipinski's rule of five, ADME properties, and molecular docking studies. Further, the highest occupied molecular orbital, lowest unoccupied molecular orbital and energy gap values were calculated for the hits compounds using density functional theory. Finally, 3 hit compounds were selected based on their good molecular interactions with key amino acids in the KHK active site, GOLD fitness score, and lowest energy gaps. PMID:26521124

  12. Water-gas Shift Reaction on oxide/Cu(111): Rational Catalyst Screening from Density Functional Theory

    SciTech Connect

    Liu, P.

    2010-11-28

    Developing improved catalysts based on a fundamental understanding of reaction mechanism has become one of the grand challenges in catalysis. A theoretical understanding and screening the metal-oxide composite catalysts for the water-gas shift (WGS) reaction is presented here. Density functional theory was employed to identify the key step for the WGS reaction on the Au, Cu-oxide catalysts, where the calculated reaction energy for water dissociation correlates well with the experimental measured WGS activity. Accordingly, the calculated reaction energy for water dissociation was used as the scaling descriptor to screen the inverse model catalysts, oxide/Cu(111), for the better WGS activity. Our calculations predict that the WGS activity increases in a sequence: Cu(111), ZnO/Cu(111) < TiO{sub 2}/Cu(111), ZrO{sub 2}/Cu(111) < MoO{sub 3}/Cu(111). Our results imply that the high performances of Au, Cu-oxide nanocatalysts in the WGS reaction rely heavily on the direct participation of both oxide and metal sites. The degree that the oxide is reduced by Cu plays an important role in determining the WGS activity of oxide/Cu catalysts. The reducible oxide can be transformed from the fully oxidized form to the reduced form due to the interaction with Cu and, therefore, the transfer of electron density from Cu, which helps in releasing the bottleneck water dissociation and, therefore, facilitating the WGS reaction on copper.

  13. High-throughput functional screening reveals low frequency of antibiotic resistance genes in DNA recovered from the Upper Mississippi River.

    PubMed

    Staley, Christopher; Gould, Trevor J; Wang, Ping; Phillips, Jane; Cotner, James B; Sadowsky, Michael J

    2015-09-01

    In this study, we determined the frequency of antibiotic resistance genes (ARGs) in the Upper Mississippi River using a high-throughput, functional, metagenomic screening procedure. Fosmid libraries containing ∼10,000 clones were screened for resistance to ampicillin, cephalothin, kanamycin, and tetracycline. We hypothesized that nutrient concentrations, land cover type, and taxonomic community composition may select for ARGs. Resistance to ampicillin, cephalothin, and kanamycin was low (<1.00%), and no resistance to tetracycline was detected. Ammonium and total dissolved solids (TDS) concentrations were correlated with kanamycin and cephalothin resistances (r=0.617 and -0.449, P=0.002 and 0.036, respectively). Cephalothin resistance was also positively correlated with the percentage of forested land cover (r=0.444, P=0.039). Only the candidate division OD1, among 35 phyla identified, was correlated with ampicillin resistance (r=0.456, P=0.033), suggesting that minority members of the community may be responsible for dissemination of ARGs in this ecosystem. Results of this study suggest that ammonium and TDS may be involved in a complex selection process for ARGs. Furthermore, we suggest that minority species, potentially contributed in low numbers from sediment and biofilm reservoirs, may be the primary carriers of ARGs in this riverine system. PMID:26322755

  14. Water-gas shift reaction on oxide/Cu(111): Rational catalyst screening from density functional theory

    NASA Astrophysics Data System (ADS)

    Liu, Ping

    2010-11-01

    Developing improved catalysts based on a fundamental understanding of reaction mechanism has become one of the grand challenges in catalysis. A theoretical understanding and screening the metal-oxide composite catalysts for the water-gas shift (WGS) reaction is presented here. Density functional theory was employed to identify the key step for the WGS reaction on the Au, Cu-oxide catalysts, where the calculated reaction energy for water dissociation correlates well with the experimental measured WGS activity. Accordingly, the calculated reaction energy for water dissociation was used as the scaling descriptor to screen the inverse model catalysts, oxide/Cu(111), for the better WGS activity. Our calculations predict that the WGS activity increases in a sequence: Cu(111), ZnO/Cu(111) < TiO2/Cu(111), ZrO2/Cu(111) < MoO3/Cu(111). Our results imply that the high performances of Au, Cu-oxide nanocatalysts in the WGS reaction rely heavily on the direct participation of both oxide and metal sites. The degree that the oxide is reduced by Cu plays an important role in determining the WGS activity of oxide/Cu catalysts. The reducible oxide can be transformed from the fully oxidized form to the reduced form due to the interaction with Cu and, therefore, the transfer of electron density from Cu, which helps in releasing the bottleneck water dissociation and, therefore, facilitating the WGS reaction on copper.

  15. 3D QSAR Pharmacophore Modeling, in Silico Screening, and Density Functional Theory (DFT) Approaches for Identification of Human Chymase Inhibitors

    PubMed Central

    Arooj, Mahreen; Thangapandian, Sundarapandian; John, Shalini; Hwang, Swan; Park, Jong Keun; Lee, Keun Woo

    2011-01-01

    Human chymase is a very important target for the treatment of cardiovascular diseases. Using a series of theoretical methods like pharmacophore modeling, database screening, molecular docking and Density Functional Theory (DFT) calculations, an investigation for identification of novel chymase inhibitors, and to specify the key factors crucial for the binding and interaction between chymase and inhibitors is performed. A highly correlating (r = 0.942) pharmacophore model (Hypo1) with two hydrogen bond acceptors, and three hydrophobic aromatic features is generated. After successfully validating “Hypo1”, it is further applied in database screening. Hit compounds are subjected to various drug-like filtrations and molecular docking studies. Finally, three structurally diverse compounds with high GOLD fitness scores and interactions with key active site amino acids are identified as potent chymase hits. Moreover, DFT study is performed which confirms very clear trends between electronic properties and inhibitory activity (IC50) data thus successfully validating “Hypo1” by DFT method. Therefore, this research exertion can be helpful in the development of new potent hits for chymase. In addition, the combinational use of docking, orbital energies and molecular electrostatic potential analysis is also demonstrated as a good endeavor to gain an insight into the interaction between chymase and inhibitors. PMID:22272131

  16. In silico structure-based screening of versatile P-glycoprotein inhibitors using polynomial empirical scoring functions

    PubMed Central

    Shityakov, Sergey; Förster, Carola

    2014-01-01

    P-glycoprotein (P-gp) is an ATP (adenosine triphosphate)-binding cassette transporter that causes multidrug resistance of various chemotherapeutic substances by active efflux from mammalian cells. P-gp plays a pivotal role in limiting drug absorption and distribution in different organs, including the intestines and brain. Thus, the prediction of P-gp–drug interactions is of vital importance in assessing drug pharmacokinetic and pharmacodynamic properties. To find the strongest P-gp blockers, we performed an in silico structure-based screening of P-gp inhibitor library (1,300 molecules) by the gradient optimization method, using polynomial empirical scoring (POLSCORE) functions. We report a strong correlation (r2=0.80, F=16.27, n=6, P<0.0157) of inhibition constants (Kiexp or pKiexp; experimental Ki or negative decimal logarithm of Kiexp) converted from experimental IC50 (half maximal inhibitory concentration) values with POLSCORE-predicted constants (KiPOLSCORE or pKiPOLSCORE), using a linear regression fitting technique. The hydrophobic interactions between P-gp and selected drug substances were detected as the main forces responsible for the inhibition effect. The results showed that this scoring technique might be useful in the virtual screening and filtering of databases of drug-like compounds at the early stage of drug development processes. PMID:24711707

  17. Screening of CACNA1A and ATP1A2 genes in hemiplegic migraine: clinical, genetic, and functional studies

    PubMed Central

    Carreño, Oriel; Corominas, Roser; Serra, Selma Angèlica; Sintas, Cèlia; Fernández-Castillo, Noèlia; Vila-Pueyo, Marta; Toma, Claudio; Gené, Gemma G; Pons, Roser; Llaneza, Miguel; Sobrido, María-Jesús; Grinberg, Daniel; Valverde, Miguel Ángel; Fernández-Fernández, José Manuel; Macaya, Alfons; Cormand, Bru

    2013-01-01

    Hemiplegic migraine (HM) is a rare and severe subtype of autosomal dominant migraine, characterized by a complex aura including some degree of motor weakness. Mutations in four genes (CACNA1A, ATP1A2, SCN1A and PRRT2) have been detected in familial and in sporadic cases. This genetically and clinically heterogeneous disorder is often accompanied by permanent ataxia, epileptic seizures, mental retardation, and chronic progressive cerebellar atrophy. Here we report a mutation screening in the CACNA1A and ATP1A2 genes in 18 patients with HM. Furthermore, intragenic copy number variant (CNV) analysis was performed in CACNA1A using quantitative approaches. We identified four previously described missense CACNA1A mutations (p.Ser218Leu, p.Thr501Met, p.Arg583Gln, and p.Thr666Met) and two missense changes in the ATP1A2 gene, the previously described p.Ala606Thr and the novel variant p.Glu825Lys. No structural variants were found. This genetic screening allowed the identification of more than 30% of the disease alleles, all present in a heterozygous state. Functional consequences of the CACNA1A-p.Thr501Met mutation, previously described only in association with episodic ataxia, and ATP1A2-p.Glu825Lys, were investigated by means of electrophysiological studies, cell viability assays or Western blot analysis. Our data suggest that both these variants are disease-causing. PMID:24498617

  18. An integrated miRNA functional screening and target validation method for organ morphogenesis

    PubMed Central

    Rebustini, Ivan T.; Vlahos, Maryann; Packer, Trevor; Kukuruzinska, Maria A.; Maas, Richard L.

    2016-01-01

    The relative ease of identifying microRNAs and their increasing recognition as important regulators of organogenesis motivate the development of methods to efficiently assess microRNA function during organ morphogenesis. In this context, embryonic organ explants provide a reliable and reproducible system that recapitulates some of the important early morphogenetic processes during organ development. Here we present a method to target microRNA function in explanted mouse embryonic organs. Our method combines the use of peptide-based nanoparticles to transfect specific microRNA inhibitors or activators into embryonic organ explants, with a microRNA pulldown assay that allows direct identification of microRNA targets. This method provides effective assessment of microRNA function during organ morphogenesis, allows prioritization of multiple microRNAs in parallel for subsequent genetic approaches, and can be applied to a variety of embryonic organs. PMID:26980315

  19. An integrative genomics screen uncovers ncRNA T-UCR functions in neuroblastoma tumours.

    PubMed

    Mestdagh, P; Fredlund, E; Pattyn, F; Rihani, A; Van Maerken, T; Vermeulen, J; Kumps, C; Menten, B; De Preter, K; Schramm, A; Schulte, J; Noguera, R; Schleiermacher, G; Janoueix-Lerosey, I; Laureys, G; Powel, R; Nittner, D; Marine, J-C; Ringnér, M; Speleman, F; Vandesompele, J

    2010-06-17

    Different classes of non-coding RNAs, including microRNAs, have recently been implicated in the process of tumourigenesis. In this study, we examined the expression and putative functions of a novel class of non-coding RNAs known as transcribed ultraconserved regions (T-UCRs) in neuroblastoma. Genome-wide expression profiling revealed correlations between specific T-UCR expression levels and important clinicogenetic parameters such as MYCN amplification status. A functional genomics approach based on the integration of multi-level transcriptome data was adapted to gain insights into T-UCR functions. Assignments of T-UCRs to cellular processes such as TP53 response, differentiation and proliferation were verified using various cellular model systems. For the first time, our results define a T-UCR expression landscape in neuroblastoma and suggest widespread T-UCR involvement in diverse cellular processes that are deregulated in the process of tumourigenesis. PMID:20383195

  20. Analysis of mammalian gene function through broad based phenotypic screens across a consortium of mouse clinics

    PubMed Central

    Adams, David J; Adams, Niels C; Adler, Thure; Aguilar-Pimentel, Antonio; Ali-Hadji, Dalila; Amann, Gregory; André, Philippe; Atkins, Sarah; Auburtin, Aurelie; Ayadi, Abdel; Becker, Julien; Becker, Lore; Bedu, Elodie; Bekeredjian, Raffi; Birling, Marie-Christine; Blake, Andrew; Bottomley, Joanna; Bowl, Mike; Brault, Véronique; Busch, Dirk H; Bussell, James N; Calzada-Wack, Julia; Cater, Heather; Champy, Marie-France; Charles, Philippe; Chevalier, Claire; Chiani, Francesco; Codner, Gemma F; Combe, Roy; Cox, Roger; Dalloneau, Emilie; Dierich, André; Di Fenza, Armida; Doe, Brendan; Duchon, Arnaud; Eickelberg, Oliver; Esapa, Chris T; El Fertak, Lahcen; Feigel, Tanja; Emelyanova, Irina; Estabel, Jeanne; Favor, Jack; Flenniken, Ann; Gambadoro, Alessia; Garrett, Lilian; Gates, Hilary; Gerdin, Anna-Karin; Gkoutos, George; Greenaway, Simon; Glasl, Lisa; Goetz, Patrice; Da Cruz, Isabelle Goncalves; Götz, Alexander; Graw, Jochen; Guimond, Alain; Hans, Wolfgang; Hicks, Geoff; Hölter, Sabine M; Höfler, Heinz; Hancock, John M; Hoehndorf, Robert; Hough, Tertius; Houghton, Richard; Hurt, Anja; Ivandic, Boris; Jacobs, Hughes; Jacquot, Sylvie; Jones, Nora; Karp, Natasha A; Katus, Hugo A; Kitchen, Sharon; Klein-Rodewald, Tanja; Klingenspor, Martin; Klopstock, Thomas; Lalanne, Valerie; Leblanc, Sophie; Lengger, Christoph; le Marchand, Elise; Ludwig, Tonia; Lux, Aline; McKerlie, Colin; Maier, Holger; Mandel, Jean-Louis; Marschall, Susan; Mark, Manuel; Melvin, David G; Meziane, Hamid; Micklich, Kateryna; Mittelhauser, Christophe; Monassier, Laurent; Moulaert, David; Muller, Stéphanie; Naton, Beatrix; Neff, Frauke; Nolan, Patrick M; Nutter, Lauryl MJ; Ollert, Markus; Pavlovic, Guillaume; Pellegata, Natalia S; Peter, Emilie; Petit-Demoulière, Benoit; Pickard, Amanda; Podrini, Christine; Potter, Paul; Pouilly, Laurent; Puk, Oliver; Richardson, David; Rousseau, Stephane; Quintanilla-Fend, Leticia; Quwailid, Mohamed M; Racz, Ildiko; Rathkolb, Birgit; Riet, Fabrice; Rossant, Janet; Roux, Michel; Rozman, Jan; Ryder, Ed; Salisbury, Jennifer; Santos, Luis; Schäble, Karl-Heinz; Schiller, Evelyn; Schrewe, Anja; Schulz, Holger; Steinkamp, Ralf; Simon, Michelle; Stewart, Michelle; Stöger, Claudia; Stöger, Tobias; Sun, Minxuan; Sunter, David; Teboul, Lydia; Tilly, Isabelle; Tocchini-Valentini, Glauco P; Tost, Monica; Treise, Irina; Vasseur, Laurent; Velot, Emilie; Vogt-Weisenhorn, Daniela; Wagner, Christelle; Walling, Alison; Weber, Bruno; Wendling, Olivia; Westerberg, Henrik; Willershäuser, Monja; Wolf, Eckhard; Wolter, Anne; Wood, Joe; Wurst, Wolfgang; Yildirim, Ali Önder; Zeh, Ramona; Zimmer, Andreas; Zimprich, Annemarie

    2015-01-01

    The function of the majority of genes in the mouse and human genomes remains unknown. The mouse ES cell knockout resource provides a basis for characterisation of relationships between gene and phenotype. The EUMODIC consortium developed and validated robust methodologies for broad-based phenotyping of knockouts through a pipeline comprising 20 disease-orientated platforms. We developed novel statistical methods for pipeline design and data analysis aimed at detecting reproducible phenotypes with high power. We acquired phenotype data from 449 mutant alleles, representing 320 unique genes, of which half had no prior functional annotation. We captured data from over 27,000 mice finding that 83% of the mutant lines are phenodeviant, with 65% demonstrating pleiotropy. Surprisingly, we found significant differences in phenotype annotation according to zygosity. Novel phenotypes were uncovered for many genes with unknown function providing a powerful basis for hypothesis generation and further investigation in diverse systems. PMID:26214591

  1. Using synthetic biology to screen for functional diversity of GH1 enzymes

    SciTech Connect

    Deutsch, Sam; Datta, Supratim; Hamilton, Matthew; Friedland, Greg; D'Haeseleer, Patrik; Chen, Jan-Fang; Chivian, Dylan; Egan, Rob; Sale, Kenneth; Simmons, Blake; Rubin, Eddy

    2011-05-31

    Advances in next-generation sequencing technologies have enabled single genomes as well as complex environmental samples (metagenomes) to be comprehensively sequenced on a routine basis. Bioinformatics analysis of the resulting sequencing data reveals a continually expanding catalogue of predicted proteins ( 14 million as of April 2011), 75 percent of which are associated with functional annotation (COG, Pfam, Enzyme, Kegg, etc). These predicted proteins cover the full spectrum of known pathways and functional activities, including many novel biocatalysts that are expected to significantly contribute to the development of clean technologies including biomass degradation, lipid transformation for biodiesel generation, intermediates for polymer production, carbon capture, and bioremediation.

  2. Screening biochars for heavy metal retention in soil: role of oxygen functional groups

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Oxygen-containing carboxyl, hydroxyl, and phenolic surface functional groups of soil organic and mineral components play central roles in binding metal ions, and biochar amendment can provide means of increasing these surface ligands in soil. In this study, positive matrix factorization (PMF) was f...

  3. Functional Identification of Tumor Suppressor Genes Through an in vivo RNA Interference Screen in a Mouse Lymphoma Model

    PubMed Central

    Bric, Anka; Miething, Cornelius; Bialucha, Carl Uli; Scuoppo, Claudio; Zender, Lars; Krasnitz, Alexander; Xuan, Zhenyu; Zuber, Johannes; Wigler, Michael; Hicks, James; McCombie, Richard W.; Hemann, Michael T.; Hannon, Gregory J.; Powers, Scott; Lowe, Scott W.

    2009-01-01

    SUMMARY Short hairpin RNAs (shRNAs) capable of stably suppressing gene function by RNA interference (RNAi) can mimic tumor suppressor gene loss in mice. By selecting for shRNAs capable of accelerating lymphomagenesis in a well-characterized mouse lymphoma model, we identified over ten candidate tumor suppressors, including Sfrp1, Numb, Mek1, and Angiopoietin 2. Several components of the DNA damage response machinery were also identified, including Rad17, which acts as a haploinsufficient tumor suppressor that responds to oncogenic stress and whose loss is associated with poor prognosis in human patients. Our results emphasize the utility of in vivo RNAi screens, identify and validate a diverse set of tumor suppressors, and have therapeutic implications. PMID:19800577

  4. Rapid Screening of Gene Function by Systemic Delivery of Morpholino Oligonucleotides to Live Mouse Embryos

    PubMed Central

    McClelland, Kathryn S.; Wainwright, Elanor N.; Bowles, Josephine; Koopman, Peter

    2015-01-01

    Traditional gene targeting methods in mice are complex and time consuming, especially when conditional deletion methods are required. Here, we describe a novel technique for assessing gene function by injection of modified antisense morpholino oligonucleotides (MOs) into the heart of mid-gestation mouse embryos. After allowing MOs to circulate through the embryonic vasculature, target tissues were explanted, cultured and analysed for expression of key markers. We established proof-of-principle by partially phenocopying known gene knockout phenotypes in the fetal gonads (Stra8, Sox9) and pancreas (Sox9). We also generated a novel double knockdown of Gli1 and Gli2, revealing defects in Leydig cell differentiation in the fetal testis. Finally, we gained insight into the roles of Adamts19 and Ctrb1, genes of unknown function in sex determination and gonadal development. These studies reveal the utility of this method as a means of first-pass analysis of gene function during organogenesis before committing to detailed genetic analysis. PMID:25629157

  5. Functional Proteomics Screen Enables Enrichment of Distinct Cell Types from Human Pancreatic Islets

    PubMed Central

    Sharivkin, Revital; Walker, Michael D.; Soen, Yoav

    2015-01-01

    The current world-wide epidemic of diabetes has prompted attempts to generate new sources of insulin-producing cells for cell replacement therapy. An inherent challenge in many of these strategies is the lack of cell-surface markers permitting isolation and characterization of specific cell types from differentiating stem cell populations. Here we introduce an iterative proteomics procedure allowing tag-free isolation of cell types based on their function. Our method detects and associates specific cell-surface markers with particular cell functionality by coupling cell capture on antibody arrays with immunofluorescent labeling. Using this approach in an iterative manner, we discovered marker combinations capable of enriching for discrete pancreatic cell subtypes from human islets of Langerhans: insulin-producing beta cells (CD9high/CD56+), glucagon-producing alpha cells (CD9- /CD56+) and trypsin-producing acinar cells (CD9- /CD56-). This strategy may assist future beta cell research and the development of diagnostic tools for diabetes. It can also be applied more generally for function-based purification of desired cell types from other limited and heterogeneous biological samples. PMID:25706282

  6. Discovery of a Dipeptide Epimerase Enzymatic Function Guided by Homology Modeling and Virtual Screening

    SciTech Connect

    Kalyanaraman, C.; Imker, H; Fedorov, A; Fedorov, E; Glasner, M; Babbitt, P; Almo, S; Gerlt, J; Jacobson, M

    2008-01-01

    We have developed a computational approach to aid the assignment of enzymatic function for uncharacterized proteins that uses homology modeling to predict the structure of the binding site and in silico docking to identify potential substrates. We apply this method to proteins in the functionally diverse enolase superfamily that are homologous to the characterized L-Ala-D/L-Glu epimerase from Bacillus subtilis. In particular, a protein from Thermotoga martima was predicted to have different substrate specificity, which suggests that it has a different, but as yet unknown, biological function. This prediction was experimentally confirmed, resulting in the assignment of epimerase activity for L-Ala-D/L-Phe, L-Ala-D/L-Tyr, and L-Ala-D/L-His, whereas the enzyme is annotated incorrectly in GenBank as muconate cycloisomerase. Subsequently, crystal structures of the enzyme were determined in complex with three substrates, showing close agreement with the computational models and revealing the structural basis for the observed substrate selectivity.

  7. Functional mutagenesis screens reveal the ‘cap structure’ formation in disulfide-bridge free TASK channels

    PubMed Central

    Goldstein, Matthias; Rinné, Susanne; Kiper, Aytug K.; Ramírez, David; Netter, Michael F.; Bustos, Daniel; Ortiz-Bonnin, Beatriz; González, Wendy; Decher, Niels

    2016-01-01

    Two-pore-domain potassium (K2P) channels have a large extracellular cap structure formed by two M1-P1 linkers, containing a cysteine for dimerization. However, this cysteine is not present in the TASK-1/3/5 subfamily. The functional role of the cap is poorly understood and it remained unclear whether K2P channels assemble in the domain-swapped orientation or not. Functional alanine-mutagenesis screens of TASK-1 and TRAAK were used to build an in silico model of the TASK-1 cap. According to our data the cap structure of disulfide-bridge free TASK channels is similar to that of other K2P channels and is most likely assembled in the domain-swapped orientation. As the conserved cysteine is not essential for functional expression of all K2P channels tested, we propose that hydrophobic residues at the inner leaflets of the cap domains can interact with each other and that this way of stabilizing the cap is most likely conserved among K2P channels. PMID:26794006

  8. A Genomewide RNAi Screen for Genes That Affect the Stability, Distribution and Function of P Granules in Caenorhabditis elegans

    PubMed Central

    Updike, Dustin L.; Strome, Susan

    2009-01-01

    P granules are non-membrane-bound organelles found in the germ-line cytoplasm throughout Caenorhabditis elegans development. Like their “germ granule” counterparts in other animals, P granules are thought to act as determinants of the identity and special properties of germ cells, properties that include the unique ability to give rise to all tissues of future generations of an organism. Therefore, understanding how P granules work is critical to understanding how cellular immortality and totipotency are retained, gained, and lost. Here we report on a genomewide RNAi screen in C. elegans, which identified 173 genes that affect the stability, localization, and function of P granules. Many of these genes fall into specific classes with shared P-granule phenotypes, allowing us to better understand how cellular processes such as protein degradation, translation, splicing, nuclear transport, and mRNA homeostasis converge on P-granule assembly and function. One of the more striking phenotypes is caused by the depletion of CSR-1, an Argonaute associated with an endogenous siRNA pathway that functions in the germ line. We show that CSR-1 and two other endo-siRNA pathway members, the RNA-dependent RNA polymerase EGO-1 and the helicase DRH-3, act to antagonize RNA and P-granule accumulation in the germ line. Our findings strengthen the emerging view that germ granules are involved in numerous aspects of RNA metabolism, including an endo-siRNA pathway in germ cells. PMID:19805813

  9. Screening of GNAL variants in Brazilian patients with isolated dystonia reveals a novel mutation with partial loss of function.

    PubMed

    Dos Santos, Camila Oliveira; Masuho, Ikuo; da Silva-Júnior, Francisco Pereira; Barbosa, Egberto Reis; Silva, Sonia Maria Cesar Azevedo; Borges, Vanderci; Ferraz, Henrique Ballalai; Rocha, Maria Sheila Guimarães; Limongi, João Carlos Papaterra; Martemyanov, Kirill A; de Carvalho Aguiar, Patricia

    2016-04-01

    GNAL was identified as a cause of dystonia in patients from North America, Europe and Asia. In this study, we aimed to investigate the prevalence of GNAL variants in Brazilian patients with dystonia. Ninety-one patients with isolated idiopathic dystonia, negative for THAP1 and TOR1A mutations, were screened for GNAL variants by Sanger sequencing. Functional characterization of the Gαolf protein variant was performed using the bioluminescence resonance energy transfer assay. A novel heterozygous nonsynonymous variant (p. F133L) was identified in a patient with cervical and laryngeal dystonia since the third decade of life, with no family history. This variant was not identified in healthy Brazilian controls and was not described in 63,000 exomas of the ExAC database. The F133L mutant exhibited significantly elevated levels of basal BRET and severely diminished amplitude of response elicited by dopamine, that both indicate substantial functional impairment of Gαolf in transducing receptor signals, which could be involved in dystonia pathophysiology. GNAL mutations are not a common cause of dystonia in the Brazilian population and have a lower prevalence than THAP1 and TOR1A mutations. We present a novel variant that results in partial Gαolf loss of function. PMID:26810727

  10. Effect of Functional Status on the Quality of Bowel Preparation in Elderly Patients Undergoing Screening and Surveillance Colonoscopy

    PubMed Central

    Kumar, Akash; Lin, Lisa; Bernheim, Oren; Bagiella, Emilia; Jandorf, Lina; Itzkowitz, Steven H.; Shah, Brijen J.

    2016-01-01

    Background/Aims Optimal bowel preparation is essential for successful screening or for surveillance colonoscopy (SC). Inadequate bowel preparation is associated with older age, the male gender, and the presence of certain comorbidities. However, the association between patients’ functional status and bowel preparation quality has not been studied. We prospectively examined the relationship between functional status, namely, the ability to perform activities of daily living (ADLs) and ambulate, and the quality of bowel preparation in elderly patients undergoing SC. Methods Before undergoing SC, 88 elderly patients were surveyed regarding their functional status, specifically regarding their ability to perform ADLs and ambulate a quarter of a mile. Gastroenterologists then determined the quality of the bowel preparation, which was classified as either adequate or inadequate. Then, the frequency of inadequate bowel preparation in patients who did or did not experience difficulty performing ADLs and ambulating was calculated. Results Difficulty ambulating (unadjusted odds ratio [OR], 4.83; p<0.001), difficulty performing ADLs (OR, 2.93; p=0.001), and history of diabetes (OR, 2.88; p=0.007) were significant univariate predictors of inadequate bowel preparation. After adjusting for the above variables, only difficulty ambulating (adjusted OR, 5.78; p=0.004) was an independent predictor of inadequate bowel preparation. Conclusions Difficulty with ambulation is a strong predictor of inadequate bowel preparation in elderly patients undergoing SC. PMID:27021501

  11. A comparative evaluation of rare-earth screen-film systems. System speed, contrast, sensitometry, RMS noise, square-wave response function, and contrast-dose-detail analysis.

    PubMed

    Fearon, T; Vucich, J; Hoe, J; McSweeney, W J; Potter, B M

    1986-08-01

    We evaluated the physical characteristics and contrast-dose-detail performance of 11 rare-earth and three calcium tungstate screen-film systems. Measurements included system speed, contrast, sensitometry, RMS noise, square-wave response function, and contrast-dose-detail analysis. The major differences in physical characteristics among systems were system speed and RMS noise. Square-wave response differences were more subtle. For contrast-dose-detail analysis, the rare-earth screen-film systems and the calcium tungstate system responses were significant over a limited subject contrast range as a function of detail diameter. Relative dose efficiency in the noise-limited region is a function of the properties of the screen only and is independent of the film. PMID:3744739

  12. Axonal and dendritic synaptotagmin isoforms revealed by a pHluorin-syt functional screen

    PubMed Central

    Dean, Camin; Dunning, F. Mark; Liu, Huisheng; Bomba-Warczak, Ewa; Martens, Henrik; Bharat, Vinita; Ahmed, Saheeb; Chapman, Edwin R.

    2012-01-01

    The synaptotagmins (syts) are a family of molecules that regulate membrane fusion. There are 17 mammalian syt isoforms, most of which are expressed in the brain. However, little is known regarding the subcellular location and function of the majority of these syts in neurons, largely due to a lack of isoform-specific antibodies. Here we generated pHluorin-syt constructs harboring a luminal domain pH sensor, which reports localization, pH of organelles to which syts are targeted, and the kinetics and sites of exocytosis and endocytosis. Of interest, only syt-1 and 2 are targeted to synaptic vesicles, whereas other isoforms selectively recycle in dendrites (syt-3 and 11), axons (syt-5, 7, 10, and 17), or both axons and dendrites (syt-4, 6, 9, and 12), where they undergo exocytosis and endocytosis with distinctive kinetics. Hence most syt isoforms localize to distinct secretory organelles in both axons and dendrites and may regulate neuropeptide/neurotrophin release to modulate neuronal function. PMID:22398727

  13. Combinatorial density functional theory-based screening of surface alloys for the oxygen reduction reaction.

    SciTech Connect

    Greeley, J.; Norskov, J.; Center for Nanoscale Materials; Technical Univ. of Denmark

    2009-03-26

    A density functional theory (DFT) -based, combinatorial search for improved oxygen reduction reaction (ORR) catalysts is presented. A descriptor-based approach to estimate the ORR activity of binary surface alloys, wherein alloying occurs only in the surface layer, is described, and rigorous, potential-dependent computational tests of the stability of these alloys in aqueous, acidic environments are presented. These activity and stability criteria are applied to a database of DFT calculations on nearly 750 binary transition metal surface alloys; of these, many are predicted to be active for the ORR but, with few exceptions, they are found to be thermodynamically unstable in the acidic environments typical of low-temperature fuel cells. The results suggest that, absent other thermodynamic or kinetic mechanisms to stabilize the alloys, surface alloys are unlikely to serve as useful ORR catalysts over extended periods of operation.

  14. A C. elegans Screening Platform for the Rapid Assessment of Chemical Disruption of Germline Function

    PubMed Central

    Allard, Patrick; Kleinstreuer, Nicole C.; Knudsen, Thomas B.

    2013-01-01

    Background: Despite the developmental impact of chromosome segregation errors, we lack the tools to assess environmental effects on the integrity of the germline in animals. Objectives: We developed an assay in Caenorhabditis elegans that fluorescently marks aneuploid embryos after chemical exposure. Methods: We qualified the predictive value of the assay against chemotherapeutic agents as well as environmental compounds from the ToxCast Phase I library by comparing results from the C. elegans assay with the comprehensive mammalian in vivo end point data from the ToxRef database. Results: The assay was highly predictive of mammalian reproductive toxicities, with a 69% maximum balanced accuracy. We confirmed the effect of select compounds on germline integrity by monitoring germline apoptosis and meiotic progression. Conclusions: This C. elegans assay provides a comprehensive strategy for assessing environmental effects on germline function. PMID:23603051

  15. In vitro screening of metal oxide nanoparticles for effects on neural function using cortical networks on microelectrode arrays.

    PubMed

    Strickland, Jenna D; Lefew, William R; Crooks, James; Hall, Diana; Ortenzio, Jayna Nr; Dreher, Kevin; Shafer, Timothy J

    2016-06-01

    Nanoparticles (NPs) may translocate to the brain following inhalation or oral exposures, yet higher throughput methods to screen NPs for potential neurotoxicity are lacking. The present study examined effects of 5 CeO2 (5- 1288 nm), and 4 TiO2 (6-142 nm) NPs and microparticles (MP) on network function in primary cultures of rat cortex on 12 well microelectrode array (MEA) plates. Particles were without cytotoxicity at concentrations ≤50 µg/ml. After recording 1 h of baseline activity prior to particle (3-50 µg/ml) exposure, changes in the total number of spikes (TS) and # of active electrodes (#AEs) were assessed 1, 24, and 48 h later. Following the 48 h recording, the response to a challenge with the GABAA antagonist bicuculline (BIC; 25 µM) was assessed. In all, particles effects were subtle, but 69 nm CeO2 and 25 nm TiO2 NPs caused concentration-related decreases in TS following 1 h exposure. At 48 h, 5 and 69 nm CeO2 and 25 and 31 nm TiO2 decreased #AE, while the two MPs increased #AEs. Following BIC, only 31 nm TiO2 produced concentration-related decreases in #AEs, while 1288 nm CeO2 caused concentration-related increases in both TS and #AE. The results indicate that some metal oxide particles cause subtle concentration-related changes in spontaneous and/or GABAA receptor-mediated neuronal activity in vitro at times when cytotoxicity is absent, and that MEAs can be used to screen and prioritize nanoparticles for neurotoxicity hazard. PMID:26593696

  16. A preliminary investigation into the relationship between functional movement screen scores and athletic physical performance in female team sport athletes.

    PubMed

    Lockie, Rg; Schultz, Ab; Callaghan, Sj; Jordan, Ca; Luczo, Tm; Jeffriess, Md

    2015-03-01

    There is little research investigating relationships between the Functional Movement Screen (FMS) and athletic performance in female athletes. This study analyzed the relationships between FMS (deep squat; hurdle step [HS]; in-line lunge [ILL]; shoulder mobility; active straight-leg raise [ASLR]; trunk stability push-up; rotary stability) scores, and performance tests (bilateral and unilateral sit-and-reach [flexibility]; 20-m sprint [linear speed]; 505 with turns from each leg; modified T-test with movement to left and right [change-of-direction speed]; bilateral and unilateral vertical and standing broad jumps; lateral jumps [leg power]). Nine healthy female recreational team sport athletes (age = 22.67 ± 5.12 years; height = 1.66 ± 0.05 m; body mass = 64.22 ± 4.44 kilograms) were screened in the FMS and completed the afore-mentioned tests. Percentage between-leg differences in unilateral sit-and-reach, 505 turns and the jumps, and difference between the T-test conditions, were also calculated. Spearman's correlations (p ≤ 0.05) examined relationships between the FMS and performance tests. Stepwise multiple regressions (p ≤ 0.05) were conducted for the performance tests to determine FMS predictors. Unilateral sit-and-reach positive correlated with the left-leg ASLR (r = 0.704-0.725). However, higher-scoring HS, ILL, and ASLR related to poorer 505 and T-test performance (r = 0.722-0.829). A higher-scored left-leg ASLR related to a poorer unilateral vertical and standing broad jump, which were the only significant relationships for jump performance. Predictive data tended to confirm the correlations. The results suggest limitations in using the FMS to identify movement deficiencies that could negatively impact athletic performance in female team sport athletes. PMID:25729149

  17. Functional Genomic Screen Identifies Klebsiella pneumoniae Factors Implicated in Blocking Nuclear Factor κB (NF-κB) Signaling*

    PubMed Central

    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.

    2015-01-01

    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. PMID:25971969

  18. Functional Genomic Screen Identifies Klebsiella pneumoniae Factors Implicated in Blocking Nuclear Factor κB (NF-κB) Signaling.

    PubMed

    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

    2015-07-01

    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. PMID:25971969

  19. Computer simulated screening of dentin bonding primer monomers through analysis of their chemical functions and their spatial 3D alignment.

    PubMed

    Vaidyanathan, J; Vaidyanathan, T K; Ravichandran, S

    2009-02-01

    Binding interactions between dentin bonding primer monomers and dentinal collagen were studied by an analysis of their chemical functions and their spatial 3D alignment. A trial set of 12 monomers used as primers in dentin adhesives was characterized to assess them for binding to a complementary target. HipHop utility in the Catalyst software from Accelrys was used for the study. Ten hypotheses were generated by HipHop procedures involving (a) conformational generation using a poling technique to promote conformational variation, (b) extraction of functions to remodel ligands as function-based structures, and (c) identification of common patterns of functional alignment displayed by low energy conformations. The hypotheses, designated as pharmacaphores, were also scored and ranked. Analysis of pharmacaphore models through mapping of ligands revealed important differences between ligands. Top-ranked poses from direct docking simulations using type 1 collagen target were mapped in a rigid manner to the highest ranked pharmacophore model. The visual match observed in mapping and associated fit values suggest a strong correspondence between direct and indirect docking simulations. The results elegantly demonstrate that an indirect approach used to identify pharmacaphore models from adhesive ligands without a target may be a simple and viable approach to assess their intermolecular interactions with an intended target. Inexpensive indirect/direct virtual screening of hydrophilic monomer candidates may be a practical way to assess their initial promise for dentin primer use well before additional experimental evaluation of their priming/bonding efficacy. This is also of value in the search/design of new compounds for priming dentin. PMID:18546179

  20. Many amino acid substitution variants identified in DNA repair genes during human population screenings are predicted to impact protein function

    SciTech Connect

    Xi, T; Jones, I M; Mohrenweiser, H W

    2003-11-03

    Over 520 different amino acid substitution variants have been previously identified in the systematic screening of 91 human DNA repair genes for sequence variation. Two algorithms were employed to predict the impact of these amino acid substitutions on protein activity. Sorting Intolerant From Tolerant (SIFT) classified 226 of 508 variants (44%) as ''Intolerant''. Polymorphism Phenotyping (PolyPhen) classed 165 of 489 amino acid substitutions (34%) as ''Probably or Possibly Damaging''. Another 9-15% of the variants were classed as ''Potentially Intolerant or Damaging''. The results from the two algorithms are highly associated, with concordance in predicted impact observed for {approx}62% of the variants. Twenty one to thirty one percent of the variant proteins are predicted to exhibit reduced activity by both algorithms. These variants occur at slightly lower individual allele frequency than do the variants classified as ''Tolerant'' or ''Benign''. Both algorithms correctly predicted the impact of 26 functionally characterized amino acid substitutions in the APE1 protein on biochemical activity, with one exception. It is concluded that a substantial fraction of the missense variants observed in the general human population are functionally relevant. These variants are expected to be the molecular genetic and biochemical basis for the associations of reduced DNA repair capacity phenotypes with elevated cancer risk.

  1. Density-Functional Theory with Screened van der Waals Interactions for the Modeling of Hybrid Inorganic/Organic Systems

    NASA Astrophysics Data System (ADS)

    Ruiz, Victor G.; Liu, Wei; Zojer, Egbert; Scheffler, Matthias; Tkatchenko, Alexandre

    2012-02-01

    The electronic properties and the function of hybrid inorganic/organic systems (HIOS) are intimately linked to their geometry, with van der Waals (vdW) interactions playing an essential role for the latter. Here we show that the inclusion of the many--body collective response of the substrate electrons inside the inorganic bulk enables us to reliably predict the HIOS geometries and energies. Specifically, dispersion-corrected density-functional theory (the DFT+vdW approach) [PRL 102, 073005 (2009)], is combined with the Lifshitz-Zaremba-Kohn theory [PRB 13, 2270 (1976)] for the non--local Coulomb screening within the bulk. Our method (DFT+vdW^surf ) includes both image-plane and interface polarization effects. We show that DFT+vdW^surf yields geometries in remarkable agreement ( 0.1 å) with normal incidence x-ray standing wave measurements for the 3,4,9,10--perylene--tetracarboxylic acid dianhydride (C24H8O6, PTCDA) molecule on Cu(111), Ag(111), and Au(111). Similarly accurate results are obtained for xenon and benzene adsorbed on metal surfaces.

  2. Density-Functional Theory with Screened van der Waals Interactions for the Modeling of Hybrid Inorganic-Organic Systems

    NASA Astrophysics Data System (ADS)

    Ruiz, Victor G.; Liu, Wei; Zojer, Egbert; Scheffler, Matthias; Tkatchenko, Alexandre

    2012-04-01

    The electronic properties and the function of hybrid inorganic-organic systems (HIOS) are intimately linked to their interface geometry. Here we show that the inclusion of the many-body collective response of the substrate electrons inside the inorganic bulk enables us to reliably predict the HIOS geometries and energies. This is achieved by the combination of dispersion-corrected density-functional theory (the DFT+ van der Waals approach) [Phys. Rev. Lett. 102, 073005 (2009)PRLTAO0031-900710.1103/PhysRevLett.102.073005], with the Lifshitz-Zaremba-Kohn theory for the nonlocal Coulomb screening within the bulk. Our method yields geometries in remarkable agreement (≈0.1Å) with normal incidence x-ray standing wave measurements for the 3, 4, 9, 10-perylene-tetracarboxylic acid dianhydride (C24O6H8, PTCDA) molecule on Cu(111), Ag(111), and Au(111) surfaces. Similarly accurate results are obtained for xenon and benzene adsorbed on metal surfaces.

  3. Identification of RING finger protein 4 (RNF4) as a modulator of DNA demethylation through a functional genomics screen.

    PubMed

    Hu, Xiaoyi V; Rodrigues, Tânia M A; Tao, Haiyan; Baker, Robert K; Miraglia, Loren; Orth, Anthony P; Lyons, Gary E; Schultz, Peter G; Wu, Xu

    2010-08-24

    DNA methylation is an important epigenetic modification involved in transcriptional regulation, nuclear organization, development, aging, and disease. Although DNA methyltransferases have been characterized, the mechanisms for DNA demethylation remain poorly understood. Using a cell-based reporter assay, we performed a functional genomics screen to identify genes involved in DNA demethylation. Here we show that RNF4 (RING finger protein 4), a SUMO-dependent ubiquitin E3-ligase previously implicated in maintaining genome stability, plays a key role in active DNA demethylation. RNF4 reactivates methylation-silenced reporters and promotes global DNA demethylation. Rnf4 deficiency is embryonic lethal with higher levels of methylation in genomic DNA. Mechanistic studies show that RNF4 interacts with and requires the base excision repair enzymes TDG and APE1 for active demethylation. This activity appears to occur by enhancing the enzymatic activities that repair DNA G:T mismatches generated from methylcytosine deamination. Collectively, our study reveals a unique function for RNF4, which may serve as a direct link between epigenetic DNA demethylation and DNA repair in mammalian cells. PMID:20696907

  4. Item analysis and differential item functioning of a brief conduct problem screen.

    PubMed

    Wu, Johnny; King, Kevin M; Witkiewitz, Katie; Racz, Sarah Jensen; McMahon, Robert J

    2012-06-01

    Research has shown that boys display higher levels of childhood conduct problems than girls, and Black children display higher levels than White children, but few studies have tested for scalar equivalence of conduct problems across gender and race. The authors conducted a 2-parameter item response theory (IRT) model to examine item characteristics of the Authority Acceptance scale from the Teacher Observation of Classroom Adaptation-Revised (AA-TOCA-R; L. Larsson-Werthamer, S. G. Kellam, & L. Wheeler, 1991) in 8,820 kindergarten children and estimated the degree of differential item functioning (DIF) by gender and race/urban status. The mean level of latent conduct problems was best represented by behaviors such as being stubborn, breaking rules, and being disobedient, whereas breaking things and taking others' property best represented the construct at one standard deviation above the mean. DIF by gender was detected, such that at equivalent levels of latent conduct problems, males received more endorsements of overt behaviors from teachers, whereas females received more endorsements of nonphysical behaviors. Moreover, overt behaviors were better discriminators of latent conduct problems for males, and nonphysical behaviors were better discriminators of latent conduct problems for females. Differences across race/urban status were not found to be conceptually meaningful. The authors' analyses also suggest that the item scaling of the AA-TOCA-R may be best represented by 5e categories instead of 6. These findings provide support for the use of IRT modeling to examine item characteristics of conduct problem scales and DIF to test for scalar equivalence across diverse subpopulations. PMID:22040514

  5. Functional Movement Screening Performance of Brazilian Jiu-Jitsu Athletes From Brazil: Differences Considering Practice Time and Combat Style.

    PubMed

    Del Vecchio, Fabrício Boscolo; Gondim, Denis Foster; Arruda, Antonio Carlos Pereira

    2016-08-01

    Boscolo Del Vecchio, F, Foster, D, and Arruda, A. Functional movement screening performance of Brazilian jiu-jitsu athletes from Brazil: differences considering practice time and combat style. J Strength Cond Res 30(8): 2341-2347, 2016-Brazilian jiu-jitsu (BJJ) is a grappling combat sport that athletes, lying (guard fighter) or kneeling (pass fighter) on the mat, attempt to force their opponents to submit. Brazilian jiu-jitsu practices may result in muscular imbalances, which increase the risk of injury. Instead, the Functional Movement Screen (FMS) is an evaluation routine that could be related to injury incidence and seeks to detect muscular imbalance and movement dysfunction. Thus, the aim of the study was to investigate the injury profile and the FMS score and their relationship, with consideration for the BJJ fight style. Sports injuries were recorded in the last 12 months of 33 BJJ athletes, and the statistical analyses were applied to a routine evaluation FMS and a score of 14 points or less was considered low performance in FMS. We used a logistic regression; the effect size (ES) was calculated, and 5% was assumed as the statistical significance level. Pass fighters showed a higher percentage of injuries on the thorax (24.24%) than did guard fighters (6.67%, p = 0.01). Upper limbs were the most injured part of the body (χ = 36.7; p < 0.001), and they were 79% of the injuries that occurred in training sessions (χ = 14.53; p < 0.001). Despite the lack of statistical differences in the FMS performance between guard and pass fighters (t = 1.97; p = 0.05), its magnitude was considered medium (ES = 0.77). There was an association between FMS and presence of injury (χ = 4.95; p = 0.03). Considering the FMS score as a predictor and the presence or absence of injury as the dependent variable, the data met a Wald coefficient of 4.55, p = 0.03 and Exp (B) = 5.71. The study found that almost half of the sample had injuries in the upper limbs and a quarter had

  6. Focus screen optimization.

    PubMed

    Plummer, W T

    1975-11-01

    Ground glass used for camera focus screens often has far from optimum diffusion properties. An analysis of the function of the focus screen shows that a screen with random (Gaussian) diffusion properties can be constructed with both brightness and focus efficiencies above 84%. These considerations have led to the design of an unusually bright and effective focus screen for the Polaroid SX-70 Land camera. PMID:20155099

  7. Why activity-based costing works.

    PubMed

    Gabram, S G; Mendola, R A; Rozenfeld, J; Gamelli, R L

    1997-01-01

    With advancing technology and the quest for delineating the true cost of a procedure or diagnostic test, cost accounting techniques are being re-explored in the health care setting. Activity-based costing (ABC), adopted from other businesses, is one such example that has applications in the health industry. The purpose of this paper is to enhance the understanding of health care costs among physician providers, emphasizing a new approach--activity-based costing. PMID:10169347

  8. Transpo-mAb display: Transposition-mediated B cell display and functional screening of full-length IgG antibody libraries.

    PubMed

    Waldmeier, Lorenz; Hellmann, Ina; Gutknecht, Chantal K; Wolter, Fabian I; Cook, Skylar C; Reddy, Sai T; Grawunder, Ulf; Beerli, Roger R

    2016-01-01

    In vitro antibody display and screening technologies geared toward the discovery and engineering of clinically applicable antibodies have evolved from screening artificial antibody formats, powered by microbial display technologies, to screening of natural, full-IgG molecules expressed in mammalian cells to readily yield lead antibodies with favorable properties in production and clinical applications. Here, we report the development and characterization of a novel, next-generation mammalian cell-based antibody display and screening platform called Transpo-mAb Display, offering straightforward and efficient generation of cellular libraries by using non-viral transposition technology to obtain stable antibody expression. Because Transpo-mAb Display uses DNA-transposable vectors with substantial cargo capacity, genomic antibody heavy chain expression constructs can be utilized that undergo the natural switch from membrane bound to secreted antibody expression in B cells by way of alternative splicing of Ig-heavy chain transcripts from the same genomic expression cassette. We demonstrate that stably transposed cells co-express transmembrane and secreted antibodies at levels comparable to those provided by dedicated constructs for secreted and membrane-associated IgGs. This unique feature expedites the screening and antibody characterization process by obviating the need for intermediate sequencing and re-cloning of individual antibody clones into separate expression vectors for functional screening purposes. In a series of proof-of-concept experiments, we demonstrate the seamless integration of antibody discovery with functional screening for various antibody properties, including binding affinity and suitability for preparation of antibody-drug conjugates. PMID:26986818

  9. piggyBac-Based Mosaic Screen Identifies a Postmitotic Function for Cohesin in Regulating Developmental Axon Pruning

    PubMed Central

    Schuldiner, Oren; Berdnik, Daniela; Levy, Jonathan Ma; Wu, Joy Sing-Yi; Luginbuhl, David; Gontang, Allison Camille; Luo, Liqun

    2008-01-01

    Summary Developmental axon pruning is widely used to refine neural circuits. We performed a mosaic screen to identify mutations affecting axon pruning of Drosophila mushroom body γ neurons. We constructed a modified piggyBac vector with improved mutagenicity and generated insertions in >2000 genes. We identified two cohesin subunits (SMC1 and SA) as essential for axon pruning. The cohesin complex maintains sister chromatid cohesion during cell division in eukaryotes. However, we show that the pruning phenotype in SMC1-/- clones is rescued by expressing SMC1 in neurons, revealing a new postmitotic function. SMC1-/- clones exhibit reduced levels of the ecdysone receptor EcR-B1, a key regulator of axon pruning. The pruning phenotype is significantly suppressed by overexpressing EcR-B1 and enhanced by reduced dosage of EcR, supporting a causal relationship. We also demonstrate a postmitotic role for SMC1 in dendrite targeting of olfactory projection neurons. We suggest that cohesin regulates diverse aspects of neuronal morphogenesis. PMID:18267091

  10. piggyBac-based mosaic screen identifies a postmitotic function for cohesin in regulating developmental axon pruning.

    PubMed

    Schuldiner, Oren; Berdnik, Daniela; Levy, Jonathan Ma; Wu, Joy S; Luginbuhl, David; Gontang, Allison Camille; Luo, Liqun

    2008-02-01

    Developmental axon pruning is widely used to refine neural circuits. We performed a mosaic screen to identify mutations affecting axon pruning of Drosophila mushroom body gamma neurons. We constructed a modified piggyBac vector with improved mutagenicity and generated insertions in >2000 genes. We identified two cohesin subunits (SMC1 and SA) as being essential for axon pruning. The cohesin complex maintains sister-chromatid cohesion during cell division in eukaryotes. However, we show that the pruning phenotype in SMC1(-/-) clones is rescued by expressing SMC1 in neurons, revealing a postmitotic function. SMC1(-/-) clones exhibit reduced levels of the ecdysone receptor EcR-B1, a key regulator of axon pruning. The pruning phenotype is significantly suppressed by overexpressing EcR-B1 and is enhanced by a reduced dose of EcR, supporting a causal relationship. We also demonstrate a postmitotic role for SMC1 in dendrite targeting of olfactory projection neurons. We suggest that cohesin regulates diverse aspects of neuronal morphogenesis. PMID:18267091

  11. Electronic bands and excited states of III-V semiconductor polytypes with screened-exchange density functional calculations

    SciTech Connect

    Akiyama, Toru; Nakamura, Kohji; Ito, Tomonori; Freeman, Arthur J.

    2014-03-31

    The electronic band structures and excited states of III-V semiconductors such as GaP, AlP, AlAs, and AlSb for various polytypes are determined employing the screened-exchange density functional calculations implemented in the full-potential linearized augmented plane-wave methods. We demonstrate that GaP and AlSb in the wurtzite (WZ) structure have direct gap while III-V semiconductors in the zinc blende, 4H, and 6H structures considered in this study exhibit an indirect gap. Furthermore, we find that inclusion of Al atoms less than 17% and 83% in the hexagonal Al{sub x}Ga{sub 1−x}P and Al{sub x}Ga{sub 1−x}As alloys, respectively, leads to a direct transition with a gap energy of ∼2.3 eV. The feasibility of III-V semiconductors with a direct gap in WZ structure offers a possible crystal structure engineering to tune the optical properties of semiconductor materials.

  12. Identification of Functional Domains in the Cohesin Loader Subunit Scc4 by a Random Insertion/Dominant Negative Screen

    PubMed Central

    Shwartz, Michal; Matityahu, Avi; Onn, Itay

    2016-01-01

    Cohesin is a multi-subunit complex that plays an essential role in genome stability. Initial association of cohesin with chromosomes requires the loader—a heterodimer composed of Scc4 and Scc2. However, very little is known about the loader’s mechanism of action. In this study, we performed a genetic screen to identify functional domains in the Scc4 subunit of the loader. We isolated scc4 mutant alleles that, when overexpressed, have a dominant negative effect on cell viability. We defined a small region in the N terminus of Scc4 that is dominant negative when overexpressed, and on which Scc2/Scc4 activity depends. When the mutant alleles are expressed as a single copy, they are recessive and do not support cell viability, cohesion, cohesin loading or Scc4 chromatin binding. In addition, we show that the mutants investigated reduce, but do not eliminate, the interaction of Scc4 with either Scc2 or cohesin. However, we show that Scc4 cannot bind cohesin in the absence of Scc2. Our results provide new insight into the roles of Scc4 in cohesin loading, and contribute to deciphering the loading mechanism. PMID:27280786

  13. Benchmarking density functional perturbation theory to enable high-throughput screening of materials for dielectric constant and refractive index

    NASA Astrophysics Data System (ADS)

    Petousis, Ioannis; Chen, Wei; Hautier, Geoffroy; Graf, Tanja; Schladt, Thomas D.; Persson, Kristin A.; Prinz, Fritz B.

    2016-03-01

    We demonstrate a high-throughput density functional perturbation theory (DFPT) methodology capable of screening compounds for their dielectric properties. The electronic and ionic dielectric tensors are calculated for 88 compounds, where the eigenvalues of the total dielectric tensors are compared with single crystal and polycrystalline experimental values reported in the literature. We find that GGA/PBE has a smaller mean average deviation from experiments (MARD=16.2 %) when compared to LDA. The prediction accuracy of DFPT is lowest for compounds that exhibit complex structural relaxation effects (e.g., octahedra rotation in perovskites) and/or strong anharmonicity. Despite some discrepancies between DFPT results and reported experimental values, the high-throughput methodology is found to be useful in identifying interesting compounds by ranking. This is demonstrated by the high Spearman correlation factor (ρ =0.92 ). Finally, we demonstrate that DFPT provides a good estimate for the refractive index of a compound without calculating the frequency dependence of the dielectric matrix (MARD=5.7 %).

  14. Functional screening of enzymes and bacteria for the dechlorination of hexachlorocyclohexane by a high-throughput colorimetric assay.

    PubMed

    Sharma, Pooja; Jindal, Swati; Bala, Kiran; Kumari, Kirti; Niharika, Neha; Kaur, Jasvinder; Pandey, Gunjan; Pandey, Rinku; Russell, Robyn J; Oakeshott, John G; Lal, Rup

    2014-04-01

    Two distinct microbial dehalogenases are involved in the first steps of degradation of hexachlorocyclohexane (HCH) isomers. The enzymes, LinA and LinB, catalyze dehydrochlorination and dechlorination reactions of HCH respectively, each with distinct isomer specificities. The two enzymes hold great promise for use in the bioremediation of HCH residues in contaminated soils, although their kinetics and isomer specificities are currently limiting. Here we report the functional screening of a library of 700 LinA and LinB clones generated from soil DNA for improved dechlorination activity by means of a high throughput colorimetric assay. The assay relies upon visual colour change of phenol red in an aqueous medium, due to the pH drop associated with the dechlorination reactions. The assay is performed in a microplate format using intact cells, making it quick and simple to perform and it has high sensitivity, dynamic range and reproducibility. The method has been validated with quantitative gas chromatographic analysis of promising clones, revealing some novel variants of both enzymes with superior HCH degrading activities. Some sphingomonad isolates with potentially superior activities were also identified. PMID:23740574

  15. Electronic bands and excited states of III-V semiconductor polytypes with screened-exchange density functional calculations

    NASA Astrophysics Data System (ADS)

    Akiyama, Toru; Nakamura, Kohji; Ito, Tomonori; Freeman, Arthur J.

    2014-03-01

    The electronic band structures and excited states of III-V semiconductors such as GaP, AlP, AlAs, and AlSb for various polytypes are determined employing the screened-exchange density functional calculations implemented in the full-potential linearized augmented plane-wave methods. We demonstrate that GaP and AlSb in the wurtzite (WZ) structure have direct gap while III-V semiconductors in the zinc blende, 4H, and 6H structures considered in this study exhibit an indirect gap. Furthermore, we find that inclusion of Al atoms less than 17% and 83% in the hexagonal AlxGa1-xP and AlxGa1-xAs alloys, respectively, leads to a direct transition with a gap energy of ˜2.3 eV. The feasibility of III-V semiconductors with a direct gap in WZ structure offers a possible crystal structure engineering to tune the optical properties of semiconductor materials.

  16. Functional screening implicates miR-371-3p and peroxiredoxin 6 in reversible tolerance to cancer drugs

    PubMed Central

    Sahu, Nisebita; Stephan, Jean-Philippe; Cruz, Darlene Dela; Merchant, Mark; Haley, Benjamin; Bourgon, Richard; Classon, Marie; Settleman, Jeff

    2016-01-01

    Acquired resistance to cancer drug therapies almost always occurs in advanced-stage patients even following a significant response to treatment. In addition to mutational mechanisms, various non-mutational resistance mechanisms have now been recognized. We previously described a chromatin-mediated subpopulation of reversibly drug-tolerant persisters that is dynamically maintained within a wide variety of tumour cell populations. Here we explore a potential role for microRNAs in such transient drug tolerance. Functional screening of 879 human microRNAs reveals miR-371-3p as a potent suppressor of drug tolerance. We identify PRDX6 (peroxiredoxin 6) as a key target of miR-371-3p in establishing drug tolerance by regulating PLA2/PKCα activity and reactive oxygen species. PRDX6 expression is associated with poor prognosis in cancers of multiple tissue origins. These findings implicate miR-371-3p as a suppressor of PRDX6 and suggest that co-targeting of peroxiredoxin 6 or modulating miR-371-3p expression together with targeted cancer therapies may delay or prevent acquired drug resistance. PMID:27484502

  17. Characterization of three new carboxylic ester hydrolases isolated by functional screening of a forest soil metagenomic library.

    PubMed

    Biver, Sophie; Vandenbol, Micheline

    2013-02-01

    Three new lipolytic genes were isolated from a forest soil metagenomic library by functional screening on tributyrin agar plates. The genes SBLip1, SBLip2 and SBLip5.1 respectively encode polypeptides of 445, 346 and 316 amino acids. Phylogenetic analyses revealed that SBLip2 and SBLip5.1 belong to bacterial esterase/lipase family IV, whereas SBLip1 shows similarity to class C β-lactamases and is thus related to esterase family VIII. The corresponding genes were overexpressed and their products purified by affinity chromatography for characterization. Analyses of substrate specificity with different p-nitrophenyl esters showed that all three enzymes have a preference for short-acyl-chain p-nitrophenyl esters, a feature of carboxylesterases as opposed to lipases. The β-lactamase activity of SBLip1, measured with the chromogenic substrate nitrocefin, was very low. The three esterases have the same optimal pH (pH 10) and remain active across a relatively broad pH range, displaying more than 60 % activity between pH 6 and 10. The temperature optima determined were 35 °C for SBLip1, 45 °C for SBLip2 and 50 °C for SBLip5.1. The three esterases displayed different levels of tolerance to salts, solvents and detergents, SBLip2 being overall more tolerant to high concentrations of solvent and SBLip5.1 less affected by detergents. PMID:23160923

  18. A Functional Yeast Survival Screen of Tumor-Derived cDNA Libraries Designed to Identify Anti-Apoptotic Mammalian Oncogenes

    PubMed Central

    Melzer, Inga Maria; Moser, Julia; Siele, Dagmar; Köhl, Ulrike; Rieker, Ralf Joachim; Wachter, David Lukas; Agaimy, Abbas; Herpel, Esther; Baumgarten, Peter; Mittelbronn, Michel; Rakel, Stefanie; Kögel, Donat; Böhm, Stefanie; Gutschner, Tony; Diederichs, Sven; Zörnig, Martin

    2013-01-01

    Yeast cells can be killed upon expression of pro-apoptotic mammalian proteins. We have established a functional yeast survival screen that was used to isolate novel human anti-apoptotic genes overexpressed in treatment-resistant tumors. The screening of three different cDNA libraries prepared from metastatic melanoma, glioblastomas and leukemic blasts allowed for the identification of many yeast cell death-repressing cDNAs, including 28% of genes that are already known to inhibit apoptosis, 35% of genes upregulated in at least one tumor entity and 16% of genes described as both anti-apoptotic in function and upregulated in tumors. These results confirm the great potential of this screening tool to identify novel anti-apoptotic and tumor-relevant molecules. Three of the isolated candidate genes were further analyzed regarding their anti-apoptotic function in cell culture and their potential as a therapeutic target for molecular therapy. PAICS, an enzyme required for de novo purine biosynthesis, the long non-coding RNA MALAT1 and the MAST2 kinase are overexpressed in certain tumor entities and capable of suppressing apoptosis in human cells. Using a subcutaneous xenograft mouse model, we also demonstrated that glioblastoma tumor growth requires MAST2 expression. An additional advantage of the yeast survival screen is its universal applicability. By using various inducible pro-apoptotic killer proteins and screening the appropriate cDNA library prepared from normal or pathologic tissue of interest, the survival screen can be used to identify apoptosis inhibitors in many different systems. PMID:23717670

  19. Screening of Target Genes and Regulatory Function of miRNAs as Prognostic Indicators for Prostate Cancer.

    PubMed

    Xiaoli, Zhang; Yawei, Wei; Lianna, Liu; Haifeng, Li; Hui, Zhang

    2015-01-01

    BACKGROUND MicroRNAs expression profiling of prostate cancer is becoming increasingly used due to its usefulness in diagnosis, staging, prognosis, and response to treatment. The aim of this study was to screen differentially expressed miRNAs in prostate cancer and analyze the functions and signal pathways of their target genes. MATERIAL AND METHODS High-throughput data of miRNAs were downloaded from The Cancer Genome Atlas (TCGA) database. A total of 551 samples (52 normal and 499 prostate cancer cases) and 1046 miRNAs expression values were selected for further analysis. Differentially expressed miRNAs between normal and prostate cancer tissues were identified using SAMR. StarBase and TargetScan software were used to predict the miRNAs' target group and target genes, respectively. GO functional and KEGG pathway analysis was conducted on up/down-regulated expressed miRNA with DAVID. Finally, survival analysis was performed to evaluate the association of differently expressed miRNAs signature and overall survival of prostate cancer patients. RESULTS A total of 162 miRNAs were differentially expressed between normal and prostate cancer samples, including 128 up-regulated and 38 down-regulated ones; hsa-mir-153-2, hsa-mir-92a-1, and hsa-mir-182 (up-regulated); and hsa-mir-29a, hsa-mir-10a, and hsa-mir-221 (down-regulated) were identified as good biomarkers. In GO and KEGG analysis, target genes of down-regulated miRNAs were significantly enriched in positive ion combination and JAK-STAT pathway annotation, respectively; the ones with up-regulated miRNAs were significantly enriched in the function of plasma membrane and MARK signaling pathway annotation, respectively. Patients were categorized into low- or high-score groups according to their risk scores from each miRNA. The patients in the low-score group had better overall survival compared with those in high-score group. CONCLUSIONS The 6 differentially expressed miRNAs and their target genes were used to define

  20. Screening of Target Genes and Regulatory Function of miRNAs as Prognostic Indicators for Prostate Cancer

    PubMed Central

    Xiaoli, Zhang; Yawei, Wei; Lianna, Liu; Haifeng, Li; Hui, Zhang

    2015-01-01

    Background MicroRNAs expression profiling of prostate cancer is becoming increasingly used due to its usefulness in diagnosis, staging, prognosis, and response to treatment. The aim of this study was to screen differentially expressed miRNAs in prostate cancer and analyze the functions and signal pathways of their target genes. Material/Methods High-throughput data of miRNAs were downloaded from The Cancer Genome Atlas (TCGA) database. A total of 551 samples (52 normal and 499 prostate cancer cases) and 1046 miRNAs expression values were selected for further analysis. Differentially expressed miRNAs between normal and prostate cancer tissues were identified using SAMR. StarBase and TargetScan software were used to predict the miRNAs’ target group and target genes, respectively. GO functional and KEGG pathway analysis was conducted on up/down-regulated expressed miRNA with DAVID. Finally, survival analysis was performed to evaluate the association of differently expressed miRNAs signature and overall survival of prostate cancer patients. Results A total of 162 miRNAs were differentially expressed between normal and prostate cancer samples, including 128 up-regulated and 38 down-regulated ones; hsa-mir-153-2, hsa-mir-92a-1, and hsa-mir-182 (up-regulated); and hsa-mir-29a, hsa-mir-10a, and hsa-mir-221 (down-regulated) were identified as good biomarkers. In GO and KEGG analysis, target genes of down-regulated miRNAs were significantly enriched in positive ion combination and JAK-STAT pathway annotation, respectively; the ones with up-regulated miRNAs were significantly enriched in the function of plasma membrane and MARK signaling pathway annotation, respectively. Patients were categorized into low- or high-score groups according to their risk scores from each miRNA. The patients in the low-score group had better overall survival compared with those in high-score group. Conclusions The 6 differentially expressed miRNAs and their target genes were used to define

  1. Screening NK-, B- and T-cell phenotype and function in patients suffering from Chronic Fatigue Syndrome

    PubMed Central

    2013-01-01

    Background Chronic Fatigue Syndrome (CFS) is a debilitating neuro-immune disorder of unknown etiology diagnosed by an array of clinical manifestations. Although several immunological abnormalities have been described in CFS, their heterogeneity has limited diagnostic applicability. Methods Immunological features of CFS were screened in 22 CFS diagnosed individuals fulfilling Fukuda criteria and 30 control healthy individuals. Peripheral blood T, B and NK cell function and phenotype were analyzed by flow cytometry in both groups. Results CFS diagnosed individuals showed similar absolute numbers of T, B and NK cells, with minor differences in the percentage of CD4+ and CD8+ T cells. B cells showed similar subset frequencies and proliferative responses between groups. Conversely, significant differences were observed in T cell subsets. CFS individuals showed increased levels of T regulatory cells (CD25+/FOXP3+) CD4 T cells, and lower proliferative responses in vitro and in vivo. Moreover, CD8 T cells from the CFS group showed significantly lower activation and frequency of effector memory cells. No clear signs of T-cell immunosenescence were observed. NK cells from CFS individuals displayed higher expression of NKp46 and CD69 but lower expression of CD25 in all NK subsets defined. Overall, T cell and NK cell features clearly clustered CFS individuals. Conclusions Our findings suggest that alterations in T-cell phenotype and proliferative response along with the specific signature of NK cell phenotype may be useful to identify CFS individuals. The striking down modulation of T cell mediated immunity may help to understand intercurrent viral infections in CFS. PMID:23514202

  2. A Functional Genomic Screen for Evolutionarily Conserved Genes Required for Lifespan and Immunity in Germline-Deficient C. elegans

    PubMed Central

    Sinha, Amit; Rae, Robbie

    2014-01-01

    The reproductive system regulates lifespan in insects, nematodes and vertebrates. In Caenorhabditis elegans removal of germline increases lifespan by 60% which is dependent upon insulin signaling, nuclear hormone signaling, autophagy and fat metabolism and their microRNA-regulators. Germline-deficient C. elegans are also more resistant to various bacterial pathogens but the underlying molecular mechanisms are largely unknown. Firstly, we demonstrate that previously identified genes that regulate the extended lifespan of germline-deficient C. elegans (daf-2, daf-16, daf-12, tcer-1, mir-7.1 and nhr-80) are also essential for resistance to the pathogenic bacterium Xenorhabdus nematophila. We then use a novel unbiased approach combining laser cell ablation, whole genome microarrays, RNAi screening and exposure to X. nematophila to generate a comprehensive genome-wide catalog of genes potentially required for increased lifespan and innate immunity in germline-deficient C. elegans. We find 3,440 genes to be upregulated in C. elegans germline-deficient animals in a gonad dependent manner, which are significantly enriched for genes involved in insulin signaling, fatty acid desaturation, translation elongation and proteasome complex function. Using RNAi against a subset of 150 candidate genes selected from the microarray results, we show that the upregulated genes such as transcription factor DAF-16/FOXO, the PTEN homolog lipid phosphatase DAF-18 and several components of the proteasome complex (rpn-6.1, rpn-7, rpn-9, rpn-10, rpt-6, pbs-3 and pbs-6) are essential for both lifespan and immunity of germline deficient animals. We also identify a novel role for genes including par-5 and T12G3.6 in both lifespan-extension and increased survival on X. nematophila. From an evolutionary perspective, most of the genes differentially expressed in germline deficient C. elegans also show a conserved expression pattern in germline deficient Pristionchus pacificus, a nematode species

  3. Genetic Screen in Drosophila Larvae Links ird1 Function to Toll Signaling in the Fat Body and Hemocyte Motility

    PubMed Central

    Schmid, Martin R.; Anderl, Ines; Vo, Hoa T. M.; Valanne, Susanna; Yang, Hairu; Kronhamn, Jesper; Rämet, Mika; Rusten, Tor Erik

    2016-01-01

    To understand how Toll signaling controls the activation of a cellular immune response in Drosophila blood cells (hemocytes), we carried out a genetic modifier screen, looking for deletions that suppress or enhance the mobilization of sessile hemocytes by the gain-of-function mutation Toll10b (Tl10b). Here we describe the results from chromosome arm 3R, where five regions strongly suppressed this phenotype. We identified the specific genes immune response deficient 1 (ird1), headcase (hdc) and possibly Rab23 as suppressors, and we studied the role of ird1 in more detail. An ird1 null mutant and a mutant that truncates the N-terminal kinase domain of the encoded Ird1 protein affected the Tl10b phenotype, unlike mutations that affect the C-terminal part of the protein. The ird1 null mutant suppressed mobilization of sessile hemocytes, but enhanced other Tl10b hemocyte phenotypes, like the formation of melanotic nodules and the increased number of circulating hemocytes. ird1 mutants also had blood cell phenotypes on their own. They lacked crystal cells and showed aberrant formation of lamellocytes. ird1 mutant plasmatocytes had a reduced ability to spread on an artificial substrate by forming protrusions, which may explain why they did not go into circulation in response to Toll signaling. The effect of the ird1 mutation depended mainly on ird1 expression in hemocytes, but ird1-dependent effects in other tissues may contribute. Specifically, the Toll receptor was translocated from the cell membrane to intracellular vesicles in the fat body of the ird1 mutant, and Toll signaling was activated in that tissue, partially explaining the Tl10b-like phenotype. As ird1 is otherwise known to control vesicular transport, we conclude that the vesicular transport system may be of particular importance during an immune response. PMID:27467079

  4. A functional screen identifies miRNAs that inhibit DNA repair and sensitize prostate cancer cells to ionizing radiation

    PubMed Central

    Hatano, Koji; Kumar, Binod; Zhang, Yonggang; Coulter, Jonathan B.; Hedayati, Mohammad; Mears, Brian; Ni, Xiaohua; Kudrolli, Tarana A.; Chowdhury, Wasim H.; Rodriguez, Ronald; DeWeese, Theodore L.; Lupold, Shawn E.

    2015-01-01

    MicroRNAs (miRNAs) have been implicated in DNA repair pathways through transcriptional responses to DNA damaging agents or through predicted miRNA regulation of DNA repair genes. We hypothesized that additional DNA damage regulating miRNAs could be identified by screening a library of 810 miRNA mimetics for the ability to alter cellular sensitivity to ionizing radiation (IR). A prostate cancer Metridia luciferase cell model was applied to examine the effects of individual miRNAs on IR sensitivity. A large percentage of miRNA mimetics were found to increase cellular sensitivity to IR, while a smaller percentage were protective. Two of the most potent IR sensitizing miRNAs, miR-890 and miR-744–3p, significantly delayed IR induced DNA damage repair. Both miRNAs inhibited the expression of multiple components of DNA damage response and DNA repair. miR-890 directly targeted MAD2L2, as well as WEE1 and XPC, where miR-744–3p directly targeted RAD23B. Knock-down of individual miR-890 targets by siRNA was not sufficient to ablate miR-890 radiosensitization, signifying that miR-890 functions by regulating multiple DNA repair genes. Intratumoral delivery of miR-890 mimetics prior to IR therapy significantly enhanced IR therapeutic efficacy. These results reveal novel miRNA regulation of DNA repair and identify miR-890 as a potent IR sensitizing agent. PMID:25845598

  5. Dehydrothyrsiferol does not modulate multidrug resistance-associated protein 1 resistance: a functional screening system for MRP1 substrates.

    PubMed

    Pec, Martina K; Aguirre, Amable; Fernández, Javier J; Souto, Maria L; Dorta, Javier F; Villar, Jesús

    2002-11-01

    We had shown previously that the novel, marine, anticancer compound dehydrothyrsiferol (DHT) does not modulate P-glycoprotein (P-gp) dependent drug efflux. Many chemotherapeutics with clinical impact are substrates for the structurally distant related membrane transport protein MRP1 (multidrug resistance-associated protein 1). Thus, we were interested in analysing the behaviour of DHT and control compounds in specific drug transport of MRP1 overexpressing cells. We established a fluorescence based drug efflux system for specific, functional detection of interference of a test compound in MRP1 mediated drug extrusion. Briefly, MRP1 overexpressing HL60/Adr cells were incubated to uptake and then efflux fluorescent 5(6)-carboxyfluorescein diacetate (CFDA), rhodamine 123 (Rh123), or 3,3-diethylocarbocyanine iodide (DiOC2), respectively. Changes in cell fluorescence intensity after coincubation with the compound of interest were determined by flow cytometry. MRP1 mediated efflux of CFDA was analysed in the presence of DHT, the known substrates genistein, probenecid, and the specific inhibitor MK-571. To exclude unknown P-gp related interference in drug transport, efflux of the fluorescent P-gp substrate DiOC2 and specific inhibition by cyclosporin A (CsA) were analysed. Cytotoxicity of DHT in resistant HL60/Adr cells was found to be even superior to that in the parental HL60 leukaemia cell line. Consequently, DHT did not interfere in MRP1 mediated drug transport. In contrast to DiOC2, rhodamine 123 was not specifically effluxed by P-gp but also by MRP1. Therefore, we propose the MRP1 specific CFDA efflux model as a screening and/or excluding system for MRP1 substrates. Together with previous data our results suggest DHT to be an interesting candidate for further investigation directed towards a drug development regimen. PMID:12373300

  6. Genetic Screen in Drosophila Larvae Links ird1 Function to Toll Signaling in the Fat Body and Hemocyte Motility.

    PubMed

    Schmid, Martin R; Anderl, Ines; Vo, Hoa T M; Valanne, Susanna; Yang, Hairu; Kronhamn, Jesper; Rämet, Mika; Rusten, Tor Erik; Hultmark, Dan

    2016-01-01

    To understand how Toll signaling controls the activation of a cellular immune response in Drosophila blood cells (hemocytes), we carried out a genetic modifier screen, looking for deletions that suppress or enhance the mobilization of sessile hemocytes by the gain-of-function mutation Toll10b (Tl10b). Here we describe the results from chromosome arm 3R, where five regions strongly suppressed this phenotype. We identified the specific genes immune response deficient 1 (ird1), headcase (hdc) and possibly Rab23 as suppressors, and we studied the role of ird1 in more detail. An ird1 null mutant and a mutant that truncates the N-terminal kinase domain of the encoded Ird1 protein affected the Tl10b phenotype, unlike mutations that affect the C-terminal part of the protein. The ird1 null mutant suppressed mobilization of sessile hemocytes, but enhanced other Tl10b hemocyte phenotypes, like the formation of melanotic nodules and the increased number of circulating hemocytes. ird1 mutants also had blood cell phenotypes on their own. They lacked crystal cells and showed aberrant formation of lamellocytes. ird1 mutant plasmatocytes had a reduced ability to spread on an artificial substrate by forming protrusions, which may explain why they did not go into circulation in response to Toll signaling. The effect of the ird1 mutation depended mainly on ird1 expression in hemocytes, but ird1-dependent effects in other tissues may contribute. Specifically, the Toll receptor was translocated from the cell membrane to intracellular vesicles in the fat body of the ird1 mutant, and Toll signaling was activated in that tissue, partially explaining the Tl10b-like phenotype. As ird1 is otherwise known to control vesicular transport, we conclude that the vesicular transport system may be of particular importance during an immune response. PMID:27467079

  7. Design and analysis of a piezoelectric material based touch screen with additional pressure and its acceleration measurement functions

    NASA Astrophysics Data System (ADS)

    Chu, Xiang-Cheng; Liu, Jia-Yi; Gao, Ren-Long; Chang, Jie; Li, Long-Tu

    2013-12-01

    Touch screens are becoming more and more prevalent in everyday environments due to their convenience and humanized operation. In this paper, a piezoelectric material based touch screen is developed and investigated. Piezoelectric ceramics arrayed under the touch panel at the edges or corners are used as tactile sensors to measure the touch positioning point similarly to conventional touch screens. However, additional touch pressure and its acceleration performance can also be obtained to obtain a higher-level human-machine interface. The piezoelectric ceramics can also be added to a traditional touch screen structure, or they can be used independently to construct a novel touch screen with a high light transmittance approach to a transparent glass. The piezoelectric ceramics were processed from PZT piezoelectric ceramic powder into a round or rectangular shape. According to the varied touch position and physical press strength of a finger, or even a gloved hand or fingernail, the piezoelectric tactile sensors will have different output voltage responses. By calculating the ratio of different piezoelectric tactile sensors’ responses and summing up all piezoelectric tactile sensors’ output voltages, the touch point position, touch pressure and touch force acceleration can be detected. A prototype of such a touch screen is manufactured and its position accuracy, touch pressure and response speed are measured in detail. The experimental results show that the prototype has many advantages such as high light transmittance, low energy cost and high durability.

  8. Evaluation of recently validated non-invasive formula using basic lung functions as new screening tool for pulmonary hypertension in idiopathic pulmonary fibrosis patients

    PubMed Central

    Ghanem, Maha K.; Makhlouf, Hoda A.; Agmy, Gamal R.; Imam, Hisham M. K.; Fouad, Doaa A.

    2009-01-01

    BACKGROUND: A prediction formula for mean pulmonary artery pressure (MPAP) using standard lung function measurement has been recently validated to screen for pulmonary hypertension (PH) in idiopathic pulmonary fibrosis (IPF) patients. OBJECTIVE: To test the usefulness of this formula as a new non invasive screening tool for PH in IPF patients. Also, to study its correlation with patients' clinical data, pulmonary function tests, arterial blood gases (ABGs) and other commonly used screening methods for PH including electrocardiogram (ECG), chest X ray (CXR), trans-thoracic echocardiography (TTE) and computerized tomography pulmonary angiography (CTPA). MATERIALS AND METHODS: Cross-sectional study of 37 IPF patients from tertiary hospital. The accuracy of MPAP estimation was assessed by examining the correlation between the predicted MPAP using the formula and PH diagnosed by other screening tools and patients' clinical signs of PH. RESULTS: There was no statistically significant difference in the prediction of PH using cut off point of 21 or 25 mm Hg (P = 0.24). The formula-predicted MPAP greater than 25 mm Hg strongly correlated in the expected direction with O2 saturation (r = −0.95, P < 0.000), partial arterial O2 tension (r = −0.71, P < 0.000), right ventricular systolic pressure measured by TTE (r = 0.6, P < 0.000) and hilar width on CXR (r = 0.31, P = 0.03). Chest symptoms, ECG and CTPA signs of PH poorly correlated with the same formula (P > 0.05). CONCLUSIONS: The prediction formula for MPAP using standard lung function measurements is a simple non invasive tool that can be used as TTE to screen for PH in IPF patients and select those who need right heart catheterization. PMID:19881164

  9. The Reliability of Functional Movement Screening and In-Season Changes in Physical Function and Performance Among Elite Rugby League Players.

    PubMed

    Waldron, Mark; Gray, Adrian; Worsfold, Paul; Twist, Craig

    2016-04-01

    This study aimed to (a) assess the reliability of the functional movement screening (FMS) protocol and (b) to establish changes in both FMS and tests of physical performance throughout a season. The reliability of the FMS components (12 in total) was assessed through a nonparametric statistical approach, based on 2 trials, separated by 1 week. Score on the FMS, strength (3 repetition maximum full squat, 1 repetition maximum [1 RM] bench press), running speed (10 and 40 m), and jump height of 12 elite male under-19 rugby league players was monitored at pre-, mid-, and late-season periods. There was no bias (p > 0.05) found between trials for the FMS, with the majority of components reaching 100% "perfect agreement," reflecting the good reliability of the FMS tool. There were no effects (p > 0.05) of season stage on any of the FMS components; however, an improvement (p ≤ 0.05) between the pre- and both mid- and late-season periods was apparent in every component of fitness, such as 1 RM bench press (112.92 ± 24.54 kg; 125.83 ± 21.41 kg; 125.98 ± 24.48 kg) and 40-m sprint time (5.69 ± 0.35 seconds; 5.62 ± 0.31 seconds; 5.64 ± 0.27 seconds). Our findings demonstrate that the FMS can be reliably administered to elite rugby league players but will not change in accordance with physical performance across a competitive season. Our findings should not necessarily deter practitioners from using the FMS but begin to question the specific qualities that are being assessed through its administration. PMID:27003450

  10. HTS-DB: an online resource to publish and query data from functional genomics high-throughput siRNA screening projects

    PubMed Central

    Saunders, Rebecca E.; Instrell, Rachael; Rispoli, Rossella; Jiang, Ming; Howell, Michael

    2013-01-01

    High-throughput screening (HTS) uses technologies such as RNA interference to generate loss-of-function phenotypes on a genomic scale. As these technologies become more popular, many research institutes have established core facilities of expertise to deal with the challenges of large-scale HTS experiments. As the efforts of core facility screening projects come to fruition, focus has shifted towards managing the results of these experiments and making them available in a useful format that can be further mined for phenotypic discovery. The HTS-DB database provides a public view of data from screening projects undertaken by the HTS core facility at the CRUK London Research Institute. All projects and screens are described with comprehensive assay protocols, and datasets are provided with complete descriptions of analysis techniques. This format allows users to browse and search data from large-scale studies in an informative and intuitive way. It also provides a repository for additional measurements obtained from screens that were not the focus of the project, such as cell viability, and groups these data so that it can provide a gene-centric summary across several different cell lines and conditions. All datasets from our screens that can be made available can be viewed interactively and mined for further hit lists. We believe that in this format, the database provides researchers with rapid access to results of large-scale experiments that might facilitate their understanding of genes/compounds identified in their own research. Database URL: http://hts.cancerresearchuk.org/db/public PMID:24122843

  11. Large-Scale Functional RNAi Screen in C. elegans Identifies TGF-β and Notch Signaling Pathways as Modifiers of CACNA1A.

    PubMed

    Pereira, Maria da Conceição; Morais, Sara; Sequeiros, Jorge; Alonso, Isabel

    2016-04-01

    Variants inCACNA1Athat encodes the pore-forming α1-subunit of human voltage-gated Cav2.1 (P/Q-type) Ca(2+)channels cause several autosomal-dominant neurologic disorders, including familial hemiplegic migraine type 1, episodic ataxia type 2, and spinocerebellar ataxia type 6. To identify modifiers of incoordination in movement disorders, we performed a large-scale functional RNAi screen, using theCaenorhabditis elegansstrain CB55, which carries a truncating mutation in theunc-2gene, the worm ortholog for the humanCACNA1A The screen was carried out by the feeding method in 96-well liquid culture format, using the ORFeome v1.1 feeding library, and time-lapse imaging of worms in liquid culture was used to assess changes in thrashing behavior. We looked for genes that, when silenced, either ameliorated the slow and uncoordinated phenotype ofunc-2, or interacted to produce a more severe phenotype. Of the 350 putative hits from the primary screen, 37 genes consistently showed reproducible results. At least 75% of these are specifically expressed in theC. elegansneurons. Functional network analysis and gene ontology revealed overrepresentation of genes involved in development, growth, locomotion, signal transduction, and vesicle-mediated transport. We have expanded the functional network of genes involved in neurodegeneration leading to cerebellar ataxia related tounc-2/CACNA1A, further confirming the involvement of the transforming growth factor β pathway and adding a novel signaling cascade, the Notch pathway. PMID:27005779

  12. Large-Scale Functional RNAi Screen in C. elegans Identifies TGF-β and Notch Signaling Pathways as Modifiers of CACNA1A

    PubMed Central

    Pereira, Maria da Conceição; Morais, Sara; Sequeiros, Jorge

    2016-01-01

    Variants in CACNA1A that encodes the pore-forming α1-subunit of human voltage-gated Cav2.1 (P/Q-type) Ca2+ channels cause several autosomal-dominant neurologic disorders, including familial hemiplegic migraine type 1, episodic ataxia type 2, and spinocerebellar ataxia type 6. To identify modifiers of incoordination in movement disorders, we performed a large-scale functional RNAi screen, using the Caenorhabditis elegans strain CB55, which carries a truncating mutation in the unc-2 gene, the worm ortholog for the human CACNA1A. The screen was carried out by the feeding method in 96-well liquid culture format, using the ORFeome v1.1 feeding library, and time-lapse imaging of worms in liquid culture was used to assess changes in thrashing behavior. We looked for genes that, when silenced, either ameliorated the slow and uncoordinated phenotype of unc-2, or interacted to produce a more severe phenotype. Of the 350 putative hits from the primary screen, 37 genes consistently showed reproducible results. At least 75% of these are specifically expressed in the C. elegans neurons. Functional network analysis and gene ontology revealed overrepresentation of genes involved in development, growth, locomotion, signal transduction, and vesicle-mediated transport. We have expanded the functional network of genes involved in neurodegeneration leading to cerebellar ataxia related to unc-2/CACNA1A, further confirming the involvement of the transforming growth factor β pathway and adding a novel signaling cascade, the Notch pathway. PMID:27005779

  13. Development and evaluation of an immunochromatographic strip for rapid screening of sildenafil-type compounds as illegal additives in functional foods.

    PubMed

    Guo, Jiebiao; Liu, Wangpei; Lan, Xianquan; Chen, Hualong; Xiao, Zijun

    2016-07-01

    Sildenafil is a phosphodiesterase-5 inhibitor (PDE-5) for the treatment of erectile dysfunction. Undeclared sildenafil and related analogues adulterated in functional foods are a threat to public health. To screen these illegal drugs rapidly in herbal samples, an immunochromatographic (IC) assay was developed based on polyclonal antibodies specific to both sildenafil and its analogues. A group that is pharmacological necessary for sildenafil and its analogues was employed as a representative hapten for the generation antibodies against the target compounds. The desired antisera showed satisfactory specificities to sildenafil and major analogues with IC50 values ranging from 19.3 to 34.6 ng ml(-1) in a referring enzyme-linked immunosorbent assay (ELISA). The optimised IC assay showed detection thresholds in the range 5.0-20 μg g(-1) for sildenafil and major analogues in herbal samples. Sixty herbal food supplements were screened and six were found to be positive using the IC strip. It was confirmed by ELISA and UPLC-PDA-MS/MS that positive samples contain target illegal additives in levels of 10-40 mg g(-1) (1-4%). In this range, sensitivity of the IC strip is adequate to screen sildenafil-type compounds in herbal commodities under a dilution ratio of 1:10(3). Thus, the current IC assay is a suitable tool for screening sildenafil and its analogues as illegal additives in herbal food supplements. PMID:27310564

  14. Identification of a farnesol analog as a Ras function inhibitor using both an in vivo Ras activation sensor and a phenotypic screening approach

    PubMed Central

    Srinivasan, Kamalakkannan; Subramanian, Thangaiah; Spielmann, H. Peter

    2013-01-01

    Mutations in Ras isoforms such as K-Ras, N-Ras, and H-Ras contribute to roughly 85, 15, and 1 % of human cancers, respectively. Proper membrane targeting of these Ras isoforms, a prerequisite for Ras activity, requires farnesylation or geranylgeranylation at the C-terminal CAAX box. We devised an in vivo screening strategy based on monitoring Ras activation and phenotypic physiological outputs for assaying synthetic Ras function inhibitors (RFI). Ras activity was visualized by the trans-location of RBDRaf1-GFP to activated Ras at the plasma membrane. By using this strategy, we screened one synthetic farnesyl substrate analog (AGOH) along with nine putative inhibitors and found that only m-CN-AGOH inhibited Ras activation. Phenotypic analysis of starving cells could be used to monitor polarization, motility, and the inability of these treated cells to aggregate properly during fruiting body formation. Incorporation of AGOH and m-CN-AGOH to cellular proteins was detected by western blot. These screening assays can be incorporated into a high throughput screening format using Dictyostelium discoideum and automated microscopy to determine effective RFIs. These RFI candidates can then be further tested in mammalian systems. PMID:24194124

  15. In vitro screening of metal oxide nanoparticles for effects on neural function using cortical networks on microelectrode arrays

    EPA Science Inventory

    Nanoparticles (NPs) may translocate to the brain following inhalation or oral exposures, yet higher throughput methods to screen NPs for potential neurotoxicity are lacking. The present study examined effects of 5 Ce02 (5- 1288 nm), and 4 Ti02 (6-142 nm) NPs and microparticles (M...

  16. Development of a Multiplex Assay for Studying Functional Selectivity of Human Serotonin 5-HT2A Receptors and Identification of Active Compounds by High-Throughput Screening.

    PubMed

    Iglesias, Alba; Lage, Sonia; Cadavid, Maria Isabel; Loza, Maria Isabel; Brea, José

    2016-09-01

    G protein-coupled receptors (GPCRs) exist as collections of conformations in equilibrium, and the efficacy of drugs has been proposed to be associated with their absolute and relative affinities for these different conformations. The serotonin 2A (5-HT2A) receptor regulates multiple physiological functions, is involved in the pathophysiology of schizophrenia, and serves as an important target of atypical antipsychotic drugs. This receptor was one of the first GPCRs for which the functional selectivity phenomenon was observed, with its various ligands exerting differential effects on the phospholipase A2 (PLA2) and phospholipase C (PLC) signaling pathways. We aimed to develop a multiplex functional assay in 96-well plates for the simultaneous measurement of the PLA2 and PLC pathways coupled to 5-HT2A receptors; this approach enables the detection of either functional selectivity or cooperativity phenomena in early drug screening stages. The suitability of the method for running screening campaigns was tested using the Prestwick Chemical Library, and 22 confirmed hits with activities of more than 90% were identified; 11 of these hits produced statistically significant differences between the two effector pathways. Thus, we have developed a miniaturized multiplex assay in 96-well plates to measure functional selectivity for 5-HT2A receptors in the early stages of the drug discovery process. PMID:27095818

  17. Structural and functional screening in human induced-pluripotent stem cell-derived cardiomyocytes accurately identifies cardiotoxicity of multiple drug types

    SciTech Connect

    Doherty, Kimberly R. Talbert, Dominique R.; Trusk, Patricia B.; Moran, Diarmuid M.; Shell, Scott A.; Bacus, Sarah

    2015-05-15

    Safety pharmacology studies that evaluate new drug entities for potential cardiac liability remain a critical component of drug development. Current studies have shown that in vitro tests utilizing human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CM) may be beneficial for preclinical risk evaluation. We recently demonstrated that an in vitro multi-parameter test panel assessing overall cardiac health and function could accurately reflect the associated clinical cardiotoxicity of 4 FDA-approved targeted oncology agents using hiPS-CM. The present studies expand upon this initial observation to assess whether this in vitro screen could detect cardiotoxicity across multiple drug classes with known clinical cardiac risks. Thus, 24 drugs were examined for their effect on both structural (viability, reactive oxygen species generation, lipid formation, troponin secretion) and functional (beating activity) endpoints in hiPS-CM. Using this screen, the cardiac-safe drugs showed no effects on any of the tests in our panel. However, 16 of 18 compounds with known clinical cardiac risk showed drug-induced changes in hiPS-CM by at least one method. Moreover, when taking into account the Cmax values, these 16 compounds could be further classified depending on whether the effects were structural, functional, or both. Overall, the most sensitive test assessed cardiac beating using the xCELLigence platform (88.9%) while the structural endpoints provided additional insight into the mechanism of cardiotoxicity for several drugs. These studies show that a multi-parameter approach examining both cardiac cell health and function in hiPS-CM provides a comprehensive and robust assessment that can aid in the determination of potential cardiac liability. - Highlights: • 24 drugs were tested for cardiac liability using an in vitro multi-parameter screen. • Changes in beating activity were the most sensitive in predicting cardiac risk. • Structural effects add in

  18. Introducing Thermal Wave Transport Analysis (TWTA): A Thermal Technique for Dopamine Detection by Screen-Printed Electrodes Functionalized with Molecularly Imprinted Polymer (MIP) Particles.

    PubMed

    Peeters, Marloes M; van Grinsven, Bart; Foster, Christopher W; Cleij, Thomas J; Banks, Craig E

    2016-01-01

    A novel procedure is developed for producing bulk modified Molecularly Imprinted Polymer (MIP) screen-printed electrodes (SPEs), which involves the direct mixing of the polymer particles within the screen-printed ink. This allowed reduction of the sample preparation time from 45 min to 1 min, and resulted in higher reproducibility of the electrodes. The samples are measured with a novel detection method, namely, thermal wave transport analysis (TWTA), relying on the analysis of thermal waves through a functional interface. As a first proof-of-principle, MIPs for dopamine are developed and successfully incorporated within a bulk modified MIP SPE. The detection limits of dopamine within buffer solutions for the MIP SPEs are determined via three independent techniques. With cyclic voltammetry this was determined to be 4.7 × 10(-6) M, whereas by using the heat-transfer method (HTM) 0.35 × 10(-6) M was obtained, and with the novel TWTA concept 0.26 × 10(-6) M is possible. This TWTA technique is measured simultaneously with HTM and has the benefits of reducing measurement time to less than 5 min and increasing effect size by nearly a factor of two. The two thermal methods are able to enhance dopamine detection by one order of magnitude compared to the electrochemical method. In previous research, it was not possible to measure neurotransmitters in complex samples with HTM, but with the improved signal-to-noise of TWTA for the first time, spiked dopamine concentrations were determined in a relevant food sample. In summary, novel concepts are presented for both the sensor functionalization side by employing screen-printing technology, and on the sensing side, the novel TWTA thermal technique is reported. The developed bio-sensing platform is cost-effective and suitable for mass-production due to the nature of screen-printing technology, which makes it very interesting for neurotransmitter detection in clinical diagnostic applications. PMID:27128891

  19. Identification of Spen as a Crucial Factor for Xist Function through Forward Genetic Screening in Haploid Embryonic Stem Cells

    PubMed Central

    Monfort, Asun; Di Minin, Giulio; Postlmayr, Andreas; Freimann, Remo; Arieti, Fabiana; Thore, Stéphane; Wutz, Anton

    2015-01-01

    Summary In mammals, the noncoding Xist RNA triggers transcriptional silencing of one of the two X chromosomes in female cells. Here, we report a genetic screen for silencing factors in X chromosome inactivation using haploid mouse embryonic stem cells (ESCs) that carry an engineered selectable reporter system. This system was able to identify several candidate factors that are genetically required for chromosomal repression by Xist. Among the list of candidates, we identify the RNA-binding protein Spen, the homolog of split ends. Independent validation through gene deletion in ESCs confirms that Spen is required for gene repression by Xist. However, Spen is not required for Xist RNA localization and the recruitment of chromatin modifications, including Polycomb protein Ezh2. The identification of Spen opens avenues for further investigation into the gene-silencing pathway of Xist and shows the usefulness of haploid ESCs for genetic screening of epigenetic pathways. PMID:26190100

  20. Identification of Spen as a Crucial Factor for Xist Function through Forward Genetic Screening in Haploid Embryonic Stem Cells.

    PubMed

    Monfort, Asun; Di Minin, Giulio; Postlmayr, Andreas; Freimann, Remo; Arieti, Fabiana; Thore, Stéphane; Wutz, Anton

    2015-07-28

    In mammals, the noncoding Xist RNA triggers transcriptional silencing of one of the two X chromosomes in female cells. Here, we report a genetic screen for silencing factors in X chromosome inactivation using haploid mouse embryonic stem cells (ESCs) that carry an engineered selectable reporter system. This system was able to identify several candidate factors that are genetically required for chromosomal repression by Xist. Among the list of candidates, we identify the RNA-binding protein Spen, the homolog of split ends. Independent validation through gene deletion in ESCs confirms that Spen is required for gene repression by Xist. However, Spen is not required for Xist RNA localization and the recruitment of chromatin modifications, including Polycomb protein Ezh2. The identification of Spen opens avenues for further investigation into the gene-silencing pathway of Xist and shows the usefulness of haploid ESCs for genetic screening of epigenetic pathways. PMID:26190100

  1. High Throughput Enzyme Inhibitor Screening by Functionalized Magnetic Carbonaceous Microspheres and Graphene Oxide-Based MALDI-TOF-MS

    NASA Astrophysics Data System (ADS)

    Liu, Yang; Li, Yan; Liu, Junyan; Deng, Chunhui; Zhang, Xiangmin

    2011-12-01

    In this work, a high throughput methodology for screening enzyme inhibitors has been demonstrated by combining enzyme immobilized magnetic carbonaceous microspheres and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) with grapheme oxide as matrix. First, model enzyme acetylcholinesterase (AChE) was immobilized onto the 3-glycidoxypropyltrimethoxysilane (GLYMO)-modified magnetic carbonaceous (MC) microspheres, displaying a high enzyme activity and stability, and also facilitating the separation of enzyme from substrate and product. The efficiency of immobilized AChE was monitored by biochemical assay, which was carried out by mixing enzyme-immobilized MC microspheres with model substrate acetylcholine (ACh), and subsequent quantitative determination of substrate ACh and product choline using graphene oxide-based MALDI-TOF-MS with no background inference. The limit of detection (LOD) for ACh was 0.25 fmol/μL, and excellent linearity (R2 = 0.9998) was maintained over the range of 0.5 and 250 fmol/μL. Choline was quantified over the range of 0.05 and 15 pmol/μL, also with excellent linearity (R2 = 0.9994) and low LOD (0.15 fmol/μL). Good accuracy and precision were obtained for all concentrations within the range of the standard curves. All together, eight compounds (four known AChE inhibitors and four control chemical compounds with no AChE inhibit effect) were tested with our promoted methodology, and the obtained results demonstrated that our high throughput screening methodology could be a great help to the routine enzyme inhibitor screening.

  2. An siRNA screen for ATG protein depletion reveals the extent of the unconventional functions of the autophagy proteome in virus replication.

    PubMed

    Mauthe, Mario; Langereis, Martijn; Jung, Jennifer; Zhou, Xingdong; Jones, Alex; Omta, Wienand; Tooze, Sharon A; Stork, Björn; Paludan, Søren Riis; Ahola, Tero; Egan, Dave; Behrends, Christian; Mokry, Michal; de Haan, Cornelis; van Kuppeveld, Frank; Reggiori, Fulvio

    2016-08-29

    Autophagy is a catabolic process regulated by the orchestrated action of the autophagy-related (ATG) proteins. Recent work indicates that some of the ATG proteins also have autophagy-independent roles. Using an unbiased siRNA screen approach, we explored the extent of these unconventional functions of ATG proteins. We determined the effects of the depletion of each ATG proteome component on the replication of six different viruses. Our screen reveals that up to 36% of the ATG proteins significantly alter the replication of at least one virus in an unconventional fashion. Detailed analysis of two candidates revealed an undocumented role for ATG13 and FIP200 in picornavirus replication that is independent of their function in autophagy as part of the ULK complex. The high numbers of unveiled ATG gene-specific and pathogen-specific functions of the ATG proteins calls for caution in the interpretation of data, which rely solely on the depletion of a single ATG protein to specifically ablate autophagy. PMID:27573464

  3. COMBINATORIAL MATERIALS SYNTHESIS AND SCREENING: An Integrated Materials Chip Approach to Discovery and Optimization of Functional Materials

    NASA Astrophysics Data System (ADS)

    Xiang, X.-D.

    1999-08-01

    Combinatorial materials synthesis methods and high throughput evaluation techniques have been developed to accelerate the process of materials discovery and optimization. Analogous to integrated circuit chips, integrated materials chips containing thousands, possibly millions, of different compounds/materials, often in the form of high-quality epitaxial thin film can be fabricated and screened for interesting physical or chemical properties. Microspot X-ray methods, various optical measurement techniques, and a novel evanescent microwave microscope have been used to characterize the structural, optical, magnetic, and electrical properties of samples on materials chips. These techniques are routinely used to discover and optimize luminescent, ferroelectric, dielectric, and magnetic materials.

  4. Airport Screening

    MedlinePlus

    ... 2011 Photo courtesy of Dan Paluska/Flickr Denver Airport Security Screening Introduction With air travel regaining popularity and increased secu- rity measures, airport security screening has become an area of interest for ...

  5. Health Screening

    MedlinePlus

    Screenings are tests that look for diseases before you have symptoms. Screening tests can find diseases early, when they're easier ... Overweight and obesity Prostate cancer in men Which tests you need depends on your age, your sex, ...

  6. MRSA Screening

    MedlinePlus

    ... be limited. Home Visit Global Sites Search Help? MRSA Screening Share this page: Was this page helpful? Formal name: Methicillin resistant Staphylococcus aureus Screening Related tests: Wound Culture At a Glance ...

  7. Screening for high-spin metal organic frameworks (MOFs): density functional theory study on DUT-8(M1,M2) (with Mi = V,…,Cu).

    PubMed

    Schwalbe, Sebastian; Trepte, Kai; Seifert, Gotthard; Kortus, Jens

    2016-03-01

    We present a first principles study of low-spin (LS)/high-spin (HS) screening for 3d metal centers in the metal organic framework (MOF) DUT-8(Ni). Various density functional theory (DFT) codes have been used to evaluate numerical and DFT related errors. We compare highly accurate all-electron implementations with the widely used plane wave approach. We present electronically and magnetically stable DUT-8(Ni) HS secondary building units (SBUs). In this work we show how to tune the magnetic and electronic properties of the original SBU only by changing the metal centers. PMID:26922864

  8. Fully Automated One-Step Production of Functional 3D Tumor Spheroids for High-Content Screening.

    PubMed

    Monjaret, François; Fernandes, Mathieu; Duchemin-Pelletier, Eve; Argento, Amelie; Degot, Sébastien; Young, Joanne

    2016-04-01

    Adoption of spheroids within high-content screening (HCS) has lagged behind high-throughput screening (HTS) due to issues with running complex assays on large three-dimensional (3D) structures.To enable multiplexed imaging and analysis of spheroids, different cancer cell lines were grown in 3D on micropatterned 96-well plates with automated production of nine uniform spheroids per well. Spheroids achieve diameters of up to 600 µm, and reproducibility was experimentally validated (interwell and interplate CV(diameter) <5%). Biphoton imaging confirmed that micropatterned spheroids exhibit characteristic cell heterogeneity with distinct microregions. Furthermore, central necrosis appears at a consistent spheroid size, suggesting standardized growth.Using three reference compounds (fluorouracil, irinotecan, and staurosporine), we validated HT-29 micropatterned spheroids on an HCS platform, benchmarking against hanging-drop spheroids. Spheroid formation and imaging in a single plate accelerate assay workflow, and fixed positioning prevents structures from overlapping or sticking to the well wall, augmenting image processing reliability. Furthermore, multiple spheroids per well increase the statistical confidence sufficiently to discriminate compound mechanisms of action and generate EC50 values for endpoints of cell death, architectural change, and size within a single-pass read. Higher quality data and a more efficient HCS work chain should encourage integration of micropatterned spheroid models within fundamental research and drug discovery applications. PMID:26385905

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

    PubMed Central

    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

  10. The activity-based anorexia mouse model.

    PubMed

    Klenotich, Stephanie J; Dulawa, Stephanie C

    2012-01-01

    Animals housed with running wheels and subjected to daily food restriction show paradoxical reductions in food intake and increases in running wheel activity. This phenomenon, known as activity-based anorexia (ABA), leads to marked reductions in body weight that can ultimately lead to death. Recently, ABA has been proposed as a model of anorexia nervosa (AN). AN affects about 8 per 100,000 females and has the highest mortality rate among all psychiatric illnesses. Given the reductions in quality of life, high mortality rate, and the lack of pharmacological treatments for AN, a better understanding of the mechanisms underlying AN-like behavior is greatly needed. This chapter provides basic guidelines for conducting ABA experiments using mice. The ABA mouse model provides an important tool for investigating the neurobiological underpinnings of AN-like behavior and identifying novel treatments. PMID:22231828

  11. Double screening

    NASA Astrophysics Data System (ADS)

    Gratia, Pierre; Hu, Wayne; Joyce, Austin; Ribeiro, Raquel H.

    2016-06-01

    Attempts to modify gravity in the infrared typically require a screening mechanism to ensure consistency with local tests of gravity. These screening mechanisms fit into three broad classes; we investigate theories which are capable of exhibiting more than one type of screening. Specifically, we focus on a simple model which exhibits both Vainshtein and kinetic screening. We point out that due to the two characteristic length scales in the problem, the type of screening that dominates depends on the mass of the sourcing object, allowing for different phenomenology at different scales. We consider embedding this double screening phenomenology in a broader cosmological scenario and show that the simplest examples that exhibit double screening are radiatively stable.

  12. Phosphoramidates as novel activity-based probes for serine proteases.

    PubMed

    Haedke, Ute R; Frommel, Sandra C; Hansen, Fabian; Hahne, Hannes; Kuster, Bernhard; Bogyo, Matthew; Verhelst, Steven H L

    2014-05-26

    Activity-based probes (ABPs) are small molecules that exclusively form covalent bonds with catalytically active enzymes. In the last decade, they have especially been used in functional proteomics studies of proteases. Here, we present phosphoramidate peptides as a novel type of ABP for serine proteases. These molecules can be made in a straightforward manner by standard Fmoc-based solid-phase peptide synthesis, allowing rapid diversification. The resulting ABPs covalently bind different serine proteases, depending on the amino acid recognition element adjacent to the reactive group. A reporter tag enables downstream gel-based analysis or LC-MS/MS-mediated identification of the targeted proteases. Overall, we believe that these readily accessible probes will provide new avenues for the functional study of serine proteases in complex proteomes. PMID:24817682

  13. A functional genome-wide genetic screening identifies new pathways controlling the G1/S transcriptional wave.

    PubMed

    Gaspa, Laura; González-Medina, Alberto; Hidalgo, Elena; Ayté, José

    2016-03-01

    The Schizosaccharomyces pombe MBF complex activates the transcription of genes required for DNA synthesis and S phase. The MBF complex contains several proteins, including the core components Cdc10, Res1 and Res2, the co-repressor proteins Yox1 and Nrm1 and the co-activator Rep2. It has recently been shown how MBF is regulated when either the DNA damage or the DNA synthesis checkpoints are activated. However, how MBF is regulated in a normal unperturbed cell cycle is still not well understood. We have set up a genome-wide genomic screen searching for global regulators of MBF. We have crossed our knock-out collection library with a reporter strain that allows the measurement of MBF activity in live cells by flow cytometry. We confirm previously known regulators of MBF and show that COP9/signalosome and tRNA methyltransferases also regulate MBF activity. PMID:26890608

  14. Lead expansion and virtual screening of Indinavir derivate HIV-1 protease inhibitors using pharmacophoric - shape similarity scoring function

    PubMed Central

    Shityakov, Sergey; Dandekar, Thomas

    2010-01-01

    Indinavir (Crivaxan®) is a potent inhibitor of the HIV (human immunodeficiency virus) protease. This enzyme has an important role in viral replication and is considered to be very attractive target for new antiretroviral drugs. However, it becomes less effective due to highly resistant new viral strains of HIV, which have multiple mutations in their proteases. For this reason, we used a lead expansion method to create a new set of compounds with a new mode of action to protease binding site. 1300 compounds chemically diverse from the initial hit were generated and screened to determine their ability to interact with protease and establish their QSAR properties. Further computational analyses revealed one unique compound with different protease binding ability from the initial hit and its role for possible new class of protease inhibitors is discussed in this report. PMID:20978602

  15. A Cell-Based Internalization and Degradation Assay with an Activatable Fluorescence–Quencher Probe as a Tool for Functional Antibody Screening

    PubMed Central

    Liu, Peter Corbett; Shen, Yang; Snavely, Marshall D.; Hiraga, Kaori

    2015-01-01

    For the development of therapeutically potent anti-cancer antibody drugs, it is often important to identify antibodies that internalize into cells efficiently, rather than just binding to antigens on the cell surface. Such antibodies can mediate receptor endocytosis, resulting in receptor downregulation on the cell surface and potentially inhibiting receptor function and tumor growth. Also, efficient antibody internalization is a prerequisite for the delivery of cytotoxic drugs into target cells and is critical for the development of antibody–drug conjugates. Here we describe a novel activatable fluorescence–quencher pair to quantify the extent of antibody internalization and degradation in the target cells. In this assay, candidate antibodies were labeled with a fluorescent dye and a quencher. Fluorescence is inhibited outside and on the surface of cells, but activated upon endocytosis and degradation of the antibody. This assay enabled the development of a process for rapid characterization of candidate antibodies potentially in a high-throughput format. By employing an activatable secondary antibody, primary antibodies in purified form or in culture supernatants can be screened for internalization and degradation. Because purification of candidate antibodies is not required, this method represents a direct functional screen to identify antibodies that internalize efficiently early in the discovery process. PMID:26024945

  16. A Cell-Based Internalization and Degradation Assay with an Activatable Fluorescence-Quencher Probe as a Tool for Functional Antibody Screening.

    PubMed

    Li, Yan; Liu, Peter Corbett; Shen, Yang; Snavely, Marshall D; Hiraga, Kaori

    2015-08-01

    For the development of therapeutically potent anti-cancer antibody drugs, it is often important to identify antibodies that internalize into cells efficiently, rather than just binding to antigens on the cell surface. Such antibodies can mediate receptor endocytosis, resulting in receptor downregulation on the cell surface and potentially inhibiting receptor function and tumor growth. Also, efficient antibody internalization is a prerequisite for the delivery of cytotoxic drugs into target cells and is critical for the development of antibody-drug conjugates. Here we describe a novel activatable fluorescence-quencher pair to quantify the extent of antibody internalization and degradation in the target cells. In this assay, candidate antibodies were labeled with a fluorescent dye and a quencher. Fluorescence is inhibited outside and on the surface of cells, but activated upon endocytosis and degradation of the antibody. This assay enabled the development of a process for rapid characterization of candidate antibodies potentially in a high-throughput format. By employing an activatable secondary antibody, primary antibodies in purified form or in culture supernatants can be screened for internalization and degradation. Because purification of candidate antibodies is not required, this method represents a direct functional screen to identify antibodies that internalize efficiently early in the discovery process. PMID:26024945

  17. Structural and electronic properties of UnOm (n=1-3,m=1-3n) clusters: A theoretical study using screened hybrid density functional theory

    NASA Astrophysics Data System (ADS)

    Yang, Yu; Liu, Haitao; Zhang, Ping

    2016-05-01

    The structural and electronic properties of small uranium oxide clusters UnOm (n=1-3, m=1-3n) are systematically studied within the screened hybrid density functional theory. It is found that the formation of U-O-U bondings and isolated U-O bonds are energetically more stable than U-U bondings. As a result, no uranium cores are observed. Through fragmentation studies, we find that the UnOm clusters with the m/n ratio between 2 and 2.5 are very stable, hinting that UO2+x hyperoxides are energetically stable. Electronically, we find that the O-2p states always distribute in the deep energy range, and the U-5f states always distribute at the two sides of the Fermi level. The U-6d states mainly hybridize with the U-5f states in U-rich clusters, while hybridizing with O-2p states in O-rich clusters. Our work is the first one on the screened hybrid density functional theory level studying the atomic and electronic properties of the actinide oxide clusters.

  18. Structural and electronic properties of UnOm (n=1-3,m=1-3n) clusters: A theoretical study using screened hybrid density functional theory.

    PubMed

    Yang, Yu; Liu, Haitao; Zhang, Ping

    2016-05-14

    The structural and electronic properties of small uranium oxide clusters UnOm (n=1-3, m=1-3n) are systematically studied within the screened hybrid density functional theory. It is found that the formation of U-O-U bondings and isolated U-O bonds are energetically more stable than U-U bondings. As a result, no uranium cores are observed. Through fragmentation studies, we find that the UnOm clusters with the m/n ratio between 2 and 2.5 are very stable, hinting that UO2+x hyperoxides are energetically stable. Electronically, we find that the O-2p states always distribute in the deep energy range, and the U-5f states always distribute at the two sides of the Fermi level. The U-6d states mainly hybridize with the U-5f states in U-rich clusters, while hybridizing with O-2p states in O-rich clusters. Our work is the first one on the screened hybrid density functional theory level studying the atomic and electronic properties of the actinide oxide clusters. PMID:27179481

  19. A gain-of-function senescence bypass screen identifies the homeobox transcription factor DLX2 as a regulator of ATM-p53 signaling.

    PubMed

    Wang, Yifan; Xu, Qikai; Sack, Laura; Kang, Chanhee; Elledge, Stephen J

    2016-02-01

    Senescence stimuli activate multiple tumor suppressor pathways to initiate cycle arrest and a differentiation program characteristic of senescent cells. We performed a two-stage, gain-of-function screen to select for the genes whose enhanced expression can bypass replicative senescence. We uncovered multiple genes known to be involved in p53 and Rb regulation and ATM regulation, two components of the CST (CTC1-STN1-TEN1) complex involved in preventing telomere erosion, and genes such as REST and FOXO4 that have been implicated in aging. Among the new genes now implicated in senescence, we identified DLX2, a homeobox transcription factor that has been shown to be required for tumor growth and metastasis and is associated with poor cancer prognosis. Growth analysis showed that DLX2 expression led to increased cellular replicative life span. Our data suggest that DLX2 expression reduces the protein components of the TTI1/TTI2/TEL2 complex, a key complex required for the proper folding and stabilization of ATM and other members of the PIKK (phosphatidylinositol 3-kinase-related kinase) family kinase, leading to reduced ATM-p53 signaling and senescence bypass. We also found that the overexpression of DLX2 exhibited a mutually exclusive relationship with p53 alterations in cancer patients. Our functional screen identified novel players that may promote tumorigenesis by regulating the ATM-p53 pathway and senescence. PMID:26833729

  20. Formulation, High Throughput In Vitro Screening and In Vivo Functional Characterization of Nanoemulsion-Based Intranasal Vaccine Adjuvants

    PubMed Central

    Wong, Pamela T.; Leroueil, Pascale R.; Smith, Douglas M.; Ciotti, Susan; Bielinska, Anna U.; Janczak, Katarzyna W.; Mullen, Catherine H.; Groom, Jeffrey V.; Taylor, Erin M.; Passmore, Crystal; Makidon, Paul E.; O’Konek, Jessica J.; Myc, Andrzej; Hamouda, Tarek; Baker, James R.

    2015-01-01

    Vaccine adjuvants have been reported to induce both mucosal and systemic immunity when applied to mucosal surfaces and this dual response appears important for protection against certain pathogens. Despite the potential advantages, however, no mucosal adjuvants are currently approved for human use. Evaluating compounds as mucosal adjuvants is a slow and costly process due to the need for lengthy animal immunogenicity studies. We have constructed a library of 112 intranasal adjuvant candidate formulations consisting of oil-in-water nanoemulsions that contain various cationic and nonionic surfactants. To facilitate adjuvant development we first evaluated this library in a series of high-throughput, in vitro assays for activities associated with innate and adaptive immune activation in vivo. These in vitro assays screened for the ability of the adjuvant to bind to mucin, induce cytotoxicity, facilitate antigen uptake in epithelial and dendritic cells, and activate cellular pathways. We then sought to determine how these parameters related to adjuvant activity in vivo. While the in vitro assays alone were not enough to predict the in vivo adjuvant activity completely, several interesting relationships were found with immune responses in mice. Furthermore, by varying the physicochemical properties of the surfactant components (charge, surfactant polar head size and hydrophobicity) and the surfactant blend ratio of the formulations, the strength and type of the immune response generated (TH1, TH2, TH17) could be modulated. These findings suggest the possibility of using high-throughput screens to aid in the design of custom adjuvants with unique immunological profiles to match specific mucosal vaccine applications. PMID:25962136

  1. A Genome-wide Functional Screen Shows MAGI-1 Is an L1CAM-Dependent Stabilizer of Apical Junctions in C. elegans

    PubMed Central

    Lynch, Allison M.; Grana, Theresa; Cox, Elizabeth; Couthier, Annabelle; Cameron, Michel; Chin-Sang, Ian; Pettitt, Jonathan; Hardin, Jeff

    2012-01-01

    Summary Background In multicellular organisms, cell-cell junctions are involved in many aspects of tissue morphogenesis. α-catenin links the cadherin-catenin complex (CCC) to the actin cytoskeleton, stabilizing cadherin-dependent adhesions. Results To identify modulators of cadherin-based cell adhesion, we conducted a genome-wide RNAi screen in C. elegans and uncovered MAGI-1, a highly conserved protein scaffold. Loss of magi-1 function in wild-type embryos results in disorganized epithelial migration and occasional morphogenetic failure. MAGI-1 physically interacts with the putative actin regulator AFD-1/afadin; knocking down magi-1 or afd-1 function in a hypomorphic α-catenin background leads to complete morphogenetic failure and actin disorganization in the embryonic epidermis. MAGI-1 and AFD-1 localize to a unique domain in the apical junction and normal accumulation of MAGI-1 at junctions requires SAX-7/L1CAM, which can bind MAGI-1 via its C-terminus. Depletion of MAGI-1 leads to loss of spatial segregation and expansion of apical junctional domains and greater mobility of junctional proteins. Conclusions Our screen is the first genome-wide approach to identify proteins that function synergistically with the CCC during epidermal morphogenesis in a living embryo. We demonstrate novel physical interactions between MAGI-1, AFD-1/afadin and SAX-7/L1CAM, which are part of a functional interactome that includes components of the core CCC. Our results further suggest MAGI-1 helps to partition and maintain a stable, spatially ordered apical junction during morphogenesis. PMID:22981773

  2. Screening for Panic Disorder

    MedlinePlus

    ... Membership Journal & Multimedia Resources Awards Consumers Screening for Panic Disorder Main navigation FAQs Screen Yourself Screening for Depression ... Screening for Obsessive-Compulsive Disorder (OCD) Screening for Panic Disorder Screening for Posttraumatic Stress Disorder (PTSD) Screening for ...

  3. Genetic Screening

    PubMed Central

    Burke, Wylie; Tarini, Beth; Press, Nancy A.; Evans, James P.

    2011-01-01

    Current approaches to genetic screening include newborn screening to identify infants who would benefit from early treatment, reproductive genetic screening to assist reproductive decision making, and family history assessment to identify individuals who would benefit from additional prevention measures. Although the traditional goal of screening is to identify early disease or risk in order to implement preventive therapy, genetic screening has always included an atypical element—information relevant to reproductive decisions. New technologies offer increasingly comprehensive identification of genetic conditions and susceptibilities. Tests based on these technologies are generating a different approach to screening that seeks to inform individuals about all of their genetic traits and susceptibilities for purposes that incorporate rapid diagnosis, family planning, and expediting of research, as well as the traditional screening goal of improving prevention. Use of these tests in population screening will increase the challenges already encountered in genetic screening programs, including false-positive and ambiguous test results, overdiagnosis, and incidental findings. Whether this approach is desirable requires further empiric research, but it also requires careful deliberation on the part of all concerned, including genomic researchers, clinicians, public health officials, health care payers, and especially those who will be the recipients of this novel screening approach. PMID:21709145

  4. Drug screens based on the newly found role of dystroglycan proteolysis and restoration of dystroglycan function thereof

    DOEpatents

    Bissell, Mina J.; Muschler, John L.

    2010-02-23

    The present invention provides methods and compositions for the diagnosis and treatment of cells lacking normal growth arresting characteristic. The present invention demonstrates that many tumor cells lack normal cell surface .alpha.-dystroglycan and thereby lack dystroglycan function. Dystroglycan can be lost from the cell surface by proteolytic shedding of a fragment of .alpha.-dystroglycan into the surrounding medium. Upon restoration of dystroglycan function and over-expression of the dystroglycan gene, the once tumorigenic cells revert to non-tumorigenic cells which polarize and arrest cell growth in the presence of basement membrane proteins, demonstrating that dystroglycan functions as a tumor marker and suppressor.

  5. Mathematical framework for activity-based cancer biomarkers

    PubMed Central

    Kwong, Gabriel A.; Dudani, Jaideep S.; Carrodeguas, Emmanuel; Mazumdar, Eric V.; Zekavat, Seyedeh M.; Bhatia, Sangeeta N.

    2015-01-01

    Advances in nanomedicine are providing sophisticated functions to precisely control the behavior of nanoscale drugs and diagnostics. Strategies that coopt protease activity as molecular triggers are increasingly important in nanoparticle design, yet the pharmacokinetics of these systems are challenging to understand without a quantitative framework to reveal nonintuitive associations. We describe a multicompartment mathematical model to predict strategies for ultrasensitive detection of cancer using synthetic biomarkers, a class of activity-based probes that amplify cancer-derived signals into urine as a noninvasive diagnostic. Using a model formulation made of a PEG core conjugated with protease-cleavable peptides, we explore a vast design space and identify guidelines for increasing sensitivity that depend on critical parameters such as enzyme kinetics, dosage, and probe stability. According to this model, synthetic biomarkers that circulate in stealth but then activate at sites of disease have the theoretical capacity to discriminate tumors as small as 5 mm in diameter—a threshold sensitivity that is otherwise challenging for medical imaging and blood biomarkers to achieve. This model may be adapted to describe the behavior of additional activity-based approaches to allow cross-platform comparisons, and to predict allometric scaling across species. PMID:26417077

  6. Mathematical framework for activity-based cancer biomarkers.

    PubMed

    Kwong, Gabriel A; Dudani, Jaideep S; Carrodeguas, Emmanuel; Mazumdar, Eric V; Zekavat, Seyedeh M; Bhatia, Sangeeta N

    2015-10-13

    Advances in nanomedicine are providing sophisticated functions to precisely control the behavior of nanoscale drugs and diagnostics. Strategies that coopt protease activity as molecular triggers are increasingly important in nanoparticle design, yet the pharmacokinetics of these systems are challenging to understand without a quantitative framework to reveal nonintuitive associations. We describe a multicompartment mathematical model to predict strategies for ultrasensitive detection of cancer using synthetic biomarkers, a class of activity-based probes that amplify cancer-derived signals into urine as a noninvasive diagnostic. Using a model formulation made of a PEG core conjugated with protease-cleavable peptides, we explore a vast design space and identify guidelines for increasing sensitivity that depend on critical parameters such as enzyme kinetics, dosage, and probe stability. According to this model, synthetic biomarkers that circulate in stealth but then activate at sites of disease have the theoretical capacity to discriminate tumors as small as 5 mm in diameter-a threshold sensitivity that is otherwise challenging for medical imaging and blood biomarkers to achieve. This model may be adapted to describe the behavior of additional activity-based approaches to allow cross-platform comparisons, and to predict allometric scaling across species. PMID:26417077

  7. NAIS: Nuclear activation-based imaging spectroscopy

    SciTech Connect

    Günther, M. M.; Britz, A.; Harres, K.; Hoffmeister, G.; Nürnberg, F.; Otten, A.; Pelka, A.; Roth, M.; Clarke, R. J.; Vogt, K.

    2013-07-15

    In recent years, the development of high power laser systems led to focussed intensities of more than 10{sup 22} W/cm{sup 2} at high pulse energies. Furthermore, both, the advanced high power lasers and the development of sophisticated laser particle acceleration mechanisms facilitate the generation of high energetic particle beams at high fluxes. The challenge of imaging detector systems is to acquire the properties of the high flux beam spatially and spectrally resolved. The limitations of most detector systems are saturation effects. These conventional detectors are based on scintillators, semiconductors, or radiation sensitive films. We present a nuclear activation-based imaging spectroscopy method, which is called NAIS, for the characterization of laser accelerated proton beams. The offline detector system is a combination of stacked metal foils and imaging plates (IP). After the irradiation of the stacked foils they become activated by nuclear reactions, emitting gamma decay radiation. In the next step, an autoradiography of the activated foils using IPs and an analysis routine lead to a spectrally and spatially resolved beam profile. In addition, we present an absolute calibration method for IPs.

  8. Activity-based costing for electric utilities

    SciTech Connect

    Croyle, D.R.; Schapiro, I.A.; Keglevic, P.M. )

    1992-08-01

    This EPRI report is a primer'' on Activity-Based Costing (ABC). ABC is a cost management aproach which can make an important contribution to understanding and controlling the changing costs in the electric utility industry. It is a method for attributing costs to activities, products and services by better understanding the underlying factors which drive those costs. ABC can help utility managers make better decisions through the application of more accurate process and product cost information and a fuller understanding of which activities add value and which do not. Armed with such information, utility managers are better equipped to address many of the strategic and operating decisions which they routinely face. The report introduces the ABC concept and approach to utility managers and offers insights into how ABC can be and is being used to control costs and improve strategic and operating decisions in electric utilities and other industries. The report (1) describes the ABC approach, (2) discusses the value of ABC to elecuic utilities, (3) identifies potential applications of ABC to current utility issues, (4) describes a step-by-step approach to developing and implementing ABC in the utility environment, and (5) presents a survey of more than 30 electric utilities and several detailed case studies of electric utilities and other companies who have adopted and are using ABC.

  9. Functional screen reveals essential roles of miR-27a/24 in differentiation of embryonic stem cells

    PubMed Central

    Ma, Yanni; Yao, Nan; Liu, Guang; Dong, Lei; Liu, Yufang; Zhang, Meili; Wang, Fang; Wang, Bin; Wei, Xueju; Dong, He; Wang, Lanlan; Ji, Shaowei; Zhang, Junwu; Wang, Yangming; Huang, Yue; Yu, Jia

    2015-01-01

    MicroRNAs play important roles in controlling the embryonic stem cell (ESC) state. Although much is known about microRNAs maintaining ESC state, microRNAs that are responsible for promoting ESC differentiation are less reported. Here, by screening 40 microRNAs pre-selected by their expression patterns and predicted targets in Dgcr8-null ESCs, we identify 14 novel differentiation-associated microRNAs. Among them, miR-27a and miR-24, restrained by c-Myc in ESC, exert their roles of silencing self-renewal through directly targeting several important pluripotency-associated factors, such as Oct4, Foxo1 and Smads. CRISPR/Cas9-mediated knockout of all miR-27/24 in ESCs leads to serious deficiency in ESC differentiation in vitro and in vivo. Moreover, depleting of them in mouse embryonic fibroblasts can evidently promote somatic cell reprogramming. Altogether, our findings uncover the essential role of miR-27 and miR-24 in ESC differentiation and also demonstrate novel microRNAs responsible for ESC differentiation. PMID:25519956

  10. [Functionalization of screen printed electrodes with organic-inorganic hybrid nano-composites for bio-sensing applications].

    PubMed

    Shumyantseva, V V; Bulko, T V; Kuzikov, A V; Khan, R; Archakov, A I

    2015-01-01

    New types of organic-inorganic hybrid nanocomposites based on nanosized Titanium (IV) oxide TiO2 (<100 nm particle size) and carbon nanotubes (CNT, outer diameter 10-15 nm, inner diamentre 2-6 nm, length 0.1-10 µm) and phosphatidilcholine were elaborated for improvement of analytical characteristics of screen printed electrodes. These nanomaterials were employed as an interface for the immobilization of skeletal myoglobin. Electrochemical behavior of myoglobin on such interfaces was characterized with cyclic voltammetry (CV) and square wave voltammetry (SWV). Direct unmediated electron transfer between myoglobin and electrodes modified with organic-inorganic hybrid nanocomposites was registered. TiO2 film and CNT film are biocompartible nanomaterials for myoglobin as was demonstrated with UV-Vis spectra. The midpoint potential of Fe3+/Fe2+ pair of myoglobin corresponded to Е1/2=-0,263 V for CNT film, and Е1/2=-0,468 V for TiO2 nanocomposite (vs. Ag/AgCl reference electrode). PMID:26350738

  11. The 'GALS' locomotor screen.

    PubMed Central

    Doherty, M; Dacre, J; Dieppe, P; Snaith, M

    1992-01-01

    The locomotor system is complex and difficult to examine. A selective clinical process to detect important locomotor abnormalities and functional disability could prove valuable. A screen based on a tested 'minimal' history and examination system is described, together with a simple method of recording. The screen is fast and easy to perform. As well as providing a useful introduction to examination of the locomotor system, the screen includes objective observation of functional movements relevant to activities of daily living. Its inclusion in the undergraduate clerking repertoire could improve junior doctors' awareness and recognition of rheumatic disease and general disability. It could also provide a valuable screening test for use in general practice. Images PMID:1444632

  12. MicroRNA Library-Based Functional Screening Identified Androgen-Sensitive miR-216a as a Player in Bicalutamide Resistance in Prostate Cancer

    PubMed Central

    Miyazaki, Toshiaki; Ikeda, Kazuhiro; Sato, Wataru; Horie-Inoue, Kuniko; Okamoto, Koji; Inoue, Satoshi

    2015-01-01

    Prostate cancer is a major hormone-dependent tumor affecting men, and is often treated by hormone therapy at the primary stages. Despite its initial efficiency, the disease eventually acquires resistance, resulting in the recurrence of castration-resistant prostate cancer. Recent studies suggest that dysregulation of microRNA (miRNA) function is one of the mechanisms underlying hormone therapy resistance. Identification of critical miRNAs involved in endocrine resistance will therefore be important for developing therapeutic targets for prostate cancer. In the present study, we performed an miRNA library screening to identify anti-androgen bicalutamide resistance-related miRNAs in prostate cancer LNCaP cells. Cells were infected with a lentiviral miRNA library and subsequently maintained in media containing either bicalutamide or vehicle for a month. Microarray analysis determined the amounts of individual miRNA precursors and identified 2 retained miRNAs after one-month bicalutamide treatment. Of these, we further characterized miR-216a, because its function in prostate cancer remains unknown. miR-216a could be induced by dihydrotestosterone in LNCaP cells and ectopic expression of miR-216a inhibited bicalutamide-mediated growth suppression of LNCaP cells. Furthermore, a microarray dataset revealed that the expression levels of miR-216a were significantly higher in clinical prostate cancer than in benign samples. These results suggest that functional screening using an miRNA expression library could be useful for identifying novel miRNAs that contribute to bicalutamide resistance in prostate cancer. PMID:26506397

  13. Comprehensive Screening of Gene Function and Networks by DNA Microarray Analysis in Japanese Patients with Idiopathic Portal Hypertension

    PubMed Central

    Kotani, Kohei; Kawabe, Joji; Morikawa, Hiroyasu; Akahoshi, Tomohiko; Hashizume, Makoto; Shiomi, Susumu

    2015-01-01

    The functions of genes involved in idiopathic portal hypertension (IPH) remain unidentified. The present study was undertaken to identify the functions of genes expressed in blood samples from patients with IPH through comprehensive analysis of gene expression using DNA microarrays. The data were compared with data from healthy individuals to explore the functions of genes showing increased or decreased expression in patients with IPH. In cluster analysis, no dominant probe group was shown to differ between patients with IPH and healthy controls. In functional annotation analysis using the Database for Annotation Visualization and Integrated Discovery tool, clusters showing dysfunction in patients with IPH involved gene terms related to the immune system. Analysis using network-based pathways revealed decreased expression of adenosine deaminase, ectonucleoside triphosphate diphosphohydrolase 4, ATP-binding cassette, subfamily C, member 1, transforming growth factor-β, and prostaglandin E receptor 2; increased expression of cytochrome P450, family 4, subfamily F, polypeptide 3, and glutathione peroxidase 3; and abnormalities in the immune system, nucleic acid metabolism, arachidonic acid/leukotriene pathways, and biological processes. These results suggested that IPH involved compromised function of immunocompetent cells and that such dysfunction may be associated with abnormalities in nucleic acid metabolism and arachidonic acid/leukotriene-related synthesis/metabolism. PMID:26549939

  14. High-throughput FACS-based mutant screen identifies a gain-of-function allele of the Fusarium graminearum adenylyl cyclase causing deoxynivalenol over-production.

    PubMed

    Blum, Ailisa; Benfield, Aurélie H; Stiller, Jiri; Kazan, Kemal; Batley, Jacqueline; Gardiner, Donald M

    2016-05-01

    Fusarium head blight and crown rot, caused by the fungal plant pathogen Fusarium graminearum, impose a major threat to global wheat production. During the infection, plants are contaminated with mycotoxins such as deoxynivalenol (DON), which can be toxic for humans and animals. In addition, DON is a major virulence factor during wheat infection. However, it is not fully understood how DON production is regulated in F. graminearum. In order to identify regulators of DON production, a high-throughput mutant screen using Fluorescence Activated Cell Sorting (FACS) of a mutagenised TRI5-GFP reporter strain was established and a mutant over-producing DON under repressive conditions identified. A gain-of-function mutation in the F. graminearum adenylyl cyclase (FAC1), which is a known positive regulator of DON production, was identified as the cause of this phenotype through genome sequencing and segregation analysis. Our results show that the high-throughput mutant screening procedure developed here can be applied for identification of fungal proteins involved in diverse processes. PMID:26932301

  15. FRET Based Quantification and Screening Technology Platform for the Interactions of Leukocyte Function-Associated Antigen-1 (LFA-1) with InterCellular Adhesion Molecule-1 (ICAM-1)

    PubMed Central

    Chakraborty, Sandeep; Núñez, David; Hu, Shih-Yang; Domingo, María Pilar; Pardo, Julian; Karmenyan, Artashes; Chiou, Arthur

    2014-01-01

    The interaction between leukocyte function-associated antigen-1(LFA-1) and intercellular adhesion molecule-1 (ICAM-1) plays a pivotal role in cellular adhesion including the extravasation and inflammatory response of leukocytes, and also in the formation of immunological synapse. However, irregular expressions of LFA-1 or ICAM-1 or both may lead to autoimmune diseases, metastasis cancer, etc. Thus, the LFA-1/ICAM-1 interaction may serve as a potential therapeutic target for the treatment of these diseases. Here, we developed one simple ‘in solution’ steady state fluorescence resonance energy transfer (FRET) technique to obtain the dissociation constant (Kd) of the interaction between LFA-1 and ICAM-1. Moreover, we developed the assay into a screening platform to identify peptides and small molecules that inhibit the LFA-1/ICAM-1 interaction. For the FRET pair, we used Alexa Fluor 488-LFA-1 conjugate as donor and Alexa Fluor 555-human recombinant ICAM-1 (D1-D2-Fc) as acceptor. From our quantitative FRET analysis, the Kd between LFA-1 and D1-D2-Fc was determined to be 17.93±1.34 nM. Both the Kd determination and screening assay were performed in a 96-well plate platform, providing the opportunity to develop it into a high-throughput assay. This is the first reported work which applies FRET based technique to determine Kd as well as classifying inhibitors of the LFA-1/ICAM-1 interaction. PMID:25032811

  16. Analysis of mammalian gene function through broad-based phenotypic screens across a consortium of mouse clinics.

    PubMed

    Hrabě de Angelis, Martin; Nicholson, George; Selloum, Mohammed; White, Jacqueline K; Morgan, Hugh; Ramirez-Solis, Ramiro; Sorg, Tania; Wells, Sara; Fuchs, Helmut; Fray, Martin; Adams, David J; Adams, Niels C; Adler, Thure; Aguilar-Pimentel, Antonio; Ali-Hadji, Dalila; Amann, Gregory; André, Philippe; Atkins, Sarah; Auburtin, Aurelie; Ayadi, Abdel; Becker, Julien; Becker, Lore; Bedu, Elodie; Bekeredjian, Raffi; Birling, Marie-Christine; Blake, Andrew; Bottomley, Joanna; Bowl, Michael R; Brault, Véronique; Busch, Dirk H; Bussell, James N; Calzada-Wack, Julia; Cater, Heather; Champy, Marie-France; Charles, Philippe; Chevalier, Claire; Chiani, Francesco; Codner, Gemma F; Combe, Roy; Cox, Roger; Dalloneau, Emilie; Dierich, André; Di Fenza, Armida; Doe, Brendan; Duchon, Arnaud; Eickelberg, Oliver; Esapa, Chris T; Fertak, Lahcen El; Feigel, Tanja; Emelyanova, Irina; Estabel, Jeanne; Favor, Jack; Flenniken, Ann; Gambadoro, Alessia; Garrett, Lilian; Gates, Hilary; Gerdin, Anna-Karin; Gkoutos, George; Greenaway, Simon; Glasl, Lisa; Goetz, Patrice; Da Cruz, Isabelle Goncalves; Götz, Alexander; Graw, Jochen; Guimond, Alain; Hans, Wolfgang; Hicks, Geoff; Hölter, Sabine M; Höfler, Heinz; Hancock, John M; Hoehndorf, Robert; Hough, Tertius; Houghton, Richard; Hurt, Anja; Ivandic, Boris; Jacobs, Hughes; Jacquot, Sylvie; Jones, Nora; Karp, Natasha A; Katus, Hugo A; Kitchen, Sharon; Klein-Rodewald, Tanja; Klingenspor, Martin; Klopstock, Thomas; Lalanne, Valerie; Leblanc, Sophie; Lengger, Christoph; le Marchand, Elise; Ludwig, Tonia; Lux, Aline; McKerlie, Colin; Maier, Holger; Mandel, Jean-Louis; Marschall, Susan; Mark, Manuel; Melvin, David G; Meziane, Hamid; Micklich, Kateryna; Mittelhauser, Christophe; Monassier, Laurent; Moulaert, David; Muller, Stéphanie; Naton, Beatrix; Neff, Frauke; Nolan, Patrick M; Nutter, Lauryl M J; Ollert, Markus; Pavlovic, Guillaume; Pellegata, Natalia S; Peter, Emilie; Petit-Demoulière, Benoit; Pickard, Amanda; Podrini, Christine; Potter, Paul; Pouilly, Laurent; Puk, Oliver; Richardson, David; Rousseau, Stephane; Quintanilla-Fend, Leticia; Quwailid, Mohamed M; Racz, Ildiko; Rathkolb, Birgit; Riet, Fabrice; Rossant, Janet; Roux, Michel; Rozman, Jan; Ryder, Edward; Salisbury, Jennifer; Santos, Luis; Schäble, Karl-Heinz; Schiller, Evelyn; Schrewe, Anja; Schulz, Holger; Steinkamp, Ralf; Simon, Michelle; Stewart, Michelle; Stöger, Claudia; Stöger, Tobias; Sun, Minxuan; Sunter, David; Teboul, Lydia; Tilly, Isabelle; Tocchini-Valentini, Glauco P; Tost, Monica; Treise, Irina; Vasseur, Laurent; Velot, Emilie; Vogt-Weisenhorn, Daniela; Wagner, Christelle; Walling, Alison; Wattenhofer-Donze, Marie; Weber, Bruno; Wendling, Olivia; Westerberg, Henrik; Willershäuser, Monja; Wolf, Eckhard; Wolter, Anne; Wood, Joe; Wurst, Wolfgang; Yildirim, Ali Önder; Zeh, Ramona; Zimmer, Andreas; Zimprich, Annemarie; Holmes, Chris; Steel, Karen P; Herault, Yann; Gailus-Durner, Valérie; Mallon, Ann-Marie; Brown, Steve D M

    2015-09-01

    The function of the majority of genes in the mouse and human genomes remains unknown. The mouse embryonic stem cell knockout resource provides a basis for the characterization of relationships between genes and phenotypes. The EUMODIC consortium developed and validated robust methodologies for the broad-based phenotyping of knockouts through a pipeline comprising 20 disease-oriented platforms. We developed new statistical methods for pipeline design and data analysis aimed at detecting reproducible phenotypes with high power. We acquired phenotype data from 449 mutant alleles, representing 320 unique genes, of which half had no previous functional annotation. We captured data from over 27,000 mice, finding that 83% of the mutant lines are phenodeviant, with 65% demonstrating pleiotropy. Surprisingly, we found significant differences in phenotype annotation according to zygosity. New phenotypes were uncovered for many genes with previously unknown function, providing a powerful basis for hypothesis generation and further investigation in diverse systems. PMID:26214591

  17. HIGH INFORMATION CONTENT TOXICITY SCREENING USING MOUSE AND HUMAN STEM CELL MODELS OF ENDOCRINE DEVELOPMENT AND FUNCTION

    EPA Science Inventory

    The project will result in the rapid assessment of chemicals for adverse effects on the development of gametes, adipocytes, and islet B-cells; and on the adipocyte and B-cell endocrine signaling function in human and murine embryonic stem cells. Based on the data, hierarchical...

  18. D-A-D-type narrow-bandgap small-molecule photovoltaic donors: pre-synthesis virtual screening using density functional theory.

    PubMed

    Gim, Yeongrok; Kim, Daekyeom; Kyeong, Minkyu; Byun, Seunghwan; Park, Yuri; Kwon, Sooncheol; Kim, Heejoo; Hong, Sukwon; Lansac, Yves; Jang, Yun Hee

    2016-06-01

    A new series of D-A-D-type small-molecule photovoltaic donors are designed and virtually screened before synthesis using time-dependent density functional theory calculations carefully validated against various polymeric and molecular donors. In this series of new design, benzodithiophene is kept as D to achieve the optimum highest-occupied molecular orbital energy level, while thienopyrroledione is initially chosen as A but later replaced by difluorinated benzodiathiazole or its selenide derivative to achieve the optimum band gap. The D-A-D core is end-capped by pyridone units which could not only enhance their self-assembly via hydrogen bonds but also play a role as an acceptor (A') to form an extended A'-D-A-D-A' small-molecule donor. PMID:27193426

  19. Noise-driven diamagnetic susceptibility of impurity doped quantum dots: Role of anisotropy, position-dependent effective mass and position-dependent dielectric screening function

    NASA Astrophysics Data System (ADS)

    Bera, Aindrila; Saha, Surajit; Ganguly, Jayanta; Ghosh, Manas

    2016-08-01

    We explore Diamagnetic susceptibility (DMS) of impurity doped quantum dot (QD) in presence of Gaussian white noise introduced to the system additively and multiplicatively. In view of this profiles of DMS have been pursued with variations of geometrical anisotropy and dopant location. We have invoked position-dependent effective mass (PDEM) and position-dependent dielectric screening function (PDDSF) of the system. Presence of noise sometimes suppresses and sometimes amplifies DMS from that of noise-free condition and the extent of suppression/amplification depends on mode of application of noise. It is important to mention that the said suppression/amplification exhibits subtle dependence on use of PDEM, PDDSF and geometrical anisotropy. The study reveals that DMS, or more fundamentally, the effective confinement of LDSS, can be tuned by appropriate mingling of geometrical anisotropy/effective mass/dielectric constant of the system with noise and also on the pathway of application of latter.

  20. Groundwater Screen

    1993-11-09

    GWSCREEN was developed for assessment of the groundwater pathway from leaching of radioactive and non radioactive substances from surface or buried sources and release to percolation ponds. The code calculates the limiting soil concentration or effluent release concentration such that, after leaching and transport to the aquifer, regulatory contaminant levels in groundwater are not exceeded. The code uses a mass conservation approach to model three processes: Contaminant release from a source volume, contaminant transport inmore » the unsaturated zone, and contaminant transport in the saturated zone. The source model considers the sorptive properties and solubility of the contaminant. Transport in the unsaturated zone is described by a plug flow model. Transport in the saturated zone is calculated with a semi-analytical solution to the advection dispersion equation in groundwater. Concentration as a function of time at a user specified receptor point and maximum concentration averaged over the exposure interval are also calculated. In addition, the code calculates transport and impacts of radioactive progeny. Input to GWSCREEN is through one, free format ASCII file. This code was designed for assessment and screening of the groundwater pathway when field data is limited. It was not intended to be a predictive tool.« less

  1. Decline of Pulmonary Function Is Associated With the Presence of Rheumatoid Factor in Korean Health Screening Subjects Without Clinically Apparent Lung Disease

    PubMed Central

    Hwang, Jiwon; Song, Jae-Uk; Ahn, Joong Kyong

    2016-01-01

    Abstract Although higher-than-normal levels of rheumatoid factor (RF) are often observed in subjects without specific medical problems, little is known about the influence of RF on pulmonary function in health screening subjects. This study aimed to determine the association between the presence of RF and decreased pulmonary function in Korean health screening subjects without any history of joint disease or clinically apparent lung disease. A total of 115,641 study subjects (age range, 18–88 years) participated in the health checkup program. We excluded subjects who did not have pulmonary function test, as well as those with abnormal chest radiographs. Subjects with medical history of arthritis including rheumatoid arthritis, and lung disease based on the self-reported questionnaire. Final analysis was performed on 94,438 Koreans (41,261 women). RF-positive subjects had a lower forced vital capacity (FVC) predicted value and forced expiratory volume in 1 s (FEV1) predicted value than RF-negative subjects (82.8 ± 11.5% vs 83.8 ± 11.4% for FVC% predicted and 83.5 ± 13.0% vs 85.1 ± 12.9% for FEV1% predicted, P < 0.001 for both). RF positivity was significantly associated with the decline of FEV1% predicted regardless of smoking history (adjusted odds ratio [OR] = 1.289 [95% confidence interval [CI] 1.163–1.429], P < 0.001 for nonsmokers and adjusted OR = 1.138 [95% CI 1.004–1.289], P < 0.001 for smokers) while the decline of FVC% predicted only in nonsmokers (adjusted OR = 1.251 [95% CI 1.133–1.382], P < 0.001). Our results suggest that the presence of RF could impact pulmonary function in apparently healthy subjects. A follow-up study to investigate serial changes in pulmonary function may reveal the actual influence of raised RF titers. PMID:27175698

  2. Decline of Pulmonary Function Is Associated With the Presence of Rheumatoid Factor in Korean Health Screening Subjects Without Clinically Apparent Lung Disease: A Cross-Sectional Study.

    PubMed

    Hwang, Jiwon; Song, Jae-Uk; Ahn, Joong Kyong

    2016-05-01

    Although higher-than-normal levels of rheumatoid factor (RF) are often observed in subjects without specific medical problems, little is known about the influence of RF on pulmonary function in health screening subjects. This study aimed to determine the association between the presence of RF and decreased pulmonary function in Korean health screening subjects without any history of joint disease or clinically apparent lung disease.A total of 115,641 study subjects (age range, 18-88 years) participated in the health checkup program. We excluded subjects who did not have pulmonary function test, as well as those with abnormal chest radiographs. Subjects with medical history of arthritis including rheumatoid arthritis, and lung disease based on the self-reported questionnaire. Final analysis was performed on 94,438 Koreans (41,261 women).RF-positive subjects had a lower forced vital capacity (FVC) predicted value and forced expiratory volume in 1 s (FEV1) predicted value than RF-negative subjects (82.8 ± 11.5% vs 83.8 ± 11.4% for FVC% predicted and 83.5 ± 13.0% vs 85.1 ± 12.9% for FEV1% predicted, P < 0.001 for both). RF positivity was significantly associated with the decline of FEV1% predicted regardless of smoking history (adjusted odds ratio [OR] = 1.289 [95% confidence interval [CI] 1.163-1.429], P < 0.001 for nonsmokers and adjusted OR = 1.138 [95% CI 1.004-1.289], P < 0.001 for smokers) while the decline of FVC% predicted only in nonsmokers (adjusted OR = 1.251 [95% CI 1.133-1.382], P < 0.001). Our results suggest that the presence of RF could impact pulmonary function in apparently healthy subjects. A follow-up study to investigate serial changes in pulmonary function may reveal the actual influence of raised RF titers. PMID:27175698

  3. Wicking of liquids in screens

    NASA Technical Reports Server (NTRS)

    Symons, E. P.

    1974-01-01

    An investigation was conducted to determine the magnitude of the wicking rates of liquids in various screens. Evaluation of the parameters characterizing the wicking process resulted in the development of an expression which defined the wicking velocity in terms of screen and system geometry, liquid properties, and gravitational effects. Experiment data obtained both in normal gravity and in weightlessness demonstrated that the model successfully predicted the functional relation of the liquid properties and the distance from the liquid source to the wicking velocity. Because the pore geometry in the screens was complex, several screen geometric parameters were lumped into a single constant which was determined experimentally for each screen.

  4. Genetic screening

    PubMed Central

    Andermann, Anne; Blancquaert, Ingeborg

    2010-01-01

    Abstract OBJECTIVE To provide a primer for primary care professionals who are increasingly called upon to discuss the growing number of genetic screening services available and to help patients make informed decisions about whether to participate in genetic screening, how to interpret results, and which interventions are most appropriate. QUALITY OF EVIDENCE As part of a larger research program, a wide literature relating to genetic screening was reviewed. PubMed and Internet searches were conducted using broad search terms. Effort was also made to identify the gray literature. MAIN MESSAGE Genetic screening is a type of public health program that is systematically offered to a specified population of asymptomatic individuals with the aim of providing those identified as high risk with prevention, early treatment, or reproductive options. Ensuring an added benefit from screening, as compared with standard clinical care, and preventing unintended harms, such as undue anxiety or stigmatization, depends on the design and implementation of screening programs, including the recruitment methods, education and counseling provided, timing of screening, predictive value of tests, interventions available, and presence of oversight mechanisms and safeguards. There is therefore growing apprehension that economic interests might lead to a market-driven approach to introducing and expanding screening before program effectiveness, acceptability, and feasibility have been demonstrated. As with any medical intervention, there is a moral imperative for genetic screening to do more good than harm, not only from the perspective of individuals and families, but also for the target population and society as a whole. CONCLUSION Primary care professionals have an important role to play in helping their patients navigate the rapidly changing terrain of genetic screening services by informing them about the benefits and risks of new genetic and genomic technologies and empowering them to

  5. Prevalence of disability in a composite ≥75 year-old population in Spain: A screening survey based on the International Classification of Functioning

    PubMed Central

    2011-01-01

    Background The prevalence and predictors of functional status and disability of elderly people have been studied in several European countries including Spain. However, there has been no population-based study incorporating the International Classification of Functioning, Disability and Health (ICF) framework as the basis for assessing disability. The present study reports prevalence rates for mild, moderate, and severe/extreme disability by the domains of activities and participation of the ICF. Methods Nine populations surveyed in previous prevalence studies contributed probabilistic and geographically defined samples in June 2005. The study sample was composed of 503 subjects aged ≥75 years. We implemented a two-phase screening design using the MMSE and the World Health Organization-Disability Assessment Schedule 2nd edition (WHO-DAS II, 12 items) as cognitive and disability screening tools, respectively. Participants scoring within the positive range of the disability screening were administered the full WHO-DAS II (36 items; score range: 0-100) assessing the following areas: Understanding and communication, Getting along with people, Life activities, Getting around, Participation in society, and Self-care. Each disability area assessed by WHO-DAS II (36 items) was reported according to the ICF severity ranges (No problem, 0-4; Mild disability, 5-24; Moderate disability, 25-49; Severe/Extreme disability, 50-100). Results The age-adjusted disability prevalence figures were: 39.17 ± 2.18%, 15.31 ± 1.61%, and 10.14 ± 1.35% for mild, moderate, and severe/extreme disability, respectively. Severe and extreme disability prevalence in mobility and life activities was three times higher than the average, and highest among women. Sex variations were minimal, although life activities for women of 85 years and over had more severe/extreme disability as compared to men (OR = 5.15 95% CI 3.19-8.32). Conclusions Disability is highly prevalent among the Spanish elderly

  6. Screening of MAMLD1 Mutations in 70 Children with 46,XY DSD: Identification and Functional Analysis of Two New Mutations

    PubMed Central

    Kalfa, Nicolas; Fukami, Maki; Philibert, Pascal; Audran, Francoise; Pienkowski, Catherine; Weill, Jacques; Pinto, Graziella; Manouvrier, Sylvie; Polak, Michel; Ogata, Totsumo; Sultan, Charles

    2012-01-01

    More than 50% of children with severe 46,XY disorders of sex development (DSD) do not have a definitive etiological diagnosis. Besides gonadal dysgenesis, defects in androgen biosynthesis, and abnormalities in androgen sensitivity, the Mastermind-like domain containing 1 (MAMLD1) gene, which was identified as critical for the development of male genitalia, may be implicated. The present study investigated whether MAMLD1 is implicated in cases of severe 46,XY DSD and whether routine sequencing of MAMLD1 should be performed in these patients. Seventy children with severe non-syndromic 46,XY DSD of unknown etiology were studied. One hundred and fifty healthy individuals were included as controls. Direct sequencing of the MAMLD1, AR, SRD5A2 and NR5A1 genes was performed. The transactivation function of the variant MAMLD1 proteins was quantified by the luciferase method. Two new mutations were identified: p.S143X (c.428C>A) in a patient with scrotal hypospadias with microphallus and p.P384L (c.1151C>T) in a patient with penile hypospadias with microphallus. The in vitro functional study confirmed no residual transactivating function of the p.S143X mutant and a significantly reduced transactivation function of the p.P384L protein (p = 0.0032). The p.P359S, p.N662S and p.H347Q variants are also reported with particularly high frequency of the p.359T- p.662G haplotype in the DSD patients. Severe undervirilization in XY newborns can reveal mutations of MAMLD1. MAMLD1 should be routinely sequenced in these patients with otherwise normal AR, SRD5A2 and NR5A1genes. PMID:22479329

  7. Density functional theory screening of gas-treatment strategies for stabilization of high energy-density lithium metal anodes

    NASA Astrophysics Data System (ADS)

    Koch, Stephan L.; Morgan, Benjamin J.; Passerini, Stefano; Teobaldi, Gilberto

    2015-11-01

    To explore the potential of molecular gas treatment of freshly cut lithium foils in non-electrolyte-based passivation of high-energy-density Li anodes, density functional theory (DFT) has been used to study the decomposition of molecular gases on metallic lithium surfaces. By combining DFT geometry optimization and Molecular Dynamics, the effects of atmospheric (N2, O2, CO2) and hazardous (F2, SO2) gas decomposition on Li(bcc) (100), (110), and (111) surfaces on relative surface energies, work functions, and emerging electronic and elastic properties are investigated. The simulations suggest that exposure to different molecular gases can be used to induce and control reconstructions of the metal Li surface and substantial changes (up to over 1 eV) in the work function of the passivated system. Contrary to the other considered gases, which form metallic adlayers, SO2 treatment emerges as the most effective in creating an insulating passivation layer for dosages ≤1 mono-layer. The substantial Li → adsorbate charge transfer and adlayer relaxation produce marked elastic stiffening of the interface, with the smallest change shown by nitrogen-treated adlayers.

  8. Loss-of-function screening to identify miRNAs involved in senescence: tumor suppressor activity of miRNA-335 and its new target CARF.

    PubMed

    Yu, Yue; Gao, Ran; Kaul, Zeenia; Li, Ling; Kato, Yoshio; Zhang, Zhenya; Groden, Joanna; Kaul, Sunil C; Wadhwa, Renu

    2016-01-01

    Significance of microRNAs (miRs), small non-coding molecules, has been implicated in a variety of biological processes. Here, we recruited retroviral insertional mutagenesis to obtain induction of an arbitrary noncoding RNAs, and coupled it with a cell based loss-of-function (5-Aza-2'-deoxycytidine (5Aza-dC)-induced senescence bypass) screening system. Cells that escaped 5-Aza-dC-induced senescence were subjected to miR-microarray analysis with respect to the untreated control. We identified miR-335 as one of the upregulated miRs. In order to characterize the functional significance, we overexpressed miR-335 in human cancer cells and found that it caused growth suppression. We demonstrate that the latter accounted for inhibition of 5-Aza-dC incorporation into the cell genome, enabling them to escape from induction of senescence. We also report that CARF (Collaborator of ARF) is a new target of miR-335 that regulates its growth suppressor function by complex crosstalk with other proteins including p16(INK4A), pRB, HDM2 and p21(WAF1). PMID:27457128

  9. Including screening in van der Waals corrected density functional theory calculations: The case of atoms and small molecules physisorbed on graphene

    SciTech Connect

    Silvestrelli, Pier Luigi; Ambrosetti, Alberto

    2014-03-28

    The Density Functional Theory (DFT)/van der Waals-Quantum Harmonic Oscillator-Wannier function (vdW-QHO-WF) method, recently developed to include the vdW interactions in approximated DFT by combining the quantum harmonic oscillator model with the maximally localized Wannier function technique, is applied to the cases of atoms and small molecules (X=Ar, CO, H{sub 2}, H{sub 2}O) weakly interacting with benzene and with the ideal planar graphene surface. Comparison is also presented with the results obtained by other DFT vdW-corrected schemes, including PBE+D, vdW-DF, vdW-DF2, rVV10, and by the simpler Local Density Approximation (LDA) and semilocal generalized gradient approximation approaches. While for the X-benzene systems all the considered vdW-corrected schemes perform reasonably well, it turns out that an accurate description of the X-graphene interaction requires a proper treatment of many-body contributions and of short-range screening effects, as demonstrated by adopting an improved version of the DFT/vdW-QHO-WF method. We also comment on the widespread attitude of relying on LDA to get a rough description of weakly interacting systems.

  10. Loss-of-function screening to identify miRNAs involved in senescence: tumor suppressor activity of miRNA-335 and its new target CARF

    PubMed Central

    Yu, Yue; Gao, Ran; Kaul, Zeenia; Li, Ling; Kato, Yoshio; Zhang, Zhenya; Groden, Joanna; Kaul, Sunil C; Wadhwa, Renu

    2016-01-01

    Significance of microRNAs (miRs), small non-coding molecules, has been implicated in a variety of biological processes. Here, we recruited retroviral insertional mutagenesis to obtain induction of an arbitrary noncoding RNAs, and coupled it with a cell based loss-of-function (5-Aza-2′-deoxycytidine (5Aza-dC)-induced senescence bypass) screening system. Cells that escaped 5-Aza-dC-induced senescence were subjected to miR-microarray analysis with respect to the untreated control. We identified miR-335 as one of the upregulated miRs. In order to characterize the functional significance, we overexpressed miR-335 in human cancer cells and found that it caused growth suppression. We demonstrate that the latter accounted for inhibition of 5-Aza-dC incorporation into the cell genome, enabling them to escape from induction of senescence. We also report that CARF (Collaborator of ARF) is a new target of miR-335 that regulates its growth suppressor function by complex crosstalk with other proteins including p16INK4A, pRB, HDM2 and p21WAF1. PMID:27457128

  11. Including screening in van der Waals corrected density functional theory calculations: the case of atoms and small molecules physisorbed on graphene.

    PubMed

    Silvestrelli, Pier Luigi; Ambrosetti, Alberto

    2014-03-28

    The Density Functional Theory (DFT)/van der Waals-Quantum Harmonic Oscillator-Wannier function (vdW-QHO-WF) method, recently developed to include the vdW interactions in approximated DFT by combining the quantum harmonic oscillator model with the maximally localized Wannier function technique, is applied to the cases of atoms and small molecules (X=Ar, CO, H2, H2O) weakly interacting with benzene and with the ideal planar graphene surface. Comparison is also presented with the results obtained by other DFT vdW-corrected schemes, including PBE+D, vdW-DF, vdW-DF2, rVV10, and by the simpler Local Density Approximation (LDA) and semilocal generalized gradient approximation approaches. While for the X-benzene systems all the considered vdW-corrected schemes perform reasonably well, it turns out that an accurate description of the X-graphene interaction requires a proper treatment of many-body contributions and of short-range screening effects, as demonstrated by adopting an improved version of the DFT/vdW-QHO-WF method. We also comment on the widespread attitude of relying on LDA to get a rough description of weakly interacting systems. PMID:24697424

  12. Screening for Functional Non-coding Genetic Variants Using Electrophoretic Mobility Shift Assay (EMSA) and DNA-affinity Precipitation Assay (DAPA).

    PubMed

    Miller, Daniel E; Patel, Zubin H; Lu, Xiaoming; Lynch, Arthur T; Weirauch, Matthew T; Kottyan, Leah C

    2016-01-01

    Population and family-based genetic studies typically result in the identification of genetic variants that are statistically associated with a clinical disease or phenotype. For many diseases and traits, most variants are non-coding, and are thus likely to act by impacting subtle, comparatively hard to predict mechanisms controlling gene expression. Here, we describe a general strategic approach to prioritize non-coding variants, and screen them for their function. This approach involves computational prioritization using functional genomic databases followed by experimental analysis of differential binding of transcription factors (TFs) to risk and non-risk alleles. For both electrophoretic mobility shift assay (EMSA) and DNA affinity precipitation assay (DAPA) analysis of genetic variants, a synthetic DNA oligonucleotide (oligo) is used to identify factors in the nuclear lysate of disease or phenotype-relevant cells. For EMSA, the oligonucleotides with or without bound nuclear factors (often TFs) are analyzed by non-denaturing electrophoresis on a tris-borate-EDTA (TBE) polyacrylamide gel. For DAPA, the oligonucleotides are bound to a magnetic column and the nuclear factors that specifically bind the DNA sequence are eluted and analyzed through mass spectrometry or with a reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blot analysis. This general approach can be widely used to study the function of non-coding genetic variants associated with any disease, trait, or phenotype. PMID:27585267

  13. Co-activation based parcellation of the human frontal pole.

    PubMed

    Ray, K L; Zald, D H; Bludau, S; Riedel, M C; Bzdok, D; Yanes, J; Falcone, K E; Amunts, K; Fox, P T; Eickhoff, S B; Laird, A R

    2015-12-01

    Historically, the human frontal pole (FP) has been considered as a single architectonic area. Brodmann's area 10 is located in the frontal lobe with known contributions in the execution of various higher order cognitive processes. However, recent cytoarchitectural studies of the FP in humans have shown that this portion of cortex contains two distinct cytoarchitectonic regions. Since architectonic differences are accompanied by differential connectivity and functions, the frontal pole qualifies as a candidate region for exploratory parcellation into functionally discrete sub-regions. We investigated whether this functional heterogeneity is reflected in distinct segregations within cytoarchitectonically defined FP-areas using meta-analytic co-activation based parcellation (CBP). The CBP method examined the co-activation patterns of all voxels within the FP as reported in functional neuroimaging studies archived in the BrainMap database. Voxels within the FP were subsequently clustered into sub-regions based on the similarity of their respective meta-analytically derived co-activation maps. Performing this CBP analysis on the FP via k-means clustering produced a distinct 3-cluster parcellation for each hemisphere corresponding to previously identified cytoarchitectural differences. Post-hoc functional characterization of clusters via BrainMap metadata revealed that lateral regions of the FP mapped to memory and emotion domains, while the dorso- and ventromedial clusters were associated broadly with emotion and social cognition processes. Furthermore, the dorsomedial regions contain an emphasis on theory of mind and affective related paradigms whereas ventromedial regions couple with reward tasks. Results from this study support previous segregations of the FP and provide meta-analytic contributions to the ongoing discussion of elucidating functional architecture within human FP. PMID:26254112

  14. Growth, differentiation capacity, and function of mesenchymal stem cells expanded in serum-free medium developed via combinatorial screening.

    PubMed

    Crapnell, Kirsten; Blaesius, Rainer; Hastings, Abel; Lennon, Donald P; Caplan, Arnold I; Bruder, Scott P

    2013-06-10

    The presence of serum in cell culture medium presents an obstacle to safe and efficient production of hMSCs for therapeutic purposes. Availability of defined medium will be crucial to elucidating the mechanism of action of hMSCs in many indications as well as a prerequisite to consistently produce cells with predictable performance characteristics. Using a bioinformatics driven approach, which we call the BD Discovery Platform, we have developed a novel serum-free medium that supports highly efficient growth while maintaining the surface markers and functional characteristics defining hMSCs. In a comparison with serum-containing and other commercially available serum-free formulations, all conditions led to expansion of cells that meet the minimal criteria for hMSCs as set by the International Society for Cellular Therapy (ISCT). However, differences in growth characteristics and gene expression patterns suggest that expansion in serum-free growth conditions can provide greater yields in a shorter time. The mRNA expression profile observed in cells grown without serum suggests upregulation of several genes implicated in hMSC function as well as downregulation of the proinflammatory cytokine IL6. PMID:23597555

  15. RET and EDNRB mutation screening in patients with Hirschsprung disease: Functional studies and its implications for genetic counseling.

    PubMed

    Widowati, Titis; Melhem, Shamiram; Patria, Suryono Y; de Graaf, Bianca M; Sinke, Richard J; Viel, Martijn; Dijkhuis, Jos; Sadewa, Ahmad H; Purwohardjono, Rochadi; Soenarto, Yati; Hofstra, Robert Mw; Sribudiani, Yunia

    2016-06-01

    Hirschsprung disease (HSCR) is a major cause of chronic constipation in children. HSCR can be caused by germline mutations in RET and EDNRB. Defining causality of the mutations identified is difficult and almost exclusively based on in silico predictions. Therefore, the reported frequency of pathogenic mutations might be overestimated. We combined mutation analysis with functional assays to determine the frequencies of proven pathogenic RET and EDNRB mutations in HSCR. We sequenced RET and EDNRB in 57 HSCR patients. The identified RET-coding variants were introduced into RET constructs and these were transfected into HEK293 cells to determine RET phosphorylation and activation via ERK. An exon trap experiment was performed to check a possible splice-site mutation. We identified eight rare RET-coding variants, one possible splice-site variant, but no rare EDNRB variants. Western blotting showed that three coding variants p.(Pr270Leu), p.(Ala756Val) and p.(Tyr1062Cys) resulted in lower activation of RET. Moreover, only two RET variants (p.(Ala756Val) and p.(Tyr1062Cys)) resulted in reduced ERK activation. Splice-site assays on c.1880-11A>G could not confirm its pathogenicity. Our data suggest that indeed almost half of the identified rare variants are proven pathogenic and that, hence, functional studies are essential for proper genetic counseling. PMID:26395553

  16. Neonatal Screening Part 2: Neonatal Screening in Canada

    PubMed Central

    Morris, Brian A.P.

    1990-01-01

    Neonatal screening is a widely accepted, cost-effective method for early detection of various inborn errors of metabolism. This series of three articles examines different aspects of neonatal screening. In the first article, the author discussed general principles of screening and its function in the spectrum of diagnostic techniques for genetic disease. In this, the second article, the author reviews the history and current practices of neonatal screening in Canada. The individual diseases for which screening is used and particular points of interest for each of these diseases are briefly described. The author also outlines the benefits of screening and treatment. In the final article of this series, the author will examine controversial topics that represent the possible future of screening. PMID:21234041

  17. High-throughput functional screening of steroid substrates with wild-type and chimeric P450 enzymes.

    PubMed

    Urban, Philippe; Truan, Gilles; Pompon, Denis

    2014-01-01

    The promiscuity of a collection of enzymes consisting of 31 wild-type and synthetic variants of CYP1A enzymes was evaluated using a series of 14 steroids and 2 steroid-like chemicals, namely, nootkatone, a terpenoid, and mifepristone, a drug. For each enzyme-substrate couple, the initial steady-state velocity of metabolite formation was determined at a substrate saturating concentration. For that, a high-throughput approach was designed involving automatized incubations in 96-well microplate with sixteen 6-point kinetics per microplate and data acquisition using LC/MS system accepting 96-well microplate for injections. The resulting dataset was used for multivariate statistics aimed at sorting out the correlations existing between tested enzyme variants and ability to metabolize steroid substrates. Functional classifications of both CYP1A enzyme variants and steroid substrate structures were obtained allowing the delineation of global structural features for both substrate recognition and regioselectivity of oxidation. PMID:25243177

  18. High-Throughput Functional Screening of Steroid Substrates with Wild-Type and Chimeric P450 Enzymes

    PubMed Central

    Truan, Gilles; Pompon, Denis

    2014-01-01

    The promiscuity of a collection of enzymes consisting of 31 wild-type and synthetic variants of CYP1A enzymes was evaluated using a series of 14 steroids and 2 steroid-like chemicals, namely, nootkatone, a terpenoid, and mifepristone, a drug. For each enzyme-substrate couple, the initial steady-state velocity of metabolite formation was determined at a substrate saturating concentration. For that, a high-throughput approach was designed involving automatized incubations in 96-well microplate with sixteen 6-point kinetics per microplate and data acquisition using LC/MS system accepting 96-well microplate for injections. The resulting dataset was used for multivariate statistics aimed at sorting out the correlations existing between tested enzyme variants and ability to metabolize steroid substrates. Functional classifications of both CYP1A enzyme variants and steroid substrate structures were obtained allowing the delineation of global structural features for both substrate recognition and regioselectivity of oxidation. PMID:25243177

  19. Get Screened

    MedlinePlus

    ... Get Ready 3 of 4 sections Take Action: Cost and Insurance What about cost? Depending on your insurance plan, you may be able to get screening tests at no cost to you. Most insurance plans, including Medicaid and ...

  20. TORCH screen

    MedlinePlus

    ... different infections in a newborn. TORCH stands for toxoplasmosis , rubella , cytomegalovirus, herpes simplex, and HIV, but it ... used to screen infants for infections such as toxoplasmosis, cytomegalovirus, herpes simplex, syphilis and others. These infections ...

  1. Developmental Screening

    MedlinePlus

    Learn More about Your Child’s Development: Developmental Monitoring and Screening Taking a first step, waving “bye-bye,” and pointing to something interesting are all developmental milestones, ...

  2. Hypertension screening

    NASA Technical Reports Server (NTRS)

    Foulke, J. M.

    1975-01-01

    An attempt was made to measure the response to an announcement of hypertension screening at the Goddard Space Center, to compare the results to those of previous statistics. Education and patient awareness of the problem were stressed.

  3. TORCH Screen

    MedlinePlus

    ... different infections in a newborn. TORCH stands for toxoplasmosis , rubella , cytomegalovirus, herpes simplex, and HIV, but it ... used to screen infants for infections such as toxoplasmosis, cytomegalovirus, herpes simplex, syphilis and others. These infections ...

  4. Newborn Screening

    MedlinePlus

    ... Pulse Oximetry Screening for CCHDs Sickle Cell Disease Laboratory SCID Quality Assurance Training and Resources For Lab Professionals Data and Reports Laboratory Reports National Birth Defects Prevention Network (NBDPN) Resources ...

  5. Genome-wide functional genetic screen with the anticancer agent AMPI-109 identifies PRL-3 as an oncogenic driver in triple-negative breast cancers.

    PubMed

    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. PMID:26909599

  6. A multi-parameter in vitro screen in human stem cell-derived cardiomyocytes identifies ponatinib-induced structural and functional cardiac toxicity.

    PubMed

    Talbert, Dominique R; Doherty, Kimberly R; Trusk, Patricia B; Moran, Diarmuid M; Shell, Scott A; Bacus, Sarah

    2015-01-01

    Ponatinib, a multi-targeted TKI and potent pan-ABL inhibitor, approved for the treatment of Ph + ALL and CML, was temporarily withdrawn from the U.S. market due to severe vascular adverse events. Cardiac-specific toxicities including myocardial infarction, severe congestive heart failure, and cardiac arrhythmias have also been shown with ponatinib. Targeted oncology agents such as ponatinib have transformed cancer treatment but often induce toxicity due to inhibition of survival pathways shared by both cancer and cardiac cells. These toxicities are often missed by the standard preclinical toxicity assessment methods, which include human Ether-à-go-go-related gene (hERG) and animal toxicity testing. In this study, we show that a multiparameter in vitro toxicity screening approach using human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) accurately predicted the cardiac toxicity potential of ponatinib. This in vitro model evaluated ponatinib's effect on the overall cell health, mitochondrial stress, and function of hiPSC-CM and also provided mechanistic insight into the signaling pathways and cellular structures altered with treatment. We show here that ponatinib rapidly inhibits prosurvival signaling pathways, induces structural cardiac toxicity (as shown by actin cytoskeleton damage, mitochondrial stress, cell death, and troponin secretion), and disrupts cardiac cell beating. Most of these effects occurred at doses between 10× and 50× ponatinib's Cmax, a dose range shown to be relevant for accurate prediction of in vivo toxicity. Together these studies show that a comprehensive in vitro screening tool in a more relevant human cardiac cell model can improve the detection of cardiac toxicity with targeted oncology agents such as ponatinib. PMID:25304212

  7. Genome-wide functional genetic screen with the anticancer agent AMPI-109 identifies PRL-3 as an oncogenic driver in triple-negative breast cancers

    PubMed Central

    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.

    2016-01-01

    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. PMID:26909599

  8. Gain-of-function microRNA screens identify miR-193a regulating proliferation and apoptosis in epithelial ovarian cancer cells

    PubMed Central

    NAKANO, HARUO; YAMADA, YOJI; MIYAZAWA, TATSUYA; YOSHIDA, TETSUO

    2013-01-01

    MicroRNAs (miRNAs) are a small class of non-coding RNAs that negatively regulate gene expression, and are considered as new therapeutic targets for treating cancer. In this study, we performed a gain-of-function screen using miRNA mimic library (319 miRNA species) to identify those affecting cell proliferation in human epithelial ovarian cancer cells (A2780). We discovered a number of miRNAs that increased or decreased the cell viability of A2780 cells. Pro-proliferative and anti-proliferative miRNAs include oncogenic miR-372 and miR-373, and tumor suppressive miR-124a, miR-7, miR-192 and miR-193a, respectively. We found that overexpression of miR-124a, miR-192, miR-193a and miR-193b inhibited BrdU incorporation in A2780 cells, indicating that these miRNAs affected the cell cycle. Overexpression of miR-193a and miR-193b induced an activation of caspase 3/7, and resulted in apoptotic cell death in A2780 cells. A genome-wide gene expression analysis with miR-193a-transfected A2780 cells led to identification of ARHGAP19, CCND1, ERBB4, KRAS and MCL1 as potential miR-193a targets. We demonstrated that miR-193a decreased the amount of MCL1 protein by binding 3′UTR of its mRNA. Our study suggests the potential of miRNA screens to discover miRNAs as therapeutic tools to treat ovarian cancer. PMID:23588298

  9. Functional Environmental Screening of a Metagenomic Library Identifies stlA; A Unique Salt Tolerance Locus from the Human Gut Microbiome

    PubMed Central

    Culligan, Eamonn P.; Sleator, Roy D.; Marchesi, Julian R.; Hill, Colin

    2013-01-01

    Functional environmental screening of metagenomic libraries is a powerful means to identify and assign function to novel genes and their encoded proteins without any prior sequence knowledge. In the current study we describe the identification and subsequent analysis of a salt-tolerant clone from a human gut metagenomic library. Following transposon mutagenesis we identified an unknown gene (stlA, for “salt tolerance locus A”) with no current known homologues in the databases. Subsequent cloning and expression in Escherichia coli MKH13 revealed that stlA confers a salt tolerance phenotype in its surrogate host. Furthermore, a detailed in silico analysis was also conducted to gain additional information on the properties of the encoded StlA protein. The stlA gene is rare when searched against human metagenome datasets such as MetaHit and the Human Microbiome Project and represents a novel and unique salt tolerance determinant which appears to be found exclusively in the human gut environment. PMID:24349412

  10. Functional MicroRNA Library Screening Identifies the HypoxaMiR MiR-24 as a Potent Regulator of Smooth Muscle Cell Proliferation and Vascularization

    PubMed Central

    Stöhr, Andrea; Gupta, Shashi Kumar; Hartmann, Dorothee; Holzmann, Angelika; Just, Annette; Hansen, Arne; Hilfiker-Kleiner, Denise; Eschenhagen, Thomas

    2014-01-01

    Abstract Smooth muscle cells (SMCs) are key components within the vasculature. Dependent on the stimulus, SMC can either be in a proliferative (synthetic) or differentiated state. Alterations of SMC phenotype also appear in several disease settings, further contributing to disease progression. Aims: Here, we asked whether microRNAs (miRNAs, miRs), which are strong posttranscriptional regulators of gene expression, could alter SMC proliferation. Results and Innovation: Employing a robotic-assisted high-throughput screening method using miRNA libraries, we identified hypoxia-regulated miR-24 as a master regulator of SMC proliferation. Proteome profiling showed a strong miR-24-dependent impact on cellular stress-associated factors, overall resulting in reduced stress resistance. In vitro, synthetic miR-24 overexpression had detrimental effects on SMC functional capacity inducing apoptosis, migration defects, enhanced autophagy, and loss of contractile marker genes. Impaired SMC function was mediated in part by the herein identified direct target gene heme oxygenase 1. Ex vivo, miR-24 was shown to inhibit the development of vasculature in a model of engineered heart tissue. Conclusion: Collectively, we report the identification of the hypoxamir-24 as an inhibitor of SMC proliferation, contributing to loss of vascularization. Antioxid. Redox Signal. 21, 1167–1176. PMID:24063572

  11. Screening and functional analysis of the peroxiredoxin specifically expressed in Bursaphelenchus xylophilus--the causative agent of pine wilt disease.

    PubMed

    Fu, Han-Yu; Ren, Jia-Hong; Huang, Lin; Li, Hao; Ye, Jian-Ren

    2014-01-01

    The pine wood nematode, Bursaphelenchus xylophilus, is the causal agent of pine wilt disease. Accurately differentiating B. xylophilus from other nematodes species, especially its related species B. mucronatus, is important for pine wood nematode detection. Thus, we attempted to identify a specific protein in the pine wood nematode using proteomics technology. Here, we compared the proteomes of B. xylophilus and B. mucronatus using Two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization -time-of-flight/time-of-flight (MALDI-TOF/TOF-MS) technologies. In total, 15 highly expressed proteins were identified in B. xylophilus compared with B. mucronatus. Subsequently, the specificity of the proteins identified was confirmed by PCR using the genomic DNA of other nematode species. Finally, a gene encoding a specific protein (Bx-Prx) was obtained. This gene was cloned and expressed in E. coli. The in situ hybridisation pattern of Bx-Prx showed that it was expressed strongly in the tail of B. xylophilus. RNAi was used to assess the function of Bx-Prx, the results indicated that the gene was associated with the reproduction and pathogenicity of B. xylophilus. This discovery provides fundamental information for identifying B. xylophilus via a molecular approach. Moreover, the purified recombinant protein has potential as a candidate diagnostic antigen of pine wilt disease, which may lead to a new immunological detection method for the pine wood nematode. PMID:24918285

  12. Screening and Functional Analysis of the Peroxiredoxin Specifically Expressed in Bursaphelenchus xylophilus—The Causative Agent of Pine Wilt Disease

    PubMed Central

    Fu, Han-Yu; Ren, Jia-Hong; Huang, Lin; Li, Hao; Ye, Jian-Ren

    2014-01-01

    The pine wood nematode, Bursaphelenchus xylophilus, is the causal agent of pine wilt disease. Accurately differentiating B. xylophilus from other nematodes species, especially its related species B. mucronatus, is important for pine wood nematode detection. Thus, we attempted to identify a specific protein in the pine wood nematode using proteomics technology. Here, we compared the proteomes of B. xylophilus and B. mucronatus using Two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization-time-of-flight/time-of-flight (MALDI-TOF/TOF-MS) technologies. In total, 15 highly expressed proteins were identified in B. xylophilus compared with B. mucronatus. Subsequently, the specificity of the proteins identified was confirmed by PCR using the genomic DNA of other nematode species. Finally, a gene encoding a specific protein (Bx-Prx) was obtained. This gene was cloned and expressed in E. coli. The in situ hybridisation pattern of Bx-Prx showed that it was expressed strongly in the tail of B. xylophilus. RNAi was used to assess the function of Bx-Prx, the results indicated that the gene was associated with the reproduction and pathogenicity of B. xylophilus. This discovery provides fundamental information for identifying B. xylophilus via a molecular approach. Moreover, the purified recombinant protein has potential as a candidate diagnostic antigen of pine wilt disease, which may lead to a new immunological detection method for the pine wood nematode. PMID:24918285

  13. Anatase TiO{sub 2} nanowires functionalized by organic sensitizers for solar cells: A screened Coulomb hybrid density functional study

    SciTech Connect

    Ünal, Hatice; Mete, Ersen; Gunceler, Deniz; Gülseren, Oğuz; Ellialtioğlu, Şinasi

    2015-11-21

    The adsorption of two different organic molecules cyanidin glucoside (C{sub 21}O{sub 11}H{sub 20}) and TA-St-CA on anatase (101) and (001) nanowires has been investigated using the standard and the range separated hybrid density functional theory calculations. The electronic structures and optical spectra of resulting dye–nanowire combined systems show distinct features for these types of photochromophores. The lowest unoccupied molecular orbital of the natural dye cyanidin glucoside is located below the conduction band of the semiconductor while, in the case of TA-St-CA, it resonates with the states inside the conduction band. The wide-bandgap anatase nanowires can be functionalized for solar cells through electron-hole generation and subsequent charge injection by these dye sensitizers. The intermolecular charge transfer character of Donor-π-Acceptor type dye TA-St-CA is substantially modified by its adsorption on TiO{sub 2} surfaces. Cyanidin glucoside exhibits relatively stronger anchoring on the nanowires through its hydroxyl groups. The atomic structures of dye–nanowire systems re-optimized with the inclusion of nonlinear solvation effects showed that the binding strengths of both dyes remain moderate even in ionic solutions.

  14. Activity-based differentiation of pathologists' workload in surgical pathology.

    PubMed

    Meijer, G A; Oudejans, J J; Koevoets, J J M; Meijer, C J L M

    2009-06-01

    Adequate budget control in pathology practice requires accurate allocation of resources. Any changes in types and numbers of specimens handled or protocols used will directly affect the pathologists' workload and consequently the allocation of resources. The aim of the present study was to develop a model for measuring the pathologists' workload that can take into account the changes mentioned above. The diagnostic process was analyzed and broken up into separate activities. The time needed to perform these activities was measured. Based on linear regression analysis, for each activity, the time needed was calculated as a function of the number of slides or blocks involved. The total pathologists' time required for a range of specimens was calculated based on standard protocols and validated by comparing to actually measured workload. Cutting up, microscopic procedures and dictating turned out to be highly correlated to number of blocks and/or slides per specimen. Calculated workload per type of specimen was significantly correlated to the actually measured workload. Modeling pathologists' workload based on formulas that calculate workload per type of specimen as a function of the number of blocks and slides provides a basis for a comprehensive, yet flexible, activity-based costing system for pathology. PMID:19399515

  15. Activity-Based Probe for Histidine Kinase Signaling

    PubMed Central

    Wilke, Kaelyn E.; Francis, Samson; Carlson, Erin E.

    2012-01-01

    Bacterial two-component systems (TCSs) are signaling pathways composed of two proteins, a histidine kinase (HK) and a response regulator (RR). Upon stimulation, the HK autophosphorylates at a conserved histidine. The phosphoryl group is subsequently transferred to an aspartate on a RR, eliciting an adaptive response, often up- or downregulation of gene expression. TCS signaling controls many functions in bacteria including development, virulence and antibiotic resistance, making the proteins involved in these systems potential therapeutic targets. Efficient methods for the profiling of HKs are currently lacking. For direct readout of HK activity, we sought to design a probe that enables detection of the phosphotransfer event; however, analysis of the phosphohistidine species is made difficult by the instability of the P-N bond. We anticipated that use of a γ-thiophosphorylated ATP analog, which would yield a thiophosphorylated histidine intermediate, could overcome this challenge. We determined that the fluorophore-conjugated probe, ATPγS-BODIPY, labels active HK proteins and is competitive for the ATP-binding site. This activity-based probe provides a new strategy for analysis of TCSs and other HK-mediated processes and will facilitate both functional studies and inhibitor identification. PMID:22606938

  16. Rhipicephalus (Boophilus) microplus tick in vitro feeding methods for functional (dsRNA) and vaccine candidate (antibody) screening.

    PubMed

    Lew-Tabor, Ala E; Bruyeres, Anthea G; Zhang, Bing; Rodriguez Valle, Manuel

    2014-09-01

    Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) ticks cause economic losses for cattle industries throughout tropical and subtropical regions of the world estimated at $US2.5 billion annually. Lack of access to efficacious long-lasting vaccination regimes and increases in tick acaricide resistance have led to the investigation of targets for the development of novel tick vaccines and treatments. In vitro tick feeding has been used for many tick species to study the effect of new acaricides on the transmission of tick-borne pathogens. Few studies have reported the use of in vitro feeding for functional genomic studies using RNA interference and/or the effect of specific anti-tick antibodies. In particular, in vitro feeding reports for the cattle tick are limited due to its relatively short hypostome. Previously published methods were further modified to broaden optimal tick sizes/weights, feeding sources including bovine and ovine serum, optimisation of commercially available blood anti-coagulant tubes, and IgG concentrations for effective antibody delivery. Ticks are fed overnight and monitored for ∼5-6 weeks to determine egg output and success of larval emergence using a humidified incubator. Lithium-heparin blood tubes provided the most reliable anti-coagulant for bovine blood feeding compared with commercial citrated (CPDA) and EDTA tubes. Although >30mg semi-engorged ticks fed more reliably, ticks as small as 15mg also fed to repletion to lay viable eggs. Ticks which gained less than ∼10mg during in vitro feeding typically did not lay eggs. One mg/ml IgG from Bm86-vaccinated cattle produced a potent anti-tick effect in vitro (83% efficacy) similar to that observed in vivo. Alternatively, feeding of dsRNA targeting Bm86 did not demonstrate anti-tick effects (11% efficacy) compared with the potent effects of ubiquitin dsRNA. This study optimises R. microplus tick in vitro feeding methods which support the development of cattle tick vaccines and

  17. Activity Based Startup Plan for Prototype Vertical Denitration Calciner

    SciTech Connect

    SUTTER, C.S.

    1999-08-31

    Testing activities on the Prototype Vertical Denitration Calciner at PFP were suspended in January 1997 due to the hold on fissile material handling in the facility. The restart of testing activities will require a review through an activity based startup process based upon Integrated Safety Management (ISM) principles to verify readiness. The Activity Based Startup Plan has been developed for this process.

  18. Improving hospital cost accounting with activity-based costing.

    PubMed

    Chan, Y C

    1993-01-01

    In this article, activity-based costing, an approach that has proved to be an improvement over the conventional costing system in product costing, is introduced. By combining activity-based costing with standard costing, health care administrators can better plan and control the costs of health services provided while ensuring that the organization's bottom line is healthy. PMID:8444618

  19. ENDOCRINE-DISRUPTING CHEMICALS: PREPUBERTAL EXPOSURES AND EFFECTS ON SEXUAL MATURATION AND THYROID FUNCTION IN THE MALE RAT. A FOCUS ON THE EDSTAC RECOMMENDATIONS. ENDOCRINE DISRUPTER SCREENING AND TESTING ADVISORY COMMITTEE

    EPA Science Inventory

    Endocrine-disrupting chemicals: prepubertal exposures and effects on sexual maturation and thyroid function in the male rat. A focus on the EDSTAC recommendations. Endocrine Disrupter Screening and Testing Advisory Committee.

    Stoker TE, Parks LG, Gray LE, Cooper RL.

  20. Activity-based protein profiling for biochemical pathway discovery in cancer

    PubMed Central

    Nomura, Daniel K.; Dix, Melissa M.; Cravatt, Benjamin F.

    2011-01-01

    Large-scale profiling methods have uncovered numerous gene and protein expression changes that correlate with tumorigenesis. However, determining the relevance of these expression changes and which biochemical pathways they affect has been hindered by our incomplete understanding of the proteome and its myriad functions and modes of regulation. Activity-based profiling platforms enable both the discovery of cancer-relevant enzymes and selective pharmacological probes to perturb and characterize these proteins in tumour cells. When integrated with other large-scale profiling methods, activity-based proteomics can provide insight into the metabolic and signalling pathways that support cancer pathogenesis and illuminate new strategies for disease diagnosis and treatment. PMID:20703252

  1. Activity-Based Profiling of a Physiologic Aglycone Library Reveals Sugar Acceptor Promiscuity of Family 1 UDP-Glucosyltransferases from Grape1[W

    PubMed Central

    Bönisch, Friedericke; Frotscher, Johanna; Stanitzek, Sarah; Rühl, Ernst; Wüst, Matthias; Bitz, Oliver; Schwab, Wilfried

    2014-01-01

    Monoterpenols serve various biological functions and accumulate in grape (Vitis vinifera), where a major fraction occurs as nonvolatile glycosides. We have screened the grape genome for sequences with similarity to terpene URIDINE DIPHOSPHATE GLYCOSYLTRANSFERASES (UGTs) from Arabidopsis (Arabidopsis thaliana). A ripening-related expression pattern was shown for three candidates by spatial and temporal expression analyses in five grape cultivars. Transcript accumulation correlated with the production of monoterpenyl β-d-glucosides in grape exocarp during ripening and was low in vegetative tissue. Targeted functional screening of the recombinant UGTs for their biological substrates was performed by activity-based metabolite profiling (ABMP) employing a physiologic library of aglycones built from glycosides isolated from grape. This approach led to the identification of two UDP-glucose:monoterpenol β-d-glucosyltransferases. Whereas VvGT14a glucosylated geraniol, R,S-citronellol, and nerol with similar efficiency, the three allelic forms VvGT15a, VvGT15b, and VvGT15c preferred geraniol over nerol. Kinetic resolution of R,S-citronellol and R,S-linalool was shown for VvGT15a and VvGT14a, respectively. ABMP revealed geraniol as the major biological substrate but also disclosed that these UGTs may add to the production of further glycoconjugates in planta. ABMP of aglycone libraries provides a versatile tool to uncover novel biologically relevant substrates of small-molecule glycosyltransferases that often show broad sugar acceptor promiscuity. PMID:25073706

  2. Colon cancer screening

    MedlinePlus

    ... screening; Sigmoidoscopy - screening; Virtual colonoscopy - screening; Fecal immunochemical test; Stool DNA test; sDNA test ... death and complications caused by colorectal cancer. SCREENING TESTS There are several ways to screen for colon ...

  3. Chemical Library Screening and Structure-Function Relationship Studies Identify Bisacodyl as a Potent and Selective Cytotoxic Agent Towards Quiescent Human Glioblastoma Tumor Stem-Like Cells

    PubMed Central

    Mameri, Samir; Dong, Jihu; Salomé, Christophe; Chen, Wanyin; El-Habr, Elias A.; Bousson, Fanny; Sy, Mohamadou; Obszynski, Julie; Boh, Alexandre; Villa, Pascal; Assad Kahn, Suzana; Didier, Bruno; Bagnard, Dominique; Junier, Marie-Pierre; Chneiweiss, Hervé; Haiech, Jacques; Hibert, Marcel; Kilhoffer, Marie-Claude

    2015-01-01

    Cancer stem-like cells reside in hypoxic and slightly acidic tumor niches. Such microenvironments favor more aggressive undifferentiated phenotypes and a slow growing "quiescent state" which preserves them from chemotherapeutic agents that essentially target proliferating cells. Our objective was to identify compounds active on glioblastoma stem-like cells, including under conditions that mimick those found in vivo within this most severe and incurable form of brain malignancy. We screened the Prestwick Library to identify cytotoxic compounds towards glioblastoma stem-like cells, either in a proliferating state or in more slow-growing "quiescent" phenotype resulting from non-renewal of the culture medium in vitro. Compound effects were assessed by ATP-level determination using a cell-based assay. Twenty active molecules belonging to different pharmacological classes have thus been identified. Among those, the stimulant laxative drug bisacodyl was the sole to inhibit in a potent and specific manner the survival of quiescent glioblastoma stem-like cells. Subsequent structure-function relationship studies led to identification of 4,4'-dihydroxydiphenyl-2-pyridyl-methane (DDPM), the deacetylated form of bisacodyl, as the pharmacophore. To our knowledge, bisacodyl is currently the only known compound targeting glioblastoma cancer stem-like cells in their quiescent, more resistant state. Due to its known non-toxicity in humans, bisacodyl appears as a new potential anti-tumor agent that may, in association with classical chemotherapeutic compounds, participate in tumor eradication. PMID:26270679

  4. Screening the dopamine D{sub 1} receptor gene in 131 schizophrenics and eight alcoholics: Identification of polymorphisms but lack of functionally significant sequence changes

    SciTech Connect

    Liu, Qiang; Sommer, S.S.; Sobell, J.L.

    1995-04-24

    To determine whether mutations in the D{sub 1} dopamine receptor (D{sub 1}DR) gene are associated with schizophrenia, the coding sequence was examined in 106 Caucasian, 11 African-American, 8 Asian, and 6 Native American patients. Approximately 350 kb of genomic sequence was screened by dideoxy fingerprinting, a method related to single strand conformational polymorphism (SSCP) analysis that detects virtually 100% of sequence changes. One polymorphism was identified in Asians and one in Caucasians, but neither altered 421 the amino acid sequence (Leu{sup 66}, and Ser{sup 421}, respectively). In addition, a previously reported polymorphism in the 5{prime} untranslated region of exon 2 at bp -48 was found to be common, with an allele frequency of approximately 40% in Caucasians of Western European descent. Based on the fact that no sequence changes of likely functional significance were identified, these data suggest that mutations affecting the structure of the D{sub 1} dopamine receptor protein are uncommon and are unlikely to contribute significantly to the genetic predisposition to schizophrenia. The D{sub 1}DR gene also was examined in eight alcoholics, including 3 African-Americans and 1 Native American, but no sequence changes were identified. 35 refs., 1 fig., 3 tabs.

  5. A strategy for screening active lead compounds and functional compound combinations from herbal medicines based on pharmacophore filtering and knockout/knockin chromatography.

    PubMed

    Song, Hui-Peng; Wu, Si-Qi; Qi, Lian-Wen; Long, Fang; Jiang, Li-Feng; Liu, Ke; Zeng, Hao; Xu, Zhi-Meng; Li, Ping; Yang, Hua

    2016-07-22

    Screening and deciphering active natural products of herbal medicines are of great importance for modern drug discovery. In this study, a novel strategy was proposed to rapidly filter ineffective compounds and target the most potential leads. The aim is to answer the key question of what components are responsible for the holistic bioactivity of an herbal product. To support the strategy, the pharmacophore-guided knockout/knockin chromatography was established for the first time. The greatest advantage of this method is that any interesting components could be automatically fished or knocked out. The method validation shows that the herbal extract was accurately reconstructed according to the experimental design. By combining with bioactivity assays, we demonstrated that "functional compound combination (FCC)", which is the core and indispensable effective part, could be discovered from an herbal medicine and suitable as marker compounds for quality control. The applicable objects of the strategy include single herbs, herbal formulas and commercially herbal preparations. As an illustrative case study, the strategy was successfully applied to simultaneously determine active leads and the FCC in Dan-Qi formula which shows excellent free radical scavenging activity. The potential mechanisms of compounds in Dan-Qi formula reacting with three different free radicals were systematically reported for the first time. This strategy was expected to unveil the mystery of herbal medicines and inspire a natural product-based drug discovery. PMID:27320377

  6. Can injury in major junior hockey players be predicted by a pre-season functional movement screen – a prospective cohort study

    PubMed Central

    Dossa, Khaled; Cashman, Glenn; Howitt, Scott; West, Bill; Murray, Nick

    2014-01-01

    Background: The Functional Movement Screen (FMS) is a tool that is commonly used to predict the occurrence of injury. Previous studies have shown that a score of 14 or less (with a maximum possible score of 21) successfully predicted future injury occurrence in athletes. No studies have looked at the use of the FMS to predict injuries in hockey players. Objective: To see if injury in major junior hockey players can be predicted by a preseason FMS. Methods: A convenience sample of 20 hockey players was scored on the FMS prior to the start of the hockey season. Injuries and number of man-games lost for each injury were documented over the course of the season. Results: The mean FMS score was 14.7+/−2.58. Those with an FMS score of ≤14 were not more likely to sustain an injury as determined by the Fisher’s exact test (one-tailed, P = 0.32). Conclusion: This study did not support the notion that lower FMS scores predict injury in major junior hockey players. PMID:25550667

  7. A Genome-Wide RNAi Screen Identifies Opposing Functions of Snai1 and Snai2 on the Nanog Dependency of Establishing Pluripotency

    PubMed Central

    Gingold, Julian A.; Fidalgo, Miguel; Guallar, Diana; Lau, Zerlina; Sun, Zhen; Zhou, Hongwei; Faiola, Francesco; Huang, Xin; Lee, Dung-Fang; Waghray, Avinash; Schaniel, Christoph; Felsenfeld, Dan P.; Lemischka, Ihor R.; Wang, Jianlong

    2014-01-01

    SUMMARY Nanog facilitates ESC self-renewal and iPSC generation during the final stage of reprogramming. From a genome-wide siRNA screen using a Nanog-GFP reporter line we discovered opposing effects of Snai1 and Snai2 depletion on Nanog promoter activity. We further discovered mutually repressive expression profiles and opposing functions of Snai1 and Snai2 during the Nanog-driven final stage of reprogramming. We found that Snai1, but not Snai2, is both a transcriptional target and protein partner of Nanog in reprogramming. Ectopic expression of Snai1 or depletion of Snai2 greatly facilitate the Nanog-driven reprogramming. Snai1 (but not Snai2) and Nanog co-bind to and transcriptionally activate pluripotency-associated genes including Lin28 and miRNA-290-295. Ectopic expression of miRNA-290-295 cluster genes partially rescues reprogramming inefficiency caused by Snai1 depletion. Our studies thus uncover a novel interplay between Nanog and mesenchymal transcription factors Snai1 and Snai2 in the transcriptional regulation of pluripotency-associated genes and miRNAs during the Nanog-driven reprogramming process. PMID:25240402

  8. Density-functional theory with screened van der Waals interactions applied to atomic and molecular adsorbates on close-packed and non-close-packed surfaces

    NASA Astrophysics Data System (ADS)

    Ruiz, Victor G.; Liu, Wei; Tkatchenko, Alexandre

    2016-01-01

    Modeling the adsorption of atoms and molecules on surfaces requires efficient electronic-structure methods that are able to capture both covalent and noncovalent interactions in a reliable manner. In order to tackle this problem, we have developed a method within density-functional theory (DFT) to model screened van der Waals interactions (vdW) for atoms and molecules on surfaces (the so-called DFT+vdWsurf method). The relatively high accuracy of the DFT+vdWsurf method in the calculation of both adsorption distances and energies, as well as the high degree of its reliability across a wide range of adsorbates, indicates the importance of the collective electronic effects within the extended substrate for the calculation of the vdW energy tail. We examine in detail the theoretical background of the method and assess its performance for adsorption phenomena including the physisorption of Xe on selected close-packed transition metal surfaces and 3,4,9,10-perylene-tetracarboxylic acid dianhydride (PTCDA) on Au(111). We also address the performance of DFT+vdWsurf in the case of non-close-packed surfaces by studying the adsorption of Xe on Cu(110) and the interfaces formed by the adsorption of a PTCDA monolayer on the Ag(111), Ag(100), and Ag(110) surfaces. We conclude by discussing outstanding challenges in the modeling of vdW interactions for studying atomic and molecular adsorbates on inorganic substrates.

  9. Deep recombination centers in C u2ZnSnS e4 revealed by screened-exchange hybrid density functional theory

    NASA Astrophysics Data System (ADS)

    Yee, Ye Sheng; Magyari-Köpe, Blanka; Nishi, Yoshio; Bent, Stacey F.; Clemens, Bruce M.

    2015-11-01

    We present a comprehensive study of the thermodynamic and electronic properties of intrinsic point defects in the solar energy conversion materials C u2ZnSnS e4 and CuInS e2 based on the screened-exchange hybrid density functional theory. A comparison between the defect transition levels for C u2ZnSnS e4 and CuInS e2 reveals that in C u2ZnSnS e4 , the S nCu and S nZn antisite defects can be recombination centers with defect states close to midgap, while the I nCu antisite defect has a shallow defect level in CuInS e2 . The resultant higher Shockley-Read-Hall recombination rate in C u2ZnSnS e4 reduces the steady-state concentration of minority carriers and quasi-Fermi level separation under illumination. This may explain the origin of the low open-circuit voltage values for C u2ZnSnS e4 solar cells compared to CuInS e2 solar cells.

  10. Screening for cancer

    SciTech Connect

    Miller, A.B.

    1985-01-01

    This book contains three sections: Fundamentals of Screening, Screening Tests, and Screening for Specific Cancer Sites. Each section consists of several chapters. Some of the chapter titles are: Principles of Screening and of the Evaluation of Screening Programs; Economic Aspects of Screening; Cervical Cytology; Screening Tests for Bladder Cancer; Fecal Occult Blood Testing; Screening for Cancer of the Cervix; Screening for Gastric Cancer; and Screening for Oral Cancer.

  11. MicroRNA-574-3p, identified by microRNA library-based functional screening, modulates tamoxifen response in breast cancer

    PubMed Central

    Ujihira, T.; Ikeda, K.; Suzuki, T.; Yamaga, R.; Sato, W.; Horie-Inoue, K.; Shigekawa, T.; Osaki, A.; Saeki, T.; Okamoto, K.; Takeda, S.; Inoue, S.

    2015-01-01

    Most primary breast cancers express estrogen receptor α and can be treated via endocrine therapy using anti-estrogens such as tamoxifen; however, acquired endocrine resistance is a critical issue. To identify tamoxifen response-related microRNAs (miRNAs) in breast cancer, MCF-7 cells infected with a lentiviral miRNA library were treated with 4-hydroxytamoxifen (OHT) or vehicle for 4 weeks, and the amounts of individual miRNA precursors that had integrated into the genome were evaluated by microarray. Compared to the vehicle-treated cells, 5 ‘dropout' miRNAs, which were downregulated in OHT-treated cells, and 6 ‘retained' miRNAs, which were upregulated in OHT-treated cells, were identified. Of the dropout miRNAs, we found that miR-574-3p expression was downregulated in clinical breast cancer tissues as compared with their paired adjacent tissues. In addition, anti-miR-574-3p reversed tamoxifen-mediated suppression of MCF-7 cell growth. Clathrin heavy chain (CLTC) was identified as a miR-574-3p target gene by in silico algorithms and luciferase reporter assay using the 3′ untranslated region of CLTC mRNA. Interestingly, loss and gain of miR-574-3p function in MCF-7 cells causes CLTC to be upregulated and downregulated, respectively. These results suggest that functional screening mediated by miRNA libraries can provide new insights into the genes essential for tamoxifen response in breast cancer. PMID:25560734

  12. Complete blood counts, liver function tests, and chest x-rays as routine screening in early-stage breast cancer: value added or just cost?

    PubMed

    Louir, Raphael J; Tonneson, Jennifer E; Gowarty, Minda; Goodney, Philip P; Barth, Richard J; Rosenkranz, Kari M

    2015-11-01

    Current National Comprehensive Cancer Network guidelines for breast cancer staging include pre-treatment complete blood count (CBC) and liver function tests (LFT) to screen for occult metastatic disease. To date, the relevance of these tests in detecting metastatic disease in asymptomatic women with early-stage breast cancer (Stage I/II) has not been demonstrated. Although chest x-rays are no longer recommended in the NCCN guidelines, many centers continue to include this imaging as part of their screening process. We aim to determine the clinical and financial impact of these labs and x-rays in the evaluation of early-stage breast cancer patients. A single institution IRB-approved retrospective chart review was conducted of patients with biopsy-proven invasive breast cancer treated from January 1, 2005–December 31, 2009. We collected patient demographics, clinical and pathologic staging, chest x-ray, CBC, and LFT results at the time of referral. Patients were stratified according to radiographic stage at the time of diagnosis. We obtained Medicare reimbursement fees for cost analysis. From 2005 to 2009, 1609 patients with biopsy-proven invasive breast cancer were treated at our institution. Of the 1082 patients with radiographic stage I/II disease, 27.3 % of patients had abnormal CBCs. No additional testing was performed to evaluate these abnormalities. In the early-stage population, 24.7 % of patients had elevated LFTs, resulting in 84 additional imaging studies. No metastatic disease was detected. The cost of CBC, LFTs and chest x-rays was $110.20 per patient, totaling $106,410.99. Additional tests prompted by abnormal results cost $58,143.30 over the five-year period. We found that pre-treatment CBCs, LFTs, and chest x-rays did not improve detection of occult metastatic disease but resulted in additional financial costs. Avoiding routine ordering of these tests would save the US healthcare system $25.7 million annually. PMID:26467045

  13. Biomarker Case-Detection and Prediction with Potential for Functional Psychosis Screening: Development and Validation of a Model Related to Biochemistry, Sensory Neural Timing and End Organ Performance

    PubMed Central

    Fryar-Williams, Stephanie; Strobel, Jörg E.

    2016-01-01

    potential for case-detection, -screening, -monitoring, and -targeted personalized management. The findings unmask unmet needs within the functional psychosis condition and suggest new biological understandings of psychosis phenomenology. PMID:27148083

  14. Biomarker Case-Detection and Prediction with Potential for Functional Psychosis Screening: Development and Validation of a Model Related to Biochemistry, Sensory Neural Timing and End Organ Performance.

    PubMed

    Fryar-Williams, Stephanie; Strobel, Jörg E

    2016-01-01

    potential for case-detection, -screening, -monitoring, and -targeted personalized management. The findings unmask unmet needs within the functional psychosis condition and suggest new biological understandings of psychosis phenomenology. PMID:27148083

  15. Hearing Screening

    ERIC Educational Resources Information Center

    Johnson-Curiskis, Nanette

    2012-01-01

    Hearing levels are threatened by modern life--headsets for music, rock concerts, traffic noises, etc. It is crucial we know our hearing levels so that we can draw attention to potential problems. This exercise requires that students receive a hearing screening for their benefit as well as for making the connection of hearing to listening.

  16. Classroom Screening.

    ERIC Educational Resources Information Center

    Alpha Plus Corp., Piedmont, CA.

    This classroom screening device was developed by the Circle Preschool First Chance Project, a government-funded program to integrate handicapped children into regular classroom activities, for use in preschools, nursery schools, Head Start centers and other agencies working with young children. It is designed to give a gross measure of a child's…

  17. Competitive Activity-Based Protein Profiling Identifies Aza-β-Lactams as a Versatile Chemotype for Serine Hydrolase Inhibition

    PubMed Central

    Zuhl, Andrea M.; Mohr, Justin T.; Bachovchin, Daniel A.; Niessen, Sherry; Hsu, Ku-Lung; Berlin, Jacob M.; Dochnahl, Maximilian; López-Alberca, María P.; Fu, Gregory C.; Cravatt, Benjamin F.

    2012-01-01

    Serine hydrolases are one of the largest and most diverse enzyme classes in Nature. Most serine hydrolases lack selective inhibitors, which are needed for assigning functions to these enzymes. We recently discovered a set of aza-β-lactams (ABLs) that act as potent and selective inhibitors of the mammalian serine hydrolase protein-phosphatase methylesterase-1 (PME-1). The ABLs inactivate PME-1 by covalent acylation of the enzyme’s serine nucleophile, suggesting that they could offer a general scaffold for serine hydrolase inhibitor discovery. Here, we have tested this hypothesis by screening ABLs more broadly against cell and tissue proteomes by competitive activity-based protein profiling (ABPP), leading to the discovery of lead inhibitors for several serine hydrolases, including the uncharacterized enzyme alpha, beta-hydrolase-10 (ABHD10). ABPP-guided medicinal chemistry yielded a compound ABL303 that potently (IC50 value ~ 30 nM) and selectively inactivated ABHD10 in vitro and in living cells. A comparison of optimized inhibitors for PME-1 and ABHD10 indicates that modest structural changes that alter steric bulk can tailor the ABL to selectively react with distinct, sequence-unrelated serine hydrolases. Our findings, taken together, designate the ABL as a versatile reactive group for creating first-in-class serine hydrolase inhibitors. PMID:22400490

  18. Competitive activity-based protein profiling identifies aza-β-lactams as a versatile chemotype for serine hydrolase inhibition.

    PubMed

    Zuhl, Andrea M; Mohr, Justin T; Bachovchin, Daniel A; Niessen, Sherry; Hsu, Ku-Lung; Berlin, Jacob M; Dochnahl, Maximilian; López-Alberca, María P; Fu, Gregory C; Cravatt, Benjamin F

    2012-03-21

    Serine hydrolases are one of the largest and most diverse enzyme classes in Nature. Most serine hydrolases lack selective inhibitors, which are valuable probes for assigning functions to these enzymes. We recently discovered a set of aza-β-lactams (ABLs) that act as potent and selective inhibitors of the mammalian serine hydrolase protein-phosphatase methylesterase-1 (PME-1). The ABLs inactivate PME-1 by covalent acylation of the enzyme's serine nucleophile, suggesting that they could offer a general scaffold for serine hydrolase inhibitor discovery. Here, we have tested this hypothesis by screening ABLs more broadly against cell and tissue proteomes by competitive activity-based protein profiling (ABPP), leading to the discovery of lead inhibitors for several serine hydrolases, including the uncharacterized enzyme α,β-hydrolase domain-containing 10 (ABHD10). ABPP-guided medicinal chemistry yielded a compound ABL303 that potently (IC(50) ≈ 30 nM) and selectively inactivated ABHD10 in vitro and in living cells. A comparison of optimized inhibitors for PME-1 and ABHD10 indicates that modest structural changes that alter steric bulk can tailor the ABL to selectively react with distinct, distantly related serine hydrolases. Our findings, taken together, designate the ABL as a versatile reactive group for creating first-in-class serine hydrolase inhibitors. PMID:22400490

  19. Activity-Based Costing Systems for Higher Education.

    ERIC Educational Resources Information Center

    Day, Dennis H.

    1993-01-01

    Examines traditional costing models utilized in higher education and pinpoints shortcomings related to proper identification of costs. Describes activity-based costing systems as a superior alternative for cost identification, measurement, and allocation. (MLF)

  20. A Native Chemical Ligation Handle that Enables the Synthesis of Advanced Activity-Based Probes: Diubiquitin as a Case Study

    PubMed Central

    Mulder, Monique P C; El Oualid, Farid; ter Beek, Jarno; Ovaa, Huib

    2014-01-01

    We present the development of a native chemical ligation handle that also functions as a masked electrophile that can be liberated during synthesis when required. This handle can thus be used for the synthesis of complex activity-based probes. We describe the use of this handle in the generation of linkage-specific activity-based deubiquitylating enzyme probes that contain substrate context and closely mimic the native ubiquitin isopeptide linkage. We have generated activity-based probes based on all seven isopeptide-linked diubiquitin topoisomers and demonstrated their structural integrity and ability to label DUBs in a linkage-specific manner. PMID:24623714

  1. Activity-based costing saves on supply distribution costs.

    PubMed

    1997-10-01

    Activity-based costing is coming, but is your organization ready? A few pioneering hospitals are already reaping the operational and economic benefits of activity-based costing in their materials management, and now the VHA purchasing alliance is offering this costing option to its 1,200 hospital members. The concept is simple, so why aren't there more takers? Here are the details on this pragmatic pricing approach that could save your facility plenty. PMID:10178010

  2. Activity Based Startup Plan for Prototype Vertical Denitration Calciner

    SciTech Connect

    SUTTER, C.S.

    1999-08-16

    Testing activities on the Prototype Vertical Denitration Calciner at Plutonium Finish Plant (PFP) were suspended in January 1997 due to the hold on fissile material handling in the facility. The restart of testing activities will require a review through an activity based startup process based upon Integrated Safety Management (ISM) principles to verify readiness. The Activity Based Startup Plan for the Prototype vertical Denitration Calciner has been developed for this process.

  3. Using the Larval Zebrafish Locomotor Asssay in Functional Neurotoxicity Screening: Light Brightness and the Order of Stimulus Presentation Affect the Outcome

    EPA Science Inventory

    We are evaluating methods to screen/prioritize large numbers of chemicals using 6 day old zebrafish (Danio rerio) as an alternative model for detecting neurotoxic effects. Our behavioral testing paradigm simultaneously tests individual larval zebrafish under sequential light and...

  4. Vision Screening

    NASA Technical Reports Server (NTRS)

    1993-01-01

    The Visi Screen OSS-C, marketed by Vision Research Corporation, incorporates image processing technology originally developed by Marshall Space Flight Center. Its advantage in eye screening is speed. Because it requires no response from a subject, it can be used to detect eye problems in very young children. An electronic flash from a 35 millimeter camera sends light into a child's eyes, which is reflected back to the camera lens. The photorefractor then analyzes the retinal reflexes generated and produces an image of the child's eyes, which enables a trained observer to identify any defects. The device is used by pediatricians, day care centers and civic organizations that concentrate on children with special needs.

  5. Small-molecule screening identifies inhibition of salt-inducible kinases as a therapeutic strategy to enhance immunoregulatory functions of dendritic cells

    PubMed Central

    Sundberg, Thomas B.; Choi, Hwan Geun; Song, Joo-Hye; Russell, Caitlin N.; Hussain, Mahmud M.; Graham, Daniel B.; Khor, Bernard; Gagnon, John; O’Connell, Daniel J.; Narayan, Kavitha; Dančík, Vlado; Perez, Jose R.; Reinecker, Hans-Christian; Gray, Nathanael S.; Schreiber, Stuart L.; Xavier, Ramnik J.; Shamji, Alykhan F.

    2014-01-01

    Genetic alterations that reduce the function of the immunoregulatory cytokine IL-10 contribute to colitis in mouse and man. Myeloid cells such as macrophages (MΦs) and dendritic cells (DCs) play an essential role in determining the relative abundance of IL-10 versus inflammatory cytokines in the gut. As such, using small molecules to boost IL-10 production by DCs–MΦs represents a promising approach to increase levels of this cytokine specifically in gut tissues. Toward this end, we screened a library of well-annotated kinase inhibitors for compounds that enhance production of IL-10 by murine bone-marrow–derived DCs stimulated with the yeast cell wall preparation zymosan. This approach identified a number of kinase inhibitors that robustly up-regulate IL-10 production including the Food and Drug Administration (FDA)-approved drugs dasatinib, bosutinib, and saracatinib that target ABL, SRC-family, and numerous other kinases. Correlating the kinase selectivity profiles of the active compounds with their effect on IL-10 production suggests that inhibition of salt-inducible kinases (SIKs) mediates the observed IL-10 increase. This was confirmed using the SIK-targeting inhibitor HG-9-91-01 and a series of structural analogs. The stimulatory effect of SIK inhibition on IL-10 is also associated with decreased production of the proinflammatory cytokines IL-1β, IL-6, IL-12, and TNF-α, and these coordinated effects are observed in human DCs–MΦs and anti-inflammatory CD11c+ CX3CR1hi cells isolated from murine gut tissue. Collectively, these studies demonstrate that SIK inhibition promotes an anti-inflammatory phenotype in activated myeloid cells marked by robust IL-10 production and establish these effects as a previously unidentified activity associated with several FDA-approved multikinase inhibitors. PMID:25114223

  6. Identification of novel histone deacetylase 1 inhibitors by combined pharmacophore modeling, 3D-QSAR analysis, in silico screening and Density Functional Theory (DFT) approaches

    NASA Astrophysics Data System (ADS)

    Choubey, Sanjay K.; Mariadasse, Richard; Rajendran, Santhosh; Jeyaraman, Jeyakanthan

    2016-12-01

    Overexpression of HDAC1, a member of Class I histone deacetylase is reported to be implicated in breast cancer. Epigenetic alteration in carcinogenesis has been the thrust of research for few decades. Increased deacetylation leads to accelerated cell proliferation, cell migration, angiogenesis and invasion. HDAC1 is pronounced as the potential drug target towards the treatment of breast cancer. In this study, the biochemical potential of 6-aminonicotinamide derivatives was rationalized. Five point pharmacophore model with one hydrogen-bond acceptor (A3), two hydrogen-bond donors (D5, D6), one ring (R12) and one hydrophobic group (H8) was developed using 6-aminonicotinamide derivatives. The pharmacophore hypothesis yielded a 3D-QSAR model with correlation-coefficient (r2 = 0.977, q2 = 0.801) and it was externally validated with (r2pred = 0.929, r2cv = 0.850 and r2m = 0.856) which reveals the statistical significance of the model having high predictive power. The model was then employed as 3D search query for virtual screening against compound libraries (Zinc, Maybridge, Enamine, Asinex, Toslab, LifeChem and Specs) in order to identify novel scaffolds which can be experimentally validated to design future drug molecule. Density Functional Theory (DFT) at B3LYP/6-31G* level was employed to explore the electronic features of the ligands involved in charge transfer reaction during receptor ligand interaction. Binding free energy (ΔGbind) calculation was done using MM/GBSA which defines the affinity of ligands towards the receptor.

  7. Screening on the differentially expressed miRNAs in zebrafish (Danio rerio) exposed to trace β-diketone antibiotics and their related functions.

    PubMed

    Li, Jieyi; Liu, Jinfeng; Zhang, Yuhuan; Wang, Xuedong; Li, Weijun; Zhang, Hongqin; Wang, Huili

    2016-09-01

    The toxicity of β-diketone antibiotics (DKAs) to larval and adult zebrafish (Danio rerio) was investigated by miRNA sequencing and bioinformatics analyses. In control and DKA-exposed groups, 215 differentially expressed miRNAs were screened, and 4076 differential target genes were predicted. Among 51 co-differentially expressed genes, 45 were annotated in KOG functional classification, and 34 in KEGG pathway analysis. The homology analysis of 20 miRNAs with human hsa-miRNAs demonstrated 17 high homologous sequences. The expression levels of 12 miRNAs by qRT-PCR were consistent with those by sRNA-seq. A regulatory network for 4 positive miRNA genes (dre-miR-10, -96, -92 and -184) was plotted, and the high-degree of connectivity between miRNA-gene pairs suggests that these miRNAs play critical roles during zebrafish development. The consistent expression of dre-miR-184 and dre-miR-96 was proved in 120-hpf zebrafish brain, gill, otoliths and lateral line neuromast by qRT-PCR, miRNA-seq, W-ISH and ISH. DKA-exposure led to vacuolation of interstitial cells, reduced number of neurons, glial cell proliferation and formation of glial scar, and the obvious abnormality of cell structure might result from abnormal expression of differentially expressed miRNAs. In general, chronic DKA-exposure resulted in comprehensively toxic effects on larval and adult zebrafish tissues, especially for nervous system. PMID:27450238

  8. Functional screening of willow alleles in Arabidopsis combined with QTL mapping in willow (Salix) identifies SxMAX4 as a coppicing response gene

    PubMed Central

    Salmon, Jemma; Ward, Sally P; Hanley, Steven J; Leyser, Ottoline; Karp, Angela

    2014-01-01

    Willows (Salix spp.) are important biomass crops due to their ability to grow rapidly with low fertilizer inputs and ease of cultivation in short-rotation coppice cycles. They are relatively undomesticated and highly diverse, but functional testing to identify useful allelic variation is time-consuming in trees and transformation is not yet possible in willow. Arabidopsis is heralded as a model plant from which knowledge can be transferred to advance the improvement of less tractable species. Here, knowledge and methodologies from Arabidopsis were successfully used to identify a gene influencing stem number in coppiced willows, a complex trait of key biological and industrial relevance. The strigolactone-related More AXillary growth (MAX) genes were considered candidates due to their role in shoot branching. We previously demonstrated that willow and Arabidopsis show similar response to strigolactone and that transformation rescue of Arabidopsis max mutants with willow genes could be used to detect allelic differences. Here, this approach was used to screen 45 SxMAX1, SxMAX2, SxMAX3 and SxMAX4 alleles cloned from 15 parents of 11 mapping populations varying in shoot-branching traits. Single-nucleotide polymorphism (SNP) frequencies were locus dependent, ranging from 29.2 to 74.3 polymorphic sites per kb. SxMAX alleles were 98%–99% conserved at the amino acid level, but different protein products varying in their ability to rescue Arabidopsis max mutants were identified. One poor rescuing allele, SxMAX4D, segregated in a willow mapping population where its presence was associated with increased shoot resprouting after coppicing and colocated with a QTL for this trait. PMID:24393130

  9. Substrate-Free High-Throughput Screening Identifies Selective Inhibitors for Uncharacterized Enzymes

    PubMed Central

    Bachovchin, Daniel A.; Brown, Steven J.; Rosen, Hugh; Cravatt, Benjamin F.

    2009-01-01

    Target-based high-throughput screening (HTS) is essential for the discovery of small-molecule modulators of proteins. Typical screening methods for enzymes rely on extensively tailored substrate assays, which are not available for targets of poorly characterized biochemical activity. Here, we report a general, substrate-free platform for HTS that overcomes this problem by monitoring the reaction of broad-spectrum, activity-based probes with enzymes using fluorescence polarization. We show that this platform is applicable to enzymes from multiple mechanistic classes, regardless of their degree of functional annotation, and can be coupled with secondary competitive activity-based proteomic assays to rapidly determine the specificity of screening hits. Using this platform, we identified the bioactive alkaloid emetine as a selective inhibitor of the uncharacterized cancer-associated hydrolase RBBP9. We furthermore show that the detoxification enzyme GSTO1, also implicated in cancer, is inhibited by several electrophilic compounds found in public libraries, some of which display high selectivity for this enzyme. PMID:19329999

  10. Sensitive high-throughput screening for the detection of reducing sugars.

    PubMed

    Mellitzer, Andrea; Glieder, Anton; Weis, Roland; Reisinger, Christoph; Flicker, Karlheinz

    2012-01-01

    The exploitation of renewable resources for the production of biofuels relies on efficient processes for the enzymatic hydrolysis of lignocellulosic materials. The development of enzymes and strains for these processes requires reliable and fast activity-based screening assays. Additionally, these assays are also required to operate on the microscale and on the high-throughput level. Herein, we report the development of a highly sensitive reducing-sugar assay in a 96-well microplate screening format. The assay is based on the formation of osazones from reducing sugars and para-hydroxybenzoic acid hydrazide. By using this sensitive assay, the enzyme loads and conversion times during lignocellulose hydrolysis can be reduced, thus allowing higher throughput. The assay is about five times more sensitive than the widely applied dinitrosalicylic acid based assay and can reliably detect reducing sugars down to 10 μM. The assay-specific variation over one microplate was determined for three different lignocellulolytic enzymes and ranges from 2 to 8%. Furthermore, the assay was combined with a microscale cultivation procedure for the activity-based screening of Pichia pastoris strains expressing functional Thermomyces lanuginosus xylanase A, Trichoderma reesei β-mannanase, or T. reesei cellobiohydrolase 2. PMID:21538898

  11. Quadruple screen test

    MedlinePlus

    ... screen; Multiple marker screening; AFP plus; Triple screen test; AFP maternal; MSAFP; 4-marker screen ... This test is most often done between the 15th and 22nd weeks of the pregnancy. It is most accurate ...

  12. The scattering of the screened Coulomb potential

    NASA Astrophysics Data System (ADS)

    Cao, Xin-Wei; Chen, Wen-Li; Li, Yuan-Yuan; Wei, Gao-Feng

    2014-08-01

    We study the scattering states of the screened Coulomb potential in the nonrelativistic frame. The explicitly calculation formula of phase shift is derived and the normalized radial wave functions of scattering states on the ^{\\prime} k/2\\pi scale^{\\prime} are presented. By studying analytical properties of scattering amplitude the screening effects on bound states are discussed numerically. It is shown that the screening effects increase with increasing screened parameter, especially for large quantum states.

  13. Early Dementia Screening.

    PubMed

    Panegyres, Peter K; Berry, Renee; Burchell, Jennifer

    2016-01-01

    As the population of the world increases, there will be larger numbers of people with dementia and an emerging need for prompt diagnosis and treatment. Early dementia screening is the process by which a patient who might be in the prodromal phases of a dementing illness is determined as having, or not having, the hallmarks of a neurodegenerative condition. The concepts of mild cognitive impairment, or mild neurocognitive disorder, are useful in analyzing the patient in the prodromal phase of a dementing disease; however, the transformation to dementia may be as low as 10% per annum. The search for early dementia requires a comprehensive clinical evaluation, cognitive assessment, determination of functional status, corroborative history and imaging (including MRI, FDG-PET and maybe amyloid PET), cerebrospinal fluid (CSF) examination assaying Aβ1-42, T-τ and P-τ might also be helpful. Primary care physicians are fundamental in the screening process and are vital in initiating specialist investigation and treatment. Early dementia screening is especially important in an age where there is a search for disease modifying therapies, where there is mounting evidence that treatment, if given early, might influence the natural history-hence the need for cost-effective screening measures for early dementia. PMID:26838803

  14. Early Dementia Screening

    PubMed Central

    Panegyres, Peter K.; Berry, Renee; Burchell, Jennifer

    2016-01-01

    As the population of the world increases, there will be larger numbers of people with dementia and an emerging need for prompt diagnosis and treatment. Early dementia screening is the process by which a patient who might be in the prodromal phases of a dementing illness is determined as having, or not having, the hallmarks of a neurodegenerative condition. The concepts of mild cognitive impairment, or mild neurocognitive disorder, are useful in analyzing the patient in the prodromal phase of a dementing disease; however, the transformation to dementia may be as low as 10% per annum. The search for early dementia requires a comprehensive clinical evaluation, cognitive assessment, determination of functional status, corroborative history and imaging (including MRI, FDG-PET and maybe amyloid PET), cerebrospinal fluid (CSF) examination assaying Aβ1–42, T-τ and P-τ might also be helpful. Primary care physicians are fundamental in the screening process and are vital in initiating specialist investigation and treatment. Early dementia screening is especially important in an age where there is a search for disease modifying therapies, where there is mounting evidence that treatment, if given early, might influence the natural history—hence the need for cost-effective screening measures for early dementia. PMID:26838803

  15. A New Activity-Based Financial Cost Management Method

    NASA Astrophysics Data System (ADS)

    Qingge, Zhang

    The standard activity-based financial cost management model is a new model of financial cost management, which is on the basis of the standard cost system and the activity-based cost and integrates the advantages of the two. It is a new model of financial cost management with more accurate and more adequate cost information by taking the R&D expenses as the accounting starting point and after-sale service expenses as the terminal point and covering the whole producing and operating process and the whole activities chain and value chain aiming at serving the internal management and decision.

  16. A Preliminary Investigation of the Reinforcement Function of Signal Detections in Simulated Baggage Screening: Further Support for the Vigilance Reinforcement Hypothesis

    ERIC Educational Resources Information Center

    Hogan, Lindsey C.; Bell, Matthew; Olson, Ryan

    2009-01-01

    The vigilance reinforcement hypothesis (VRH) asserts that errors in signal detection tasks are partially explained by operant reinforcement and extinction processes. VRH predictions were tested with a computerized baggage screening task. Our experiment evaluated the effects of signal schedule (extinction vs. variable interval 6 min) and visual…

  17. Application of Targeted Functional Assays to Assess a Putative Vascular Disruption Developmental Toxicity Pathway Informed By ToxCast High-Throughput Screening Data

    EPA Science Inventory

    Chemical perturbation of vascular development is a putative toxicity pathway which may result in developmental toxicity. EPA’s high-throughput screening (HTS) ToxCast program contains assays which measure cellular signals and biological processes critical for blood vessel develop...

  18. Using the Larval Zebrafish Locomotor Assay in Functional Neurotoxicity Screening: Light Intensity and the Order of Stimulus Presentation Affect the Outcome

    EPA Science Inventory

    The U.S. Environmental Protection Agency is evaluating methods to screen and prioritize large numbers of chemicals using 6 day old zebrafish (Danio rerio) as an alternative test model for detecting neurotoxic chemicals. We use a behavioral testing paradigm that simultaneously tes...

  19. Newborn Screening

    PubMed Central

    Pitt, James J

    2010-01-01

    Early detection of many disorders, mainly inherited, is feasible with population-wide analysis of newborn dried blood spot samples. Phenylketonuria was the prototype disorder for newborn screening (NBS) and early dietary treatment has resulted in vastly improved outcomes for this disorder. Testing for primary hypothyroidism and cystic fibrosis (CF) was later added to NBS programs following the development of robust immunoassays and molecular testing. Current CF testing usually relies on a combined immunoreactive trypsin/mutation detection strategy. Multiplex testing for approximately 25 inborn errors of metabolism using tandem mass spectrometry is a relatively recent addition to NBS. The simultaneous introduction of many disorders has caused some re-evaluation of the traditional guidelines for NBS, because very rare disorders or disorders without good treatments can be included with minimal effort. NBS tests for many other disorders have been developed, but these are less uniformly applied or are currently considered developmental. This review focuses on Australasian NBS practices. PMID:20498829

  20. Lunar Dust Mitigation Screens

    NASA Astrophysics Data System (ADS)

    Knutson, Shawn; Holloway, Nancy

    being developed in a collaborative effort between Langley Research Center and Kennedy Space Center. The screens typically consist of spiral shaped conductive traces patterned on high dielectric substrates (i.e. glass, quartz, polyimide film, etc.). Two broad categories of substrate materials are being investigated for the screens. One category consists of transparent substrates (i.e. glass, quartz, sapphire, etc.), and the other non-transparent sub-strates (Kapton, polyimide films, metals, etc.). The transparent screens utilize patterns made from indium tin oxide (ITO), a transparent conductive material, on clear substrates while the non-transparent screens use copper patterns on a transluscent or opaque substrates. Further, the screen is coated with a high dielectric polyimide cover layer to protect the screen pattern. One promising cover layer material that is currently being investigated is Langley Research Center-Soluble Imide (LaRC-SI), a NASA LaRC developed polyimide. Lastly, a top-coat of hard, inorganic material is evaporated onto the cover layer for protection from scratches due to abrasive nature of the dust. Of note, several top-coat materials are under investigation and include: aluminum oxide, silicon dioxide, titanium oxide, yttrium oxide, zirconium oxide, and zinc sulfide. The electrostatic dust mitigation screens function when a high voltage (700V or greater) is applied to the screen electrodes, thus creating an electromagnetic wave across the surface of the screen that repels the dust. Lunar dust typically contains a high positive charge; therefore, the screens are charged with a higher positive charge that effectively repels dust from the surface (i.e. like charges repel, unlike charges attract). It is anticipated that full development and maturation of this technology will enable humans to sustain a long term presence on the moon, and other planets where dust may have negative implications.

  1. Activity-Based Intervention Practices in Special Education

    ERIC Educational Resources Information Center

    Ozen, Arzu; Ergenekon, Yasemin

    2011-01-01

    Teaching practices in natural settings such as activity-based intervention (ABI) are suggested as alternatives to be used in effective early childhood education. As a multidisciplinary model, ABI consists of four components, which are choosing activities according to the child's interests; teaching generalizable goals embedded in routines and…

  2. Activity-Based Costing: A Cost Management Tool.

    ERIC Educational Resources Information Center

    Turk, Frederick J.

    1993-01-01

    In college and university administration, overhead costs are often charged to programs indiscriminately, whereas the support activities that underlie those costs remain unanalyzed. It is time for institutions to decrease ineffective use of resources. Activity-based management attributes costs more accurately and can improve efficiency. (MSE)

  3. Activity Based Curriculum for Elementary Education. Additional Activities, K-6.

    ERIC Educational Resources Information Center

    Wichita Public Schools, KS.

    This elementary curriculum is a vehicle to provide manipulative activities that reinforce academic skills through meaningful, relevant, activity-based awareness of modern society. The twenty-six activity plans included in the curriculum place a major emphasis upon realistic or concrete experiences that deal with the manipulation and exploration of…

  4. Solution structure and functional ligand screening of HI0719, a highly conserved protein from bacteria to humans in the YjgF/YER057c/UK114 family.

    PubMed

    Parsons, Lisa; Bonander, Nicklas; Eisenstein, Edward; Gilson, Michael; Kairys, Visvaldas; Orban, John

    2003-01-14

    HI0719 belongs to a large family of highly conserved proteins with no definitive molecular function and is found in organisms ranging from bacteria to humans. We describe the NMR structure of HI0719, the first solution structure for a member of this family. The overall fold is similar to the crystal structures of two homologues, YabJ from Bacillus subtilis and YjgF from Escherichia coli, and all three structures are similar to that of chorismate mutase, although there is little sequence homology and no apparent functional connection. HI0719 is a homotrimer with a distinct cavity located at the subunit interface. Six of the seven invariant residues in the high identity group of proteins are located in this cavity, suggesting that this may be a binding site for small molecules. Using previously published observations about the biological role of HI0719 family members as a guide, over 100 naturally occurring small molecules or structural analogues were screened for ligand binding using NMR spectroscopy. The targeted screening approach identified six compounds that bind to HI0719 at the putative active site. Five of these compounds are either alpha-keto acids or alpha,beta-unsaturated acids, while the sixth compound is structurally similar. Previous studies have proposed that some HI0719 homologues may act on small molecules in the isoleucine biosynthetic path and, if this is correct, the ligand screening results presented here suggest that the interaction most likely occurs with 2-ketobutyrate and/or its unstable enamine precursor. PMID:12515541

  5. Rapid, Optimized Interactomic Screening

    PubMed Central

    Hakhverdyan, Zhanna; Domanski, Michal; Hough, Loren; Oroskar, Asha A.; Oroskar, Anil R.; Keegan, Sarah; Dilworth, David J.; Molloy, Kelly R.; Sherman, Vadim; Aitchison, John D.; Fenyö, David; Chait, Brian T.; Jensen, Torben Heick; Rout, Michael P.; LaCava, John

    2015-01-01

    We must reliably map the interactomes of cellular macromolecular complexes in order to fully explore and understand biological systems. However, there are no methods to accurately predict how to capture a given macromolecular complex with its physiological binding partners. Here, we present a screen that comprehensively explores the parameters affecting the stability of interactions in affinity-captured complexes, enabling the discovery of physiological binding partners and the elucidation of their functional interactions in unparalleled detail. We have implemented this screen on several macromolecular complexes from a variety of organisms, revealing novel profiles even for well-studied proteins. Our approach is robust, economical and automatable, providing an inroad to the rigorous, systematic dissection of cellular interactomes. PMID:25938370

  6. A barcode-free combinatorial screening platform for matrix metalloproteinase screening.

    PubMed

    Rane, Tushar D; Zec, Helena C; Wang, Tza-Huei

    2015-02-01

    Application of droplet microfluidics to combinatorial screening applications remains elusive because of the need for composition-identifying unique barcodes. Here we propose a barcode-free continuous flow droplet microfluidic platform to suit the requirements of combinatorial screening applications. We demonstrate robust and repeatable functioning of this platform with matrix metalloproteinase activity screening as a sample application. PMID:25543856

  7. A Barcode-Free Combinatorial Screening Platform for Matrix Metalloproteinase Screening

    PubMed Central

    2015-01-01

    Application of droplet microfluidics to combinatorial screening applications remains elusive because of the need for composition-identifying unique barcodes. Here we propose a barcode-free continuous flow droplet microfluidic platform to suit the requirements of combinatorial screening applications. We demonstrate robust and repeatable functioning of this platform with matrix metalloproteinase activity screening as a sample application. PMID:25543856

  8. A draft genome sequence and functional screen reveals the repertoire of type III secreted proteins of Pseudomonas syringae pathovar tabaci 11528

    PubMed Central

    Studholme, David J; Ibanez, Selena Gimenez; MacLean, Daniel; Dangl, Jeffery L; Chang, Jeff H; Rathjen, John P

    2009-01-01

    Background Pseudomonas syringae is a widespread bacterial pathogen that causes disease on a broad range of economically important plant species. Pathogenicity of P. syringae strains is dependent on the type III secretion system, which secretes a suite of up to about thirty virulence 'effector' proteins into the host cytoplasm where they subvert the eukaryotic cell physiology and disrupt host defences. P. syringae pathovar tabaci naturally causes disease on wild tobacco, the model member of the Solanaceae, a family that includes many crop species as well as on soybean. Results We used the 'next-generation' Illumina sequencing platform and the Velvet short-read assembly program to generate a 145X deep 6,077,921 nucleotide draft genome sequence for P. syringae pathovar tabaci strain 11528. From our draft assembly, we predicted 5,300 potential genes encoding proteins of at least 100 amino acids long, of which 303 (5.72%) had no significant sequence similarity to those encoded by the three previously fully sequenced P. syringae genomes. Of the core set of Hrp Outer Proteins that are conserved in three previously fully sequenced P. syringae strains, most were also conserved in strain 11528, including AvrE1, HopAH2, HopAJ2, HopAK1, HopAN1, HopI, HopJ1, HopX1, HrpK1 and HrpW1. However, the hrpZ1 gene is partially deleted and hopAF1 is completely absent in 11528. The draft genome of strain 11528 also encodes close homologues of HopO1, HopT1, HopAH1, HopR1, HopV1, HopAG1, HopAS1, HopAE1, HopAR1, HopF1, and HopW1 and a degenerate HopM1'. Using a functional screen, we confirmed that hopO1, hopT1, hopAH1, hopM1', hopAE1, hopAR1, and hopAI1' are part of the virulence-associated HrpL regulon, though the hopAI1' and hopM1' sequences were degenerate with premature stop codons. We also discovered two additional HrpL-regulated effector candidates and an HrpL-regulated distant homologue of avrPto1. Conclusion The draft genome sequence facilitates the continued development of P

  9. 49 CFR 1544.411 - Continuing qualifications of screening personnel.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 49 Transportation 9 2010-10-01 2010-10-01 false Continuing qualifications of screening personnel... Screening § 1544.411 Continuing qualifications of screening personnel. (a) Impairment. No individual may perform a screening function if he or she shows evidence of impairment, such as impairment due to...

  10. Advantages of the Quadruple Screen over noninvasive prenatal testing.

    PubMed

    Keller, Nathan A; Rijshinghani, Asha

    2016-03-01

    Noninvasive prenatal testing (NIPT) is becoming increasingly popular with some offering it as a primary screen option in all patients in place of serum screening. Serum screening offers insight into placental function, which NIPT does not. Abnormal levels of analytes in the serum screen have been associated with pregnancy complications. PMID:27014443

  11. Screening for Specific Phobias

    MedlinePlus

    ... Screening for Posttraumatic Stress Disorder (PTSD) Screening for Social Anxiety Disorder Screening for Specific Phobias Screening for an Anxiety Disorder: Children Screening for an Anxiety Disorder: Family Member Self-Help Strategies: Webinars to Calm Anxious Minds "Triumph" E-News ...

  12. [Comparison of screens and screen-film-systems (author's transl)].

    PubMed

    Maurer, H J; Goos, F

    1979-06-01

    Important details are to be payed attention in comparison of different scrreens resp. screen-film-systems: 1. Physical characteristics of different groups of luminescent materials: f.i. calcium tung-state, rare-earths compounds, double halogenides. - 2. Different types of screens: highest details up to highest speed intensifying screens, have to be defined more specifically and differentiated against to each other too. - 3. Besides intensification, resolution has to be included into consideration since one of these dates alone does not allow any statement on the total function of a screen or a screen-film-system. - 4. The technical methodological conditions of apparatuses, object and its positioning have to be defined, f.i. X-ray quality, distances, grid, and in automatically controlled exposition, if necessary, position of ionization chamber as well as absorption of cassetts and screen. - 5. Considering these points gradation curves have to include the whole necessary or interesting diagnostic range. - 6. Due to functional correlation between intensification and resolution, the resolution has to be taken in consideration due to application; its interdependence of density and object (f.i. scattered radiation) is often not taken enough in consideration. PMID:461776

  13. Chemical screening with zebrafish embryos.

    PubMed

    Zhong, Hanbing; Lin, Shuo

    2011-01-01

    Functional chemicals are very useful tools for molecular biology studies. Due to its small size, large progeny clutch, and embryonic transparency, zebrafish serves as a superb in vivo animal model for chemical compound screens and characterization. During zebrafish embryogenesis, multiple developmental phenotypes can be easily examined under the microscope, therefore allowing a more comprehensive evaluation for identifying novel functional chemicals than cell-based assays. Ever since the first zebrafish-based chemical screen was conducted in the year 2000, many functional chemicals have been discovered using this strategy. In this chapter, we describe how to perform a typical zebrafish-based chemical screen and discuss the details of the protocol by using the example of the identification and characterization of two new Smo inhibitors with a Gli:GFP transgenic line. PMID:21318908

  14. Time-Driven Activity-Based Costing in Emergency Medicine.

    PubMed

    Yun, Brian J; Prabhakar, Anand M; Warsh, Jonathan; Kaplan, Robert; Brennan, John; Dempsey, Kyle E; Raja, Ali S

    2016-06-01

    Value in emergency medicine is determined by both patient-important outcomes and the costs associated with achieving them. However, measuring true costs is challenging. Without an understanding of costs, emergency department (ED) leaders will be unable to determine which interventions might improve value for their patients. Although ongoing research may determine which outcomes are meaningful, an accurate costing system is also needed. This article reviews current costing mechanisms in the ED and their pitfalls. It then describes how time-driven activity-based costing may be superior to these current costing systems. Time-driven activity-based costing, in addition to being a more accurate costing system, can be used for process improvements in the ED. PMID:26365921

  15. Health Screenings and Immunizations

    MedlinePlus

    ... your primary doctor. Blood Tests – A Common Screening Method (Source: National Heart, Lung, and Blood Institute) Click ... tests, see What Are Blood Tests? Other Screening Methods Doctors can't screen for all diseases and ...

  16. Screen time and children

    MedlinePlus

    "Screen time" is a term used for activities done in front of a screen, such as watching TV, working on a computer, or playing video games. Screen time is sedentary activity, meaning you are being physically ...

  17. What Is Carrier Screening?

    MedlinePlus

    ... you want to learn. Search form Search Carrier screening You are here Home Testing & Services Testing for ... help you make the decision. What Is Carrier Screening? Carrier screening checks if a person is a " ...

  18. RBC Antibody Screen

    MedlinePlus

    ... be limited. Home Visit Global Sites Search Help? RBC Antibody Screen Share this page: Was this page ... Screen Related tests: Direct Antiglobulin Test ; Blood Typing ; RBC Antibody Identification ; Type and Screen; Crossmatch All content ...

  19. The utilization of activity-based cost accounting in hospitals.

    PubMed

    Emmett, Dennis; Forget, Robert

    2005-01-01

    Healthcare costs are being examined on all fronts. Healthcare accounts for 11% of the gross national product and will continue to rise as the "babyboomers" reach retirement age. While ascertaining costs is important, most research shows that costing methods have not been implemented in hospitals. This study is concerned with the use of costing methods; particularly activity-based cost accounting. A mail survey of CFOs was undertaken to determine the type of cost accounting method they use. In addition, they were asked whether they were aware of activity-based cost accounting and whether they had implemented it or were planning to implement it. Only 71.8% were aware of it and only 4.7% had implemented it. In addition, only 52% of all hospitals report using any cost accounting systems. Education needs to ensure that all healthcare executives are cognizant of activity-based accounting and its importance in determining costs. Only by determining costs can hospitals strive to contain them. PMID:16201419

  20. Putting the Screen in Screening

    PubMed Central

    Harris, Sion Kim; Knight, John R.

    2014-01-01

    Alcohol is strongly linked to the leading causes of adolescent and adult mortality and health problems, making medical settings such as primary care and emergency departments important venues for addressing alcohol use. Extensive research evidence supports the effectiveness of alcohol screening and brief interventions (SBIs) in medical settings, but this valuable strategy remains underused, with medical staff citing lack of time and training as major implementation barriers. Technology-based tools may offer a way to improve efficiency and quality of SBI delivery in such settings. This review describes the latest research examining the feasibility and efficacy of computer- or other technology-based alcohol SBI tools in medical settings, as they relate to the following three patient populations: adults (18 years or older); pregnant women; and adolescents (17 years or younger). The small but growing evidence base generally shows strong feasibility and acceptability of technology-based SBI in medical settings. However, evidence for effectiveness in changing alcohol use is limited in this young field. PMID:26259001

  1. What's My Substrate? Computational Function Assignment of Candida parapsilosis ADH5 by Genome Database Search, Virtual Screening, and QM/MM Calculations.

    PubMed

    Dhoke, Gaurao V; Ensari, Yunus; Davari, Mehdi D; Ruff, Anna Joëlle; Schwaneberg, Ulrich; Bocola, Marco

    2016-07-25

    Zinc-dependent medium chain reductase from Candida parapsilosis can be used in the reduction of carbonyl compounds to pharmacologically important chiral secondary alcohols. To date, the nomenclature of cpADH5 is differing (CPCR2/RCR/SADH) in the literature, and its natural substrate is not known. In this study, we utilized a substrate docking based virtual screening method combined with KEGG, MetaCyc pathway, and Candida genome databases search for the discovery of natural substrates of cpADH5. The virtual screening of 7834 carbonyl compounds from the ZINC database provided 94 aldehydes or methyl/ethyl ketones as putative carbonyl substrates. Out of which, 52 carbonyl substrates of cpADH5 with catalytically active docking pose were identified by employing mechanism based substrate docking protocol. Comparison of the virtual screening results with KEGG, MetaCyc database search, and Candida genome pathway analysis suggest that cpADH5 might be involved in the Ehrlich pathway (reduction of fusel aldehydes in leucine, isoleucine, and valine degradation). Our QM/MM calculations and experimental activity measurements affirmed that butyraldehyde substrates are the potential natural substrates of cpADH5, suggesting a carbonyl reductase role for this enzyme in butyraldehyde reduction in aliphatic amino acid degradation pathways. Phylogenetic tree analysis of known ADHs from Candida albicans shows that cpADH5 is close to caADH5. We therefore propose, according to the experimental substrate identification and sequence similarity, the common name butyraldehyde dehydrogenase cpADH5 for Candida parapsilosis CPCR2/RCR/SADH. PMID:27387009

  2. High throughput screening of biologically functional small molecules for modulating the expression of FGFR1OP2/wit3.0 in fibroblasts.

    PubMed

    Cheng, William; Nishimura, Ichiro

    2012-12-01

    Oral wounds heal rapidly without scarring through yet unknown molecular mechanisms. A small cytoskeleton molecule identified in oral wound fibroblasts, FGFR1OP2/wit3.0, has been shown to accelerate wound closure in vitro and in vivo. The objective of this study was to elucidate the transcriptional mechanism of FGFR1OP2/ wit3.0 in fibroblasts using a high throughput drug-screening platform. This pilot study identified chemical compounds that could effectively modulate the FGFR1OP2/wit3.0 expression for future studies on effective wound management. PMID:23362665

  3. Screened-exchange density functional theory description of the electronic structure and phase stability of the chalcopyrite materials AgInSe2 and AuInSe2

    NASA Astrophysics Data System (ADS)

    Kim, Namhoon; Martin, Pamela Peña; Rockett, Angus A.; Ertekin, Elif

    2016-04-01

    We present a systematic assessment of the structural properties, the electronic density of states, the charge densities, and the phase stabilities of AgInSe2 and AuInSe2 using screened-exchange hybrid density functional theory, and compare their properties to those of CuInSe2. For AgInSe2, hybrid density functional theory properly captures several experimentally measured properties, including the increase in the band gap and the change in the direction of the lattice distortion parameter u in comparison to CuInSe2. While the electronic properties of AuInSe2 have not yet been experimentally characterized, we predict it to be a small gap (≈0.15 eV) semiconductor. We also present the phase stability of AgInSe2 and AuInSe2 according to screened-exchange density functional theory, and compare the results to predictions from conventional density functional theory, results tabulated from several online materials data repositories, and experiment (when available). In comparison to conventional density functional theory, the hybrid functional predicts phase stabilities of AgInSe2 in better agreement with experiment: discrepancies in the calculated formation enthalpies are reduced by approximately a factor of 3, from ≈0.20 eV/atom to ≈0.07 eV/atom, similar to the improvement observed for CuInSe2. We further predict that AuInSe2 is not a stable phase, and can only be present under nonequilibrium conditions.

  4. Unbiased Cell-based Screening in a Neuronal Cell Model of Batten Disease Highlights an Interaction between Ca2+ Homeostasis, Autophagy, and CLN3 Protein Function.

    PubMed

    Chandrachud, Uma; Walker, Mathew W; Simas, Alexandra M; Heetveld, Sasja; Petcherski, Anton; Klein, Madeleine; Oh, Hyejin; Wolf, Pavlina; Zhao, Wen-Ning; Norton, Stephanie; Haggarty, Stephen J; Lloyd-Evans, Emyr; Cotman, Susan L

    2015-06-01

    Abnormal accumulation of undigested macromolecules, often disease-specific, is a major feature of lysosomal and neurodegenerative disease and is frequently attributed to defective autophagy. The mechanistic underpinnings of the autophagy defects are the subject of intense research, which is aided by genetic disease models. To gain an improved understanding of the pathways regulating defective autophagy specifically in juvenile neuronal ceroid lipofuscinosis (JNCL or Batten disease), a neurodegenerative disease of childhood, we developed and piloted a GFP-microtubule-associated protein 1 light chain 3 (GFP-LC3) screening assay to identify, in an unbiased fashion, genotype-sensitive small molecule autophagy modifiers, employing a JNCL neuronal cell model bearing the most common disease mutation in CLN3. Thapsigargin, a sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) Ca(2+) pump inhibitor, reproducibly displayed significantly more activity in the mouse JNCL cells, an effect that was also observed in human-induced pluripotent stem cell-derived JNCL neural progenitor cells. The mechanism of thapsigargin sensitivity was Ca(2+)-mediated, and autophagosome accumulation in JNCL cells could be reversed by Ca(2+) chelation. Interrogation of intracellular Ca(2+) handling highlighted alterations in endoplasmic reticulum, mitochondrial, and lysosomal Ca(2+) pools and in store-operated Ca(2+) uptake in JNCL cells. These results further support an important role for the CLN3 protein in intracellular Ca(2+) handling and in autophagic pathway flux and establish a powerful new platform for therapeutic screening. PMID:25878248

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

    PubMed

    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

    2015-08-01

    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 3 pre-mRNA processing factors (PRPF6, PRPF8 and PRPF31) mutated in autosomal dominant retinitis pigmentosa. The PRPFs localize 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, also known as CEP90, and C21orf2, also known as 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

  6. Unbiased Cell-based Screening in a Neuronal Cell Model of Batten Disease Highlights an Interaction between Ca2+ Homeostasis, Autophagy, and CLN3 Protein Function*

    PubMed Central

    Chandrachud, Uma; Walker, Mathew W.; Simas, Alexandra M.; Heetveld, Sasja; Petcherski, Anton; Klein, Madeleine; Oh, Hyejin; Wolf, Pavlina; Zhao, Wen-Ning; Norton, Stephanie; Haggarty, Stephen J.; Lloyd-Evans, Emyr; Cotman, Susan L.

    2015-01-01

    Abnormal accumulation of undigested macromolecules, often disease-specific, is a major feature of lysosomal and neurodegenerative disease and is frequently attributed to defective autophagy. The mechanistic underpinnings of the autophagy defects are the subject of intense research, which is aided by genetic disease models. To gain an improved understanding of the pathways regulating defective autophagy specifically in juvenile neuronal ceroid lipofuscinosis (JNCL or Batten disease), a neurodegenerative disease of childhood, we developed and piloted a GFP-microtubule-associated protein 1 light chain 3 (GFP-LC3) screening assay to identify, in an unbiased fashion, genotype-sensitive small molecule autophagy modifiers, employing a JNCL neuronal cell model bearing the most common disease mutation in CLN3. Thapsigargin, a sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) Ca2+ pump inhibitor, reproducibly displayed significantly more activity in the mouse JNCL cells, an effect that was also observed in human-induced pluripotent stem cell-derived JNCL neural progenitor cells. The mechanism of thapsigargin sensitivity was Ca2+-mediated, and autophagosome accumulation in JNCL cells could be reversed by Ca2+ chelation. Interrogation of intracellular Ca2+ handling highlighted alterations in endoplasmic reticulum, mitochondrial, and lysosomal Ca2+ pools and in store-operated Ca2+ uptake in JNCL cells. These results further support an important role for the CLN3 protein in intracellular Ca2+ handling and in autophagic pathway flux and establish a powerful new platform for therapeutic screening. PMID:25878248

  7. New glucosidase activities identified by functional screening of a genomic DNA library from the gut microbiota of the termite Reticulitermes santonensis.

    PubMed

    Mattéotti, Christel; Thonart, Philippe; Francis, Frédéric; Haubruge, Eric; Destain, Jacqueline; Brasseur, Catherine; Bauwens, Julien; De Pauw, Edwin; Portetelle, Daniel; Vandenbol, Micheline

    2011-12-20

    β-Glucosidases are widely distributed in living organisms and play a major role in the degradation of wood, hydrolysing cellobiose or cello-oligosaccharides to glucose. Termites are among the rare animals capable of digesting wood, thanks to enzyme activities of their own and to enzymes produced by their gut microbiota. Many bacteria have been identified in the guts of lower termites, some of which possess cellulolytic or/and hemicellulolytic activity, required for digesting wood. Here, having isolated bacterial colonies from the gut of Reticulitermes santonensis, we constructed in Escherichia coli a genomic DNA library corresponding to all of the colonies obtained and screened the library for clones displaying β-glucosidase activity. This screen revealed 8 positive clones. Sequence analysis with the BLASTX program revealed putative enzymes belonging to three glycoside hydrolase families (GH1, GH3 and GH4). Agar-plate tests and enzymatic assays revealed differences between the GH1- and GH3-type enzymes (as regards substrate specificity and regulation) and a difference in substrate specificity within the GH3 group. The substrate specificities and characteristic activities of these enzymes suggest that they may intervene in the depolymerisation of cellulose and hemicellulose. PMID:21324659

  8. Clinical and molecular spectra in galactosemic patients from neonatal screening in northeastern Italy: structural and functional characterization of new variations in the galactose-1-phosphate uridyltransferase (GALT) gene.

    PubMed

    Viggiano, E; Marabotti, A; Burlina, A P; Cazzorla, C; D'Apice, M R; Giordano, L; Fasan, I; Novelli, G; Facchiano, A; Burlina, A B

    2015-04-01

    Classical galactosemia is an autosomal recessive inborn error of metabolism due to mutations of the GALT gene leading to toxic accumulation of galactose and derived metabolites. With the benefit of early diagnosis by neonatal screening and early therapy, the acute presentation of classical galactosemia can be prevented. However, despite early diagnosis and treatment, the long term outcome for these patients is still unpredictable because they may go on to develop cognitive disability, speech problems, neurological and/or movement disorders and, in females, ovarian dysfunction. The objectives of the current study were to report our experience with a group of galactosemic patients identified through the neonatal screening programs in northeastern Italy during the last 30years. No neonatal deaths due to galactosemia complications occurred after the introduction of the neonatal screening program. However, despite the early diagnosis and dietary treatment, the patients with classical galactosemia showed one or more long-term complications. A total of 18 different variations in the GALT gene were found in the patient cohort: 12 missense, 2 frameshift, 1 nonsense, 1 deletion, 1 silent variation, and 1 intronic. Six (p.R33P, p.G83V, p.P244S, p.L267R, p.L267V, p.E271D) were new variations. The most common variation was p.Q188R (12 alleles, 31.5%), followed by p.K285N (6 alleles, 15.7%) and p.N314D (6 alleles, 15.7%). The other variations comprised 1 or 2 alleles. In the patients carrying a new mutation, the biochemical analysis of GALT activity in erythrocytes showed an activity of <1%. In silico analysis (SIFT, PolyPhen-2 and the computational analysis on the static protein structure) showed potentially damaging effects of the six new variations on the GALT protein, thus expanding the genetic spectrum of GALT variations in Italy. The study emphasizes the difficulty in establishing a genotype-phenotype correlation in classical galactosemia and underlines the importance of

  9. Using activity-based costing to guide strategic decision making.

    PubMed

    Dowless, R M

    1997-06-01

    Activity-based costing (ABC) is not widely used in the healthcare industry. Some healthcare provider organizations are considering ABC, however, because of its potential to improve resource management and thereby maximize efficiency. ABC supports better pricing practices through more accurate costing and can be used to identify underutilized resources as well as associated costs that can be reduced. ABC can be a useful tool for determining the cost of unused capacity and for making strategic management decisions that will reduce costs. PMID:10167847

  10. Applying activity-based costing in long-term care.

    PubMed

    Wodchis, W P

    1998-01-01

    As greater numbers of the elderly use health services, and as health care costs climb, effective financial tracking is essential. Cost management in health care can benefit if costs are linked to the care activities where they are incurred. Activity-based costing (ABC) provides a useful approach. The framework aligns costs (inputs), through activities (process), to outputs and outcomes. It allocates costs based on client care needs rather than management structure. The ABC framework was tested in a residential care facility and in supportive housing apartments. The results demonstrate the feasibility and advantages of ABC for long term care agencies, including community-based care. PMID:10339203

  11. A genome-scale in vivo loss-of-function screen identifies Phf6 as a lineage-specific regulator of leukemia cell growth

    PubMed Central

    Meacham, Corbin E.; Lawton, Lee N.; Soto-Feliciano, Yadira M.; Pritchard, Justin R.; Joughin, Brian A.; Ehrenberger, Tobias; Fenouille, Nina; Zuber, Johannes; Williams, Richard T.; Young, Richard A.

    2015-01-01

    We performed a genome-scale shRNA screen for modulators of B-cell leukemia progression in vivo. Results from this work revealed dramatic distinctions between the relative effects of shRNAs on the growth of tumor cells in culture versus in their native microenvironment. Specifically, we identified many “context-specific” regulators of leukemia development. These included the gene encoding the zinc finger protein Phf6. While inactivating mutations in PHF6 are commonly observed in human myeloid and T-cell malignancies, we found that Phf6 suppression in B-cell malignancies impairs tumor progression. Thus, Phf6 is a “lineage-specific” cancer gene that plays opposing roles in developmentally distinct hematopoietic malignancies. PMID:25737277

  12. Functional Screening of the Cronobacter sakazakii BAA-894 Genome reveals a role for ProP (ESA_02131) in carnitine uptake

    PubMed Central

    Feeney, Audrey; Sleator, Roy D

    2015-01-01

    Cronobacter sakazakii is a neonatal pathogen responsible for up to 80% of fatalities in infected infants. Low birth weight infants and neonates infected with C. sakazakii suffer necrotizing enterocolitis, bacteraemia and meningitis. The mode of transmission most often associated with infection is powdered infant formula (PIF) which, with an aw of ∼0.2, is too low to allow most microorganisms to persist. Survival of C. sakazakii in environments subject to extreme hyperosmotic stress has previously been attributed to the uptake of compatible solutes including proline and betaine. Herein, we report the construction and screening of a C. sakazakii genome bank and the identification of ProP (ESA_02131) as a carnitine uptake system. PMID:25915804

  13. Plasmodium falciparum CTP:phosphocholine cytidylyltransferase possesses two functional catalytic domains and is inhibited by a CDP-choline analog selected from a virtual screening.

    PubMed

    Contet, Alicia; Pihan, Emilie; Lavigne, Marina; Wengelnik, Kai; Maheshwari, Sweta; Vial, Henri; Douguet, Dominique; Cerdan, Rachel

    2015-04-13

    Phosphatidylcholine is the major lipid component of the malaria parasite membranes and is required for parasite multiplication in human erythrocytes. Plasmodium falciparum CTP:phosphocholine cytidylyltransferase (PfCCT) is the rate-limiting enzyme of the phosphatidylcholine biosynthesis pathway and thus considered as a potential antimalarial target. In contrast to its mammalian orthologs, PfCCT contains a duplicated catalytic domain. Here, we show that both domains are catalytically active with similar kinetic parameters. A virtual screening strategy allowed the identification of a drug-size molecule competitively inhibiting the enzyme. This compound also prevented phosphatidylcholine biosynthesis in parasites and exerted an antimalarial effect. This study constitutes the first step towards a rationalized design of future new antimalarial agents targeting PfCCT. PMID:25771858

  14. Post-docking virtual screening of diverse binding pockets: comparative study using DOCK, AMMOS, X-Score and FRED scoring functions.

    PubMed

    Pencheva, Tania; Soumana, Oumarou Samna; Pajeva, Ilza; Miteva, Maria A

    2010-06-01

    Most of the benchmark studies on docking-scoring methods reported in the last decade conclude that no single scoring function performs well across different protein targets. In this study a comparison of thirteen commonly used force field and empirical scoring functions as implemented in DOCK, AMMOS, X-Score and FRED is carried out on five proteins with diverse binding pockets. The performance is analyzed in relation to the physicochemical properties of the binding sites. The solvation effects are considered via the Generalized Born/Surface Area (GBSA) solvation method for one of the assessed scoring functions. We examined the ability of these scoring functions to discriminate between active and inactive compounds over receptor-based focused libraries. Our results demonstrated that the employed here empirical scoring functions were more appropriate for the pocket of predominant hydrophobic nature while the force field scoring functions performed better on the mixed or polar pockets. PMID:20227800

  15. Defining responsibility for screening.

    PubMed

    Sifri, R; Wender, R

    1999-10-01

    Patients commonly receive medical care from multiple providers and confusion as to who is responsible for cancer screening undoubtedly contributes to inadequate recommendations. Effective screening requires successful implementation of a series of steps that begin with the initial discussion of a screening test and proceed through obtaining results and instituting appropriate follow-up. Clear definition of generalist and specialist physician roles are necessary to optimally screen the public. This article explores the differences in how generalists and specialists approach screening, describes models of care that facilitate shared responsibility for screening, and suggests strategies on how to improve communication between physicians to maximize screening performance. PMID:10452930

  16. Noise Reduction in High-Throughput Gene Perturbation Screens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Motivation: Accurate interpretation of perturbation screens is essential for a successful functional investigation. However, the screened phenotypes are often distorted by noise, and their analysis requires specialized statistical analysis tools. The number and scope of statistical methods available...

  17. Environmental Test Screening Procedure

    NASA Technical Reports Server (NTRS)

    Zeidler, Janet

    2000-01-01

    This procedure describes the methods to be used for environmental stress screening (ESS) of the Lightning Mapper Sensor (LMS) lens assembly. Unless otherwise specified, the procedures shall be completed in the order listed, prior to performance of the Acceptance Test Procedure (ATP). The first unit, S/N 001, will be subjected to the Qualification Vibration Levels, while the remainder will be tested at the Operational Level. Prior to ESS, all units will undergo Pre-ESS Functional Testing that includes measuring the on-axis and plus or minus 0.95 full field Modulation Transfer Function and Back Focal Length. Next, all units will undergo ESS testing, and then Acceptance testing per PR 460.

  18. Plasma Screen Floating Mount

    DOEpatents

    Eakle, Robert F.; Pak, Donald J.

    2004-10-26

    A mounting system for a flat display screen, particularly a plasma display screen, suspends the screen separately in each of the x-, y- and z-directions. A series of frames located by linear bearings and isolated by springs and dampers allows separate controlled movement in each axis. The system enables the use of relatively larger display screens in vehicles in which plasma screen are subject to damage from vibration.

  19. Advancing understanding of microbial bioenergy conversion processes by activity-based protein profiling

    SciTech Connect

    Liu, Yun; Fredrickson, James K.; Sadler, Natalie C.; Nandhikonda, Premchendar; Smith, Richard D.; Wright, Aaron T.

    2015-09-25

    Here, the development of renewable biofuels is a global priority, but success will require novel technologies that greatly improve our understanding of microbial systems biology. An approach with great promise in enabling functional characterization of microbes is activity-based protein profiling (ABPP), which employs chemical probes to directly measure enzyme function in discrete enzyme classes in vivo and/or in vitro, thereby facilitating the rapid discovery of new biocatalysts and enabling much improved biofuel production platforms. We review general design strategies in ABPP, and highlight recent advances that are or could be pivotal to biofuels processes including applications of ABPP to cellulosic bioethanol, biodiesel, and phototrophic production of hydrocarbons. We also examine the key challenges and opportunities of ABPP in renewable biofuels research. The integration of ABPP with molecular and systems biology approaches will shed new insight on the catalytic and regulatory mechanisms of functional enzymes and their synergistic effects in the field of biofuels production.

  20. Activity-based analyses lead to better decision making.

    PubMed

    Player, S

    1998-08-01

    Activity-based costing (ABC) and activity-based management (ABM) are cost-management tools that are relatively new to the healthcare industry. ABC is used for strategic decision making. It assesses the costs associated with specific activities and resources and links those costs to specific internal and external customers of the healthcare enterprise (e.g., patients, service lines, and physician groups) to determine the costs associated with each customer. This cost information then can be adjusted to account for anticipated changes and to predict future costs. ABM, on the other hand, supports operations by focusing on the causes of costs and how costs can be reduced. It assesses cost drivers that directly affect the cost of a product or service, and uses performance measures to evaluate the financial or nonfinancial benefit an activity provides. By identifying each cost driver and assessing the value the element adds to the healthcare enterprise, ABM provides a basis for selecting areas that can be changed to reduce costs. PMID:10182280

  1. Risks of Breast Cancer Screening

    MedlinePlus

    ... of Breast & Gynecologic Cancers Breast Cancer Screening Research Breast Cancer Screening (PDQ®)–Patient Version What is screening? Go ... cancer screening: Cancer Screening Overview General Information About Breast Cancer Key Points Breast cancer is a disease in ...

  2. Luminescent screen composition for cathode ray tubes

    NASA Technical Reports Server (NTRS)

    Hilborn, E. H.

    1968-01-01

    Screen composition for cathode ray tubes exhibits differential color of emission as a function of beam current variation at a constant accelerating voltage. The screen consists of a mixture of phosphors which emit different hues, have different current saturation values and exhibit a nonlinear current-brightness characteristic.

  3. RNAi Screening in Spodoptera frugiperda.

    PubMed

    Ghosh, Subhanita; Singh, Gatikrushna; Sachdev, Bindiya; Kumar, Ajit; Malhotra, Pawan; Mukherjee, Sunil K; Bhatnagar, Raj K

    2016-01-01

    RNA interference is a potent and precise reverse genetic approach to carryout large-scale functional genomic studies in a given organism. During the past decade, RNAi has also emerged as an important investigative tool to understand the process of viral pathogenesis. Our laboratory has successfully generated transgenic reporter and RNAi sensor line of Spodoptera frugiperda (Sf21) cells and developed a reversal of silencing assay via siRNA or shRNA guided screening to investigate RNAi factors or viral pathogenic factors with extraordinary fidelity. Here we describe empirical approaches and conceptual understanding to execute successful RNAi screening in Spodoptera frugiperda 21-cell line. PMID:27581295

  4. A Functional Genomic Screen Combined with Time-Lapse Microscopy Uncovers a Novel Set of Genes Involved in Dorsal Closure of Drosophila Embryos

    PubMed Central

    Jankovics, Ferenc; Henn, László; Bujna, Ágnes; Vilmos, Péter; Kiss, Nóra; Erdélyi, Miklós

    2011-01-01

    Morphogenesis, the establishment of the animal body, requires the coordinated rearrangement of cells and tissues regulated by a very strictly-determined genetic program. Dorsal closure of the epithelium in the Drosophila melanogaster embryo is one of the best models for such a complex morphogenetic event. To explore the genetic regulation of dorsal closure, we carried out a large-scale RNA interference-based screen in combination with in vivo time-lapse microscopy and identified several genes essential for the closure or affecting its dynamics. One of the novel dorsal closure genes, the small GTPase activator pebble (pbl), was selected for detailed analysis. We show that pbl regulates actin accumulation and protrusion dynamics in the leading edge of the migrating epithelial cells. In addition, pbl affects dorsal closure dynamics by regulating head involution, a morphogenetic process mechanically coupled with dorsal closure. Finally, we provide evidence that pbl is involved in closure of the adult thorax, suggesting its general requirement in epithelial closure processes. PMID:21799798

  5. Reverse genetic screen for loss-of-function mutations uncovers a frameshifting deletion in the melanophilin gene accountable for a distinctive coat color in Belgian Blue cattle.

    PubMed

    Li, Wanbo; Sartelet, Arnaud; Tamma, Nico; Coppieters, Wouter; Georges, Michel; Charlier, Carole

    2016-02-01

    In the course of a reverse genetic screen in the Belgian Blue cattle breed, we uncovered a 10-bp deletion (c.87_96del) in the first coding exon of the melanophilin gene (MLPH), which introduces a premature stop codon (p.Glu32Aspfs*1) in the same exon, truncating 94% of the protein. Recessive damaging mutations in the MLPH gene are well known to cause skin, hair, coat or plumage color dilution phenotypes in numerous species, including human, mice, dog, cat, mink, rabbit, chicken and quail. Large-scale array genotyping undertaken to identify p.Glu32Aspfs*1 homozygous mutant animals revealed a mutation frequency of 5% in the breed and allowed for the identification of 10 homozygous mutants. As expression of a colored coat requires at least one wild-type allele at the co-dominant Roan locus encoded by the KIT ligand gene (KITLG), homozygous mutants for p.Ala227Asp corresponding with the missense mutation were excluded. The six remaining colored calves displayed a distinctive dilution phenotype as anticipated. This new coat color was named 'cool gray'. It is the first damaging mutation in the MLPH gene described in cattle and extends the already long list of species with diluted color due to recessive mutations in MLPH and broadens the color palette of gray in this breed. PMID:26582259

  6. Screening for cholesterol-lowering probiotic based on deoxycholic acid removal pathway and studying its functional mechanisms in vitro.

    PubMed

    Guo, Chun-Feng; Zhang, Lan-Wei; Han, Xue; Yi, Hua-Xi; Li, Jing-Yan; Tuo, Yan-Feng; Zhang, Ying-Chun; Du, Ming; Shan, Yu-Juan; Yang, Lin

    2012-10-01

    Elevated serum cholesterol in humans is generally a risk factor correlated with the development of coronary heart disease (CHD). Reducing deoxycholic acid (DCA) content in the intestine can reduce serum cholesterol levels, which reduce the incidence of CHD. A total of 150 strains of lactic acid bacteria and bifidobacteria were isolated from human fecal samples. The DCA removal ability of these strains was evaluated. Results showed that 9 strains displayed above 10% DCA removal rate. The probiotic potentials of the 9 strains were evaluated. The strain Lactobacillus casei F0822 was screened out due to the stronger adhesion to HT-29 cells and tolerance to bile and acid. DCA removal for this strain resulted from that the S-layer protein locating the cell surface bound DCA. The FTIR spectra showed that the carboxyl group in DCA was the principal group by which DCA was bound to the S-layer protein of L. casei F0822. These findings suggested that L. casei F0822 is a better candidate probiotic strain, which has the potential to reduce human serum cholesterol levels. PMID:22926345

  7. Functional, chemical genomic, and super-enhancer screening identify sensitivity to cyclin D1/CDK4 pathway inhibition in Ewing sarcoma

    PubMed Central

    Crompton, Brian; Cowley, Glenn; Vazquez, Francisca; Weir, Barbara A.; Tsherniak, Aviad; Parasuraman, Sudha; Kim, Sunkyu; Alexe, Gabriela; Stegmaier, Kimberly

    2015-01-01

    Ewing sarcoma is an aggressive bone and soft tissue tumor in children and adolescents, with treatment remaining a clinical challenge. This disease is mediated by somatic chromosomal translocations of the EWS gene and a gene encoding an ETS transcription factor, most commonly, FLI1. While direct targeting of aberrant transcription factors remains a pharmacological challenge, identification of dependencies incurred by EWS/FLI1 expression would offer a new therapeutic avenue. We used a combination of super-enhancer profiling, near-whole genome shRNA-based and small-molecule screening to identify cyclin D1 and CDK4 as Ewing sarcoma-selective dependencies. We revealed that super-enhancers mark Ewing sarcoma specific expression signatures and EWS/FLI1 target genes in human Ewing sarcoma cell lines. Particularly, a super-enhancer regulates cyclin D1 and promotes its expression in Ewing sarcoma. We demonstrated that Ewing sarcoma cells require CDK4 and cyclin D1 for survival and anchorage-independent growth. Additionally, pharmacologic inhibition of CDK4 with selective CDK4/6 inhibitors led to cytostasis and cell death of Ewing sarcoma cell lines in vitro and growth delay in an in vivo Ewing sarcoma xenograft model. These results demonstrated a dependency in Ewing sarcoma on CDK4 and cyclin D1 and support exploration of CDK4/6 inhibitors as a therapeutic approach for patients with this disease. PMID:26337082

  8. Confirming target engagement for reversible inhibitors in vivo by kinetically tuned activity-based probes.

    PubMed

    Adibekian, Alexander; Martin, Brent R; Chang, Jae Won; Hsu, Ku-Lung; Tsuboi, Katsunori; Bachovchin, Daniel A; Speers, Anna E; Brown, Steven J; Spicer, Timothy; Fernandez-Vega, Virneliz; Ferguson, Jill; Hodder, Peter S; Rosen, Hugh; Cravatt, Benjamin F

    2012-06-27

    The development of small-molecule inhibitors for perturbing enzyme function requires assays to confirm that the inhibitors interact with their enzymatic targets in vivo. Determining target engagement in vivo can be particularly challenging for poorly characterized enzymes that lack known biomarkers (e.g., endogenous substrates and products) to report on their inhibition. Here, we describe a competitive activity-based protein profiling (ABPP) method for measuring the binding of reversible inhibitors to enzymes in animal models. Key to the success of this approach is the use of activity-based probes that show tempered rates of reactivity with enzymes, such that competition for target engagement with reversible inhibitors can be measured in vivo. We apply the competitive ABPP strategy to evaluate a newly described class of piperazine amide reversible inhibitors for the serine hydrolases LYPLA1 and LYPLA2, two enzymes for which selective, in vivo active inhibitors are lacking. Competitive ABPP identified individual piperazine amides that selectively inhibit LYPLA1 or LYPLA2 in mice. In summary, competitive ABPP adapted to operate with moderately reactive probes can assess the target engagement of reversible inhibitors in animal models to facilitate the discovery of small-molecule probes for characterizing enzyme function in vivo. PMID:22690931

  9. Confirming Target Engagement for Reversible Inhibitors In Vivo by Kinetically Tuned Activity-Based Probes

    PubMed Central

    Adibekian, Alexander; Martin, Brent R.; Chang, Jae Won; Hsu, Ku-Lung; Tsuboi, Katsunori; Bachovchin, Daniel A.; Speers, Anna E.; Brown, Steven J.; Spicer, Timothy; Fernandez-Vega, Virneliz; Ferguson, Jill; Hodder, Peter S.; Rosen, Hugh; Cravatt, Benjamin F.

    2012-01-01

    The development of small-molecule inhibitors for perturbing enzyme function requires assays to confirm that the inhibitors interact with their enzymatic targets in vivo. Determining target engagement in vivo can be particularly challenging for poorly characterized enzymes that lack known biomarkers (e.g., endogenous substrates and products) to report on their inhibition. Here, we describe a competitive activity-based protein profiling (ABPP) method for measuring the binding of reversible inhibitors to enzymes in animal models. Key to the success of this approach is the use of activity-based probes that show tempered rates of reactivity with enzymes, such that competition for target engagement with reversible inhibitors can be measured in vivo. We apply the competitive ABPP strategy to evaluate a newly described class of piperazine amide reversible inhibitors for the serine hydrolases LYPAL1 and LYPLA2, two enzymes for which selective, in vivo-active inhibitors are lacking. Competitive ABPP identified individual piperazine amides that selectively inhibit LYPLA1 or LYPLA2 in mice. In summary, competitive ABPP adapted to operate with moderately reactive probes can assess the target engagement of reversible inhibitors in animal models to facilitate the discovery of small-molecule probes for characterizing enzyme function in vivo. PMID:22690931

  10. Neurobiology driving hyperactivity in activity-based anorexia.

    PubMed

    Adan, R A H; Hillebrand, J J G; Danner, U N; Cardona Cano, S; Kas, M J H; Verhagen, L A W

    2011-01-01

    Hyperactivity in anorexia nervosa is difficult to control and negatively impacts outcome. Hyperactivity is a key driving force to starvation in an animal model named activity-based anorexia (ABA). Recent research has started unraveling what mechanisms underlie this hyperactivity. Besides a general increase in locomotor activity that may be an expression of foraging behavior and involves frontal brain regions, the increased locomotor activity expressed before food is presented (food anticipatory behavior or FAA) involves hypothalamic neural circuits. Ghrelin plays a role in FAA, whereas decreased leptin signaling is involved in both aspects of increased locomotor activity. We hypothesize that increased ghrelin and decreased leptin signaling drive the activity of dopamine neurons in the ventral tegmental area. In anorexia nervosa patients, this altered activity of the dopamine system may be involved not only in hyperactivity but also in aberrant cognitive processing related to food. PMID:21243479

  11. Activity-based intelligence tipping and cueing using polarimetric sensors

    NASA Astrophysics Data System (ADS)

    Lewis, Christian M.; Messinger, David; Gartley, Michael G.

    2014-05-01

    Activity Based Intelligence (ABI) is the derivation of information from the composite of a series of individual actions being recorded over a period of time. Due to its temporal nature, ABI is usually developed from Motion Imagery (MI) or Full Motion Video (FMV) taken of a given scene. One of today's common issues is sifting through such large volumes of temporal data. Here we propose using a technique known as tipping an cueing to alleviate the need to manually sift through said data. Being able to tip the analysts or automated algorithm towards a particular person or object in the data is useful in reducing search time. We propose using a polarimetric sensor to identify objects of interest, in a scene where their signature would be unusual. Once identified, this data will be used to cue a FMV RGB sensor to track the object and determine the activities being executed by the person bringing the object into the scene.

  12. Activity-Based Probe for N-Acylethanolamine Acid Amidase.

    PubMed

    Romeo, Elisa; Ponzano, Stefano; Armirotti, Andrea; Summa, Maria; Bertozzi, Fabio; Garau, Gianpiero; Bandiera, Tiziano; Piomelli, Daniele

    2015-09-18

    N-Acylethanolamine acid amidase (NAAA) is a lysosomal cysteine hydrolase involved in the degradation of saturated and monounsaturated fatty acid ethanolamides (FAEs), a family of endogenous lipid signaling molecules that includes oleoylethanolamide (OEA) and palmitoylethanolamide (PEA). Among the reported NAAA inhibitors, α-amino-β-lactone (3-aminooxetan-2-one) derivatives have been shown to prevent FAE hydrolysis in innate-immune and neural cells and to reduce reactions to inflammatory stimuli. Recently, we disclosed two potent and selective NAAA inhibitors, the compounds ARN077 (5-phenylpentyl-N-[(2S,3R)-2-methyl-4-oxo-oxetan-3-yl]carbamate) and ARN726 (4-cyclohexylbutyl-N-[(S)-2-oxoazetidin-3-yl]carbamate). The former is active in vivo by topical administration in rodent models of hyperalgesia and allodynia, while the latter exerts systemic anti-inflammatory effects in mouse models of lung inflammation. In the present study, we designed and validated a derivative of ARN726 as the first activity-based protein profiling (ABPP) probe for the in vivo detection of NAAA. The newly synthesized molecule 1 is an effective in vitro and in vivo click-chemistry activity based probe (ABP), which is able to capture the catalytically active form of NAAA in Human Embryonic Kidney 293 (HEK293) cells overexpressing human NAAA as well as in rat lung tissue. Competitive ABPP with 1 confirmed that ARN726 and ARN077 inhibit NAAA in vitro and in vivo. Compound 1 is a useful new tool to identify activated NAAA both in vitro and in vivo and to investigate the physiological and pathological roles of this enzyme. PMID:26102511

  13. Scientific and Regulatory Policy Committee (SRPC) Points to Consider*: Histopathology Evaluation of the Pubertal Development and Thyroid Function Assay (OPPTS 890.1450, OPPTS 890.1500) in Rats to Screen for Endocrine Disruptors

    PubMed Central

    Keane, Kevin A.; Parker, George A.; Regan, Karen S.; Picut, Catherine; Dixon, Darlene; Creasy, Dianne; Giri, Dipak; Hukkanen, Renee R.

    2015-01-01

    The U.S. Environmental Protection Agency Endocrine Disruptor Screening Program (EDSP) is a multitiered approach to determine the potential for environmental chemicals to alter the endocrine system. The Pubertal Development and Thyroid Function in Intact Juvenile/Peripubertal Female and Male Rats (OPPTS 890.1450, 890.1500) are 2 of the 9 EDSP tier 1 test Guidelines, which assess upstream mechanistic pathways along with downstream morphological end points including histological evaluation of the kidneys, thyroid, and select male/female reproductive tissues (ovaries, uterus, testes, and epididymides). These assays are part of a battery of in vivo and in vitro screens used for initial detection of test article endocrine activity. In this Points to Consider article, we describe tissue processing, evaluation, and nomenclature to aid in standardization of assay results across laboratories. Pubertal assay end points addressed include organ weights, estrous cyclicity, clinical pathology, hormonal assays, and histological evaluation. Potential treatment-related findings that may indicate endocrine disruption are reviewed. Additional tissues that may be useful in assessment of endocrine disruption (vagina, mammary glands, and liver) are discussed. This Points to Consider article is intended to provide information for evaluating peripubertal tissues within the context of individual assay end points, the overall pubertal assay, and tier I assays of the EDSP program. PMID:25948506

  14. Kinome wide functional screen identifies role of Polo-like kinase 1 (PLK1) in hormone-independent, ER-positive breast cancer

    PubMed Central

    Bhola, Neil; Jansen, Valerie M.; Bafna, Sangeeta; Giltnane, Jennifer M.; Balko, Justin M.; Estrada, Mónica V.; Meszoely, Ingrid; Mayer, Ingrid; Abramson, Vandana; Ye, Fei; Sanders, Melinda; Dugger, Teresa C.; Van Allen, Eliezer; Arteaga, Carlos L.

    2014-01-01

    Estrogen receptor α (ER)-positive breast cancers initially respond to antiestrogens but eventually become estrogen-independent and recur. ER+ breast cancer cells resistant to long-term estrogen deprivation (LTED) exhibit hormone-independent ER transcriptional activity and growth. A kinome-wide siRNA screen using a library targeting 720 kinases identified Polo-like kinase 1 (PLK1) as one of the top genes whose downregulation resulted in inhibition of estrogen-independent ER transcriptional activity and growth of LTED cells. High PLK1 mRNA and protein correlated with a high Ki67 score in primary ER+ breast cancers after treatment with the aromatase inhibitor letrozole. RNAi-mediated knockdown of PLK1 inhibited ER expression, estrogen-independent growth and ER transcription in MCF7 and HCC1428 LTED cells. Pharmacological inhibition of PLK1 with volasertib, a small molecule ATP-competitive PLK1 inhibitor, decreased LTED cell growth, ER transcriptional activity and ER expression. Volasertib in combination with the ER antagonist, fulvestrant, decreased MCF7 xenograft growth in ovariectomized mice more potently than each drug alone. JUNB, a component of the AP-1 complex, was expressed 16-fold higher in MCF7/LTED compared to parental MCF7 cells. Further, JUNB and BCL2L1 (which encodes anti-apoptotic BCL-xL) mRNA levels were markedly reduced upon volasertib treatment in MCF7/LTED cells while they were increased in parental MCF7 cells. Finally, JUNB knockdown decreased ER expression and transcriptional activity in MCF7/LTED cells, suggesting that PLK1 drives ER expression and estrogen-independent growth via JUNB. These data support a critical role of PLK1 in acquired hormone-independent growth of ER+ human breast cancer and is therefore a promising target in tumors that have escaped estrogen deprivation therapy. PMID:25480943

  15. A functional family-wide screening of SP/KLF proteins identifies a subset of suppressors of KRAS-mediated cell growth.

    PubMed

    Fernandez-Zapico, Martin E; Lomberk, Gwen A; Tsuji, Shoichiro; DeMars, Cathrine J; Bardsley, Michael R; Lin, Yi-Hui; Almada, Luciana L; Han, Jing-Jing; Mukhopadhyay, Debabrata; Ordog, Tamas; Buttar, Navtej S; Urrutia, Raul

    2011-04-15

    SP/KLF (Specificity protein/Krüppel-like factor) transcription factors comprise an emerging group of proteins that may behave as tumour suppressors. Incidentally, many cancers that display alterations in certain KLF proteins are also associated with a high incidence of KRAS (V-Ki-ras2 Kirsten rat sarcoma viral oncogene homologue) mutations. Therefore in the present paper we investigate whether SP/KLF proteins suppress KRAS-mediated cell growth, and more importantly, the potential mechanisms underlying these effects. Using a comprehensive family-wide screening of the 24 SP/KLF members, we discovered that SP5, SP8, KLF2, KLF3, KLF4, KLF11, KLF13, KLF14, KLF15 and KLF16 inhibit cellular growth and suppress transformation mediated by oncogenic KRAS. Each protein in this subset of SP/KLF members individually inhibits BrdU (5-bromo-2-deoxyuridine) incorporation in KRAS oncogenic-mutant cancer cells. SP5, KLF3, KLF11, KLF13, KLF14 and KLF16 also increase apoptosis in these cells. Using KLF11 as a representative model for mechanistic studies, we demonstrate that this protein inhibits the ability of cancer cells to form both colonies in soft agar and tumour growth in vivo. Molecular studies demonstrate that these effects of KLF11 are mediated, at least in part, through silencing cyclin A via binding to its promoter and leading to cell-cycle arrest in S-phase. Interestingly, similar to KLF11, KLF14 and KLF16 mechanistically share the ability to modulate the expression of cyclin A. Collectively, the present study stringently defines a distinct subset of SP/KLF proteins that impairs KRAS-mediated cell growth, and that mechanistically some members of this subset accomplish this, at least in part, through regulation of the cyclin A promoter. PMID:21171965

  16. Screening recommendations for the elderly.

    PubMed Central

    Beers, M H; Fink, A; Beck, J C

    1991-01-01

    BACKGROUND. Studies have documented the potential contributions of preventive health care programs. Yet little is known about which screening tests should be included in public health programs for older persons. This study offers recommendations regarding these tests. METHODS. The recommendations come from synthesizing the findings of the US Preventive Services Task Force, the literature, and the consensus of experts in geriatrics, gerontology, and health policy research. The literature was evaluated to identify methodologically sound studies of the prevalence of selected disorders and benefits and availability of screening procedures for those disorders. Experts from various fields specializing in the care of the elderly formed panels to assist in evaluating the literature and providing further information from gerontological and public health perspectives. RESULTS. We recommend vision testing for refractive error; inspection of the skin surface for fungal infection and skin cancer, drug eruptions, and xerosis; a history for symptoms of xerosis; audiometric testing for presbycusis; surveys for hearing loss; otoscopic inspection for cerumen impaction; dental examination for caries; measurement of blood pressure for hypertension; and breast examination and mammography for cancer. CONCLUSIONS. Our study suggests that these screening procedures are useful for public health screening programs. More information is needed on the effects of screening services on the health and functioning of older persons. PMID:1951823

  17. Development of bestatin-based activity-based probes for metallo-aminopeptidases

    PubMed Central

    Harbut, Michael B.; Velmourougane, Geetha; Reiss, Gilana; Chandramohanadas, Rajesh; Greenbaum, Doron C.

    2009-01-01

    A novel set of activity-based probes (ABPs) for functionally profiling metallo-aminopeptidases was synthesized based on the bestatin inhibitor scaffold, the first synthesis of bestatin analogues using solid-phase techniques. These ABPs were shown to label metallo-aminopeptidases, using both a biotin and a fluorophore reporter, in an activity-dependent manner. This probe class was also shown to be amenable to ‘click’ chemistry labeling for possible use in live cells. Finally, we demonstrate that the ABPs are able to label an aminopeptidase in a complex proteome. Thus, these bestatin-based probes should have wide utility to functionally profile aminopeptidases in many biological systems. PMID:18823778

  18. Examining the Screen for Child Anxiety-Related Emotional Disorder-71 as an Assessment Tool for Anxiety in Children with High-Functioning Autism Spectrum Disorders

    ERIC Educational Resources Information Center

    van Steensel, Francisca J. A.; Deutschman, Amber A. C. G.; Bögels, Susan M.

    2013-01-01

    The psychometric properties of a questionnaire developed to assess symptoms of anxiety disorders (SCARED-71) were compared between two groups of children: children with high-functioning autism spectrum disorder and comorbid anxiety disorders (ASD-group; "n" = 115), and children with anxiety disorders (AD-group; "n" = 122).…

  19. Functional requirements specification and data modeling for a PDA-based decision support system for the screening and management of obesity.

    PubMed

    Lee, Nam-Ju; John, Ritamarie; Bakken, Suzanne

    2006-01-01

    The purposes of this study were to identify the functional requirements of and to build the data model for a PDA-DSS. Through Use Case Analysis and UML modeling, seven Use Cases were documented, and Use Case Diagrams, Activity Diagrams, and Sequence Diagrams related to the management of were developed. PMID:17238621

  20. Screening for Prostate Cancer

    MedlinePlus

    ... of Internal Medicine Summaries for Patients Screening for Prostate Cancer: A Guidance Statement From the Clinical Guidelines Committee ... Physicians The full report is titled “Screening for Prostate Cancer: A Guidance Statement From the Clinical Guidelines Committee ...

  1. Prostate cancer screenings

    MedlinePlus

    ... this page: //medlineplus.gov/ency/patientinstructions/000846.htm Prostate cancer screenings To use the sharing features on this ... present it is not clear if screening for prostate cancer is helpful for most men. For this reason, ...

  2. Stomach (Gastric) Cancer Screening

    MedlinePlus

    ... Treatment Stomach Cancer Prevention Stomach Cancer Screening Research Stomach (Gastric) Cancer Screening (PDQ®)–Patient Version What is ... These are called diagnostic tests . General Information About Stomach (Gastric) Cancer Key Points Stomach cancer is a ...

  3. Screening for Gestational Diabetes

    MedlinePlus

    ... Task Force learned about the potential benefits and harms of screening for gestational diabetes: (1) All women ... not enough evidence to judge the benefits and harms of screening women before 24 weeks of pregnancy. ...

  4. Hearing Loss: Screening Newborns

    MedlinePlus

    ... of this page please turn JavaScript on. Feature: Hearing Loss Screening Newborns Past Issues / Spring 2015 Table of ... of newborns in the U.S. are screened for hearing loss before they leave the hospital. Research improves the ...

  5. Video Screen Capture Basics

    ERIC Educational Resources Information Center

    Dunbar, Laura

    2014-01-01

    This article is an introduction to video screen capture. Basic information of two software programs, QuickTime for Mac and BlueBerry Flashback Express for PC, are also discussed. Practical applications for video screen capture are given.

  6. Screening for Breast Problems

    MedlinePlus

    f AQ FREQUENTLY ASKED QUESTIONS FAQ178 GYNECOLOGIC PROBLEMS Mammography and Other Screening Tests for Breast Problems • What ... used to screen for breast problems? • What is mammography? • Why is mammography done? • When should I start ...

  7. Breast cancer screenings

    MedlinePlus

    ... this page: //medlineplus.gov/ency/patientinstructions/000837.htm Breast cancer screenings To use the sharing features on this page, please enable JavaScript. Breast cancer screenings can help find breast cancer early, before ...

  8. Health Screenings at School

    MedlinePlus

    ... Listen Español Text Size Email Print Share Health Screenings at School Page Content Article Body In most ... child's ability to learn. In some states these screening tests are mandated by law and may also ...

  9. Human Papillomavirus (HPV) Screening

    MedlinePlus

    ... Diseases HPV-Associated Cancers Gynecologic Cancers Redirect CDC - Screening Recommend on Facebook Tweet Share Compartir You are being redirected to the HPV Cancer Screening page. Please update your bookmarks to the link ...

  10. Screening Tests and Vaccines

    MedlinePlus

    ... Contact Us Text size | Print | Screening Tests and Vaccines This information in Spanish ( en español ) Getting important screening tests and vaccines can save your life. Check this section of ...

  11. Stretching Screens and Imaginations.

    ERIC Educational Resources Information Center

    Douthwaite, Shelaugh

    1983-01-01

    Secondary students utilize a simplified technique to make silk screen prints, which can be printed onto T-shirts. The only materials needed from art suppliers are a few squeegees and a few yards of polyester screen mesh. (RM)

  12. Cervical Cancer Screening

    MedlinePlus

    ... Cancer found early may be easier to treat. Cervical cancer screening is usually part of a woman's health ... may do more tests, such as a biopsy. Cervical cancer screening has risks. The results can sometimes be ...

  13. Oral Cancer Screening

    MedlinePlus

    ... Prevention Oral Cavity and Oropharyngeal Cancer Screening Research Oral Cavity and Oropharyngeal Cancer Screening (PDQ®)–Patient Version What ... These are called diagnostic tests . General Information About Oral Cavity and Oropharyngeal Cancer Key Points Oral cavity and ...

  14. Colon cancer screening

    MedlinePlus

    ... screening; Virtual colonoscopy - screening; Fecal immunochemical test; Stool DNA test; sDNA test ... called the fecal immunochemical test (FIT) and stool DNA test (sDNA). Sigmoidoscopy : This test uses a small ...

  15. Transfer functions

    NASA Technical Reports Server (NTRS)

    Taback, I.

    1979-01-01

    The vulnerability of electronic equipment to carbon fibers is studied. The effectiveness of interfaces, such as filters, doors, window screens, and cabinets, which affect the concentration, exposure, or deposition of carbon fibers on both (internal and external) sides of the interface is examined. The transfer function of multilayer aluminum mesh, wet and dry, polyurethane foam, and window screen are determined as a function of air velocity. FIlters installed in typical traffic control boxes and air conditioners are also considered.

  16. Pyrethroid activity-based probes for profiling cytochrome P450 activities associated with insecticide interactions

    PubMed Central

    Ismail, Hanafy M.; O’Neill, Paul M.; Hong, David W.; Finn, Robert D.; Henderson, Colin J.; Wright, Aaron T.; Cravatt, Benjamin F.; Hemingway, Janet; Paine, Mark J. I.

    2013-01-01

    Pyrethroid insecticides are used to control diseases spread by arthropods. We have developed a suite of pyrethroid mimetic activity-based probes (PyABPs) to selectively label and identify P450s associated with pyrethroid metabolism. The probes were screened against pyrethroid-metabolizing and nonmetabolizing mosquito P450s, as well as rodent microsomes, to measure labeling specificity, plus cytochrome P450 oxidoreductase and b5 knockout mouse livers to validate P450 activation and establish the role for b5 in probe activation. Using PyABPs, we were able to profile active enzymes in rat liver microsomes and identify pyrethroid-metabolizing enzymes in the target tissue. These included P450s as well as related detoxification enzymes, notably UDP-glucuronosyltransferases, suggesting a network of associated pyrethroid-metabolizing enzymes, or “pyrethrome.” Considering the central role P450s play in metabolizing insecticides, we anticipate that PyABPs will aid in the identification and profiling of P450s associated with insecticide pharmacology in a wide range of species, improving understanding of P450–insecticide interactions and aiding the development of unique tools for disease control. PMID:24248381

  17. Pyrethroid Activity-Based Probes for Profiling Cytochrome P450 Activities Associated with Insecticide Interactions

    SciTech Connect

    Ismail, Hanafy M.; O'Neill, Paul M.; Hong, David; Finn, Robert; Henderson, Colin; Wright, Aaron T.; Cravatt, Benjamin; Hemingway, Janet; Paine, Mark J.

    2014-01-18

    Pyrethroid insecticides are used to control a diverse spectrum of diseases spread by arthropods. We have developed a suite of pyrethroid mimetic activity based probes (PyABPs) to selectively label and identify P450s associated with pyrethroid metabolism. The probes were screened against pyrethroid metabolizing and non-metabolizing mosquito P450s, as well as rodent microsomes to measure labeling specificity, plus CPR and b5 knockout mouse livers to validate P450 activation and establish the role for b5 in probe activation. Using a deltamethrin mimetic PyABP we were able to profile active enzymes in rat liver microsomes and identify pyrethroid metabolizing enzymes in the target tissue. The most reactive enzyme was a P450, CYP2C11, which is known to metabolize deltamethrin. Furthermore, several other pyrethroid metabolizers were identified (CYPs 2C6, 3A4, 2C13 and 2D1) along with related detoxification enzymes, notably UDP-g’s 2B1 - 5, suggesting a network of associated pyrethroid metabolizing enzymes, or ‘pyrethrome’. Considering the central role that P450s play in metabolizing insecticides, we anticipate that PyABPs will aid the identification and profiling of P450s associated with insecticide pharmacology in a wide range of species, improving understanding of P450-insecticide interactions and aiding the development of new tools for disease control.

  18. Screening Tests for Women

    MedlinePlus

    ... questions you have. Make sure to ask about: Alcohol use Depression Weight Screening tests Screening tests Screening tests Ages ... for high cholesterol, and ways to improve cholesterol levels through lifestyle changes. ... Sheets - Alcohol Use and Health - This fact sheet talks about ...

  19. iScreen: Image-Based High-Content RNAi Screening Analysis Tools.

    PubMed

    Zhong, Rui; Dong, Xiaonan; Levine, Beth; Xie, Yang; Xiao, Guanghua

    2015-09-01

    High-throughput RNA interference (RNAi) screening has opened up a path to investigating functional genomics in a genome-wide pattern. However, such studies are often restricted to assays that have a single readout format. Recently, advanced image technologies have been coupled with high-throughput RNAi screening to develop high-content screening, in which one or more cell image(s), instead of a single readout, were generated from each well. This image-based high-content screening technology has led to genome-wide functional annotation in a wider spectrum of biological research studies, as well as in drug and target discovery, so that complex cellular phenotypes can be measured in a multiparametric format. Despite these advances, data analysis and visualization tools are still largely lacking for these types of experiments. Therefore, we developed iScreen (image-Based High-content RNAi Screening Analysis Tool), an R package for the statistical modeling and visualization of image-based high-content RNAi screening. Two case studies were used to demonstrate the capability and efficiency of the iScreen package. iScreen is available for download on CRAN (http://cran.cnr.berkeley.edu/web/packages/iScreen/index.html). The user manual is also available as a supplementary document. PMID:25548139

  20. Screening Hofmann Compounds as CO 2 Sorbents: Nontraditional Synthetic Route to Over 40 Different Pore-Functionalized and Flexible Pillared Cyanonickelates

    SciTech Connect

    Culp, Jeffrey T.; Madden, Catherine; Kauffman, Kristi; Shi, Fan; Matranga, Christopher

    2013-04-15

    A simple reaction scheme based on the heterogeneous intercalation of pillaring ligands (HIPLs) provides a convenient method for systematically tuning pore size, pore functionality, and network flexibility in an extended series of pillared cyanonickelates (PICNICs), commonly referred to as Hofmann compounds. The versatility of the approach is demonstrated through the preparation of over 40 different PICNICs containing pillar ligands ranging from 4 to 15 Å in length and modified with a wide range of functional groups, including fluoro, aldehyde, alkylamine, alkyl, aryl, trifluoromethyl, ester, nitro, ether, and nonmetalated 4,4'-bipyrimidine. The HIPL method involves reaction of a suspension of preformed polymeric sheets of powdered anhydrous nickel cyanide with an appropriate pillar ligand in refluxing organic solvent, resulting in the conversion of the planar [Ni{sub 2}(CN){sub 4}]{sub n} networks into polycrystalline three-dimensional porous frameworks containing the organic pillar ligand. Preliminary investigations indicate that the HIPL reaction is also amenable to forming Co(L)Ni(CN){sub 4}, Fe(L)Ni(CN){sub 4}, and Fe(L)Pd(CN){sub 4} networks. The materials show variable adsorption behavior for CO{sub 2} depending on the pillar length and pillar functionalization. Several compounds show structurally flexible behavior during the adsorption and desorption of CO{sub 2}. Interestingly, the newly discovered flexible compounds include two flexible Fe(L)Ni(CN){sub 4} derivatives that are structurally related to previously reported porous spin-crossover compounds. The preparations of 20 pillar ligands based on ring-functionalized 4,4'-dipyridyls, 1,4-bis(4-pyridyl)benzenes, and N-(4-pyridyl)isonicotinamides are also described.

  1. Screening Hofmann Compounds as CO{sub 2} Sorbents: Nontraditional Synthetic Route to Over 40 Different Pore-Functionalized and Flexible Pillared Cyanonickelates

    SciTech Connect

    Culp, Jeffrey T; Madden, Catherine; Kauffman, Kristi; Shi, Fan; Matranga, Christopher

    2013-03-29

    A simple reaction scheme based on the heterogeneous intercalation of pillaring ligands (HIPLs) provides a convenient method for systematically tuning pore size, pore functionality, and network flexibility in an extended series of pillared cyanonickelates (PICNICs), commonly referred to as Hofmann compounds. The versatility of the approach is demonstrated through the preparation of over 40 different PICNICs containing pillar ligands ranging from ∼4 to ∼15 Å in length and modified with a wide range of functional groups, including fluoro, aldehyde, alkylamine, alkyl, aryl, trifluoromethyl, ester, nitro, ether, and nonmetalated 4,4′-bipyrimidine. The HIPL method involves reaction of a suspension of preformed polymeric sheets of powdered anhydrous nickel cyanide with an appropriate pillar ligand in refluxing organic solvent, resulting in the conversion of the planar [Ni{sub 2}(CN){sub 4}]{sub n} networks into polycrystalline three-dimensional porous frameworks containing the organic pillar ligand. Preliminary investigations indicate that the HIPL reaction is also amenable to forming Co(L)Ni(CN){sub 4}, Fe(L)Ni(CN){sub 4}, and Fe(L)Pd(CN){sub 4} networks. The materials show variable adsorption behavior for CO{sub 2} depending on the pillar length and pillar functionalization. Several compounds show structurally flexible behavior during the adsorption and desorption of CO{sub 2}. Interestingly, the newly discovered flexible compounds include two flexible Fe(L)Ni(CN){sub 4} derivatives that are structurally related to previously reported porous spin-crossover compounds. The preparations of 20 pillar ligands based on ring-functionalized 4,4′-dipyridyls, 1,4-bis(4- pyridyl)benzenes, and N-(4-pyridyl)isonicotinamides are also described.

  2. Analyzing the Hidden Curriculum of Screen Media Advertising

    ERIC Educational Resources Information Center

    Mason, Lance E.

    2015-01-01

    This media literacy article introduces a questioning framework for analyzing screen media with students and provides an example analysis of two contemporary commercials. Investigating screen conventions can help students understand the persuasive functions of commercials, as well as how the unique sensory experience of screen viewing affects how…

  3. Cancer Screening: How Do Screening Tests Become Standard Tests?

    MedlinePlus

    ... cancer symptoms. There are different kinds of screening tests. Screening tests include the following: Physical exam and ... are linked to some types of cancer. Screening tests have risks. Not all screening tests are helpful ...

  4. Ab initio and density functional theoretical design and screening of model crown ether based ligand (host) for extraction of lithium metal ion (guest): effect of donor and electronic induction.

    PubMed

    Boda, Anil; Ali, Sk Musharaf; Rao, Hanmanth; Ghosh, Sandip K

    2012-08-01

    The structures, energetic and thermodynamic parameters of model crown ethers with different donor, cavity and electron donating/ withdrawing functional group have been determined with ab initio MP2 and density functional theory in gas and solvent phase. The calculated values of binding energy/ enthalpy for lithium ion complexation are marginally higher for hard donor based aza and oxa crown compared to soft donor based thia and phospha crown. The calculated values of binding enthalpy for lithium metal ion with 12C4 at MP2 level of theory is in good agreement with the available experimental result. The binding energy is altered due to the inductive effect imparted by the electron donating/ withdrawing group in crown ether, which is well correlated with the values of electron transfer. The role of entropy for extraction of hydrated lithium metal ion by different donor and functional group based ligand has been demonstrated. The HOMO-LUMO gap is decreased and dipole moment of the ligand is increased from gas phase to organic phase because of the dielectric constant of the solvent. The gas phase binding energy is reduced in solvent phase as the solvent molecules weaken the metal-ligand binding. The theoretical values of extraction energy for LiCl salt from aqueous solution in different organic solvent is validated by the experimental trend. The study presented here should contribute to the design of model host ligand and screening of solvent for metal ion recognition and thus can contribute in planning the experiments. PMID:22318713

  5. Screening for colorectal cancer.

    PubMed

    He, Jin; Efron, Jonathan E

    2011-01-01

    March is national colorectal cancer awareness month. It is estimated that as many as 60% of colorectal cancer deaths could be prevented if all men and women aged 50 years or older were screened routinely. In 2000, Katie Couric's televised colonoscopy led to a 20% increase in screening colonoscopies across America, a stunning rise called the "Katie Couric Effect". This event demonstrated how celebrity endorsement affects health behavior. Currently, discussion is ongoing about the optimal strategy for CRC screening, particularly the costs of screening colonoscopy. The current CRC screening guidelines are summarized in Table 2. Debates over the optimum CRC screening test continue in the face of evidence that 22 million Americans aged 50 to 75 years are not screened for CRC by any modality and 25,000 of those lives may have been saved if they had been screened for CRC. It is clear that improving screening rates and reducing disparities in underscreened communities and population subgroups could further reduce colorectal cancer morbidity and mortality. National Institutes of Health consensus identified the following priority areas to enhance the use and quality of colorectal cancer screening: Eliminate financial barriers to colorectal cancer screening and appropriate follow-up of positive results of colorectal cancer screening. Develop systems to ensure the high quality of colorectal cancer screening programs. Conduct studies to determine the comparative effectiveness of the various colorectal cancer screening methods in usual practice settings. Encouraging population adherence to screening tests and allowing patients to select the tests they prefer may do more good (as long as they choose something) than whatever procedure is chosen by the medical profession as the preferred test. PMID:21954677

  6. ENGAGE: Guided Activity-Based Gaming in Neurorehabilitation after Stroke: A Pilot Study

    PubMed Central

    Reinthal, Ann; Szirony, Kathy; Clark, Cindy; Swiers, Jeffrey; Kellicker, Michelle; Linder, Susan

    2012-01-01

    Introduction. Stroke is a leading cause of disability in healthy adults. The purpose of this pilot study was to assess the feasibility and outcomes of a novel video gaming repetitive practice paradigm, (ENGAGE) enhanced neurorehabilitation: guided activity-based gaming exercise. Methods. Sixteen individuals at least three months after stroke served as participants. All participants received concurrent outpatient therapy or took part in a stroke exercise class and completed at least 500 minutes of gaming. Primary baseline and posttest outcome measures included the Wolf motor function test (WMFT) and the Fugl-Meyer assessment (FMA). ENGAGE uses a game selection algorithm providing focused, graded activity-based repetitive practice that is highly individualized and directed. The Wilcoxon signed ranks test was used to determine statistical significance. Results. There were improvements in the WMFT (P = 0.003) and the FMA (P = 0.002) that exceeded established values of minimal clinically important difference. Conclusions. ENGAGE was feasible and an effective adjunct to concurrent therapy after stroke. PMID:22593835

  7. Suite of Activity-Based Probes for Cellulose-Degrading Enzymes

    SciTech Connect

    Chauvigne-Hines, Lacie M.; Anderson, Lindsey N.; Weaver, Holly M.; Brown, Joseph N.; Koech, Phillip K.; Nicora, Carrie D.; Hofstad, Beth A.; Smith, Richard D.; Wilkins, Michael J.; Callister, Stephen J.; Wright, Aaron T.

    2012-12-19

    Microbial glycoside hydrolases play a dominant role in the biochemical conversion of cellulosic biomass to high-value biofuels. Anaerobic cellulolytic bacteria are capable of producing multicomplex catalytic subunits containing cell-adherent cellulases, hemicellulases, xylanases, and other glycoside hydrolases to facilitate the degradation of highly recalcitrant cellulose and other related plant cell wall polysaccharides. Clostridium thermocellum is a cellulosome producing bacterium that couples rapid reproduction rates to highly efficient degradation of crystalline cellulose. Herein, we have developed and applied a suite of difluoromethylphenyl aglycone, N-halogenated glycosylamine, and 2-deoxy-2-fluoroglycoside activity-based protein profiling (ABPP) probes to the direct labeling of the C. thermocellum cellulosomal secretome. These activity-based probes (ABPs) were synthesized with alkynes to harness the utility and multimodal possibilities of click chemistry, and to increase enzyme active site inclusion for LC-MS analysis. We directly analyzed ABP-labeled and unlabeled global MS data, revealing ABP selectivity for glycoside hydrolase (GH) enzymes in addition to a large collection of integral cellulosome-containing proteins. By identifying reactivity and selectivity profiles for each ABP, we demonstrate our ability to widely profile the functional cellulose degrading machinery of the bacterium. Derivatization of the ABPs, including reactive groups, acetylation of the glycoside binding groups, and mono- and disaccharide binding groups, resulted in considerable variability in protein labeling. Our probe suite is applicable to aerobic and anaerobic cellulose degrading systems, and facilitates a greater understanding of the organismal role associated within biofuel development.

  8. A Suite of Activity-Based Probes for Cellulose Degrading Enzymes

    PubMed Central

    Chauvigné-Hines, Lacie M.; Anderson, Lindsey N.; Weaver, Holly M.; Brown, Joseph N.; Koech, Phillip K.; Nicora, Carrie D.; Hofstad, Beth A.; Smith, Richard D.; Wilkins, Michael J.; Callister, Stephen J.; Wright, Aaron T.

    2012-01-01

    Microbial glycoside hydrolases play a dominant role in the biochemical conversion of cellulosic biomass to high-value biofuels. Anaerobic cellulolytic bacteria are capable of producing multicomplex catalytic subunits containing cell-adherent cellulases, hemicellulases, xylanases, and other glycoside hydrolases to facilitate the degradation of highly recalcitrant cellulose and other related plant cell wall polysaccharides. Clostridium thermocellum is a cellulosome producing bacterium that couples rapid reproduction rates to highly efficient degradation of crystalline cellulose. Herein, we have developed and applied a suite of difluoromethylphenyl aglycone, N-halogenated glycosylamine, and 2-deoxy-2-fluoroglycoside activity-based protein profiling (ABPP) probes to the direct labeling of the C. thermocellum cellulosomal secretome. These activity-based probes (ABPs) were synthesized with alkynes to harness the utility and multimodal possibilities of click chemistry, and to increase enzyme active site inclusion for LC-MS analysis. We directly analyzed ABP-labeled and unlabeled global MS data, revealing ABP selectivity for glycoside hydrolase (GH) enzymes, in addition to a large collection of integral cellulosome-containing proteins. By identifying reactivity and selectivity profiles for each ABP, we demonstrate our ability to widely profile the functional cellulose degrading machinery of the bacterium. Derivatization of the ABPs, including reactive groups, acetylation of the glycoside binding groups, and mono- and disaccharide binding groups, resulted in considerable variability in protein labeling. Our probe suite is applicable to aerobic and anaerobic microbial cellulose degrading systems, and facilitates a greater understanding of the organismal role associated with biofuel development. PMID:23176123

  9. Activity-based kinase profiling of approved tyrosine kinase inhibitors.

    PubMed

    Kitagawa, Daisuke; Yokota, Koichi; Gouda, Masaki; Narumi, Yugo; Ohmoto, Hiroshi; Nishiwaki, Eiji; Akita, Kensaku; Kirii, Yasuyuki

    2013-02-01

    The specificities of nine approved tyrosine kinase inhibitors (imatinib, dasatinib, nilotinib, gefitinib, erlotinib, lapatinib, sorafenib, sunitinib, and pazopanib) were determined by activity-based kinase profiling using a large panel of human recombinant active kinases. This panel consisted of 79 tyrosine kinases, 199 serine/threonine kinases, three lipid kinases, and 29 disease-relevant mutant kinases. Many potential targets of each inhibitor were identified by kinase profiling at the K(m) for ATP. In addition, profiling at a physiological ATP concentration (1 mm) was carried out, and the IC(50) values of the inhibitors against each kinase were compared with the estimated plasma-free concentration (calculated from published pharmacokinetic parameters of plasma C(trough) and C(max) values). This analysis revealed that the approved kinase inhibitors were well optimized for their target kinases. This profiling also implicates activity at particular off-target kinases in drug side effects. Thus, large-scale kinase profiling at both K(m) and physiological ATP concentrations could be useful in characterizing the targets and off-targets of kinase inhibitors. PMID:23279183

  10. Activity-Based Costing in the After Press Services Industry

    NASA Astrophysics Data System (ADS)

    Shevasuthisilp, Suntichai; Punsathitwong, Kosum

    2009-10-01

    This research was conducted to apply activity-based costing (ABC) in an after press service company in Chiang Mai province, Thailand. The company produces all of its products by one-stop service (such as coating, stitching, binding, die cutting, and gluing). All products are made to order, and have different sizes and patterns. A strategy of low price is used to compete in the marketplace. After cost analysis, the study found that the company has high overhead (36.5% of total cost). The company's problem is its use of traditional cost accounting, which has low accuracy in assigning overhead costs. If management uses this information when pricing customer orders, losses may occur because real production costs may be higher than the selling price. Therefore, the application of ABC in cost analysis can help executives receive accurate cost information; establish a sound pricing strategy; and improve the manufacturing process by determining work activities which have excessively high production costs. According to this research, 6 out of 56 items had a production cost higher than the selling price, leading to losses of 123,923 baht per year. Methods used to solve this problem were: reducing production costs; establishing suitable prices; and creating a sales promotion with lower prices for customers whose orders include processes involving unused capacity. These actions will increase overall sales of the company, and allow more efficient use of its machinery.

  11. Interfacial activation-based molecular bioimprinting of lipolytic enzymes.

    PubMed Central

    Mingarro, I; Abad, C; Braco, L

    1995-01-01

    Interfacial activation-based molecular (bio)-imprinting (IAMI) has been developed to rationally improve the performance of lipolytic enzymes in nonaqueous environments. The strategy combinedly exploits (i) the known dramatic enhancement of the protein conformational rigidity in a water-restricted milieu and (ii) the reported conformational changes associated with the activation of these enzymes at lipid-water interfaces, which basically involves an increased substrate accessibility to the active site and/or an induction of a more competent catalytic machinery. Six model enzymes have been assayed in several model reactions in nonaqueous media. The results, rationalized in light of the present biochemical and structural knowledge, show that the IAMI approach represents a straightforward, versatile method to generate manageable, activated (kinetically trapped) forms of lipolytic enzymes, providing under optimal conditions nonaqueous rate enhancements of up to two orders of magnitude. It is also shown that imprintability of lipolytic enzymes depends not only on the nature of the enzyme but also on the "quality" of the interface used as the template. PMID:7724558

  12. Activity-based costing for electric utilities. Final report

    SciTech Connect

    Croyle, D.R.; Schapiro, I.A.; Keglevic, P.M.

    1992-08-01

    This EPRI report is a ``primer`` on Activity-Based Costing (ABC). ABC is a cost management aproach which can make an important contribution to understanding and controlling the changing costs in the electric utility industry. It is a method for attributing costs to activities, products and services by better understanding the underlying factors which drive those costs. ABC can help utility managers make better decisions through the application of more accurate process and product cost information and a fuller understanding of which activities add value and which do not. Armed with such information, utility managers are better equipped to address many of the strategic and operating decisions which they routinely face. The report introduces the ABC concept and approach to utility managers and offers insights into how ABC can be and is being used to control costs and improve strategic and operating decisions in electric utilities and other industries. The report (1) describes the ABC approach, (2) discusses the value of ABC to elecuic utilities, (3) identifies potential applications of ABC to current utility issues, (4) describes a step-by-step approach to developing and implementing ABC in the utility environment, and (5) presents a survey of more than 30 electric utilities and several detailed case studies of electric utilities and other companies who have adopted and are using ABC.

  13. Applying activity-based costing to the nuclear medicine unit.

    PubMed

    Suthummanon, Sakesun; Omachonu, Vincent K; Akcin, Mehmet

    2005-08-01

    Previous studies have shown the feasibility of using activity-based costing (ABC) in hospital environments. However, many of these studies discuss the general applications of ABC in health-care organizations. This research explores the potential application of ABC to the nuclear medicine unit (NMU) at a teaching hospital. The finding indicates that the current cost averages 236.11 US dollars for all procedures, which is quite different from the costs computed by using ABC. The difference is most significant with positron emission tomography scan, 463 US dollars (an increase of 96%), as well as bone scan and thyroid scan, 114 US dollars (a decrease of 52%). The result of ABC analysis demonstrates that the operational time (machine time and direct labour time) and the cost of drugs have the most influence on cost per procedure. Clearly, to reduce the cost per procedure for the NMU, the reduction in operational time and cost of drugs should be analysed. The result also indicates that ABC can be used to improve resource allocation and management. It can be an important aid in making management decisions, particularly for improving pricing practices by making costing more accurate. It also facilitates the identification of underutilized resources and related costs, leading to cost reduction. The ABC system will also help hospitals control costs, improve the quality and efficiency of the care they provide, and manage their resources better. PMID:16102243

  14. 49 CFR 1544.411 - Continuing qualifications of screening personnel.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ...) TRANSPORTATION SECURITY ADMINISTRATION, DEPARTMENT OF HOMELAND SECURITY CIVIL AVIATION SECURITY AIRCRAFT OPERATOR... screening function; (2) Has a satisfactory record of performance and attention to duty based on...

  15. 49 CFR 1544.411 - Continuing qualifications of screening personnel.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ...) TRANSPORTATION SECURITY ADMINISTRATION, DEPARTMENT OF HOMELAND SECURITY CIVIL AVIATION SECURITY AIRCRAFT OPERATOR... screening function; (2) Has a satisfactory record of performance and attention to duty based on...

  16. 49 CFR 1544.411 - Continuing qualifications of screening personnel.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ...) TRANSPORTATION SECURITY ADMINISTRATION, DEPARTMENT OF HOMELAND SECURITY CIVIL AVIATION SECURITY AIRCRAFT OPERATOR... screening function; (2) Has a satisfactory record of performance and attention to duty based on...

  17. Federal Renewable Energy Screening Assistant

    SciTech Connect

    Shelpuk, B; Walker, A

    1994-10-01

    The Federal Renewable Energy Screening Assistant is a software tool to be used by energy auditors to prioritize future studies of potentially cost-effective renewable energy applications at federal facilities. This paper describes the structure and function of the tool, gives an inventory of renewable energy technologies represented in the tool, and briefly describes the algorithms used to rank opportunities by the savings-to-investment ratio.

  18. Ghrelin treatment prevents development of activity based anorexia in mice.

    PubMed

    Legrand, Romain; Lucas, Nicolas; Breton, Jonathan; Azhar, Saïda; do Rego, Jean-Claude; Déchelotte, Pierre; Coëffier, Moïse; Fetissov, Sergueï O

    2016-06-01

    Stimulation of feeding is necessary for treatment of pathological conditions of chronic malnutrition due to anorexia. Ghrelin, a hunger hormone, is one of the candidate for pharmacological treatments of anorexia, but because of its instability in plasma has limited efficacy. We previously showed that plasmatic IgG protect ghrelin from degradation and that IgG from obese subjects and mice may increase ghrelin׳s orexigenic effect. In this study we tested if ghrelin alone or combined with IgG may improve feeding in chronically food-restricted mice with or without physical activity-based anorexia (ABA) induced by free access to a running wheel. Mice received a single daily intraperitoneal injection of ghrelin (1nM) together or not with total IgG (1nM) from obese ob/ob or lean mice before access to food during 8 days of 3h/day feeding time. We found that both ghrelin and ghrelin combined with IgG from obese, but not lean mice, prevented ABA, however, they were not able to diminish body weight loss. Physical activity was lower during the feeding period and was increased shortly after feeding in mice receiving ghrelin together with IgG from obese mice. In food-restricted mice without ABA, ghrelin treatments did not have significant effects on food intake. Thus, this study supports pharmacological use of ghrelin or ghrelin combined with IgG from obese animals for treatment of anorexia accompanied by elevated physical activity. The utility of combining ghrelin with protective IgG should be further determined in animal models of anorexia with unrestricted access to food. PMID:27052473

  19. Lung Cancer Screening Update.

    PubMed

    Ruchalski, Kathleen L; Brown, Kathleen

    2016-07-01

    Since the release of the US Preventive Services Task Force and Centers for Medicare and Medicaid Services recommendations for lung cancer screening, low-dose chest computed tomography screening has moved from the research arena to clinical practice. Lung cancer screening programs must reach beyond image acquisition and interpretation and engage in a multidisciplinary effort of clinical shared decision-making, standardization of imaging and nodule management, smoking cessation, and patient follow-up. Standardization of radiologic reports and nodule management will systematize patient care, provide quality assurance, further reduce harm, and contain health care costs. Although the National Lung Screening Trial results and eligibility criteria of a heavy smoking history are the foundation for the standard guidelines for low-dose chest computed tomography screening in the United States, currently only 27% of patients diagnosed with lung cancer would meet US lung cancer screening recommendations. Current and future efforts must be directed to better delineate those patients who would most benefit from screening and to ensure that the benefits of screening reach all socioeconomic strata and racial and ethnic minorities. Further optimization of lung cancer screening program design and patient eligibility will assure that lung cancer screening benefits will outweigh the potential risks to our patients. PMID:27306387

  20. Cervical cancer screening.

    PubMed Central

    Katz, A.

    1998-01-01

    OBJECTIVE: To review the role of family physicians in screening for cancer of the cervix, to review the evidence for screening, in particular, frequency and technique for screening, and to review the reasons cervical cancer has not been prevented and the role of family physicians in addressing these failures. QUALITY OF EVIDENCE: The value of screening has been established with level II evidence. Many of the unresolved issues are not supported either way by good evidence; level II and III evidence predominates. MAIN FINDINGS: In Canada, 1350 women were predicted to be diagnosed with cancer of the cervix in 1996. Most of these women had not been screened. Minority, rural, low-income, and older women face important barriers to screening. Family physicians have a role in reaching out to these women to provide effective health care, including cancer screening. When cancer screening is performed, it should conform to recommended techniques with appropriate follow up of abnormal test results. CONCLUSIONS: Family physicians have an important role in preventing cancer of the cervix. Efforts should be concentrated on encouraging a greater proportion of eligible women to be screened. Criteria are suggested for effective screening. PMID:9721422